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Abstract

Currently, there has been an increasing socioeconomic impact of zoonotic pathogens transmitted from animals to humans worldwide. Recently, in the Arabian Peninsula, including in Saudi Arabia, epidemiological data indicated an actual increase in the number of emerging and/or reemerging cases of several viral zoonotic diseases. Data presented in this review are very relevant because Saudi Arabia is considered the largest country in the Peninsula. We believe that zoonotic pathogens in Saudi Arabia remain an important public health problem; however, more than 10 million Muslim pilgrims from around 184 Islamic countries arrive yearly at Makkah for the Hajj season and/or for the Umrah. Therefore, for health reasons, several countries recommend vaccinations for various zoonotic diseases among preventive protocols that should be complied with before traveling to Saudi Arabia. However, there is a shortage of epidemiological data focusing on the emerging and reemerging of zoonotic pathogens transmitted from animal to humans in different densely populated cities and/or localities in Saudi Arabia. Therefore, further efforts might be needed to control the increasing impacts of zoonotic viral disease. Also, there is a need for a high collaboration to enhance the detection and determination of the prevalence, diagnosis, control, and prevention as well as intervention and reduction in outbreaks of these diseases in Saudi Arabia, particularly those from other countries. Persons in the health field including physicians and veterinarians, pet owners, pet store owners, exporters, border guards, and people involved in businesses related to animal products have adopted various preventive strategies. Some of these measures might pave the way to highly successful prevention and control results on the different transmission routes of these viral zoonotic diseases from or to Saudi Arabia. Moreover, the prevention of these viral pathogens depends on socioeconomic impacts, available data, improved diagnosis, and highly effective therapeutics or prophylaxis.

Introduction

Rudolf Virchow , one of the foremost 19th century German leaders in medicine and pathology [1] , noted a relationship between human diseases and animals and then introduced the term "zoonosis" (plural: zoonoses) in 1880 [2] . Later, the World Health Organization (WHO) in 1959 specified that "zoonoses are those diseases and infections which are naturally transmitted between vertebrate animals and man" [3] . Venkatesan and co-authors reported that the term zoonosis is derived from the Greek word "zoon" = animal and "noso" = disease [4] . Zoonotic pathogens causing different kinds of diseases are of major public health issues worldwide [5] . These zoonotic diseases include

Viral Zoonoses

Frequent mixing of different animal species in the markets in densely populated areas, and the human intrusions into the natural habitats of animals, have facilitated the emergence of novel viruses. The most important zoonotic viral diseases of which eight were diagnosed (in dead or diseased animals or through antibody detection) on the Arabian Peninsula over the last years include rabies, Middle East Respiratory Syndrome (MERS-CoV), influenza virus (IFV), Alkhurma hemorrhagic fever, Crimean-Congo hemorrhagic fever (CCHF), Rift Valley fever (RVF), West Nile fever (WNV), and dengue fever virus. Among these eight zoonotic viral diseases, two (Alkhurma and MERS-CoV) were first reported in a patient in 1994 and 2012, respectively in Saudi Arabia [33, 34] . These two were transmitted later to several other countries, not only in the Middle East but also to Africa, Asia, and Europe.

Rabies

Rabies is an almost invariably fatal zoonotic disease, which belongs to the genus Lyssavirus of the RNA family Rhabdoviridae. Rabies virus is considered an endemic viral infectious disease in animals in Saudi Arabia. Recent scientific data on rabies cases reported in camels at Al-Qassim region (one of the thirteen administrative regions of Saudi Arabia) showed that there is an increasing number of this fatal virus disease [35] . However, the most significant animal bites which have been recorded in Saudi Arabia were caused by different species of animals including dogs, cats, rodents, and foxes [36] . Later, Al-Dubaib reported rabies in dromedaries in Saudi Arabia in 2007 and suggested an incidence of about 0.2% for rabies that was reported among 48 camel herdsmen looking after more than 4000 animals [35] .

Middle East Respiratory Syndrome

The MERS-CoV infection is considered to be a new respiratory disease with a dire global concern [46] . MERS-CoV infections are caused by a newly emerging coronavirus (CoV), belonging to the designated lineage C of Betacoronavirus of the RNA family Coronaviridae. With respect to viral origin and transmission, bats are thought to be the reservoir host of Betacoronaviruses, and the African Neoromicia bats in particular are the natural reservoir of MERS-CoV [47, 48] .
Since its emergence in 2012 in Saudi Arabia, when an elderly patient (60 years old) with respiratory illness died after admission to a hospital in Jeddah [34] , the disease was subsequently reported to have been transmitted to several countries worldwide, and has affected more than 1000 patients with over 35% fatality [46, [49] [50] [51] .

Influenza

Influenza viruses are considered to be important infectious viral diseases, which is caused by three virus types (A, B, and C) [68] . Due to their zoonotic spread, influenza type A infects both humans and animals, and causes moderate to severe illness, with more likelihood of fatalities in young children and the elderly [69, 70] . Other types of influenza, including type B and C, infect only humans [71] . Furthermore, influenza A viruses, members of the RNA family Orthomyxoviridae, are further classified into human, swine, and avian influenza viruses. However, during the 1918 influenza pandemic, swine influenza virus infected one-third of the world's population (an estimated 500 million people) and caused approximately 50 million deaths [72] .
It is well-known that the influenza drugs, antiviral agents, and the current seasonal influenza vaccines are effective in reducing the incidence and severity of the disease, sickness, and/or complications. However, the important strategy for influenza management includes the provision of prophylaxis and treatment [78] . However, it is possible for widespread drug resistance against antiviral agents or vaccines to emerge in patients who extensively abused the drugs, in addition to those who have never received such treatment, globally [82] [83] [84] . Furthermore, influenza viruses pose a challenge to vaccine developers and manufacturers due to the fact that these viruses are continually changing in nature, including hemagglutinin and neuraminidase [78, 85] . Moreover, while resistance to neuraminidase inhibitors (e.g., oseltamivir and zanamivir) have been reported to sporadically occur, the resistance to oseltamivir has been widely reported since 2007, with a worldwide spread [86] . This highlights why there is an urgent need for the public health system to monitor continuously via globally active influenza surveillance programs. Furthermore, there is need to monitor the circulating influenza viruses strains, as well as the occurrence of any resistance, using appropriate diagnostic methods. This is considered highly essential in Saudi Arabia. Interestingly, survey data has shown an increasing report of the viral infection from Egypt, since Hajj Egyptians has ranked in the top 10 list of countries with the highest number of Mecca pilgrims in the last 10 years [81] . Influenza is highly susceptible to antiviral drugs such as oseltamivir, according to a more recent epidemiological study [87] . Although millions of Muslims, globally, travel annually to Saudi Arabia to perform Hajj and/or Umrah in the holy places including both Makkah and Al-Madinah for very limited period (~10 days), this gathering could play a major role in the introduction of new influenza viruses, not only to Saudi Arabia but also to the rest of the world [83, 87] . Unfortunately, there is no such influenza surveillance program in Saudi Arabia, thus this pose a serious public health concern.

Alkhurma Hemorrhagic Fever Virus

In humans, this zoonotic disease may present with clinical features ranging from subclinical or asymptomatic features to severe complications. It is related to Kyasanur Forest disease virus, which is localized in Karnataka, India [106, 107] . However, epidemiologic findings suggest another wider geographic location for the disease in western (including Jeddah and Makkah) and southern (Najran) parts of Saudi Arabia, and the virus infections mostly occur in humans [96, 101, 102] . A study was conducted by Alzahrani et al. in the southern part of Saudi Arabia particularly in the city of Najran (with populations of~250,000), an agricultural city in Saudi Arabia, where domestic animals are reared at the backyard of owners. After the initial virus identification, from January 2006 through April 2009, 28 persons with positive serologic test results were identified. Infections were suspected if a patient had an acute febrile illness for at least two days; when all other causes of fever have been ruled out [101] . Additionally, data analysis indicated that patients infected with the virus were either in contact with their domestic animals, involved in slaughtering of the animals, handling of meat products, drinking of unpasteurized milk, and/or were bitten by ticks or mosquitoes. Symptoms consistent with AHFV infection-including fever, bleeding, rash, urine, color change of the feces, gum bleeding, or neurologic signs-then develop [95] . Fortunately, infected patients responded to supportive care (including intravenous fluid administration and antimicrobial drugs when indicated), with no fatal cases.
In summary, AHFV is a zoonotic disease with clinical features ranging from subclinical or asymptomatic features to severe complications. Another study highlighted different characteristics of the exposure to the blood or tissue of infected animals in the transmission of AHFV to humans. Of the 233 patients confirmed with infections, 42% were butchers, shepherds, and abattoir workers, or were involved in the livestock industry [108] . More recently, a study on infection using C57BL/6J mice cells showed that the clinical symptoms of the disease were similar to the presentations in humans [109] . However, Alkhurma disease resulted in meningoencephalitis and death in Wistar rats, when high titers to the infection occurred [98] . In addition, exposures to mosquito bites are regarded as potential sources of transmissions of the infection; however, very few available data support this [97] . Although, available data shows that Alkhurma virus has been isolated following mosquito bites [102] . However, another study suggested that mosquitoes may play a role only as a vector in the transmission of the disease [100].

CCHF

CCHF is a zoonotic viral disease from the Bunyaviridae family, and the principal vector for the disease is ticks of the genus Hyalomma. It is most commonly endemic in Africa, Middle East, Asia, and Eastern Europe [110, 111] . It is an acute, highly-contagious, and life-threatening vector-borne disease responsible for severe hemorrhagic fever during outbreaks, and a fatality rate of up to 40% [112, 113] . The infectious disease was recognized first in the Crimean Peninsula in 1944, and it was named Crimean hemorrhagic fever virus because the virus was isolated for the first time from a febrile child in 1956 from Stanleyville (now Kisangani), Democratic Republic of Congo [114] .

RVF

RVF is a common arbovirus zoonotic disease caused by the RVF virus. The virus belongs to the genus Phlebovirus and family Bunyaviridae. It is most common in domestic animals, and causes mild to life-threatening infections in humans. The name of the disease was derived from the Great Rift Valley of Kenya, when the disease was described for the first time in 1912 [125] . Epidemiological tests have since been described after a highly fatal epizootic occurred there in 1930 [126] .
Saudi Arabia has many of the world's mosquito vectors of parasitic and arboviral diseases. However, few studies have addressed their geographic distribution and larval habitat characteristics [128] . There are complex interactions between these factors that significantly impact mosquitoes ecological fitness and vectorial capacity for disease transmission, with important implications for vector management and control at the local and regional levels [129, 130] . Therefore, studying these factors for different mosquito fauna will help in monitoring potential modifications of larval habitats due to rains, global climate change, or man-made activities. Previous studies on the ecology, distribution, and abundance of mosquito species in Kingdom of Saudi Arabia are generally few and sporadic; and most of these studies were conducted in the western and southern regions. These studies were conducted in the Asir Province in 1993-1995 and 1999-2001 [131,132] [142, 143] . These studies reported the presence of many species from many genera, the most important of which are Anopheles, Aedes, and Culex. Among these studies, only a few provided the description of habitats of the larvae of these vectors. Even fewer studies provided evidence on the active role of some species on disease transmission; the existing ones were mainly for Anopheles vectors of malaria [138, 144, 145] , as well as Aedes and Culex vectors of arboviruses such as Sindbis and dengue fever [141, 146, 147] .
Surprisingly, in 2000, Jup et al. found the mosquito species that was identified as a potential vector, which led to the assumption that the zoonotic viral disease in Saudi Arabia was transmitted by Culex tritaeniorhynchus [161] . Other species of mosquitoes were implicated in the transmission of this viral disease in other countries closer to Saudi Arabia [162] [163] [164] . Furthermore, another study reported the unexplained RVF virus infection among people from Saudi Arabia, with isolation and genetic virus characterization associated with illness in livestock, along the southwestern border of Saudi Arabia in September 2000 [164] . The study reported that vertical transmission of the virus in the epidemic mosquito vector occurred in Saudi Arabia. In addition, the study stated that the most abundant culicine mosquitoes collected were Aedes vexans arabiensis, Culex pipiens complex, and Culex tritaeniorhynchus, which were considered to be the most important epidemic and epizootic vectors of RVF virus in Saudi Arabia [164, 165] . However, the same study, focusing on a very important issue which occurred during the rainy seasons; suggested that Aedes vexans arabiensis has the potential to be an important epidemic and epizootic vector because of the tremendous numbers of individual mosquitoes produced after a flood [164] .

Dengue Hemorrhagic Fever

Dengue hemorrhagic fever (DHF) viral disease is a serious global mosquito-borne infection. The clinical manifestation ranges from mild febrile illness to severe sickness which may include dengue shock syndrome [171] . The DHF virus belongs to the genus Flavivirus in the Flaviviridae family, which can usually be spread by mosquitoes of the genus Aedes aegypti, but less often through the genus Aedes albopictus [172, 173] . Also, this virus is a single-stranded positive-sense RNA virus that exists as four different serotypes (DEN-1, DEN-2, DEN-3, and DEN-4) [174] .
In Saudi Arabia, the disease is limited to the western and southwestern regions, such as Jeddah and Makkah where Aedes aegypti exists. However, all DHF cases in Saudi Arabia presented as a mild disease [171, 175] . In fact, the first experience of DHF virus isolation from Saudi Arabia was recorded during an outbreak of the virus in 1994 [176] , where the 289 confirmed cases reported in Jeddah were caused by DENV-2 [176] [177] [178] . However, during this first outbreak, in both summer and rainy season, at the end of the year, both DENV-2 and DENV-1 were isolated. In 1997, during the rainy season in Jeddah, there was an emergence of the DENV-3 virus [179] . In subsequent years, from 1997-2004; the emergence of DHF occurred with the three identified serotypes (DENV-1, DENV-2, and DENV-3) isolated in Jeddah [171] . Khan [171, 181] . However, Egger suggested that the reemergence of the disease in Saudi Arabia might be explained by the growing levels of urbanization, international trade, and travels [182] .

West Nile Fever

WNV is known to cause neurological disease in both humans and horses. However, the clinical manifestations of the disease in horses include ataxia, paralysis of the limbs, recumbency, hyperexcitability, and hyperesthesia. In Al-Ahsa, Saudi Arabia, a study was performed on 63 horses to test the incidence of the virus using the clinical examination and serologic ELISA test. However, from this previous study, while clinical examination for neurologic signs detected no significant findings, WNV antibodies were positively identified at serology among 33.3% of the tested population [202] .

Prevention and Control

Currently, in this review, some aspects of the most common viral diseases of zoonotic importance in Saudi Arabia were summarized; these are presented in Table 1 . However, data regarding emerging and reemerging zoonotic viral diseases are reported as they occur from time to time from the same, new, and/or different localities from Saudi Arabia. While other viral zoonotic infections occur in other countries, which are considered to be close to Saudi Arabia, some infections spread to some localities within Saudi Arabia because of the geographical proximity as shown in Figure 1 . Interestingly, some of these zoonotic viral pathogens were first exotic to Saudi Arabia (e.g., MERS-CoV and AHFV) and should be of more concern when reported in prevalence studies, and whenever they are detected by Saudi authorities. Epidemiological data should be focused more on both the trade routes and wildlife migration across the region, since these are potential risks for Saudi Arabia (e.g., from Yemen, Egypt, Gulf areas, and Sudan). Fortunately, there are many ways and/or approaches to improve the control of such different zoonotic pathogens in animals and humans in Saudi Arabia. However, the control measures of these viral zoonotic pathogens will not only benefit Saudi Arabia or Arabian Peninsula but will also be of high benefit to other countries, especially those with low prevalence, by stopping or controlling the spread of the epidemic worldwide. Prevention, control, and management of several zoonotic diseases usually require several important measures including the following. Having vaccination protocols for all suspected animal species by the use of up to date vaccines and compliance with the standards needed for all animals. Taking into account the highly needed and important investigation for these zoonotic viral diseases vectors, including vector breeding control (including vectors, hosts, and arthropods), and control of the animals (livestock) movements, with respect to trade and export [212, 213] . Because an intensive livestock trade exists between Saudi Arabia and its neighboring countries, there may be increased risk of reemerging viral diseases of all kinds [214, 215] . This is supported by several previous studies concerned with the route of livestock trade between Saudi Arabia and the neighboring countries (e.g., rabies through Yemen and/or Oman [36, 216] Interestingly, some of these zoonotic viral pathogens were first exotic to Saudi Arabia (e.g., MERS-CoV and AHFV) and should be of more concern when reported in prevalence studies, and whenever they are detected by Saudi authorities. Epidemiological data should be focused more on both the trade routes and wildlife migration across the region, since these are potential risks for Saudi Arabia (e.g., from Yemen, Egypt, Gulf areas, and Sudan). Fortunately, there are many ways and/or approaches to improve the control of such different zoonotic pathogens in animals and humans in Saudi Arabia. However, the control measures of these viral zoonotic pathogens will not only benefit Saudi Arabia or Arabian Peninsula but will also be of high benefit to other countries, especially those with low prevalence, by stopping or controlling the spread of the epidemic worldwide. Prevention, control, and management of several zoonotic diseases usually require several important measures including the following. Having vaccination protocols for all suspected animal species by the use of up to date vaccines and compliance with the standards needed for all animals. Taking into account the highly needed and important investigation for these zoonotic viral diseases vectors, including vector breeding control (including vectors, hosts, and arthropods), and control of the animals (livestock) movements, with respect to trade and export [212, 213] . Because an intensive livestock trade exists between Saudi Arabia and its neighboring countries, there may be increased risk of reemerging viral diseases of all kinds [214, 215] . This is supported by several previous studies concerned with the route of livestock trade between Saudi Arabia and the neighboring countries (e.g., rabies through Yemen and/or Oman [36, 216] ; RVF through Kenya, Djibouti, and/or Egypt [127, 149, 212] ; CCHF through Sudan [121] ; influenza through Oman and Egypt [71, 87, 121, [217] [218] [219] ; WNV through Emirates, Egypt, Jordan, and Israel [196, 197, 199, 203, 208] ; and DHFV through Egypt [190] ; as well as MERS-CoV and AHFV viral infections, which originated and are transmitted globally from Saudi Arabia) [34, 52, 53, 97, 98] .
Therefore, it is clear that a huge gap still exists in the sharing of published data about the acknowledged epidemiology of zoonotic diseases in Saudi Arabia, which rigorously prohibits speculations about the health burden of people. Currently, there are surveillance activities for some viral diseases-such as rabies, MERS-CoV, and influenza-but these are still being weakly addressed or neglected, especially at the human-animal interface. The important role of vaccination both in the prevention and control of animal diseases and the need to check the human sources in food or water must not be neglected. Also, management of animals, both outdoors and indoors must be taken seriously. However, owners of pets clinics and pets stores should be held responsible in ensuring that they keep their pets' vaccination protocols up to date, and prevent any kind of animal behavior that might result in zoonotic risks to humans through bites or scratches by pets. Therefore, pet clinics and/or pets stores should be always considered a serious public health issue and vaccination should be obligatory. Therefore, the importance of the annual vaccination routine programs for all stray dogs against rabies, and regular investigation of other animals, should be considered.
By enhancing biosecurity and management in animal farms, the risk of reemerging pathogens particularly responsible for zoonotic diseases caused by viruses, can be reduced. This is a matter of economic importance; in view of the large livestock trade existing or that existed between countries in the Indian Ocean and Eastern Africa countries where several zoonotic diseases are endemic. However, a phylogenetic study strongly suggests that some zoonotic infections have been introduced into Saudi Arabia through ruminant trade [212, 213] . Furthermore, following the adoption of the recommended guidelines of the World Organization for Animal Health through its Office International des Epizooties (OIE) Code, if such policies regarding the exportation and/or importation of animals are exactly followed, these would greatly limit the extent of this risk [214] .
Furthermore, an emphasis should be made on surveillance to detect any sign of zoonotic disease that might occur in any animal kept directly in a quarantine station in any country of origin for 30 days prior to shipment to another country to ensure no clinical sign develops during that period. In addition, the longer quarantine periods or restriction of imported animals-particularly pets (e.g., dogs, cats, rodents, and monkeys) or goats, sheep, and camels-from endemic countries may be effective in reducing the introduction of zoonotic viruses. Of such measures, the control of vectors (e.g., ticks and mosquitoes), particularly the intermediate hosts and animal reservoirs, should be key components in the intervention strategy for zoonoses in Saudi Arabia. While the improving, enhancing, providing, and upgrading of laboratory techniques and/or testing in both veterinary and human medicines are fundamental to early detection and containing of any zoonotic disease or transmitted infection.
Central to the profound worldwide changes in religious beliefs and activities is the birth of a new era of both emerging and reemerging diseases that could be arranged under the umbrella of social movements, along with its own role in the spread of zoonotic diseases. Thus, any prevention and/or control strategies against any zoonotic pathogen have to take this point of view into account. Furthermore, annually, Saudi Arabia hosts the largest international gathering of Hajj where many millions gather in a small geographical area. This puts Saudi Arabia in the front line of threats of pandemic diseases [215] . Thus, Saudi Arabia must keep a high level of alertness in monitoring the situation of these pathogens, particularly in view of the potential for global spread of pandemic viruses especially during winter and around the Hajj season (e.g., MERS-CoV infections, AHFV, and influenza viruses). Therefore, there is need to prevent further spread of the virus locally, regionally, and internationally. Interestingly, with WNV outbreaks, the Israeli-like WNV that was isolated in white storks in Egypt in 1997-2000 suggests that migrating birds do play a crucial role in the geographical spread of the virus [225] . Recently, the same fact was again suggested in 2017, when the same infection by this virus was introduced into Turkey at the time of the outbreaks in Saudi Arabia and Yemen; it was stated that the WNV virus might have been introduced via unlawful entry of the viremic domestic or wild animals through the borders, or by vectors carrying the virus to Turkey [209] .

Therefore, the following recommendations are suggested in order to improve public awareness and/or health education of zoonotic viral diseases in Saudi Arabia:
Based on findings of previous studies, health education strategies could enhance the awareness of the Saudi population regarding viral zoonotic diseases through health education program experiences of other countries, particularly during Hajj and Umrah seasons. This response can draw on the availability of several studies on how to improve, control, and prevent the spread of several zoonoses in both animals and humans, worldwide [78, [96] [97] [98] [99] .
Enhancing of self-awareness among people through health education programs or other strategies for the prevention of viral zoonotic diseases, which require vectors (such as mosquitoes, ticks, and fleas) for their transmission; are important issues on which the Saudi population should be educated. They should also be educated about the adverse effects of arbitrary application of insecticides without prior knowledge on dose, resistance, and side effects. Increasing the knowledge about the biology and ecology of the animal vectors in society is also crucial. Furthermore, the Saudi Ministry of Culture and Information should establish intensive health education programs on television channels, radio, and newspapers to increase public awareness and to maintain hygiene conditions within the Kingdom and in Saudi houses. The Saudi Ministry of Agriculture could play a big role by regularly controlling the application of vaccinations and/or antibiotics on animals which used in the veterinary sector, and also accounting the misuse of such agents following other developed and developing countries on controlling and/or accounting drug strategies [87, 228, 229] . Thus, veterinary regulations of animal antibiotics-including overuse of drugs and their application-must be enforced to alleviate the serious public health problems.

Rabies

Interestingly, another survey was conducted between 1997 and 2006 in the Al-Qassim region of central Saudi Arabia among 4124 camels and showed that about 0.2% of clinical rabies incidence is caused by dogs (may be cause it highly used as a perfect guard for camels), followed by foxes; furthermore, the diagnosis of viral rabies in that region was confirmed among 26 dogs, 10 foxes, 8 camels, and 7 cats [35] . Lately, the relevant government authorities (the MOH and Ministry of Agriculture in Saudi Arabia) in an updated report between 2007 and 2009 showed that there were a total of 11,069 animal bites to humans in Saudi Arabia [36] . Furthermore, most cases of animal bites were caused by dogs (49.5%) and cats (26.6%), followed by mice and rats, camels, foxes, monkeys, and wolves [36] . Moreover, dogs, particularly feral dogs and foxes, are considered the most important host for rabies virus; however, bats are also considered as reservoirs of this disease. Humans can become rabid by direct contact with animal mucosal surfaces via bites.
In September 2016, a 60-year-old Saudi man, presented with different clinical features-such as nausea, vomiting, and epigastric pain, with significant features suggestive of gastritis-at Makkah hospital. His past medical history was significant for hypertension and diabetes type 2. During the clinical diagnostic procedure of this case, he developed respiratory distress and tachycardia, for which he was transferred to the intensive care unit [41] . Because, his case worsened with chest pain and ventricular tachycardia he was referred to the King Abdullah Medical City in Makkah for further management. The written diagnostic report indicated that he had acute anteroseptal myocardial infarction, had coronary angiogram which suggested that two-vessels were diseased with left main involvement, and surgical intervention was planned. After the decision for surgery, he was found to have leukocytosis and severe retching while attempting to drink water (hydrophobic behavior), which necessitated further review by the infectious disease consultants based on the patient's clinical symptoms. The consultant team discovered the history of an unprovoked scratch on the patient's face by a dog in Morocco a month prior to the admission at the hospital. Also, the patient stated that he only received tetanus vaccine. All diagnostic tests including neurologic examination were unremarkable and his saliva polymerase chain reaction (PCR) test confirmed rabies virus. He was administered Verorab rabies vaccine and human hyperimmune rabies immunoglobulin (20 IU/kg) intramuscularly (IM) [41] . In addition, he had troponin I (4.65 ng/mL), creatine kinase isoenzyme MB (CKMB) was found (30.08 ng/mL), and serum glucose (200 mg/dL). On the fifth day of hospital, he had recurrent episodes of ventricular tachycardia, progressively worsening of hemodynamic parameters, and he succumbed to his infection on that day. There is no vaccine against rabies recommended for travelers from/to Saudi Arabia, and no rabies treatment is offered to pet dogs. However, vaccination is given to dogs before they are infected; otherwise they are euthanized if infected.
According to a previous study, most patient injuries from animal bites in Saudi Arabia showed some variations due to the monthly incidence and/or, according to the animal species [36] . Bites by dogs and cats were reported frequently throughout the year, with a decrease in April and between August and October. However, bites by foxes increase between August and September while camel bites were more frequent between December and March of the subsequent year. The same previous study suggest that these seasonal variations of injuries might be due to the Saudi population habits, with people going to the desert for leisure activities during good weather periods. Laboratory diagnosis of rabies viral disease occur with the use of the rabies virus direct fluorescent antibody test (DFAT) on brain samples and hippocampal tissue [44] .

Middle East Respiratory Syndrome

Moreover, a 60-year-old Saudi man was admitted to a private hospital in Jeddah, Saudi Arabia in June 2012 with a history of fever, severe acute respiratory syndrome with cough, expectoration, and shortness of breath. He did not smoke; and for the disease, which was suggested to be due to an animal transmission of coronaviruses, he was treated with oseltamivir, levofloxacin, and piperacillin-tazobactam. On day 11, he died [34] . After this, a 61-year-old Saudi male with hypertension and diabetes with no history of smoking, reported for surgery. At the time of admission, he was asymptomatic. He was initially screened using nasopharyngeal swab, endotracheal aspirate, and serum sample for MERS-CoV per protocol with the MERS RRT-PCR assay. The results confirmed MERS-CoV infection. He died three days after admission. It was discovered that the patient owned a dromedary camel barn in Saudi Arabia, and had a history of close contact with camels, as well as a habit of raw milk consumption of an unknown duration [51] .
Two studies have suggested a relationship between the infection and contact with dromedary camels [52, 53] . In addition to this, serological diagnostic methods have been used to confirm MERS-CoV infections in dromedary camels for at least 2-3 decades and has thus confirmed camels as an intermediate host for this virus [54, 55] . Thus, in 2012, a novel coronavirus (MERS-CoV) was isolated from two fatal human cases in Saudi Arabia and Qatar; and since then, more than 1400 clinical cases of MERS-CoV have been identified, and the great majority of the cases were from Saudi Arabia [56] . This previous report author raised a thoughtful comment related to the emerging viral diseases "Why We Need to Worry about Bats, Camels, and Airplanes" [56] . Moreover, another study suggested that MERS-CoV infection is usually transmitted from human's direct contact with dromedary camels, especially when people drink the milk or use camel's urine for medicinal purposes [57] . More recently, a metagenomics sequencing analysis of nasopharyngeal swab samples from 108 MERS-CoV-positive live dromedary camels marketed in Abu Dhabi, United Arab Emirates, showed at least two recently identified camel coronaviruses, which were detected in 92.6% of the camels in that study [58] . However, limited human-to-human infections have been reported.
Furthermore, this study also showed that the incidence of MERS-CoV infections was highest among elderly people aged ≥60 years [34, 59] ; with speculation that there might be certain conditions or factors involved. It is considered that MERS-CoV infection might have a peculiar gender predisposition [60] . Recent data examined the mortality in patients with MERS-CoV and the gender relationships, looking at the survival of cases among females and males. It was suggested that males have a higher risk of death [61, 62] ; however, this was contradicted by the findings from two other studies which suggested that males have a low risk of death [63] ; while another survey which examined the influence of gender on 3-day and 30-day survival, found a low risk of death especially in the older age group [64] . On the other hand, Badawi et al., suggested that MERS-CoV infections could be mild and may only result in death among patients suffering from any kind of immune system disorder and/or any chronic disease [46] .

Influenza

Since 2006, several infections with this virus have been recorded from various areas worldwide, including Saudi Arabia [73] . At the end of April 2009, an outbreak of a new type of influenza, A/H1N1, started in Mexico and the USA [69] . The WHO declared the pandemic influenza A (H1N1) as a "public health emergency of international concern" following the first few initial cases in Mexico, and subsequently in the USA [69, 74] . In Saudi Arabia, the epidemiological data for influenza virus were collected using a predesigned questionnaire with the first 114 confirmed pandemics influenza A (H1N1) cases identified by the Infectious Diseases Department from the MOH, and the database during the period covered from 1 June to 3 July 2009 [72] . However, according to the Saudi MOH data, the number of laboratory-confirmed cases of the virus in Saudi Arabia as at 30 December 2009 was 15,850, with 124 deaths [72] . The virus later spread worldwide, causing a pandemic, and the most recorded cases then, as reported by the WHO in the Middle East, were in Saudi Arabia with 14,500 cases, followed by Kuwait [69] , Egypt, and Oman; with less number of infected patients [68, 75] . Nevertheless, between 1979 and 1980, a serosurveillance outcome of swine influenza virus from Egypt provided evidence of laboratory diagnosis and very early confirmation of the virus in human patients [76] . In Saudi Arabia, the influenza surveillance system has been established since 2004. Moreover, among people with certain chronic medical diseases or conditions, a trivalent influenza vaccine (TIV), which contains inactivated antigens for two different subtypes of influenza viruses (types A and B), became available in Saudi Arabia [72] .
Indeed, H1N1 is now in the post-pandemic period and has become a seasonal influenza virus that continues to circulate with localized outbreaks of varying magnitude in Saudi Arabia [77] . A previous data was collected using a predesigned questionnaire for the first 100 cases of pandemic influenza A (H1N1) from different hospitals in Saudi Arabia. The age of patients enlisted in the data ranged from 1 to 56 years. The age groups with the highest percentage of cases were between: 20 and 30 years (35%), and 1 and 10 years (22%). There were 45 males and 55 females, and 53% patients had some contacts with infected persons within Saudi Arabia while about 47% had history of travels into Saudi Arabia and/or the Philippines [72] . These facts are similar to the previous relationship noted between the occurrence of zoonotic viral diseases and the gender of patients and/or their ages, as reported for another viral (e.g., MERS-CoV) infection; provided certain conditions are met [60] [61] [62] [63] . Interestingly, among elderly patients, influenza cases were higher in females than males. This relationship with viral infection occurring particularly with respiratory viral diseases, might pave the way and play a big role of more significant importance in the detection of these diseases, taking into account the influence of climate change and the different environmental factors [69, 77, 78] .
In contrast, several epidemic zoonotic cases of influenza H5N1 have been reported in domestic cats in several countries in Asia, Europe, the USA, and Italy [87] . Moreover, the epidemic of influenza in dogs might be related to a serious public health issue and could be shown to have resulted from zoonotic diseases from pets, similar to the avian influenza H3N2 outbreak reported in pet dogs in South Korea in 2007 [92] . Nevertheless, a recent study has shown that the role of pets, particularly cats and dogs in the epidemic of influenza as a source of human infection seems limited. However, cats were shown to be fully susceptible to experimental infection, and infected cats were able to infect naive cats [87] . In 2009, pandemic H1N1 infection in a domestic cat in the USA from Iowa was diagnosed by a novel PCR assay; thus, human-to-cat transmission was presumed [93] . Despite this prior evidence, the role of pets including cats and dogs seem even more limited in the dispersal of avian influenza to humans. Rather, humans may be the source of pet infection, as suggested for influenza H1N1 and/or H3N2 virus infections [87, [92] [93] [94] .
Most importantly, epidemic zoonotic cases of influenza among pets has highlighted the importance of circulating influenza viruses globally; especially, to ensure the effective use of antivirals for the prophylaxis and treatment of influenza, in particular, with the increase in the number of pets stores in Saudi Arabia, especially in Riyadh [78, 90] . Surprisingly, previous data focused on the occurrence of zoonotic infection of different influenza virus types, and particularly, the transmission of avian influenza virus H3N2 to domestic dogs [92] . Several studies have examined and confirmed the occurrence of zoonotic infection of the influenza A virus H1N1 pandemic, especially in domestic cats [93, 94] . Nevertheless, epidemiological studies on different zoonotic infections among the pets in Saudi Arabia including cats, dogs, and/or baboons are very rare. However, a previous case report confirmed a relationship between some zoonotic diseases causing respiratory symptoms, such as influenza, among pets [95] . This study suggests that severe lung infection with dry cough and severe anemia should lead to the suspicion of a secondary infection with zoonotic balantidiasis, which infected a hamadryas baboon from Saudi Arabia in a research center for pets in Riyadh [95] . Furthermore, two other epidemiological zoonotic study on Balantidium coli protozoan zoonotic infection in camel was reported from Riyadh [31] .

Alkhurma Hemorrhagic Fever Virus

Alkhurma hemorrhagic fever virus (AHFV) in humans was discovered in 1994 [33] . The first case reported in a butcher from the city of Alkhurma, a district south of Jeddah in Saudi Arabia, died of hemorrhagic fever after slaughtering a sheep. The viral infection has a reported fatality rate of up to 25% [96] . Interestingly, one of the previous reports regarding this disease showed a misunderstanding of the real name of this infection, called Alkhurma, not Alkhumra [97, 98] . Because subsequent cases were diagnosed in patients from the small town known as Alkhurma in Jeddah from where the virus got its scientific name; the name was accepted by the International Committee on Taxonomy of Viruses [99] . Thus, based on evidence, the first case was confirmed to be the butcher, following the slaughtered sheep [100] . Therefore, a study was conducted among affected patients to address this disease as a public health issue. Blood samples were collected from household contacts of patients with laboratory-confirmed virus for follow-up testing by enzyme-linked immunosorbent serologic assay (ELISA) for AHFV-specific immunoglobulin (Ig) G. Samples from persons seeking medical care were tested by ELISA for AHFV-specific IgM and IgG using AHFV antigen. Viral-specific sequence was performed by reverse transcription PCR (TiBMolbiol, LightMix kit; Roche Applied Science, Basel, Switzerland). A total of 11 cases were identified through persons seeking medical care, whose illnesses met the case definition for AHFV, and another 17 cases were identified through follow-up testing of household contacts [100] .
Subsequently, the virus was isolated from six other butchers of different ages (between 24 and 39 years) from the city of Jeddah, with two deaths. The diagnosis was established from their blood sample tests. The serological tests later confirmed four other patients with the disease [101] . From 2001 to 2003, the study on the virus initial identification in the city of Alkhurma again identified 37 other suspected cases; with laboratory confirmation of the disease in 20 (~55%) of them. Among the 20, 11 (55%) had hemorrhagic manifestations and 5 (25%) died [102] . The virus was later identified in three other locations: from the Western Province of Saudi Arabia (Ornithodoros savignyi and Hyalomma dromedarii were found by reverse transcription in ticks) and from samples collected from camels in Najran [103, 104] . AHFV virus was considered as one of the zoonotic diseases; however, the mode of transmission is not yet clear. Recently, it was suggested that the disease reservoir hosts may include both camels and sheep. The virus might also be transmitted as a result of skin wounds contaminated with the blood or body fluids of an infected sheep; through the bite of an infected tick, and through drinking of unpasteurized or contaminated milk from camels [101, 105] .

CCHF

In 1990, the CCHF virus (CCHFV) caused an outbreak involving seven individuals in Makkah, although the virus had not been reported previously in Saudi Arabia. Therefore, a study on the epidemiology of this virus was carried out in Makkah, Jeddah, and Taif from 1991-1993. About 10 out of 13 different species of ticks that were capable of transmitting the disease were collected from camels, cattle, sheep, and goats, but camels had the highest rate of tick infestation (97%), and H. dromedarii was the commonest tick (70%). An investigation in Makkah between 1989 and 1990, which included a serological survey of abattoir workers in contact with sheep blood or tissue, identified 40 human cases of confirmed or suspected CCHF with 12 fatalities [120] . The report from the investigation stated that the virus might have been introduced to Saudi Arabia through the Jeddah seaport via infected ticks on imported sheep; since then, it has been endemic in the Western Province of Saudi [120, 121] . In addition, another previous study confirmed that the highest seropositivity rate of the virus in Saudi Arabia localities was associated with animals imported from Sudan [121] .
Furthermore, the WHO reported 22 countries with CCHF including Saudi Arabia; however, all the remaining countries are either close to Saudi Arabia or are Islamic countries with high numbers of Muslims who travel annually to Saudi Arabia for Hajj pilgrimage. The same WHO epidemiological data suggest that in these 22 countries including Saudi Arabia, in recent years, there has been report of steadily increasing number of sporadic human cases, incidence, and outbreaks of the virus [122] . Furthermore, another study by WHO investigating CCHFV in the Eastern Mediterranean Region (EMR) stated that CCHF is a clear and growing health threat in the WHO EMR. Cases are being reported in new areas, showing a geographical extension of the disease that is probably linked to the livestock trade and the spread of infected ticks by migratory birds. According to ecological models, the increase in temperature and decreased rainfall in the WHO EMR could have resulted in the sharp increase in distribution of suitable habitats for Hyalomma ticks and the subsequent drive of CCHFV infection northwards [123] .

RVF

RVF is not considered a major type in the arboviruses family, which mostly are adapted to a narrow range of vectors; however, among this family, the RVF infection has a very wide range of vector including mosquitoes such as Aedes and Culex, flies, and often, ticks [148] . Interestingly, for different RVF species, RVF vectors have special roles about how they sustain the transmission of the disease ecologically to humans [149] . In some cases, the impact of rainfall, soil type, water, the persistence of breeding, and often wind, have significant effect on vector distribution [150] . Epizootics studies indicate that RVF disease follows unusually severe rainy seasons, a situation that may likely favor the breeding of a very large insect population, needed as a vector prerequisite.
Globally, RVF epidemiology was first reported in Africa with the 1989 RVF epizootics in Kenya when laboratory test reports confirmed virus isolation [151] [152] [153] . In 2000, the disease, for the first time, affected humans and livestock outside Africa, with the larger RVF disease incidence following outbreaks, reported in Saudi Arabia [154] and Yemen. Lately, RVF infections have been associated with minimal genetic diversity, epidemiologically; which has lately been considered to be a newly introduced single lineage of RVF viral disease [155] . Epidemiological reports from both Saudi Arabia and Yemen showed that the outbreak, which occurred in 2000, resulted in about 2171 human infections, and 245 deaths [156] . Furthermore, the fatality rate reported in southern Saudi Arabia then, reached 14%, and was considered the most severe epidemic in that area ever since [157] . Moreover, the disease outbreak was thought to have been transmitted in countries such as Saudi Arabia by infected imported ruminants from East Africa via the port of Djibouti and probably from Kenya and/or Sudan [121] . However, the fact remains that the RVF epidemic has been around for more than 70 years, with infections occurring at prolonged intervals in Eastern and Southern Africa [158, 159] . Consistent with this, another report showed that the same virus strain was implicated in the 1997-1998 RVF outbreaks in Kenya and the 2000 outbreaks in Saudi Arabia and Yemen [130] . The outbreaks in Kenya later resulted in about 89,000 human infected with about 478 patients deaths [127, 160] .
Characteristically, once the virus is introduced into permissive ecologies, it becomes zoonotic; thus, they are able to enhance vulnerability of the area to periodic outbreaks, with the potential to spread further into non-endemic environments with favorable conditions [166, 167] . Saudi Arabia is considered a region where RVF virus has circulated actively. Noticeable data regarding zoonotic infection from animal to human from the Arabian Peninsula including Saudi Arabia has recently showed that it may be due to the consumption of unpasteurized camel milk [32, 159, 168] . Wernery reported Camelus dromedarius as the animal host and/or reservoir of RVF zoonotic infection, which was diagnosed in the Arabian Peninsula [23] . Due to the scientific data regarding RVF disease, it is quite clear that globalization of trade and altered weather patterns are a concern for the future spread of more infections, since the causative agent of this viral disease is capable of utilizing a wide range of vectors for its transmission. Thus, this poses a significant challenge to outbreak prediction, with inherently complex methods of infection control; therefore, mitigation and management of the virus will require concerted efforts [121, 169, 170] .

Dengue Hemorrhagic Fever

From Makkah City, the reported epidemiological study identified 63.4% of DHF infection cases among Saudi nationals [188] . Similarly, a later study puts the estimate at more than 70% of Saudi nationals [189] . These previously published studies suggest that differences in proportions may exist between Saudi nationals infected with DHF virus in Jeddah and Makkah City. Contrary to previous data from Jeddah, in Makkah, it was clear that the majority of patients presenting with clinically significant DHF were Saudi nationals. Therefore, these results emphasized the fact that Saudi nationals are at greater risk of DHF infection. The awareness of these results is considered a cornerstone to enhancing the ability of healthcare professionals' identification of the disease; and this might play an important role in the development of effective eradication strategies for the disease in Saudi Arabia localities.
Furthermore, the first cases of the virus, confirmed in Al-Madinah in 2008, showed that the isolated virus serotypes were DENV-1 and DENV-2 [190] . In 2009, the MOH in Saudi Arabia reported a total of 3350 cases of the DHF infection, with an estimated case fatality rate of about 4.6 per thousand in Saudi Arabia [171] . In August 2017, several countries in Asia, including Malaysia, Singapore, and Pakistan reported about 60,000, 1877, and 738 dengue cases including deaths, respectively. In the same period (2017), Saudi Arabia reported 39 confirmed dengue cases in Makkah, 19 of which occurred in August 2017, 60 suspected cases, and 15 cases pending laboratory confirmations. From these epidemic data indicating the reemergence of DHF infection in Saudi Arabia; Jeddah, Makkah, and Al-Madinah were shown to be the more susceptible areas, for this infectious disease, and this could be due to the fact that these cities are the sites of both the annual Hajj pilgrimage and/or the minor Umrah pilgrimage, which draw millions of Muslims to Saudi Arabia [171, 190] .

West Nile Fever

West Nile fever is one of the emerging zoonotic infections, which is caused by an arthropod-borne virus belonging to the genus Flavivirus, of the RNA family Flaviviridae. The virus' main reservoir, which is responsible for the transmission of the disease, is the genus Culex mosquitoes [193, 194] . The West Nile virus (WNV) derived the name from the site where the first case was isolated in 1937, from the blood of a woman with mild febrile illness living in the West Nile District of Uganda [195] . The first outbreak, in 1951-1952, was reported in Israel [196] . This constituted a turning point in the epidemiology of the virus, because it was thought to have originated from Israel following the introduction from Africa, and later introduced to the USA in 1999 [197, 198] . Subsequently, the infection was documented across the globe [199] , with the exception of Antarctica [194] , in various species of vertebrates, including humans, mammals, non-human primates, birds, rodents, reptiles, and amphibians [200] . However, birds are considered as one of the main reservoirs of the virus [201] . Saudi Arabia is geographically close to several of the countries where WNV had circulated actively or had been reported; thus, there is a high risk of the disease being introduced into Saudi Arabia.
Camels play an important role in public health issues regarding zoonosis and they have been involved in most of the zoonotic infections which occurred in Saudi Arabia in the last three decades. They are reported as sources of infections-including rabies, MERS-CoV, Alkhurma virus, CCHFV, and RVF virus [52, 94, 101, 108, 120, 124, 210] -via direct physical contacts with camels and/or indirectly by having camels within or near the household in Saudi Arabia. However, some zoonotic infections among camels are sometimes asymptomatic; thus, they play a vital role in the mechanism of transmission of various diseases [211] . Furthermore, Wernery et al. reported that WNV can be transmitted by mosquito bites in different species including to humans, horses, camelids, and many other mammalian species as well as reptiles and birds [159, 200, 201] . To the best of our knowledge, there is still no extensive surveillance data regarding this disease among wildlife animals in Saudi Arabia. Strikingly, several of the human zoonotic cases that involve camels-which included different viral, bacterial, and parasitic infections on the Arabian Peninsula-have recently been highlighted as being caused by the consumption of unpasteurized camel milk [168] .

Prevention and Control

In addition to this, pet clinics and stores should monitor pets' health records, and their owners should be held fully responsible in ensuring that their animals remain healthy and fully vaccinated. This will guarantee for them and their neighbors a zoonotic disease-free environment (e.g., against rabies virus particularly in dogs). This is particularly important in view of the case of human rabies reported in March 2018 from a Makkah hospital. This involved a 60-year-old Saudi man who was admitted to the hospital with a history of an unprovoked scratch on his face by a dog. A month after his admission, his saliva PCR test confirmed rabies virus [41] . Nevertheless, rabies is endemic in animals in the Arabian Peninsula, with increasing numbers of reported cases form certain countries in the area including Saudi Arabia, Yemen, and Oman [36, 41] . Kuwait, Qatar, and the United Arab Emirates are considered to be rabies-free, whereas there is no available information about Bahrain [216, 220] . Furthermore, animal rabies cycle and cases reported in these endemic countries including Saudi Arabia are characterized by different animal species such as camels, cattle, goats, and sheep; however, the majority of cases are reported in feral dogs [36, 216] .
Indeed, epidemiologic evidence should be linked with the seasonal time during the year for different zoonoses, and/or with any symptoms related to zoonotic infections that occur on the mainland a few years earlier. Up to date ecological factors on evolutionary issues, social movements, economic, and epidemiological mechanisms affecting zoonotic pathogens' or their persistence and emergence, are not yet well understood. However, studies on the ecological, socioeconomic, and health issues are needed to assess the sustainability and acceptability of measures by breeders, as well as information that ensures appropriate slaughtering or consumption practices, which will decrease the risk of infection to humans [200] . Due to these facts about the ecological cascade and evolutionary perspectives, authorities can provide valuable insights into pathogen ecology and can inform zoonotic disease control programs; and thus evaluate their global effect in terms of actual disease and its socioeconomic correlations.
On the other hand, few studies have been done in this area to identify the relationship between different gatherings and the occurrence of signs and clinical symptoms of viral infections, especially among humans of different ages and gender. However, there are several suggestions and information regarding zoonoses (e.g., influenza and MERS-CoV infections) in Saudi Arabia among the elderly, based on age and gender [63, 222] . More recently, increased availability of limited public health data on the prevalence of some zoonotic diseases and associated risk factors or data that identifies the relationship between different zoonotic pathogen antibodies in pregnant women, are of importance [223, 224] .
Lastly, increased zoonotic pathogens surveillance, particularly influenza, during the Hajj season, increased infection control interventions, screening, and quarantine of suspected cases, provision of adequate medical treatment, sustainable awareness, increased education and training of target groups at high risk (e.g., doctors, nurses, veterinarians, and animal workers such as farmers and abattoir workers, etc.) are of great importance to reduce the burden of zoonoses among Saudi Arabian localities. Fortunately, in collaboration with three organizations-including the MOH in Saudi Arabia, the USA Centers for Disease Control and prevention, and the WHO-a successful preparedness plan during the Hajj season was put in place to vaccinate all pilgrims before leaving their home countries [68] . Altogether, there is an urgent need for collaborative surveillance and intervention plans for the control of zoonotic pathogens in Saudi Arabia.

A zoonotic pathogen outbreak could be dramatically decreased among the annual Saudi pilgrims if we take into account the fact that: Jeddah Governorate, the main seaport in Saudi Arabia is considered to be the main entry point for over 2 million pilgrims coming for Hajj or Umrah annually. All these numbers of pilgrims arrive through the Jeddah Islamic Port before going on to Makkah, for the start of their Umrah and/or Hajj. Surprisingly, the current review showed that during an outbreak, each of these eight most zoonotic viruses (rabies, MERS-CoV, influenza, AHFV, CCHFV, RVFV, DHFV, and WNV) which occurred and/or cases confirmed in Saudi Arabia particularly from (Jeddah and/or Makkah) areas with at least one or all of these eight zoonotic viral pathogenic diseases [33, 44, 46, 78, [96] [97] [98] [99] 121, 130, 156, 171] .
The spread could also have been due to the fact that Jeddah is the main port for animal importation to Saudi Arabia. At the same time, it is the closest area to several countries where some zoonotic outbreaks were reported. To enhance this spread, the role of the active circulation of zoonotic viruses, during their natural transmission cycle, has been reported, however, an importation might increase risk of disease introduction to Saudi Arabia. • Almost annually, from the more than 7 million pilgrims who come to Makkah and Madinah from different countries worldwide during Hajj and Umrah, the Kingdom's revenue in 2012 was put at more than 62 billion Saudi Riyals (~about 16.5 billion US Dollars), 10% up from the 2011 figures. This Hajj revenue accounted for 3% of the gross domestic product for the Kingdom of Saudi Arabia. To avert all that number of health hazards from zoonotic diseases in view of economic facts, the global community and particularly the pilgrims need more gift items made in Saudi Arabia to control and prevent the spread of zoonotic diseases which could be transmitted among Hajj and Umrah pilgrims.
Public health authorities must highlight the importance of promoting health education and facilitate the outcomes of studies for reducing patient cases in Saudi Arabia. The Saudi authorities and government bodies such as the MOH should also launch different programs and workshops to increase public awareness about these zoonotic infections. This should involve the cooperation of the Saudi regime, and the private and public sectors. Different activities may be needed in Saudi Arabia-such as the practice of self-protection against these diseases, adult control strategies, control activities, and regular workshops-to achieve control and prevention.

Rabies

While rabies is considered nearly 100% fatal, it is also 100% preventable, and thus vaccination to pets is the key element to prevent the risk of rabies zoonotic infection [45] . Reports of the epidemiology of rabies virus worldwide, and particularly in Saudi Arabia, suggest that it is on the increase, thus the implication of this virus' potential to spread across borders from high to low prevalence countries was highlighted [23] .

Middle East Respiratory Syndrome

The prevalence of MERS-CoV infections worldwide still remains unclear. In addition to this, the WHO reported about 1797 cases of these infections since June 2012, with about 687 deaths in 27 different countries, worldwide. Recently, a study was conducted from June 2012 to July 2016, during which samples were collected from MERS-CoV infected individuals, from the National Guard Hospital in Riyadh (the Saudi Arabian capital city), the MOH in Saudi Arabia, and other Gulf Corporation Council countries, to determine the prevalence of MERS-CoV [59] . The epidemiologic data that were collected, showed that the highest number of cases (about 1441 of 1797 patients) were reported from Saudi Arabia (~93%). Among the 1441 MERS-CoV cases from Saudi Arabia, Riyadh was the worst-hit area with 756 infected cases (52.4%), followed by the western region of Makkah where 298 cases (20.6%) were reported [59] .

Influenza

In addition, another previous data confirmed the occurrence of Toxocara canis zoonotic infection based on respiratory symptoms reported at the pet clinics in Saudi Arabia (and also in Riyadh where the symptoms occurred in dogs) [30] . Still, more such studies are needed to highlight the important issues and/or provide clearer pictures of the zoonotic pathogens among pets in Saudi Arabia; however, pet ownership has been growing rapidly as well as the number of pet stores among the Saudi population.

Dengue Hemorrhagic Fever

In keeping with the findings of most previous studies, the epidemiological occurrence of DHF infection using the Saudi's national data indicated that the majority (68%) of patients with dengue virus infection were Saudi nationals [183] . On the contrary, from the epidemiological report based on Saudi's national data in previous publications, an estimated 15% of patients with DHF presented in Jeddah [184] . Kholedi [186] . In yet another recent study, the virus was reported as 38% in Saudi patients [187] . All of these Saudi studies were conducted in Jeddah.
Currently, there are few epidemiological studies on DHF virus infection in Saudi Arabia. A study by Al-Azraqi et al. was conducted in 30 hospitals and 387 primary healthcare centers in two cities in the southern province of Saudi Arabia, particularly in Jizan, and Aseer. The study, which was limited to the seroprevalence among clinically suspected hospital-based patients, detected about 31.7% positive cases of dengue virus IgG among 965 randomly selected patients attending the outpatient clinics for any reason. The associated risk factors were male gender, younger age (15-29 years) , lack of electricity, and having water basins in the house [191] . The authors suggested that the virus may occur in sporadic cases in Jizan, due to the nature of the city. Jizan is relatively flat and located at sea level [191] ; thus the likelihood of the formation of small stagnant water following the rainfall in the city is high [171] .

West Nile Fever

In 1999, Lanciotti et al. found this virus to be responsible for an outbreak of encephalitis in two fatal human cases from northeastern USA in late summer; and suggest a closely relation between this outbreak in the USA to a WNV infection which was isolated from a dead goose in Israel in 1998 [197] . The first cases of WNV in horses was identified in Egypt and France in the 1960s [203] ; ever since, WNV has had significant public health impact worldwide due to its resurgence and dynamic epidemiologic features in humans and animals. Between 2008 and 2009, a study in Iran identified WNV antibodies in horses, and the results confirmed the highest activity of the virus reported in the Western and Southern Provinces with seroprevalences of up to 88% in some areas of Iran [204] . Although human cases and/or animal infections with WNV including horses have also been reported in Jordan and Lebanon (direct and close neighbors of Saudi Arabia) between 2000 and 2014 [205] [206] [207] ; however, the reported WNV in patients or horses in these areas might have circulated in natural transmission cycles with close relationship to the WNV isolated from human and horses in Jordan, Lebanon, and Iran in 2000, 2010, and 2014, respectively. Humans and horses (incidental hosts), are unable to develop sufficient viremia to infect mosquitoes, hence, they are not included in the WNV lifecycle [203] .

Prevention and Control

Fortunately, studies about pets with different zoonotic infections from pet clinics and/or pet stores in Saudi Arabia have been rarely detected among cats, dogs, and baboons. However, there was a previous study, which reported the occurrence of Toxocara canis infection in pets (dogs) in Riyadh, Saudi Arabia [30] . There were also two previous reports regarding a protozoan zoonotic infection of some pets with clinical manifestation, particularly in Papio hamadryas baboon in Riyadh [95, 221] . In addition to this, another report highlighted the protozoan zoonotic infection in camels, in Riyadh [31] ; however, more of these kind of studies are needed, because, they provide important opportunities to present a clear picture about indoor and outdoor animals and zoonotic pathogens such as viral, bacterial, fungal, etc. which involved, in Saudi Arabia.
Enhancing biosecurity and management in the treatments of various zoonotic infections may result in appropriate use of vaccinations, drugs, and antibiotics, however, the overuse of these agents result in various types of resistance. Furthermore, regardless of the influenza virus resistance level to treatment, according to a serosurveillance, the enzootic influenza virus H5N1 in Egypt is endemic [87] . The same result to oseltamivir-resistant influenza viruses are reported globally, with a high susceptibility to these antiviral drugs among all reported cases of the virus from Egypt. Resistance was also found in most infected viral cases that are usually acquired in humans through intensive contacts, particularly with backyard birds, among women and children [82, 83, 86, 87] . Therefore, drug regimens in Saudi must include vaccines against this virus during Hajj and Umrah seasons, for Egyptians.

Middle East Respiratory Syndrome

More recently, data regarding the mortality in patients with MERS-CoV have been published. According to Saudi Arabia's MOH daily statements, dated from February 26 through March 3, laboratory-confirmed new cases of MERS-CoV and 2 deaths occurred [65] . Recently, on February 26, patients infected while hospitalized at Riyadh included two men (23 and 59 years old) in stable condition, who were not healthcare workers. According to a February 27 update, a new case involved a 71-year-old man from the city of Buraydah who later died. Meanwhile, on March 1, another MERS-CoV infection in a Riyadh hospital patient, a 64-year-old man who was listed in critical condition and who likewise had contact with camels, as the other two patients, was reported. Thus, the MOH stated that the spillover from camels is thought to be the main source of MERS-CoV in Saudi Arabia, since all these patients were exposed to the animals before reporting ill [65] .
Furthermore, an 83-year-old patient from Riyadh, and other two patients who had camel contacts from Hail city in the north central part of Saudi Arabia were listed in critical condition. The illness in these patients was reported on March 1. According to a March 3 statement, another patient, a 74-year-old man from Najran located in southern Saudi Arabia, was reported. The man was listed in a stable condition. Of these new cases, only one death, involving the 83-year-old man from Riyadh, according to the March 3 MOH statement, was reported [65] . Still, much work is needed to detect the MERS-CoV infection risk in Saudi Arabia, because data showed increasing number of cases exist among the eight countries including Saudi Arabia. Thus, the emergence of MERS-CoV in the region and its continuing transmission from 2012-2017, currently poses one of the biggest threats to global health security [66] . Most cases (over 85%) reported to date have been from countries in the region (e.g., Egypt) notably from Saudi Arabia, with 1527 cases including 624 deaths [67] .

Influenza

Nevertheless, between September 2013 and October 2014, about 406 samples were taken from several patients presenting with respiratory symptoms to King Abdulaziz University Hospital, Jeddah, Saudi Arabia. However, during this study conducted to detect the susceptibility to the influenza viruses circulating in the western part of Saudi Arabia, out of all the tested samples, 25 (6.2%) respiratory samples were positive for influenza H1N1 virus, 1 (0.25%) was positive for influenza H3N2 virus, and 7 (1.7%) were positive for influenza B virus [78] . Furthermore, H1N1, now in the post-pandemic period, has become a seasonal influenza virus that continues to circulate with localized outbreaks of varying magnitude [71] . Interestingly, the presentation of influenza virus infections in humans usually vary from mild, self-limiting respiratory-like illness, to severe cases that may result in death [70, 79] . Nevertheless, a recent study has shown that subclinical infection in human exists, as revealed by the serological surveillance [76, 80] . Therefore, the epidemiological surveillance of influenza in Saudi Arabia is highly important especially with the fact that influenza cases have also been highly reported can spread globally [78] . Thus, geographic influences on influenza virus infection in Saudi Arabia must be of concern [78] . This is relevant because a remarkably high number of Egyptian Muslims visit Saudi Arabia yearly to participate in the Umrah and/or the Hajj pilgrimage; in addition, the impact of the poultry industry in Egypt is also worth considering, with an estimated 1 billion birds and several millions of engaged laborers with/without surveillance [81] .
Recently, in a study of 1600 pilgrims screened on arrival at the 2010 Hajj season in Saudi Arabia, 120 (7.5%) had influenza A virus (9 out of the 120 had H1N1 virus) [88] . Additionally, the epidemiological data showed that the pilgrims had the potential not only to introduce these viruses to Saudi Arabia, but also to export the influenza virus back to their home countries [78, 89] . This can occur in Saudi Arabia, despite the availability of a TIV containing inactivated antigens for influenza virus types A and B, which can protect against the influenza virus infection [72] .

CCHF

Currently, the virus infects both humans and animals following tick bites [115] . However, a human can be infected by the animal through contact with the blood or tissues of the infected animal, in particular, exposures at the abattoirs are common. Therefore, workers in contact with animals (e.g., veterinarians, farmers' and workers in slaughterhouses) form a high percentage of those affected [87] . Also, different species of infected animals-such as camels, cattle, sheep, goats, and ostriches-might be infected with no clinical signs [83] . In addition, human-to-human transmission is also documented, mostly through a form of nosocomial or in-house infection [113, [116] [117] [118] .
Jazan Province, the Red Sea port city on Saudi Arabia's southern border with Yemen, serves as the east-west portal from sub-Saharan Africa at Djibouti and the south-north route across the Yemeni frontier. It is a heavily traveled corridor for humans and animals entering Saudi Arabia, particularly during the annual Hajj pilgrimage. In November 2009, a total of 197 (19%) enrolled soldiers reported symptomatic illness during deployment, 49 (25%) of whom were hospitalized. Reported signs and symptoms included fever (n = 81), rash (n = 50), and musculoskeletal complaints (n = 128). A surveillance study was conducted to detect the causes of the several outbreaks through that area, which was reported as endemic over a wide geographic range. From the surveillance, serologic testing for CCHFV, AHFV, DENV, and RVF was completed for 1024 Saudi military units from several Saudi Arabian provinces. These units were previously stationed in other parts of the country, and were deployed to Jazan Province; the initial screening for IgG of each of these viruses was conducted by IgM testing for all IgG-reactive samples. Among the samples from all military forces, the study identified 40 reactive serum samples with a combined seroprevalence of 3.9 cases/100 soldiers tested. A confirmed serologic status of 1024 soldiers who were evaluated for IgG and IgM ELISA reactivity against CCHFV, RVF, AHFV, and DENV infections were positive for 6, 20, 13, and 1 sample, respectively [124] .

Dengue Hemorrhagic Fever

More recently in 28 January 2018, the MOH began an intensive campaign to eradicate the DHF virus from Saudi Arabian cities, to enhance public health awareness, and facilitate a change in hygiene behavior of citizens and residents. This resulted in a 50.7% reduction in the number of DHF infection among inpatient cases in Jeddah when compared to the same period in the previous year. However, the overall drop in DHF cases reached 38% in 2017, compared to the previous year [192] . Furthermore, recently, it is well-known that in Saudi Arabia, the DHF infection has been limited to the western and southwestern regions such as Jeddah and Makkah where Aedes aegypti exists. However, all DHF cases in Jeddah, Saudi Arabia, were mostly mild cases [171, 175, 192] and the prospect of dengue virus control lies with vector control, health education, and possibly vaccine use.

West Nile Fever

More recently, in 2016, using standard procedures, the Central Veterinary Research Laboratory in Dubai, the United Arab Emirates, described the first WNV isolation in a dromedary calf; and this supports the conclusion that WNV is present in the country [208] . The WNV zoonotic infection was probably transmitted through the human-animal interface; that is through the well-known contact with infected Arabian camels in Saudi Arabia. Interestingly, dromedary are exported from the United Arab Emirates to Saudi Arabia and vice versa; due to the closely related WNVs genes and their circulation through the natural transmission cycles worldwide, a complete genome sequencing for more WNVs strains, as well as comparative genomic and phylogenetic studies in Saudi Arabia, are needed to ascertain whether the dromedary infection with WNV exists in the country or not. However, the same facts have been suggested recently (2017), when it was suggested that WNV infection was introduced into Turkey at the time of the outbreaks in Saudi Arabia and Yemen. It was further suggested that the virus may have been introduced via unlawful entrance of viremic domestic or wild animals through the borders or through vectors that carry the virus into Turkey [209] .

Summary and Conclusions

With Saudi Arabia, the focal point of the ongoing zoonotic pathogens outbreak could be due to the large number of religious pilgrims congregating annually particularly in Makkah, Jeddah, and Al-Madinah, the main three cities for Hajj and Umrah, which drastically increased the potential for uncontrolled global spread of zoonotic infections [168] .

Rabies

According to the MOH and Ministry of Agriculture data in Saudi Arabia, pets are responsible for most animal bites in humans [36] , and it is well-known that insufficient vaccination coverage of pets are among the most common hallmarks of the endemic status of rabies worldwide [37] . More recently, many Saudi and expatriate families are keeping pets; however, there are limited number of specialized veterinary clinics (~5) within the Kingdom of Saudi Arabia that have fully licensed veterinary laboratories with state of the art technologies and veterinary staff.

CCHF

Lately, antibodies to the virus have been detected in different animal species, as reported in 1976, in Egyptians animals' sera [119] . The preliminary seroepidemiological survey detected antibodies to the virus in 8.8% of camels' sera and 23.1% of sheep sera, but no antibody was detected against the virus in the sera of other animals such as donkeys, horses and mules, pigs, cows, and buffaloes [119] . The epidemiology and distribution of CCHF in Saudi Arabia are unclear, but there are reports of CCHF as a result of the trading and importing of infected livestock from neighboring countries to Saudi Arabia [120] .

RVF

RVF is a viral zoonosis with evidence of widespread occurrence in humans and animals in Africa and the Arabian Peninsula. The epidemiology of this virus in Saudi Arabia might be closely related to the ecological factors that are prevalent, as shown from another area, along the Great Rift Valley, which traverses Ethiopia and Kenya to northern Tanzania with the drainage ecosystems [127] .

Rabies

Globally, almost 95% of all human deaths caused by rabies occur in Africa and Asia [38] . However, Saudi Arabia, as one of the Asian countries, has scarce publications and epidemic data on rabies status [38, 39] . Moreover, Memish et al., between 2005 and in Saudi Arabia, reported the histologic detection of the virus by identifying Negri bodies in the brain samples of 40 animal rabies cases. The study showed that among the 40 suspected rabies cases, 37 (~92.5% of all cases) were found to be positive; thus confirming rabies cases among 11 dogs, 6 foxes, 6 sheep, 5 camels, 4 goats, 3 wolves, and 2 cows [36] .
43 section matches

Abstract

To study disease pathogenesis and to develop efficient and safe therapies, the use of an appropriate animal model is a critical concern. Adult mice with gene knockouts of the interferon α/β (IFN-α/β) receptor (IFNAR(−/−)) have been described as a model of arbovirus infections. Studies with the natural hosts of these viruses are limited by financial and ethical issues, and in some cases, the need to have facilities with a biosafety level 3 with sufficient space to accommodate large animals. Moreover, the number of animals in the experiments must provide results with statistical significance. Recent advances in animal models in the last decade among other gaps in knowledge have contributed to the better understanding of arbovirus infections. A tremendous advantage of the IFNAR(−/−) mouse model is the availability of a wide variety of reagents that can be used to study many aspects of the immune response to the virus. Although extrapolation of findings in mice to natural hosts must be done with care due to differences in the biology between mouse and humans, experimental infections of IFNAR(−/−) mice with several studied arboviruses closely mimics hallmarks of these viruses in their natural host. Therefore, IFNAR(−/−) mice are a good model to facilitate studies on arbovirus transmission, pathogenesis, virulence, and the protective efficacy of new vaccines. In this review article, the most important arboviruses that have been studied using the IFNAR(−/−) mouse model will be reviewed.
Arboviruses are arthropod-borne viruses that exhibit worldwide distribution and are a constant threat, not only for public health but also for wildlife, domestic animals, and even plants.

Introduction

Arboviruses are arthropod-borne viruses that exhibit worldwide distribution and are a constant threat, not only for the public health but also for wildlife, domestic animals, and even plants. The rise in global travel and trade as well as the changes in the global climate conditions are facilitating the expansion of the vector transmitters, including mosquitoes, ticks, sandflies, and midges among other arthropods, from endemic to new areas, augmenting the number of outbreaks around the world at an unprecedented rate. Arboviruses need multiple hosts to complete their cycle (i.e., host and vector), making it possible to impact disease by targeting either the arthropod vector and/or the pathogen. For some of these pathogens, efficient antivirals or vaccines are not available, in some cases due to the genetic variability of these viruses. Moreover, there are a limited availability of animal models to study infections, and some of them display a poor immunogenicity and some others viral infections cause neglected diseases that have not been deeply studied. Transmission between the vector and the host occurs when the vector feeds on the blood of the host by biting. However, the vector does not act as a simple vehicle that passively transfer viruses from one individual to another. Instead, arthropod-derived factors found in their saliva have an important role in infection and disease, modulating (positively and negatively) replication and dissemination within the host [1, 2] . In addition, the inflammatory response that the host mounts against these vector molecules can enhance the severity of arbovirus infection [3, 4] .

Zika Virus

Another member of this family with high outbreak potential is Zika virus. Zika virus infections in humans have sporadically occurred in Africa and Asia, and new outbreaks were registered in small island countries located in the Pacific Ocean, such as Yap Island [87] , French Polynesia [88] , and Easter Island [89] . In 2015, an epidemic of ZIKV originating from Brazil, spread through most of North and South America and the Caribbean, as well as thousands of imported cases from travelers returning to their home countries after visiting outbreak areas [90] [91] [92] . The ZIKV infections are typically asymptomatic, but in some cases the disease courses with fever, joint pain, maculopapular rash, and red eyes [93] . While no deaths have been reported from ZIKV infections, mother-to-child transmission during pregnancy may result in congenital Zika syndrome with abnormalities in the central nervous system (microcephaly, intellectual development, seizures, and vision impairment) [94] . Zika virus infections in adults is associated with Guillain-Barré syndrome [95] . Distinct from other flavivirus infections, sexual transmission of ZIKV from male-to-male, male-to-female, and female-to-male have been documented [96] [97] [98] [99] . Wild-type mice are refractory to Zika infection with strain MP1751 (Zika virus targets human STAT2 to inhibit type I interferon signaling, but not murine STAT2 [55] , as was observed with YFV), while IFNAR(−/−) mice succumbed to disease at 6 dpi with 20% body weight loss with a challenge of 10 6 PFU sc. Viral RNA was observed at 3 and 7 dpi in blood by RT-qPCR, as well as high levels of virus in spleen, brain, ovary, and liver of these animals. Pathology studies show that inflammatory and degenerative changes could be detected in the brain [50] . More studies have been performed using alternative strains/doses and different ages, as H/PF/2013 strain from French Polynesia and the original Ugandan ZIKV strain MR 766. Five-to 6-week-old mice sc infected with 10 2 focus-forming units (FFUs) began to lose weight by five days after infection, and by day seven, when they began to succumb to infection, animals had lost between 15% and 25% of their starting body weight. Ten and 13 days after infection, mice exhibited 100% and 80% lethality with ZIKV H/PF/2013 and MR 766, respectively. When mice were challenged intravenously, an increase of 60% in the survival rate was observed in MR 766 infected mice [51] . In older IFNAR(−/−) mice (3-, 4-, and 6-month-old), infection with 10 3 FFU of ZIKV (H/PF/2013) reduced the weight in all animals, with ∼30% of starting weight lost by nine days after infection, and a mortality of 60-20% were observed [51] . Interestingly, the lethality in 10-12-week-old animals was abolished when using 10 5 PFUs of ZIKV FSS13025 strain from Asian lineage (being a 100% and 50% of lethality in 3-and 5-week-old mice) [52] , but not for ZIKV H/PF/2013 infection (100% of deaths) [53] . Taken together, these results indicate that the disease caused by ZIKV infection in these animals was age and strain-dependent. Surprisingly, another strain associated with microcephaly case, ZIKV-Paraiba, caused weight lost in 5-8-week-old IFNAR(−/−) mice inoculated sc with 10 2 or 10 4 PFUs at days 6 and 7 post-infection, independent of the dose of ZIKV [53] . Approximately 50% of the mice succumbed to disease or were euthanized between days 9 and 11 due to development of neurological signs such as hind limb paralysis. Viral RNA was detected in many tissues as mandibular lymphonode, salivary gland, lung, heart, liver spleen, kidney, bladder, gonad, spinal cord, brain, cerebellum, and blood at different time points (3 and 8 days) . The route of inoculation does not seem to be significant among subcutaneous, intraperitoneal, and footpad administration, but in this study, only ip resulted in uniform lethality in young IFNAR(−/−) mice [53] . Sexual and maternal transmission are the most important concerns in Zika disease due to the consequences derived of ZIKV infection in the fetus. In IFNAR(−/−) males, high levels of viral RNA and antigen within the epididymal lumen (where sperm is stored) and within surrounding epithelial cells was observed. Moreover, serum testosterone levels were markedly decreased at 8 dpi and also observed was a reduction in the size of the testes at 21 days post-infection [100, 101] . In females, vaginal infection with high doses of ZIKV was lethal. Vaginal ZIKV infection of pregnant female mice at various gestational time points led to fetal growth restriction. High levels of local ZIKV replication were observed starting on 2 dpi, and ZIKV continued to replicate in the vaginal tissue through 7 dpi, suggesting that type I IFN play a critical role in blocking ZIKV replication in the vaginal mucosa [102] . The role of type IFN I during pregnancy in infected mothers have been assessed using IFNAR(−/−) mice [103] , and the findings highlight the detrimental impact of type I IFN on the developing placenta and fetus by demonstrating that only the fetuses with a functional copy of IFNAR are resorbed after ZIKV infection, whereas their IFNAR(−/−) littermates continue to develop, even having higher ZIKV titers in their placentas. These results implicate type I IFNs as a possible mediator of pregnancy complications, including spontaneous abortions and growth restriction, in the context of congenital viral infections. New generation vaccines have been shown effective against ZIKV in IFNAR(−/−) mice based on VSV viral vector expressing pRM and E ZIKV proteins, enhancing ZIKV-specific IgG with neutralizing activity, and providing protection within three days of vaccination [104] . A vaccinia-based single vector that encodes the structural polyprotein cassettes of both Zika (and chikungunya) viruses from different loci has also been recently developed. A single vaccination of mice induces neutralizing antibodies and prevent viremia and fetal/placental infection in female IFNAR(−/−) mice and testes infection and pathology in male IFNAR(−/−) mice [105] . The IFNAR(−/−) mice model has also been used to demonstrate how salivary factors expressed by the vector Aedes aegypti modulates ZIKV infectivity. Administration of the salivary factor LTRIN caused a substantial loss in body weight in IFNAR(−/−) mice up to 10-15% of their starting body weight by day 6, indicating that the administration of LTRIN exacerbated ZIKV's pathogenesis in IFNAR(−/−) mice [106] .

Chikungunya Virus

Wild-type C57BL/6 mice infected with 10 4 cell culture infectious dose 50 (CCID 50 ) of CHIKV (Asian or the Reunion isolates) produced a measurable self-limiting perimetatarsal foot swelling with clear histological signs of acute and persistent inflammatory disease [121] . In IFNAR(−/−) mice, the susceptibility as well as the role of IFNAR receptors in CHIKV control and clearance have been studied. A dose of 10 2 PFU injected intradermally (id) was sufficient to kill the IFNAR(−/−) mice between days 2.5 and 4 post-infection, and injection of 10 6 PFU resulted in even faster death, with all animals succumbing to infection between days 2-3 post-infection [122] . Similar to what was observed in highly viremic humans [123] , the viral load in IFNAR(−/−) mice infected with 10 6 PFU at 2 dpi was >10 8 tissue culture infectious dose 50 (TCID 50 )/mL. In contrast, wild-type animals cleared the infection with undetectable serum viral titers at all timepoints tested. CHIKV exhibits a marked tropism for skeletal muscles, joints and skin, which constitute the classical symptomatic organs in the human disease. Fibroblasts constitute the principal CHIKV cell target in all these organs. Before reaching its target organs, CHIKV undergoes an early burst of viral replication in the liver, where CHIKV antigens are primarily detected in sinusoidal capillary endothelial cells and to a lesser extent in Kupffer cells. At 3 dpi, there is a sharp increase in viremia, with CHIKV antigens detectable in the red pulp of the spleen. In the case of severe CHIKV infection, CHIKV disseminates to the CNS, as is observed in human [124] , via the choroid plexus route, and undergoes viral replication at the ependyma and leptomeningeal levels, not being detected at the brain micro-vessel and parenchyma. Maternal-fetal transmission of CHIKV in pregnant IFNAR(−/−) mice has also been analyzed. However, CHIKV is unable to cross the placental barrier from the mother to the fetus in the mice [58] and humans, with some exceptions, as the three cases reported in the second trimester of gestation, which CHIKV infection has been associated with antepartum fetal deaths without clear evidence for the mechanism [125] . Some vaccines and therapeutic measures against CHIKV infection have been evaluated in this model. Mouse anti-CHIKV monoclonal antibodies (MAbs), selected for their ability to inhibit infection of all three CHIKV genotypes, have been tested using IFNAR(−/−) mice. Four neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) that have been mapped to distinct epitopes on the E1 and E2 structural proteins, provided complete protection against a lethal challenge. CHK-15, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102 + CHK-152 or CHK-166 + CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 h before CHIKV-induced death, so the use of these highly neutralizing MAbs may be a promising treatment option for CHIKV in humans [126] .

Conclusions

During the last decades, arboviruses have expanded their geographic range and caused an increasing number of outbreaks along all continents, enhanced by factors like climate warming, urbanization, global trade, travel, and changes in land uses [166] . Arboviruses incorporate a vast collection of genetically diverse viral pathogens. These viruses are peculiar as many of them are zoonotic and are transmitted by arthropod vectors, an added difficulty, being a serious harm to the society and animal welfare. In order to understand the arbovirus biology during infection and to develop an effective treatment against them, an adequate animal model for these studies is required. Mouse models deficient in IFN signaling are used to overcome the natural resistance of immunocompetent mice against non-mouse-specific viral infections, due to their inability to generate a complete immune response. Their use requires careful interpretation of results due to differences in the immunological state between wild-type and IFNAR(−/−) mice, and in the biology between mice and humans or large animals. However, there is no doubt about the utility of IFNAR(−/−) mouse models in the field of virology research, pathogenesis, immunobiology of the infections, arbovirus transmission, and vaccine testing.
The IFNAR knockout mice have served to study the role of some non-structural proteins as NSs of RFVF in the evasion of the type I IFN response, antagonizing IFN function. In this case, two attenuated RVFV strains with mutations in the NSs gene, MP12 and clone 13, are highly virulent in IFNAR(−/−) mice, but remain attenuated in IFN-γ receptor-deficient mice and immunocompetent mice. The IFNAR(−/−) mouse model has also been used to study viral pathogenesis. In some cases, infection in this model leads with non-specific signs as ataxia or weight loss, or the severity of the infection is strain-and age-dependent as occur with ZIKV infections. In contrast, there are some examples that closely mimics hallmarks of natural host disease such as the case of CCFHV infections, where proinflammatory host responses, severe thrombocytopenia, and coagulopathy are observed; BTV infection, that leads to damage in lung and lymphoid organs and alteration in the level of blood parameters; or CHIKV, that exhibits a marked tropism for skeletal muscles, joints and skin, that constitute the classical symptomatology and organ affectation in the natural hosts. In some other cases, this model was useful to study various important phenomena of disease, as the role of type I IFN responses to control the access to the CNS, as the case of WNV, the study of the sexual and vertical transmission of ZIKV or the antibody dependent enhancement mediated by sub-neutralizing antibodies during secondary DENV infections.
This review has summarized the characterization studies of relevant arboviruses in knockout out IFNAR mice to provide a small animal model for studying pathogenesis and control strategies. Experimental infections of IFNAR(−/−) mice with many of the studied arbovirus closely mimics hallmarks of these viruses in their natural hosts, although extrapolation of the results obtained must be done with care due to differences in the biology between mouse and humans or large animals and the immunosuppressed state of this model. Taking all these points together, the use of IFNAR(−/−) mice as a model to study arbovirus transmission, pathogenesis, virulence, and protective efficacy of new antiviral strategies and new generation marker vaccines has been widely demonstrated, being an adequate model in the initial steps of arbovirus research.

Introduction

To study disease pathogenesis and to develop efficient and safe therapies to prevent (vaccines) or treat (antivirals) viral infections, the use of an appropriate animal model is a critical concern. The use of mice as small animal models to study immunity, pathogenesis, as well as to test candidate vaccines and antivirals against a largely variety of viral diseases is widely spread. They are cost effective, being affordable for most of research laboratories. They reproduce quickly, are easy to handle, do not require specialized facilities to house, and multiple inbred strains of genetically identical mice are available. In many cases such as Crimean Congo Hemorrhagic Fever (CCHFV), Bluetongue (BTV), Middle East respiratory syndrome (MERS), or Ebola (EBoV) viruses, the pathogenesis of disease in humans is also partially mimicked. Furthermore, optimal reagents have been developed for in vivo and in vitro studies in mice, a fact which allows the study of other animal viruses apart from those which are human specific [5] [6] [7] [8] . Also, it is possible to manipulate the mouse genome and generate transgenic, knock-out, knock-in, humanized, and conditionally mutant strains to interrogate protein function in physiological and pathological signs.

IFNAR(−/−) Mice

The role of interferons (IFNs) against viral diseases has been widely studied, as well as the strategies evolved by viruses to antagonize the effects of IFNs. Both type I and type II IFNs have been implicated in the host antiviral defense and in the immunomodulatory functions that are critical during virus infection, not only limiting virus replication and initiating an appropriate antiviral immune response, but to also negatively regulating this response to minimize tissue damage ( Figure 1 ) [14, 15] .
The IFNAR(−/−) knock-out receptor mouse model has been used to study infection, disease, pathogenesis and vaccine testing against multiple arbovirus families such as Togaviridae, Bunyaviridae, Flaviviridae, Rhabdoviridae, Orthomyxoviridae, and Reoviridae (Table 1 ). In this review, animal arbovirus families known to have been studied using the IFNAR(−/−) mice model are mentioned, describing briefly some examples, in which different aspects of biology, immunology, pathology, and vaccine design against these pathogens are exposed.

Dengue Virus

Maybe the best-known and most widespread member of this family is dengue virus. Dengue Fever virus is the etiologic agent of the self-limited febrile illness dengue fever (DF), as well as the potentially lethal severe dengue disease (dengue hemorrhagic fever and dengue shock syndrome, DHF/DSS). Symptomatic infections are characterized by: fever, retro-orbital headache, muscle, joint and bone pain, nausea, vomiting, abdominal pain, mucosal bleeding, and thrombocytopenia. In the most severe form of the disease, severe bleeding, organ dysfunction, vascular permeability, and shock can occur. Replication of DENV has been tested in immunocompetent mice [82] . C57BL/6 mice infected with DENV-1 strain Mochizuki presented some signs of dengue disease such as thrombocytopenia, hemorrhage, liver damage, and increase production of IFNγ and tumor necrosis factor alpha (TNFα) cytokines. However, no changes in CD4 and CD8 populations were observed comparing infected and mock infected groups. In addition, this strain was propagated in newborn (1 to 2 days old) Swiss mice, by intracerebral (ic) inoculation of infected cell culture supernatant. This propagation method resulted in a neurological disease phenotype that is unlike the multi-organ involvement typically observed in clinical dengue infections [44] . Although this DENV strain induce detectable viremia in C57BL/6 strain, the overwhelming majority of immunocompetent mouse models do not result in clinical signs of dengue infection [44] . To overcome this issue, a pathological analysis were performed in IFNAR(−/−) mice. It has been shown that mortality rates depend on the DENV serotype and strain used [44] . A severe dengue-like disease is observed when animals are infected with sufficiently high DENV2 challenge doses and clearance of DENV from the central nervous system (CNS) and prevention of paralysis in this mouse model has been confirmed to be dependent of CD8+ T cells and IFN-γ response [45] . Most primary DENV infections with any serotype are asymptomatic or lead to the self-limited febrile illness DF, in patients infected with DENV. However, secondary infection with a different DENV serotype leads to increased risk of developing severe dengue disease [47] . This increase in severity upon secondary infection is thought to be mediated in part via antibody-dependent enhancement (ADE), whereby interaction between antibodies generated during a prior infection and the current infecting serotype can lead to increased uptake of virus via Fc receptors expressed on susceptible myeloid cells [46] . This phenomena was observed also in IFNAR(−/−) mice, with a dramatic increase in the mortality rate in individuals intraperitonially (ip) injected with anti-E mAb 4G2 24 h before challenge [48] . Additionally, another study published in 2009 revealed the important role for CD8+ T cells in the host defense against DENV, demonstrating that the anti-DENV CD8+ T cell response can be enhanced by immunization. This study identified DENV-specific CD8 T cell epitopes, and peptide vaccination with these epitopes resulted in enhanced control of DENV infection and viral load [83] . Another immunization study has been performed in this model using live attenuated dengue vaccine 2 -o-methyltransferase mutants, eliciting a strong adaptive immune response [84] .

Yellow Fever Virus

Yellow Fever virus produced one of the most dangerous infectious diseases of the 18th and 19th centuries, resulting in mass casualties in Africa and the Americas [85] . Inoculation of wild-type 129 mice subcutaneously (sc) in each rear footpad with 10 4 PFU of YFV did not result in any weight loss or death, whereas challenged 3-4 week old IFNAR(−/−) mice (129 background) challenged with YFV strains Asibi or Angola73 developed disease under the same conditions. During infection, non-structural protein 5 (NS5) protein inhibits IFN signaling by binding to STAT2 protein and promoting its degradation [86] . In mouse infection, NS5 was not able to bind murine STAT2, allowing IFN-mediated clearance of the virus. The IFNAR(−/−) mice were shown to be susceptible to the challenge, with death occurring between 7-9 dpi. Additionally, the mice developed viscerotropic disease with virus dissemination to the visceral organs, spleen, and liver, in which severe damage can be observed with gross pathological examination and hematoxylin/eosin staining. Moreover, elevated levels of MCP-1 and IL-6 in these organs were detected, suggesting an unleashing of "cytokine storm" [49] .

West Nile Virus and Japanese Encephalitis Virus

West Nile virus is known to cause disease and death in wild type mice, but studies using IFNAR(−/−) mice have been performed to elucidate the early mechanisms in the IFN immune response. In this study, the authors showed the high susceptibility of IFNAR(−/−) mice to WNV infection. The 8-10 week old IFNAR(−/−) mice (129Sv/Ev background) challenged with 10 0 , 10 1 or 10 2 PFU (strain 3000.0259) via footpad inoculation showed severe clinical symptoms by 3 dpi, including hunched posture, ruffled fur, and reduced activities, regardless of dose. Death (100%) occurred within 12-48 h after the onset of symptoms, and the mean time to death was 4.6 ± 0.7 and 3.8 ± 0.5 dpi for IFNAR(−/−) mice in the 10 0 and 10 2 PFU groups, respectively. Infectious virus was detected in the muscle, heart, lung, kidney, and liver [56] . Also, an altered cellular tropism was observed in IFNAR(−/−) mice, with increased infection in macrophages, B cells, and T cells in the spleen, compared with wild-type mice [56] . Another feature of WNV is its capability of infecting the CNS, causing fatal encephalitis. In vivo, IFNAR(−/−) mice exhibited enhanced BBB permeability and TJ dysregulation after WNV infection, triggered by pattern recognition receptors-mediated cytokine expression. These results suggest that local CNS type I IFN responses may act on the BBB to mitigate the access of WNV to the CNS parenchyma [28] . Regarding vaccine development in this model, a novel single-cycle flavivirus vaccine has been tested, with a significant increase in the level of WNV-specific CD8+ T cells compared to the wild-type [111] .
Japanese encephalitis, whose causal agent is JEV, is considered as one of the most important encephalitic arthropod-borne diseases. An estimated 3 billion people live in countries where the disease is endemic and 30,000-50,000 cases and 10,000-15,000 deaths are reported annually [112, 113] . Wild-type mice are susceptible to the sc JEV infection, with survival rates that vary between 10-40%, being not dose-dependent [57] . In the same study, inoculations were repeated in 5-6 week old IFNAR(−/−) mice at the same doses, being highly susceptible to the challenge, with uniform, dose-dependent death occurring between 64-120 h. Viral replication could be detected in the spleens and brains of infected animals, with peak titers at 48 h [57] .

Bluetongue Virus

Bluetongue virus is the type species of this genus that can cause a severe hemorrhagic disease in ruminants, particularly in sheep. Other susceptible species are camelids and alpacas. Bluetongue virus has been responsible for important outbreaks all over the world affecting sheep, cattle, and deer, and resulting in huge economic losses. The study of many aspects of BTV infection and the evaluation of vaccines has long been hampered by the lack of a small animal model that supports this virus. While BTV is lethal in newborn mice, two-week old mice are largely refractory to infection. The first characterization of BTV infection in IFNAR(−/−) mice was developed after inoculation with serotypes 4 and 8 [6] . Afterwards, multiple serotypes and strains have been demonstrated to induce clinical signs, viremia, and mortality in this mouse model. The IFNAR(−/−) mice with a C57BL/6 and 129Sv/Ev genetic background exhibit the same level of susceptibility to BTV infection and no differences are found between subcutaneous and intravenous administration in the survival rates and appearance of disease [134, 135] . The clinical manifestations that are found in IFNAR(−/−) mice inoculated with a lethal dose of BTV comprise ocular discharges, apathy, an increased respiratory rate and hunching [6] . Notably, these are some of the clinical signs, among others, that BTV infected ruminants may display [136] . Studies of viral progression in IFNAR(−/−) mice showed that infectious virus is recovered from the spleen, lung, thymus, lymph nodes, and blood. Thus, BTV disseminates via blood and lymph as it does in the natural hosts [6] . In mice, infected thymus exhibits a profound lymphoid depletion, a loss of thymic architecture as the medulla and the cortex are hardly distinguishable and large areas of the parenchyma with necrosis. In addition, a severe distortion of normal histology together with lymphoid depletion are observed in lymph nodes [6, 66] . When virus infects spleen in IFNAR(−/−) mice, this shows a marked lymphoid depletion with severe white pulp lymphocytolisis and infiltration of neutrophilic infiltrates in the margin between the red and white pulp [66] . In these studies, a reduction in CD3 and CD79 (T and B cell markers, respectively) reactivity was observed in the spleen and thymus of BTV-infected mice that confirms the lymphopenia. This has been described commonly in BTV-infected sheep [137, 138] . Moreover, lungs from infected mice reveal a diffuse interstitial pneumonia with hyperemia, increased septum size, a moderate edema in the alveolar cavity and infiltration of lymphocytes, macrophages, and neutrophils [66] . All these data indicate that the lesions found in BTV-infected IFNAR(−/−) mice are similar to those found in the natural hosts [65, 139] . Changes in hematology including thrombocytopenia, neutrophilia, and lymphopenia have been determined after infection of IFNAR(−/−) mice with a high virulent strain of BTV-4 [66] , observations similar to those described in experimental BTV infections [65, 140] .

African Horse Sickness Virus and Epizootic Hemorrhagic Disease Virus

African Horse Sickness Virus caused a severe disease in equids, where mortality could reach 90% in susceptible horses. Dogs can be also infected after feeding contaminated horsemeat and experimental infections have been established. On the other hand, a neurotropic vaccine strain can cause encephalitis and retinitis in humans, although no infections after contact with field strains have been described [155] .
Epizootic hemorrhagic disease virus (EHDV) infects ruminants and causes severe disease mainly in deer [164] . An animal laboratory model would facilitate the studies and evaluation of vaccines against this virus. The IFNAR(−/−) mice has also been proposed as a mouse model to study EHDV infection with promising results [69] . Previously, the virus was shown to fatally infect newborn Swiss outbred mice after intracerebral inoculation [165] ; however, newborn mice cannot be used for vaccination experiments.

Rift Valley Fever Virus (Family Phenuiviridae)

Rift Valley fever virus (RVFV), a phlebovirus transmitted mainly by Aedes (Stegomya) mosquitoes, causing Rift Valley fever, a zoonotic disease of ruminants, has been confined to Sub-Saharan Africa for many decades. In the last years, a spectacular increase in the number of outbreaks, including a more northward geographic spread has been documented. This zoonosis is associated with "abortion storms" in domesticated sheep flocks and high mortality rates in newborn livestock (lambs and calves) [70] . Rift Valley fever virus is one of the major public health threats in sub-Saharan Africa, where human infection leads to a wide spectrum of clinical signs and symptoms that range from a "flu-like" illness with fever and myalgia to severe encephalitis, retinitis, and fatal hepatitis with hemorrhagic fever (1-2% of the cases) [71] . The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. Although RVFV is able to infect and replicate in wild-type mice [72] , some studies using the IFNAR(−/−) mouse model have been also performed to study the role of type I IFN signaling and the mechanism of RVFV to evade the IFN response during the course of the infection. Bouloy and colleagues brought to the light the ability of RVFV to inhibit IFN-α/β synthesis, demonstrating that IFN type I production correlates with virulence and suggesting that the accessory non-structural protein NSs is an IFN antagonist factor that prevents IFNs-α/β from being induced early during the course of RVFV infection. Also, these authors showed how two RVFV strains, MP12 and clone 13, are attenuated in immunocompetent mice and in IFN-γ receptor-deficient mice but fully lethal in IFNAR(−/−) mice [29] . These observations suggested the use of IFNAR(−/−) mice as a candidate model for testing the efficacy of experimental vaccines and/or therapeutics against RVFV under a BSL-2 containment environment, using these attenuated strains. Thus, the efficacy of DNA vaccines encoding different RVFV antigens was tested in this model, showing several degrees of protection upon a lethal challenge [30, 31] . The antiviral activity of silver nanoparticles was also tested in these mice showing reduction of viremia and delayed mortality after lethal challenge [32] . Finally, an important aspect that arose from the efficacy studies of MVA-vectored RVFV vaccines in IFNAR(−/−) mice was related to the opposite efficacy outcomes observed in IFNAR(−/−) and wild-type mice, providing important clues to dissect the role of cell-mediated immune responses in protection [33] .

Crimean Congo Fever Virus (Family Nairoviridae)

Another emerging pathogen with epidemic potential is Crimean Congo Fever Virus (CCHFV), typically spread by tick bites of the Hyalomma genus, or by contact with blood or tissues of infected livestock (whose are usually asymptomatic) or patients [73] . Susceptibility of wild-type 129 Sv/Ew and IFNAR(−/−) mice to CCHFV was studied, showing viremia and viral titer in several organs as spleen, liver, kidney, brain, and heart in both immunocompetent and immunocompromised mice, but with high viral burden and developing an acute disease with fatal outcome, with a profound liver affectation in the case of IFNAR(−/−) mice [34] . In this mice, disease progression closely mimics hallmarks of human CCHF disease as marked proinflammatory host responses, severe thrombocytopenia and coagulopathy, making IFNAR(−/−) mice a good model to assess medical countermeasures [5] . Among them, formali-inactivated cell culture CCHF alum-adjuvanted vaccines, VLPs, DNA or viral vector vaccines (modified vaccinia virus Ankara (MVA) and adenovirus) expressing nucleocapsid protein or glycoproteins conferred different rates of protection in immunized animals [35] [36] [37] [38] [39] and administration of Favipiravir after infection (twice daily) suppressed the infection and the clinical signs in treated mice [40] .

Family Flaviviridae

Flaviviridae are a family of positive, single-stranded, enveloped RNA viruses. They are transmitted by mosquitoes and ticks and cause morbidity and mortality throughout the world. Some of them are known to produce hemorrhagic diseases, such as Dengue Fever virus (DENV), Yellow Fever virus (YFV), and Zika virus (ZIKV), and other members are also responsible of encephalitis diseases: West Nile virus (WNV), Japanese encephalitis virus (JEV), Powassan virus (PV), Langat encephalitis virus (LGTV) or Tick-borne Encephalitis (TBE).

West Nile Virus and Japanese Encephalitis Virus

West Nile virus (WNV) is generally transmitted by Culex mosquitos and the natural host are birds. In addition, bites from infected mosquitos can infect humans and other mammals as horses. However, they are "dead end" hosts because they do not develop high levels of virus in their bloodstream, and cannot generally pass the virus on to other biting mosquitoes [107] . West Nile virus is endemic in Africa, Asia, Europe, and Australia, and has spread into Canada and the United States (U.S.) [108] . West Nile virus infection of humans can be characterized as asymptomatic or as a mild, febrile illness termed West Nile fever. However, a significant increase in the global incidence of severe neurological disease (associated with WNV lineage I infections) arose in the mid-1990s, culminating in the U.S. outbreak in 2003, which included 9862 reported cases and 264 deaths [109] . After its introduction in New York City in 1999, WNV rapidly spread across the continent and now appears to have firmly established itself in the ecology of North America. The rapid emergence of WNV and its virulence within a naïve population suggest that epidemic forms of the virus may encode mechanisms to evade host immunity [110] .

Family Togaviridae

The Togaviridae family are composed for linear, non-segmented, single-stranded, positive sense RNA viruses. Among this family, only the genus alphavirus, are transmitted by arthropod vectors. Sindbis, Semliki, chikungunya, Mayaro, O'nyong-nyong or Ross River alphaviruses are known to cause human diseases in which rheumatic complaints are a major feature, while eastern equine encephalitis, and Venezuelan equine encephalitis viruses can cause arthritis disease and encephalomyelitis, a potentially fatal inflammatory disease of the CNS with frequent long-term neurological deficits in survivors [116] [117] [118] [119] [120] .

Other Alphavirus

The role of the IFNAR receptor has been assessed in other members of this family. The prototypic alphavirus, Sindbis virus strain AR339, was isolated by ic inoculation of three-day-old mice with a mosquito homogenate collected near Sindbis, Egypt. In wild-type mice, the infection courses asymptomatic, while IFNAR(−/−) mice inoculated sc with 10 2 PFUs of TR339 succumbed to the infection within 3-4 dpi. By 24 hpi, a high-titer serum viremia had seeded infectious virus systemically, coincident with the systemic induction of the proinflammatory cytokines IL-12 p40, IFN-gamma, TNFα, and IL-6. Replicating virus was located in macrophage-dendritic cell (DC)-like cells at 24 hpi in the draining lymph node and in the splenic marginal zone. By 72 hpi virus replication was widespread in macrophage-DC-like cells in the spleen, liver, lung, thymus, and kidney and in fibroblast-connective tissue and periosteum, with sporadic neuroinvasion. Thus, type I IFN protects the normal adult host from viral infection by rapidly conferring an antiviral state on otherwise permissive cell types, both locally and systemically. Ablation of the type I IFN system alters the apparent cell and tissue tropism of the virus and renders macrophage-DC-lineage cells permissive to infection [59] . IFNAR(−/−) mice infected with Venezuelan equine encephalitis (VEE) also exhibit progressively increasing signs of infection characterized by pronounced hunching, lethargy, prostration, and death. Accelerated VEE dissemination to serum, spleen, and brain was observed in these mice compared with wild-type animals, and is associated with the upregulation of proinflammatory genes [60] . O'nyong-nyong (ONNV) infected mice exhibited 50-55% mortality after a sc dose of 10 3 PFU. Mortality increased to 100% when the ONNV dose was increased to 10 4 PFU. The ONNV was present in the brain and skeletal muscle of IFNAR(−/−) mice, and the presence of virus in the heart could be a function of myocyte tropism as has been reported in CHIKV infection [129] . It is of interest that the inflammatory infiltrate seen in the tissues of mice was composed predominantly of monocytes and myositis/tenosynovitis, but not the neurologic disease was observed in infected animals. In addition, IFNAR(−/−) mice generated a viremia peaking on days 2-3 post-infection that waned by day 5, which is typical of alphavirus infections in humans.

Family Rhabdoviridae

Rhabdoviridae is a virus family with a very broad host range that are capable of infecting plants, and invertebrate and vertebrate animals. Rhabdoviruses have a non-segmented, linear, negative-sense, single-stranded RNA genome. This RNA molecule codes for five viral proteins and its complete genome is approximately 11 kbp-15 kbp. Rhabdoviridae contains six genera: Lyssavirus, Ephemerovirus, Norvirhabdovirus, Cytorhabdovirus, Nucleorabdovirus, and Vesiculovirus, being the last the only transmitted by arthropods in animals. The prototype of Vesiculovirus genus is VSV, an arthropod-borne virus that primarily affects rodents, cattle, swine and horses. It can induce mild symptoms upon infection in humans and other species and may also cause severe foot-and mouth-like disease in cattle and pigs. Vesicular stomatitis virus replicates rapidly, developing high levels of progenies in a minimum amount of time and strongly interferes with the host's cell metabolism. Infection by rhabdoviruses induces a cellular response through the activation of pattern recognition receptors (PRRs) that causes the production and secretion of IFN and pro-inflammatory cytokines. The virus replication is highly sensitive to the inhibitory action of IFN therefore IFNAR(−/−) mice are highly susceptible to VSV pathogenesis [61, 62] . Interferon plays a critical role for virus control after a VSV infection, although the concrete mechanisms are unknown. Several studies have been carried out in IFNAR(−/−) mice to discover these mechanisms and whether IFN expression play a role in determining viral tropism. In fact, a study carried out by Detje et al. [61] has shown that IFN triggering within the periglomerular cells of the olfactory bulb is required to protect against lethal disease.

Family Reoviridae

The members of the genus Orbivirus, within the family Reovidae, can infect a wide range of hosts such as equids, ruminants, camelids, marsupials, seabirds, batsm and in some cases humans. The more relevant orbiviruses in animal health are Bluetongue virus (BTV), African horse sickness virus (AHSV), and Epizootic hemorrhagic disease virus (EHDV).

Bluetongue Virus

A number of experimental vaccines for BTV have been tested in the mouse model based on IFNAR(−/−) mice. First characterization of these kind of vaccination trials was done using a commercial inactivated vaccine that has been used in the field, demonstrating that this vaccine prevent clinical disease in IFNAR(−/−) mice as it does in the natural host [6] . Then, the efficacy of novel recombinant subunit, DNA, and viral vector vaccines have been tested in the IFNAR(−/−) mouse model ( Table 2) . * ND: Non-determined.

African Horse Sickness Virus and Epizootic Hemorrhagic Disease Virus

Earlier attempts to develop a mouse model to evaluate vaccines for AHSV were not successful using BALB/c mice, since AHSV vaccine strains were in most cases more virulent for mice that the wild-type strains [156] . Another study determined that although sc inoculation did not cause disease, intranasal inoculation of AHSV in immunocompetent mice increase the clinical fatality [157] . This could be explained by two hypothesis, the neurotropism acquired after intracerebral passages in mice [156] and the retrograde neuroinvasion through the olfactory pathway [158] . Nevertheless, a sc infection is a more similar route of inoculation comparing with the bite of Culicoides midges in nature and IFNAR(−/−) mice inoculated sc with AHSV are highly susceptible to the virus. Thus, this mouse model has been used to study virulence, pathology, and to evaluate vaccines with satisfactory results.

Introduction

Immunocompetent wild-type mice are susceptible to infections with a number of viral pathogens such as influenza virus [9] ; severe acute respiratory syndrome coronavirus (SARS-CoV) [10] ; and Rift Valley fever virus (RVFV) [11] . Unfortunately, immunocompetent mice are not susceptible to many other viruses with outbreak potential, and thus alternative strategies are needed.

IFNAR(−/−) Mice

In the early 90s, Muller and colleagues [12] generated mice deficient in the type I IFN (IFN-α/β) receptor (IFNAR(−/−)) by homologous recombination in embryonic stem cells. While these transgenic mice did not show any overt abnormalities by six months of age and were fertile, the animals were entirely unresponsive to the effects of type I IFNs. To monitor the response to type I IFN in vivo, they analyzed the induction of the Mx-1 gene, a strictly type I IFN-specific response marker in mouse cells [13] . Mice infected with vesicular stomatitis virus (VSV), Semliki Forest virus (SFV), vaccinia virus (VV), or lymphocytic choriomeningitis virus (LCMV) showed a completely abrogated IFN type I response and an enhanced infection susceptibility, resulting in either higher viral organ titers compared to wild-type mice and death in case of VSV and SFV challenges [12] .

West Nile Virus and Japanese Encephalitis Virus

Other flaviviral encephalities are being studied using this mouse model of infection, as that caused by Langat encephalitis virus (LGTV), showing type I IFN as a critical factor to control LGTV infection, as LGTV RNA was found in all organs in the absence of IFNAR, whereas in wild-type mice only low viral burdens can be detected in the olfactory bulb [114, 115] .

African Horse Sickness Virus and Epizootic Hemorrhagic Disease Virus

Although the infection of mice with ASHV-9 (PAKrrah/09) is not fatal in IFNAR(−/−) mice, clinical signs and viremia are present in the animals. The level of viremia was similar in animals infected with serotypes 4 and 9; however, the period of viremia was shorter when animals were infected with serotype 9 [68] . Recent studies to characterized AHSV serotype 3, revealed a higher virulence in the mouse model, since a low dose of 10 2 PFUs per mouse killed all animals (unpublished data). Studies comparing other serotypes showed that mice infected with AHSV-4 had significantly higher AHSV RNA levels than mice infected with AHSV-1, suggesting that AHSV1 represents a less virulent serotype [160] .
The IFNAR(−/−) mice are susceptible to the infection with EHDV, in a dose-dependent manner. Animals displayed clinical signs similar to those observed in BTV-infected IFNAR(−/−) mice with the exception of conjunctivitis. A dose of 5 × 10 5 PFUs killed all mice and they presented enlarged spleens and multiple necrotic foci in the liver as well as large amounts of EHDV RNA in spleen [69] . These are some of the organs where virus can be found in viremic deer (OIE 2014). More work is needed to continue characterizing aspects of the pathology of different serotypes of EHDV and to evaluate potential vaccines in the mouse model.

IFNAR(−/−) Mice

Type I IFNs are well known for their ability to directly induce an antiviral response within infected and surrounding cells, displaying autocrine and paracrine activities through the upregulation of molecules that can antagonize with multiple stages of virus replication, as the interferon stimulated genes (ISGs). Nearly all types of cells are capable of producing IFN-α/β, which are the best-defined and most broadly expressed type I IFNs; however, during the course of an infection, specialized immune cells known as plasmacytoid dendritic cells (pDCs) produce the vast majority of IFN-α [16] . As they are produced rather early on during an infection, type I IFNs are also essential for activating the antiviral innate immune response, such as natural killer (NK) cell effector functions [17] [18] [19] . Transcription of IFN genes is induced rapidly in response to viral infection. Cells sense viruses using multiple signaling pathways that ultimately will activate several transcription factors and their subsequent translocation into the nucleus, resulting in the activation of type I IFN (IFNα/β) genes. In WT mice, the released type I IFN is bound by the specific receptors IFNAR1/IFNAR2 trigging phosphorylation of JAK1/TYK2 kinases that activate STAT1 and STAT2. Phosphorylated STAT1/STAT2 heterodimers bind IRF9 and the complex is translocated to the nucleus where it induces expression of ISGs with ISRE-dependent promoters. The expression of ISGs will induce an antiviral state to prevent viral infection. However, in IFNAR1−/− mice, the antiviral state is not created, and cells are more susceptible to be infected. JAK , Janus activated kinase; TYK2, tyrosine kinase 2, ISRE, IFN-stimulated response element; ISG, IFN-stimulated gene; OAS, oligoadenylate synthetase; MX, myxovirus resistance; ISG15, IFN-stimulated gene factor 15; TRIM, tripartite motifcontaining proteins; IFITM, IFN-induced transmembrane proteins; IRF, IFN-regulatory factors; STAT, signal transducer and activator of transcription; NF-κ B, nuclear factor of kappa light polypeptide gene enhancer in B-cells.
In addition to type I IFN effects related to the antiviral state and innate immunity activation, the IFN system is linked to a variety of effector responses of the adaptive immune systems. Cytotoxic T cells (CTLs) are one of the two major effector cell populations regulated by type I IFNs (with NK). Type I IFNs have been shown to facilitate cross-presentation by DCs of viral antigens to CD8+ T cells [20] . The recruitment of cytotoxic cells to the site of infection mediated by chemokine production has been shown, as well as the induction of cytokines from CTLs that positively regulate cytotoxic cell populations and activities, as interleukin (IL)-15 type I IFN-induced production, which plays a critical role in proliferation and maintenance of NK cells and memory CD8+ T cells [19] . On the another hand, some reports showed how IFN-I exert an antiproliferative effect on anti-CD3-stimulated CD4 T cells during in vitro culture [21, 22] , but the opposite result is found when IFN-I produced in response to LCMV immunization act directly on virus-specific CD4 T cells, contributing to their clonal expansion [23] . The role of type I IFN production during apoptosis has also been studied. Given that viruses require host cell machinery to replicate, elimination of the infected cell would shut down this machinery, preventing viral spread. It is known that cells treated with type I IFNs sensitizes them to apoptosis upon subsequent viral infection, and some mechanisms of sensitization have been elucidated, such those that involve PKR or p53 [24, 25] . Type I IFNs are also important during neurotropic viral infections, where they play multifaceted roles at the blood brain barrier (BBB). Type I IFN treatment decreases BBB permeability, enhances tight junction (TJ) integrity, and restricts Transcription of IFN genes is induced rapidly in response to viral infection. Cells sense viruses using multiple signaling pathways that ultimately will activate several transcription factors and their subsequent translocation into the nucleus, resulting in the activation of type I IFN (IFNα/β) genes. In WT mice, the released type I IFN is bound by the specific receptors IFNAR1/IFNAR2 trigging phosphorylation of JAK1/TYK2 kinases that activate STAT1 and STAT2. Phosphorylated STAT1/STAT2 heterodimers bind IRF9 and the complex is translocated to the nucleus where it induces expression of ISGs with ISRE-dependent promoters. The expression of ISGs will induce an antiviral state to prevent viral infection. However, in IFNAR1−/− mice, the antiviral state is not created, and cells are more susceptible to be infected. JAK , Janus activated kinase; TYK2, tyrosine kinase 2, ISRE, IFN-stimulated response element; ISG, IFN-stimulated gene; OAS, oligoadenylate synthetase; MX, myxovirus resistance; ISG15, IFN-stimulated gene factor 15; TRIM, tripartite motif-containing proteins; IFITM, IFN-induced transmembrane proteins; IRF, IFN-regulatory factors; STAT, signal transducer and activator of transcription; NF-κ B, nuclear factor of kappa light polypeptide gene enhancer in B-cells.
In addition to type I IFN effects related to the antiviral state and innate immunity activation, the IFN system is linked to a variety of effector responses of the adaptive immune systems. Cytotoxic T cells (CTLs) are one of the two major effector cell populations regulated by type I IFNs (with NK). Type I IFNs have been shown to facilitate cross-presentation by DCs of viral antigens to CD8+ T cells [20] . The recruitment of cytotoxic cells to the site of infection mediated by chemokine production has been shown, as well as the induction of cytokines from CTLs that positively regulate cytotoxic cell populations and activities, as interleukin (IL)-15 type I IFN-induced production, which plays a critical role in proliferation and maintenance of NK cells and memory CD8+ T cells [19] . On the another hand, some reports showed how IFN-I exert an antiproliferative effect on anti-CD3-stimulated CD4 T cells during in vitro culture [21, 22] , but the opposite result is found when IFN-I produced in response to LCMV immunization act directly on virus-specific CD4 T cells, contributing to their clonal expansion [23] . The role of type I IFN production during apoptosis has also been studied. Given that viruses require host cell machinery to replicate, elimination of the infected cell would shut down this machinery, preventing viral spread. It is known that cells treated with type I IFNs sensitizes them to apoptosis upon subsequent viral infection, and some mechanisms of sensitization have been elucidated, such those that involve PKR or p53 [24, 25] . Type I IFNs are also important during neurotropic viral infections, where they play multifaceted roles at the blood brain barrier (BBB). Type I IFN treatment decreases BBB permeability, enhances tight junction (TJ) integrity, and restricts leukocyte migration across the BBB into the central nervous system (CNS) parenchyma [26] [27] [28] . The induction of type I IFN expression following detection of viral pathogens such as West Nile virus (WNV) acts directly on BBB endothelium to preserve the formation of TJ and limit BBB permeability, antagonizing with the effect promoted by Th1 cytokines also secreted during WNV infection [28] .

Schmallenberg Virus (Family Peribunyaviridae)

One non-zoonotic virus of this group with outbreak potential among domestic animals is Schmallenberg virus (SBV). The SBV causes congenital malformations and stillbirths in cattle, sheep, goat, and possibly in alpaca. Schmallenberg virus infection of susceptible pregnant animals can be associated with musculoskeletal and central nervous system malformations in stillborn or newborn lambs and calves [74] . Schmallenberg virus has spread throughout the European continent, spanning from Ireland to Turkey [75] , since its discovery in Germany in 2011, and it has been shown to be transmitted by biting midges. Its close relation to Akabane virus (AKAV) suggests that much of what is known about that virus might also be applicable to SBV. Akabane virus propagation in mice was first described in 1976 [76] , but it requires the intracranial injection of newborns. It has been shown that 2, 10, and 18-day old newborn NIH-Swiss mice intracerebrally inoculated with 400 plaque-forming units (PFUs) of SBV are also highly susceptible to the infection, with a 100% mortality rate, but this inoculation route does not resemble the natural route of infection [77] . In 2012, IFNAR(−/−) mice were shown to be susceptible to SBV infection, although clinical signs were not as evident. After SBV infection, mice showed primarily decreased weight loss, ataxia, apathy, but limited mortality [41] . In recent studies, it has been demonstrated that SBV virulence occurs as early as three days post-infection (dpi) and it becomes more severe at day six post-infection as observed by the significant weight loss and viremia [42] . The relation between type I IFN and viral spreading has been investigated using seve day old IFNAR(−/−) mice intracerebrally injected with SBV and an NSs deletion mutant, confirming the role of the NSs protein as a modulator, at least indirectly, of the IFN response in vivo [77] . This mouse model has been used to validate attenuated strains as potential vaccines [41] , and to test protective immunity induced by the Gc-ecto1 domain and nucleocapsid protein of the virus, showing that these could be valid candidates for the development of subunit vaccines [42] . Wernike et al. [43] have also demonstrated the suitability of using Gc as an efficient vaccine in this murine model.
Other viruses of this family with remarkable impact in human health and livestock have been studied using this murine model. For instance, severe fever with thrombocytopenia syndrome virus (SFTSV) (with case fatality rates up to 30% in humans), Bunyamwera virus (BUNV), Dugbe virus (DUBV), and the Simbu virus (SIMV) [43, [78] [79] [80] [81] , where the effect of the host IFN system and IFN-related genes on the outcome of infection, model suitability, and vaccination have been investigated in IFNAR(−/−) mice.

Family Orthomyxoviridae

Orthomyxoviridae is a family of enveloped viruses, generally rounded but that can be filamentous. Eight ssRNA segmented and negative-sense linear molecules compose its genome (13.5 Kb), which is encapsidated by a nucleoprotein (NP) constituted layer and encodes 11 proteins. They present a global distribution, are more common in winter, and they are characterized by causing an acute infection of the respiratory tract. Within this family are the genera Influenza virus (type A, B, C, and D), Thogotovirus, Isavirus, and Quaranjavirus, where Thogotovirus and some species of Quaranjavirus are the unique genus transmitted by arthropods (mainly ticks) within this family.
Thogoto virus (THOV), is the prototype of tick-transmitted orthomyxoviruses and shares structural and genetic similarities with its relative, influenza virus. In contrast to influenza virus infection, which is mediated via the respiratory system and thus acting locally, THOV, as a tick-mediated virus, is acting systemically. Moreover, for THOV but not influenza virus, mice are an important natural host [130, 131] . It has been shown that THOV induces type I IFN responses in several cell lines and mouse embryonic fibroblasts [132, 133] in vivo. Using the IFNAR(−/−) mice model, it has been possible to determine how THOV infection of mice leads to an unexpected strong and long-lasting mode of type I IFN expression that is most likely dominated by IPS-1-dependent IFN production of infected myeloid dendritic cells (mDC), but not plasmacytoid pDC cells [64] . Using replication-incompetent THOV-derived virus-like particles, the authors demonstrated that an infected host can use alternative pathways to induce type I IFN responses, independently of type I IFN receptor, induced by viral polymerase activity, but being largely independent of viral replication. This fact has an important relevance to understand how type I IFN can be produced in large amounts in specialized cell types independently of the IFNAR-dependent enhancement, broaden our view of host strategies to fight viral pathogens [63] .

Bluetongue Virus

Furthermore, this mouse model has been used to study the determinants of virulence of BTV field strains. Viruses were maintained in cell culture at low or high passage number and its virulence were evaluated in IFNAR(−/−) mice. The low passaged viruses BTV-2 and BTV-4 were lethal for mice, while the viruses that were extensively passaged become attenuated [141] . Interestingly, BTV-9 with a small number of passages were less pathogenic than the other strains tested, which correlates with the lower morbidity and mortality of this strain circulating in Italy in the early 2000s. Other studies compared the different degree of virulence in IFNAR(−/−) mice between a North European BTV-8 strain (BTV-8NET2006), that were highly virulent in the field, and a BTV-8 strain isolated in Italy in 2008 (BTV-8IT2008) that did not caused clinical signs. Experiments in mice reveal that mice inoculated with BTV-8NET2006 succumbed earlier to the infection than BTV-8IT2008 infected mice [142] . These data in a whole indicate that IFNAR(−/−) mice could be an adequate animal model to investigate the determinants of BTV virulence, factors of host interaction and pathogenesis.

Conclusions

For vaccine testing, the following animal model features are desirable: robust, reproducible viremia, immuno-competent, and pathology and clinical signs similar to those found in the host. Unfortunately, there is no model that fulfills all these criteria. The IFNAR(−/−) mice have defective innate immune responses, which can lead to limited adaptive immunity [20, [167] [168] [169] . In contrast, many studies have shown the viability of this model to test vaccines and to study the adaptive response induced by them. They were able to trigger strong humoral and cellular immune responses comparable with those achieved in the immunocompetent model, as it has been shown in this review for RVFV, CCHFV, DENV, ZIKV, CHICK, BTV or AHSV, where high levels of neutralizing antibodies that block the virions or cytotoxic CD8 T cell responses, able to clear the infection, were induced using different platforms (inactivated vaccines, attenuated-replication defective, subunit vaccines, DNA or viral vector based vaccines) and vaccination strategies (single dose, prime-boost).

Families Included in the Order Bunyavirales

This large and diverse group has been more formally organized (https://talk.ictvonline.org/ files/ictv_official_taxonomy_updates_since_the_8th_report/m/plant-official/6694). It comprises ten different families that include segmented negative strand virus species infecting plants, arthropods, and vertebrates. This group includes tri-segmented negative-strand RNA viruses, commonly known as bunyaviruses of which several members are important pathogens of animals and humans.
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Abstract

Hantaan virus (HTNV) and Puumala virus (PUUV) are rodent-borne hantaviruses that are the primary causes of hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia. The development of well characterized animal models of HTNV and PUUV infection is critical for the evaluation and the potential licensure of HFRS vaccines and therapeutics. In this study we present three animal models of HTNV infection (hamster, ferret and marmoset), and two animal models of PUUV infection (hamster, ferret). Infection of hamsters with a~3 times the infectious dose 99% (ID 99 ) of HTNV by the intramuscular and~1 ID 99 of HTNV by the intranasal route leads to a persistent asymptomatic infection, characterized by sporadic viremia and high levels of viral genome in the lung, brain and kidney. In contrast, infection of hamsters with~2 ID 99 of PUUV by the intramuscular or~1 ID 99 of PUUV by the intranasal route leads to seroconversion with no detectable viremia, and a transient detection of viral genome. Infection of ferrets with a high dose of either HTNV or PUUV by the intramuscular route leads to seroconversion and gradual weight loss, though kidney function remained unimpaired and serum viremia and viral dissemination to organs was not detected. In marmosets a 1,000 PFU HTNV intramuscular challenge led to robust seroconversion and neutralizing antibody production. Similarly to the ferret model of HTNV infection, no renal impairment, serum viremia or viral dissemination to organs was detected in marmosets. This is the first report of hantavirus infection in ferrets and marmosets.

Introduction

Ferrets (Mustela putorius furo) have become a popular animal model for a number of respiratory pathogens including influenza [12] , coronavirus [13] , Nipah virus [14] , and morbillivirus [15] , due to the similarity in lung physiology to humans. In addition, they have recently been described as a disease model of two hemorrhagic fever viruses, Bundibugyo virus and Ebola virus [16, 17] , supporting viral replication without prior adaptation. Most hantavirusrelated human disease occurs by aerosolized transmission of the virus from the excreta or secreta of infected rodents [18, 19] , a model of viral infection for which the ferret is well suited. In this study we demonstrate that ferrets are capable of being infected by high titers of HTNV and PUUV, though aside from gradual weight loss infected animals exhibit no clinical symptoms or impaired renal function.
Hantaviruses are negative-sense RNA viruses transmitted to humans from small animal hosts. Different viral species are associated with one of two disease syndromes: hemorrhagic fever with renal syndrome (HFRS), or hantavirus pulmonary syndrome (HPS) [1] . Hantaan virus a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 (HTNV), primarily found in Asia, is among the most prevalent HFRS-causing hantaviruses with a case fatality rate of between 1-15% [2] . Puumala virus (PUUV) causes most HFRS cases in Europe, though its case fatality rate is lower at <1% [3, 4] . There are currently no FDA licensed vaccines or therapeutics for either HFRS or HPS [5] .
The Syrian hamster (Mesocricetus auratus) is the typical animal used to model hantavirus infection and disease. Andes virus (ANDV), an HPS-causing hantavirus, causes lethal disease in immunocompetent hamsters [6] , while numerous other HPS-causing hantaviruses including Sin Nombre Virus (SNV) and Choclo virus cause lethal disease in hamsters immunosuppressed with dexamethasone and cyclophosphamide [7, 8] . In contrast to HPS-causing hantaviruses, exposure of hamsters to HFRS-causing hantaviruses such as HTNV, PUUV, Dobrava (DOBV) and Seoul (SEOV) leads to asymptomatic infection, despite viral dissemination, even when immunosuppressed (Hooper Lab, unpublished data) [8] [9] [10] [11] . In these studies hamsters were exposed to high doses of HTNV and PUUV, far exceeding the infectious dose 99% (ID 99 ) for the virus. Development and characterization of a uniformly infective, low-dose challenge model, enhances the hamster model's usefulness in vaccine and therapeutic testing. In this report we present a low-dose hamster infection model for both HTNV and PUUV infected animals.
It has been established that infection of rhesus macaques (Macaca mulatta) with HFRScausing hantaviruses (DOBV, SEOV, HTNV, and PUUV) leads to asymptomatic infection and seroconversion [9] , while infection of cynomolgus macaques (Macaca fascicularis) with PUUV leads to a mild disease characterized by lethargy, mild proteinuria and hematuria, and kidney pathology, similar to mild HFRS in humans [20] . However, the macaques' large size and cost limits their usefulness in therapeutic studies, especially when test article availability is limited, as is often the case in passive transfer studies. The common marmoset (Callithrix jacchus) is becoming more popular for infectious disease studies. Its genetic similarity to humans, cost, relative safety, and small size make it an attractive alternative to traditional non-human primate species [21] . Marmosets have been used as a disease model for other viral agents including Dengue virus [22] , Hepatitis C virus [23] , influenza virus [24] , Lassa fever virus [25] , orthopox viruses [26] [27] [28] , Rift Valley Fever virus [29] , Eastern Equine Encephalitis virus [30] , and filoviruses [31] . In this study we demonstrate that exposure of marmosets to HTNV leads to asymptomatic infection characterized by high levels of neutralizing antibodies. This is the first report of hantavirus infection in marmosets.
Medical countermeasures are products including biologics (e.g., vaccines and antibodies) and small molecule drugs that can be used to prevent or combat infectious disease outbreaks. This study presents three animal models of HTNV infection, and two models of PUUV infection that can be used to evaluate the efficacy of medical countermeasure that are intended to prevent or mitigate infection (e.g., vaccines) by these viruses through induction of sterile immunity.

Ferret sample size justification

When disease occurs independently in each of four ferrets with 50% probability, the experiment will have odds about 9:1 in favor of producing at least one diseased ferret. Conversely failure to observe any diseased ferret in a group of four will yield a 95% confidence interval extending from 0-50%. That is, with 95% confidence it will be admitted that the true disease rate may be 50% or less. For this reason groups of four ferrets were used for the experiments. For each experiment pre-sera from animals served as a negative control.

Calculation of ID 50 and ID 99 for hamster model of HTNV and PUUV infection

We have previously demonstrated that Syrian hamsters are capable of being infected by HFRScausing hantaviruses, but they do not develop any signs of clinical disease [9] . To develop standard models of HTNV and PUUV infection for future evaluation of vaccines and medical countermeasures, groups of between 10 and 20 hamsters were exposed to serial ten-fold dilutions of either HTNV or PUUV by either the i.m. or i.n. route (from 2-20,000 PFU HTNV or 0.2-20,000 PFU PUUV). Between 28-35 days post infection, hamsters were terminally bled with infection status monitored by N-ELISA titers (Fig 1) . From these data the doses required to infect 50% (ID 50 ) and 99% (ID 99 ) were calculated ( Table 1 ).

Marmoset infection model

Despite not exhibiting clinical signs of disease, the model's robust antibody response (as measured by PRNT, PsVNA and N-ELISA) make it a useful tool for evaluating vaccines and pre-or post-exposure therapeutics.

Conclusion

This paper has explored the use of three laboratory animal species as possible infection and disease models for HFRS-causing hantaviruses: the hamster, the ferret, and the marmoset. These models, especially the hamster model and marmoset model, will be useful for evaluating medical countermeasures with the potential to induce sterile immunity. The marmosets should be particularly useful for the evaluation of passively transferred protective human antibodies because of the relative genetic similarities between species in the Order Primates, and the small size of marmosets, allowing testing with smaller volumes of material than would be required for larger species such as macaques. . Heart, lung, liver, spleen, kidney, and intestine were collected and assayed by plaque assay for the presence of infectious virus (A). To confirm lack of virus recovered was not due to inhibitors, virus was spiked into serial dilutions of organ homogenate to confirm no inhibitor was present (B). For a standard plaque assay the limit of detection, 1.7 log 10 , is depicted as a dashed line in (A). In (B) the dashed line is amount of HTNV plaques obtained when spiked into media rather than organ homogenate. (TIF) S10 Fig. Immunosuppression of uninfected ferrets leads to rapid weight loss and secondary bacterial infection. Uninfected ferrets were administered 10mg/kg Cyp, and the antibiotic enrofloxicin, according to the schedule in (A). Weight (B) and temperature (C) are shown. b.i.d indicates antibody was administered twice daily, and q.d indicates antibiotic was administered daily. (TIF) S11 Fig. PUUV infected ferrets had no infectious virus in the organs. Ferrets were infected with 94,000 PFU PUUV Beaumont i.m. Heart, lung, liver, spleen, kidney, intestine, brain, eye, and adrenal gland were collected on day 35 post infection and assayed for infectious virus by plaque assay (A). Virus was spiked in to confirm no inhi bitor was present (B). For a standard plaque assay the limit of detection, 1.7 log 10 , is depicted as a dashed line in (A). In (B) the mean ± SEM is depicted in all spiked groups and the dashed line is amount of HTNV plaques obtained when spiked into media rather than organ homogenate. To confirm no inhibitors were present, virus was spiked into samples (B). For a standard plaque assay the limit of detection, 1.7 log 10 , is depicted as a dashed line in (A). In (B) the mean ± SEM is displayed for all spiked groups, and the dashed line is amount of HTNV plaques obtained when spiked into media rather than organ homogenate. (TIF)

Refinement of low-dose HTNV and PUUV infection hamster models

No infectious virus was detected in the urine for any hamster tested, even those that were RT-PCR positive for HTNV viral genome. In human ANDV infected patients with ANDV antigen positive urine, samples had to be cultured in Vero E6 cells for between 16-22 days post infection before infectious virus was detected [44] . Patients infected with HFRS hantaviruses exhibit leukocytosis and thrombocytopenia during infection [45, 46] . At every time point post infection, EDTA-treated whole blood from HTNV and PUUV exposed hamsters was evaluated to determine if changes in white blood cell count or platelets occurred. No changes were observed (S4 Fig). To further characterize disease, tissue sections from HTNV i.m. infected hamsters were analyzed by IHC and H&E to assess viral localization and any pathologic changes (S5 Fig). No significant histopathological findings were noted in the kidney or brain. Splenic follicular lymphoid hyperplasia and hepatic extramedullary hematopoiesis in the liver were each noted in three hamsters. Both are seen in animals from later time points in the study (days 17-28) and likely represent a reaction to HTNV infection, though other unidentified antigenic stimuli cannot be ruled out. Five hamsters between days 4 and 17 post infection exhibited a minimal to mild inflammation of the pericardium, characterized by a mixed lymphoplasmacytic histiocytic and neutrophilic infiltrate. Occasional macrophages in the inflammatory infiltrate within the pericardium are immunopositive suggesting a possible association with HTNV; however, no evidence of cardiac tissue injury is associated with the presence of HTNV antigen. Beginning on Day 4 post infection 66% (12/18) hamsters exhibit minimal (likely subclinical) respiratory lesions consisting of interstitial neutrophilic and histiocytic infiltrates in the lungs, with 44% (8/18) also exhibiting minimal amounts of alveolar edema. An additional hamster had minimal alveolar edema but not pulmonary infiltrates. Such findings suggest a response to antigenic stimulus and the presence of immunopositive endothelial cells, pneumocytes and alveolar macrophages suggest a response to HTNV infection.

Discussion

To date, the use of adult animal models to evaluate anti-HTNV and anti-PUUV medical counter measures has been limited. Recombinant protein, vaccinia virus-vectored, and DNA vaccines have been tested in the high dose HTNV hamster model [9, 37, 50] . Additionally, the ability of passively administered neutralizing antibodies has been evaluated in the high-dose hamster model of HTNV and PUUV and in PUUV challenged cynomolgus macaques PUUV [50, 51] . Both of these models have limitations; the size of the macaque requires large quantities of passive transfer material, and the high dose of the hamster model, with challenge doses of~650 ID 99 , could require prohibitively large volumes of test article to neutralize the high initial dose. Suckling mice, which present with a disseminated disease not reminiscent of HFRS, have been used to evaluate HTNV therapeutics as well [52] [53] [54] [55] . In this paper we present three adult animal models of HFRS-causing hantavirus infection than can be used for future evaluation of therapeutics, biologics and vaccines.

Ferret infection model

The ferret has been used as an experimental model for numerous hemorrhagic fever viruses, and respiratory viruses [12] [13] [14] [15] [16] [17] , though no published reports exist examining its susceptibility to hantavirus infection. In comparison to the hamster, the ferret is far more resistant to HTNV and PUUV infection. Exposure of ferrets to 2,000 PFU i.n. (greater than the ID 99 for both viruses in hamsters), failed to result in a productive infection and seroconversion. Instead i.m. challenge doses of~100,000-200,000 PFU were needed (Fig 5) . Initially ferrets were exposed to PUUV K27, a commonly used laboratory isolate that has been in cell culture for over 15 years. Repeated passaging of hantavirus is known to cause mutations [63] [64] [65] . In contrast PUUV Beaumont and Seloignes are relatively recent isolates, with no more than 3 passages in cell culture post isolation. These strains were used for all subsequent experiments to maximize the likelihood of PUUV to cause disease by eliminating possible attenuation of the laboratory strain of the virus. Despite the high challenge dose and use of recent isolates, no elevation in white blood cells was observed over the course of the experiment, no pathology or organ burden was detected at the conclusion of the experiment, and the N-ELISA specific OD sum, PRNT 50 , and PsVNA 50 titers remained low (Figs 6 and 8 and S9 Fig) . This outcome is almost identical to that of Marburg and Ravn virus infection in ferrets, where the development of neutralizing antibody titers was the only sign that productive infection occurred [66] . Previous experiments in hamsters, have demonstrated that 2x10 5 PFU of gamma-irradiated ANDV and SNV are not sufficient to cause seroconversion, and neither is 1x10 4 gamma-irradiated PFU PUUV [6, 57] . Thus the seroconversion observed in ferrets, though low, is not likely to be due to a reaction to the large quantity of antigen administered, but to a productive infection.
The lack of detectable virus is most surprising given the gradual weight loss infected animals exhibit (Figs 5 and 6 ). The animals gained weight until~3 days post challenge, at which point a gradual weight loss occurs, regardless of if HTNV or PUUV was the challenge virus. While weight loss is a feature of other ferret models of infectious diseases, the pattern we observed was unique: ferrets infected with morbillivirus, avian influenza and filoviruses rapidly lose weight during the first week to two weeks post infection, while infection with severe acute respiratory syndrome virus results in no significant weight loss [16, 17, [75] [76] [77] .

Plaque Reduction Neutralization test (PRNT)

PRNT assays were performed as previously described with minor modifications [35-37]. HTNV-infected monolayers were fixed 7 days post-infection, while PUUV-infected monolayers were fixed 10 days post infection by 2 mL of 10% formalin per well. Immunostaining was performed as previously described [38] . All sera samples were assayed in duplicate beginning at a 1:20 final dilution. PRNT 50 values represent the reciprocal dilution at which the serum neutralizes 50% of the virus.

Refinement of low-dose HTNV and PUUV infection hamster models

To further characterize a low-dose standard hamster infection model for HFRS-causing hantaviruses a hamster serial sacrifice study was performed. Hamsters were challenged with either 10 PFU (~3 ID 99 ) HTNV i.m., 500 PFU (~1.5 ID 99 ) HTNV i.n., 1,000 PFU (~1 ID 99 ) PUUV i.m., or 1,000 PFU (~1.5 ID 99 ) PUUV i.n. On various days post infection, groups of three hamsters were euthanized to monitor viral and serological parameters.
Seroconversion occurred, at least partially, by Day 17 post HTNV infection and Day 24 post PUUV infection. Seroconversion on Day 28 post HTNV i.m. infection was incomplete, though viral genome was recovered from all hamsters euthanized that day indicating a productive HTNV infection occurred (Fig 2) . To confirm seroconversion, all hamsters euthanized on days 17, 24, and 28 were assayed for neutralizing antibodies by PRNT regardless of N-ELISA seroconversion status. All HTNV infected hamsters with N-ELISA titers had neutralizing antibodies as measured by PRNT 50 , with all but one having full neutralization of virus at a 1:20 dilution of sera. (S1A and S1B Fig) . Similarly, all PUUV hamsters with N-ELISA titers had neutralizing antibodies as measured by PRNT 50 , though three of the five PUUV i.n. challenged hamsters did not have complete neutralization of the virus at a 1:20 dilution of sera (S1C and S1D Fig) . Two of three PUUV i.m. challenged hamsters that had not seroconverted on Day 17 post infection had low levels of neutralizing antibodies, while none of the PUUV i.n. challenged hamsters that were seronegative by N-ELISA had neutralizing antibodies. For both HTNV and PUUV infected hamsters, infection via the i.m. route lead to a more robust neutralizing antibody response than then i.n.route.
The kinetics of HTNV and PUUV infection in the heart, lung, liver, spleen, kidney, and brain were monitored by both RT-PCR and plaque assay (Figs 3 and 4) . HTNV infected by either the i.m. or i.n. route resulted in a persistent infection. High levels of viral genome were detected in the heart, lung, kidney and brain of HTNV infected hamsters beginning on either Day 11 or 17 post infection (Fig 3A, 3B , 3E and 3F). Infection of the kidney and brain was found in all examined hamsters beginning on either Day 17 or 24 post infection, while the high titers of viral genome detected in the heart and lung were present in only one or two hamsters at each time point. Low levels of viral genome were detected between days 11 and 24 post infection in the liver of HTNV i.m. but not i.n. infected hamsters (Fig 3C) . Hardly any viral genome was detected in the spleen (Fig 3D) . To confirm the lack of viral genome in the spleen was not due to the presence of inhibitors all spleen samples from i.m. and i.n. infected hamsters were spiked with HTNV prior to RT-PCR. No significant inhibition of the spiked RNA was noted, indicating that HTNV infection does not result in virus dissemination to the spleen (S2A and S2B Fig) .
The detection of PUUV in organs was transient after i.m. infection, occurring between Day 11-17 for the heart, liver, kidney and brain, with no virus detected in the spleen (Fig 3A, 3C , 3E and 3F). Viral genome was more persistent in the lung where it was detected in at least one of three hamsters on/after Day 11 post infection (Fig 3B) . A small amount of viral genome detected in the brain of a PUUV i.n. infected hamster, 28 days post challenge, is the only viral genome detected in any organ at any time point post PUUV i.n. infection (Fig 3F) . Serum viremia was detected in hamsters challenged with HTNV i.m. between 11 and 28 days post infection, though the presence of virus was sporadic except for Day 24. Serum viremia was only detected in two hamsters challenged with HTNV i.n., one on Day 11 and one on Day 17. No serum viremia was detected in hamsters challenged with PUUV by either route (Fig 3G) . Similarly, the presence of viral genome was sporadically detected in the urine of Three HFRS-associated hantavirus infection models HTNV infected hamsters between days 17-28, but was not detected in PUUV challenged hamsters ( Fig 3H) .
In HTNV i.m. infected hamsters, infectious virus was first detected in the liver beginning at Day 11, and in the lung, liver, spleen, and kidney at Day 17 post infection (Fig 4B, 4C, 4D and 4E). With the exception of the kidney, in which infectious virus was recovered from every hamster after 17 days post infection, infectious virus was recovered from the lung, liver, brain and spleen in only a portion of hamsters at each time point. No infectious virus was detected in the heart at any time point (Fig 4A) . Virus recovery from HTNV i.n. infected hamsters was markedly lower, with infectious virus being detected only in the kidney on Day17 and 28 post Three HFRS-associated hantavirus infection models infection (Fig 4E) . No infectious virus was recovered from the organ of any PUUV infected hamster (Fig 4) .

Ferret model of HTNV and PUUV infection

No published studies detail if ferrets are susceptible to hantavirus infection. To examine this, four ferrets were exposed to either 2,000 PFU HTNV or PUUV K27 i.n. No seroconversion occurred within 35 days post infection. The same animals were re-exposed to either 200,000 PFU HTNV, 94,000 PFU PUUV Beaumont (a human PUUV isolate) or 164,000 PFU PUUV Seloignes (a vole PUUV isolate) by i.m. Prior to the re-exposure one of the seronegative ferrets in the HTNV group was removed for health concerns (rapid weight loss) unrelated to the study, and it's cage mate was subsequently removed for behavioral health reasons before the completion of the study. Data from those two ferrets are not shown. As soon as three days post infection ferrets began to lose weight with HTNV infected ferrets losing between 5-12% of peak body weight as did PUUV infected ferrets (Fig 5A-5C ). By Day 35 post infection all animals had developed antibodies against all strains of the virus as measured by N-ELISA assay (Fig 5D-5F ). Neutralizing antibody development began as early as Day 14 post infection and all ferrets developed neutralizing antibodies by Day 28 post infection (Fig 5G-5L) . EDTA The organs of infected ferrets were analyzed for viral load by RT-PCR and for the presence of infectious virus by plaque assay. The lung, liver, spleen, intestine and urine of HTNV infected ferrets were negative for viral genome, with small amounts (<2 log 10 ) detected in the heart and spleen of a single animal (S7B Fig). The heart, lung, kidney, intestine and urine of PUUV infected ferrets were negative for viral genome, though small amounts were detected in the liver (2/4) and spleen (1/4) of PUUV infected ferrets (S7C and S7D Fig) . Viral genome was spiked into the assay to confirm that the lack of signal was not due to the presence of inhibitors. All organs except for the intestine (4/6) had no inhibition of RT-PCR product. Similarly, no infectious virus was found in the organ of any ferret by plaque assay, despite spiked-in virus exhibiting no significant inhibition (S9 Fig). To confirm that immunosuppression of uninfected ferrets did not result in rapid weight loss, four healthy ferrets were immunosuppressed with a loading dose of 30 mg/kg Cyp followed by 10 mg/kg Cyp maintenance doses every other day (S10A Fig). Five days post immunosuppression ferrets exhibited rapid weight loss, fever, and lethargy due to secondary infection (S10B & S10C Fig) . Immunosupression was discontinued and 5 mg/kg enrofloxicin (a broad spectrum antibiotic) was given twice daily for a week. During this time, ferrets began to gain weight and their fever diminished. On Day 11 immunosupression resumed for two doses with prophylactic enrofloxicin given once daily. Even with prophylactic antibiotics two ferrets spiked fevers within a few days post the second round of immunosuppressive treatment, though they did not lose weight (S10B & S10C Fig) . Based on these results the rapid weight loss and clinical signs observed upon immunosuppression of HTNV and PUUV infected ferrets was most likely due to secondary infection. Due to the inability to completely manage secondary infection with prophylactic antibiotic treatment, no further immunosuppression studies were carried out in ferrets.
To refine the HFRS-causing hantavirus ferret infection model four ferrets were challenged with 94,000 PFU PUUV Beaumont i.m. on Day 0. Weight and temperature were recorded daily, while twice weekly blood draws and urine collection was used to monitor kidney function. As with the pilot experiment, ferrets slowly lost between 7-11% of peak body weight, recapitulating our previous findings (Fig 6A) . No elevated temperatures were observed (Fig 6B) . Ferrets developed a robust antibody response beginning on Day 10 post infection (Fig 6C and 6D) . Neutralizing antibodies developed early as Day 14 post infection, and were present in all ferrets by Day 28 post infection, as measured by PsVNA and PRNT ( Fig 6E and 6F) .
Despite the slow weight loss no signs of renal impairment were observed. Proteinuria and hematuria are hallmarks of PUUV infection occurring in between 94-100% (proteinuria) and 58-85% (hematuria) of human clinical cases [46] . No prolonged proteinuria or hematuria was observed (Fig 7A and 7B ) in infected ferrets. Similarly, blood urea nitrogen and creatinine levels in the sera, both of which are elevated due to kidney failure in PUUV patients [47] remained unchanged in PUUV infected ferrets (Fig 7C and 7D) [48, 49] . No infectious virus or viral genome was detected in the brain, heart, lung, liver, spleen, kidney, intestine, or eye (S11 and S12 Figs). No changes in other serologic or urologic parameters were noted (S13 and S14 Figs).
No gross pathological changes or significant lesions associated with PUUV infection were noted in the ferrets (S15 Fig). In the lungs, one ferret had mild neutrophilic and histiocytic inflammation centered on the bronchioles and expanded alveolar septa. Given that the inflammation was centered around bronchioles and not the vasculature, it is unlikely to be in response to PUUV infection. A number of other common or age-associated lesions were observed in the ferrets. Two of four ferrets had proliferative cortical cells in either the adrenal capsule or adrenal cortex that were likely clinically silent as they lack clinical signs consistent with adrenal-associated endocrinopathy. Additionally, alveolar mineralization was noted in all four ferrets as was eosinophilic and lymphoplasmacytic enteritis, and hepatitis. Two ferrets had fibromyxomatous degeneration of the atrioventricular valve. The spleen, brain, kidney, and pituitary gland were normal in all ferrets examined.

Marmoset model of HTNV infection

As with ferrets, there is no literature on the susceptibility of marmosets to hantavirus infection. To test this, three male marmosets were exposed to 1,000 PFU HTNV i.m. Blood was collected weekly to measure seroconversion, serum viremia, as well as serum parameters relating to renal function. All three animals seroconverted by Day 21 post infection (Fig 8A and 8B ) despite displaying no clinical signs of illness. Neutralizing antibody production began around the same time, and was robust by Day 30 post infection (14, 557 by PsVNA 50 and 10,240-20,480 by PRNT 50 ) (Fig 8C and 8D ). Due to low volumes of blood drawn at each time point serum viremia could not be examined for each individual animal, however a pool of sera from all three animals was evaluated at each time point post infection. Low levels of serum viremia were detected between days 14 and 28 post infection by RT-PCR ( Fig 8G) . As with the ferret infection model of HFRS-causing hantavirus, no renal injury as measured by changes noted in blood urea nitrogen or serum creatinine were observed over the course of infection, nor were changes in any of the other serum parameters monitored (S16 Fig). Animals were euthanized on Day 30 post infection to examine organs for viral dissemination. No infectious virus or viral genome was detected in the heart, lung, liver, spleen, kidney, intestine or brain (S17 and S18 Figs). Lymphoid hyperplasia was noted in all three animals by histology, though the location and intensity varied between the spleen, various lymph nodes and gut associated lymphoid tissue. Such hyperplasia is indicative of a response to antigenic stimulation and was most likely caused by the viral challenge. Mild to moderate congestion Three HFRS-associated hantavirus infection models was also seen in the lungs of each marmoset; however, this was an acute change and was most likely associated with terminal anesthesia and euthanasia. No other significant histological lesions were noted in any of the three animals (S19 Fig). Due to the negative virology results and the lack of significant histologic changes, IHC to detect the presence of HTNV antigen in tissues was not performed.

Low-dose hamster model of HFRS-hantavirus infection

Lethal infection of hamsters with ANDV leads to extensive organ dissemination, with infectious virus recovered in the lung, liver, kidney, spleen and heart [6] . Asymptomatic infection of hamsters with SNV repeatedly passaged through hamsters has a similar organ distribution [58] . The organ distribution of both those viruses is similar to the low dose HTNV hamster model, with two notable exceptions. First, the HTNV model has low, transient levels of virus in the liver and hardly any virus in the spleen (Figs 3 and 4) . This dissimilarity between the models led us to confirm the lack of virus in the spleen was not due to the presence of inhibitors by spiking either infectious virus or viral RNA into samples prior to evaluation (S2 Fig). Second, in the ANDV model, the presence of virus was determined by plaque assay, indicating the virus was infectious and replication competent. In the HTNV low dose hamster model, while there is detection of high levels of viral genome (and in the case of the HTNV i.m. model, by pathology) by RT-PCR, recovery of infectious virus is sporadic, typically occurring at low levels, in only a few hamsters per time point (Figs 3 and 4 , Table 1 ). The discrepancy between RT-PCR/pathology and plaque assay is notable. The hamsters in this study were not perfused, and given the appearance of neutralizing antibodies as early as day XX post infection, the presence of these antibodies could be impairing out ability to recover live virus via the plaque assay. It is also possible that viral packaging is somehow impaired in the hamster, leading to a larger amounts of nucleocapsid protein and viral genome than live virus. Further studies need to be undertaken to elucidate.
The low dose HTNV hamster model also mimics the infection pattern of the virus in its host species, the striped field mouse (Apodemus agrarius), with viral genome being detected in the lung, liver and kidney but not the spleen (the heart and brain were not examined) [32, [59] [60] [61] .
In contrast to the HTNV low dose model, the organ distribution of virus in the PUUV low dose model is transient. In PUUV i.m. infected hamsters virus is detectable by RT-PCR around Day 11 post infection, with the virus being cleared from all organs except the lung by Day 24. No infectious virus was recovered at any time point examined. Even less virus was detected in the PUUV i.n. model; only a small amount in the brain of one hamster on Day 28 post infection. In neither case was serum viremia observed (Figs 3 and 4) . This is most similar to the SNV models involving low passage virus in immunocompetent hamsters: the virus is transiently detected in the lung by PCR, and then sporadically found in organs 12 to 14 days post infection using immunohistochemistry [41, 58] . The distribution of PUUV in the hamster differs somewhat from its host species the bank vole, where it is found to persist in the lung, spleen and kidney, and was not detected in the heart or the brain [62] .
Seroconversion of hamsters post viral exposure remains the best way to measure infection, and should be a considered the primary endpoint for efficacy studies. Though the PRNT assay was slightly more sensitive than the N-ELISA assay, detecting neutralizing antibodies in all animals with N-ELISA titers, and in two PUUV animals that did not have N-ELISA titers, the increased time, sample, and biosafety requirements necessary for a PRNT assay make the N-ELISA a better choice (Fig 2, S1 Fig) . For a 10 PFU HTNV i.m. challenge, given lower titer and specific OD sum values as compared with higher challenge doses, and the fact that one hamster with significant viral genome in its organs at Day 28 post infection (7.1 log 10 in the brain, and 6.8 log 10 in the kidney) did not seroconvert, waiting until Day 35 post infection to monitor seroconversion is advisable (Figs 1-3 ). Viral load in the brain, kidney, and lungs as measured by RT-PCR need to be evaluated at Day 35 post low-dose challenge to determine their usefulness as secondary endpoints. Recovery of infectious virus in any organ, and viremia are too sporadic to serve as proxy markers for infection.

Ferret infection model

In the hamster, infection with SNV is asymptomatic unless the hamster is immunosuppressed. When ferrets were immunosuppressed on Day 42 post infection, rapid weight loss and lethargy ensured (Fig 5) . Given that these clinical signs were also observed in unchallenged control animals (S10 Fig) , this could likely be the result of a secondary infection or drug toxicity. The use of Cyp is well documented in ferrets, primarily given at a high dose as an emetic, and no dosage for long term immunosuppression was found [78] . The dosages used in this study (between 10-30 mg/kg) successfully reduced white blood cell levels, in ferrets and demonstrate that Cyp can be used to induce long-term immunosuppression, if antibiotics are given to control for secondary infection (S8 Fig) .

Marmoset infection model

In this study we have demonstrated the susceptibility of marmosets to HTNV infection. Marmosets represent an attractive model for testing vaccines and therapeutics against HFRS-causing hantaviruses due to genetic similarities to humans and small size. Also, the model has a simple read-out of infection, i.e. robust antibody production as measured by N-ELISA and PsVNA, making the determination of protection by vaccine or passive transfer material, straightforward. Further optimization of the model, namely to determine the ID 99 , could prove important as a 1,000 PFU challenge dose could be excessively high and prohibit therapeutic effects of candidate medical countermeasures.
Overall the marmoset model is more similar to the ferret HFRS-causing hantavirus infection model than the hamster, though there are key differences. Like the ferret, no significant pathological abnormalities were noted, and no signs of renal failure were observed (S16 and S19 Figs). Serum chemistry values do not differ from the normal range with the exception of albumin, total bilirubin, and amylase. While these values fell outside the normal range, they did not change over the course of infection indicating the problem may lie in the reference values used. The Piccolo general chemistry panel used to evaluate the parameters is optimized for human testing, and therefore may be less than optimal for evaluating the marmoset, especially those parameters. Additionally, no infectious virus or viral genome was recovered from any organ at Day 30 post infection [79, 80] . This is not surprising, given the high levels of neutralizing antibodies present as early as 21 days post infection (Fig 8) . Unlike the ferret, however, marmosets develop exceptionally high neutralizing antibody titers (10,240-20,480 by PRNT 50 and 14,866-221,557 by PsVNA 50 ), and display low-level serum viremia between two and four weeks post infection (Fig 8) . The serum viremia is significantly lower than in hamsters infected with HTNV, where some animals displayed RT-PCR titers of >7log 10 , and in hamsters infected with ANDV, where infectious virus titers prior to death are > 6log 10 [41].

Hamster procedures

confidence intervals reaching out to 0.7log 10 (approximately 5-fold) under monotome assumptions of response profiles for intermediate doses. As our initial selection of dosages did not meet the desired infection rate span, some dosage groups were repeated leading to 20 hamsters per group. In the serial euthanasia study three hamsters per group were used. This is the minimum number required to provide collection of sufficient samples for detection of antibodies and viral kinetics in tissues. For each experiment pre-sera from animals served as negative control.

Cell culture amplification of infectious virus from urine

T-25 flasks of one week old Vero E6 cells were infected with 50 μL of urine plus an additional 450 μL of cEMEM media. After a 1 hr adsorption at 37˚C with 5% CO 2 , the volume was raised to 3.5 mL. On Day 4 post infection supernatant was collected and frozen down, 500 μL of which was used to infected fresh Vero E6 cells at a later time point. After a 1 hr adsorption at 37˚C with 5% CO 2 , the volume was raised to 3.5 mL. On days 7, 11, 14, 17, 21 and 28 1.2 mL of culture supernatant was collected and frozen down. The volume of cEMEM in the flask was raised to 3.5 mL with fresh media.

Plaque assay

Approximately 200 μg of organ tissue were homogenized in 1 mL of cEMEM media using M tubes on the gentleMACS dissociation system on the RNA setting. Plaque assays using urine, sera, or organ homogenate were then performed beginning at the 1:10 dilution as described in [6, 41] with minor modifications. For spiked plaque assays the protocol was identical except for equivalent amounts of virus being spiked into either media alone (control), or the 1:10-1:1,000 dilution of organ homogenate. HTNV-infected monolayers were fixed 7 days postinfection, while PUUV-infected monolayers were fixed 10 days post infection by 2 mL of 10% formalin per well. Immunostaining was performed as previously described [38] .

Immunosuppression with cyclophosphamide (Cyp)

On the indicated days, anesthetized ferrets were injected i.p. with water soluble Cyp (Baxter Health Care Corporation, Deerfield, IL) with the indicated dosages per body weight of drug diluted in sterile phosphate-buffered saline (PBS), pH 7.4. In the first experiment ferrets were administered a loading dose of 30 mg/kg on Day 41 post infection, with maintenance doses of 30 mg/kg administered every other day until euthanasia. In the second experiment, ferrets were administered a loading dose of 30 mg/kg on Day -1, and a maintenance dose of 10 mg/kg on Day 1, 3, 11, and 13. Administration of Cyp was discontinued between days 3 and 11 due to secondary infection. To combat the infection (rapid onset of fever and weight loss), ferrets were treated with 5mg/kg i.m. enrofloxicin(Norbrook Laboratories, Overland Park, KS) twice daily per veterinarian instructions starting on Day 4. Beginning on Day 11 with the resumption of immunosuppression ferrets were treated prophylactically with 10 mg/kg i.m. enrofloxicin once daily).

Refinement of low-dose HTNV and PUUV infection hamster models

To confirm that the lack of infectious virus in the spleen was not due to the presence of inhibitors select samples were spiked with HTNV virus prior to a plaque assay. No significant inhibition of the spiked RNA was noted, confirming the RT-PCR results that that HTNV infection does not result in viral dissemination to the spleen (S2C and S2D Fig) .

Low-dose hamster model of HFRS-hantavirus infection

The ID 50 's for HTNV and PUUV determined in this report are similar to the lethal dose 50% (LD 50 ) calculated for SNV and ANDV, <3 PFU via the i.m. route [6, 7, 41] . Also similar is that the challenge dose of ANDV required to infect/kill 50% of hamsters by the i.n. or intragastric route is~10-30 fold higher [56] . Previous reports also demonstrate that PUUV is capable of infecting hamsters by the intragastric route, with an ID 50 of >10,000, making it a much less effective route of infection [57] . Despite similarities in their ID 50 's, the ID 99 's of HTNV and PUUV greatly diverge, with~200 times less HTNV required to infect hamsters via the i.m. route, and~2 fold less required to infect via the i.n. route ( Table 1 ). The lower ID 99 doses, coupled with viral persistence in HTNV infected animals as opposed to viral clearance (Fig 3) , suggest that HTNV is more infectious than PUUV in the hamster. The mechanism for this difference between these closely related hantaviruses remains unknown.

Ferret infection model

PUUV antigen, viral genome, or infectious virus has been found in the brain, pituitary gland, lung, heart, liver, kidney, spleen, cerebrospinal fluid, and gastrointestinal tract of human patients with clinical symptoms of NE, though the pattern of viral dissemination varies between individuals [67] [68] [69] [70] . Acute kidney injury and vision disturbances including blurred vision, myopic shift, and lens thickening, while pulmonary involvement including pleural effusion and vascular congestion, and renal failure occurred less frequently [47, [71] [72] [73] [74] . Given the lack of high neutralizing antibody titers, which could have aided in viral clearance, the lack of viral genome in any of the ferret organs examined is rather surprising (S6, S7, S11 and S12 Figs). Furthermore, the lack of viral antigen and pathology in the organs tested suggest either a transient infection cleared prior to Day 35 post infection, levels of virus so low as to be undetectable by the tests used, or a viral reservoir outside of the organs tested.
Though susceptible to both HTNV and PUUV, the ferret has limited usefulness for studies involving medical countermeasure efficacy testing. The ferret's large mass, even as an adolescent, makes the amount of test article needed also prohibitively large. The large challenge dose required for infection could potentially obscure the protective effect of drugs of vaccines, due to the overwhelming amount of virus administered. Moreover, although the animals are infected the resultant neutralizing antibody titers are small, resulting in potential sensitivity issues with the model. Husbandry and handling of the animals under ABSL-3 procedures is also substantially more difficult than hamsters, and they lack the genetic similarity to humans that marmosets possess.

Hamster procedures

Female Syrian hamsters 6-8 wks of age (Envigo, Indianapolis, IN) were anesthetized by inhalation of vaporized isoflurane using an IMPAC 6 veterinary anesthesia machine. Once anesthetized, animals were injected with the indicated concentration of virus diluted in PBS. Intramuscular (i.m.) injections (in the caudal thigh) consisted of 0.2 ml delivered with a 1ml syringe with a 25-gauge, 5/8 in needle. Intranasal (i.n.) instillation consisted of 50 μl total volume delivered as 25 μl per nare with a plastic pipette tip. Blood sampling from the vena cava occurred under previously stated methods of anesthesia, and was limited to 7% of a hamster's total blood volume per week. At time of arrival animals were randomized into experimental groups. Animals were housed in small animal pans, not exceeding four animals to a pan, in a climate and humidity controlled animal biosafety level 3 (ABSL-3) with a 12-hour light/dark cycle. Animals had pelleted food and water provided ad libitum. Enrichment in the form of toys, nesting material and supplemental treats was provided. Humane endpoint conditions were established as decreased mobility (inability to obtain food and water) and subdued response to stimulation, and animals were monitored daily during the experiment. As infected animals did not become ill, these criteria were not met and animals were euthanized by terminal blood collection from the heart after administration of Ketamine-acepromazine-xylazine (KAX)(USAMRIID, Fort Detrick MD) and prior to intracardiac injection with pentobarbital sodium (USAMRIID) at the end of the study. Due to lack of illness no pain relief, aside from anesthesia during procedures, was required.

Ferret procedures

Adult, female neutered and descented ferrets (Marshall Farms, North Rose, NY), were anesthetized by inhalation of vaporized isoflurane using an IMPAC 6 veterinary anesthesia machine, or i.m. injection of Telazol (Zoetis, Parsippany, NJ). Injections (i.m. and i.n.) and blood sampling were conducted under the same condition as hamsters. Intraperitoneal (i.p.) injections consisted of 1 ml delivered with a 3 mL syringe and a 23-guage needle. Microchips (BMDS, Seaford, DE) were used to identify and ascertain temperature during ferret experiments. In the first ferret challenge study faulty chips lead to inaccurate temperature readings and were only used for identification purposes. Animals were randomized upon receipt into experimental groups. Ferrets were socially housed in metal caging, two to a cage, with sight lines to additional animals in the study, in a climate and humidity controlled ABSL-3 with 12/hour light and dark cycles. Each cage had a nesting box with bedding material, and numerous tubes and shelfs for play. Ferrets had access to pelleted food supplemented with treats and potable water, through an automated watering system. Enrichment in the form of manipulada (tubes, balls, mirrors) and food was provided. Animals were observed daily by trained personnel in addition to general husbandry assessments. Humane euthanasia criteria, defined as both dyspnea, loss of mobility (to obtain food and water) and >20% weight loss. At the end of the experiment, terminal blood samples were collected from the heart after administration of KAX and prior to intracardiac injection with pentobarbital sodium. No pain relief, aside from anesthesia during procedures, was used.

Marmoset procedures

Adult marmosets weighing over 300g were anesthetized by inhalation of vaporized isoflurane using an IMPAC 6 veterinary anesthesia machine. Once anesthetized, animals were injected with the indicated concentration of virus diluted in PBS. I.m. injections (in the caudal thigh) consisted of 0.2 ml delivered with a 1ml syringe with a 25-gauge, 5/8 in needle. Blood sampling from a femoral vein occurred under previously stated methods of anesthesia, and were limited to 7% of each marmoset's total blood volume per week. At time of euthanasia, terminal blood samples were collected from the heart after anesthetization by i.m. injection of Telazol and prior to intracardiac administration of pentobarbital sodium. Animals were housed in containment as previously described [26] . In brief, animals were singly housed in metal cages meeting current standards in a climate and humidity controlled room. Animals were fed pelleted food supplemented with fruits and treats daily, and were provided potable water through an automatic watering system ad libitum. Enrichment in the form of manipulada (i.e. toys, metal mirrors), foraging devices, treats, and fruit were provided daily. Animals were observed daily by trained personnel in addition to general husbandry assessments. Animals were observed daily by trained personnel in addition to general husbandry assessments. Animals found moribund (defined as labored breathing, decreased food consumption, persistent prostration and moderate unresponsiveness) would be euthanized under humane endpoint criteria, however, as animals did not become ill during the study this criteria was not met. No additional pain relief, aside from anesthesia during procedures, was necessary. All work was performed in an ABSL-3 laboratory.

Marmoset sample size justification

The marmoset study requires a sample size of 3 for adequate power to determine if the incidence of seroconversion is significantly greater than that which would be expected in the population. This sample size will allow the experimenter to detect seroconversion in at least 2 of 3 animals (66%) versus the expected population constant of <1% at a 95% confidence level using a one-tailed binomial test for proportions.

Post mortem procedures

Following euthanasia, necropsies were performed. Samples were collected aseptically for the virology studies described above. For the hamsters and ferrets, samples of the following were collected: heart, lung, liver, spleen, kidney, brain, and urine. In addition, ferrets had samples of intestine, adrenal gland, pituitary gland, and eye were collected. Samples of the following were collected from the marmosets: heart, lung, liver, spleen, kidney, intestine, and brain. After the virology samples had been collected, all major internal organs in each animal were also sampled for histology.

Ferret infection model

Despite not being able to detect infectious virus or viral genome in the kidney, we hypothesized that the weight loss we observed could be due to kidney failure. Individuals with HFRS exhibit proteinuria and hematuria, both of which can indicate kidney damage [46, 47] . Additionally, serum blood urea nitrogen and creatinine levels are both elevated in HFRS patients and provide a second way to measure kidney function [46, 47, 73] . Decreased platelet levels also characterize clinical HFRS in humans, impairing coagulation [46, 47, 73] . In a second experiment designed to monitor kidney parameters, PUUV infected ferrets exhibited the same gradual weight loss that characterized the first experiment. However, no prolonged signs of clinical kidney failure were observed: blood urea nitrogen and creatinine levels did not dramatically increase over the five week study period. Only one animal exhibited proteinuria (day 35), and two exhibited hematuria (one on Day 4, and one on Day 21) (Fig 7) . Similarly to parameters monitoring renal failure, no thrombocytopenia occurred in PUUV infected ferrets (S9 and S13 Figs). The cause of the weight loss remains undetermined.
40 section matches

Introduction

Nonhuman primate (NHP) models of SARS-CoV infection have yielded absent to moderate observable disease that has not replicated the severity of human SARS [20] [21] [22] [23] [24] [25] . Fever was notably absent in all studies, except for one African green monkey on day 3 postinfection [20] . All studies detected SARS-CoV replication in one or several monkey species and documented seroconversion, thereby confirming established infection. Aside from observable clinical symptoms, these studies relied on virus shedding and histopathology specimens from necropsy as objective markers of disease. Most studies euthanized animals during the course of infection to document histopathological disease. Only two studies followed animals for more than 14 d after infection [20, 22] . No study has examined radiographic evidence of pulmonary disease, which is one of the most prominent features of SARS in humans.
In adult humans, SARS presents as a severe febrile pneumonia [1] . It has been characterized as a three-phase illness: a first phase consisting of a flu-like illness, followed by a phase of lower respiratory tract disease, with a third phase of clinical deterioration in a process resembling adult respiratory distress syndrome [26] . Disease progression can be somewhat slow, with onset of severe respiratory disease occurring anywhere from 1 to 2 wk after initial symptoms [27] . Pulmonary radiographic abnormalities are almost universally reported in SARS cases [28] . However, early radiographs may be normal, and there is clear evidence of infection without radiographic abnormality in a small number of cases [29, 30] . Multifocal disease is present in 30-50% of initial radiographs, and the majority of persons progress to multifocal disease that peaks between 8 and 14 d after symptom onset [28, [31] [32] [33] [34] . Severe disease develops in up to 30% of patients, with the most ill developing diffuse or confluent airspace consolidation consistent with adult respiratory distress syndrome [28, 31, 33] .
In contrast to adults, SARS in young children tends to be a relatively mild disease [35] . Adolescents can experience significant respiratory disease similar to adults, but younger children generally do not [36] [37] [38] [39] . Constitutional symptoms such as myalgias, chills, and headache that are common in adults are usually absent in children [35, 40] . Children have a shorter course of illness, most being afebrile by 7 d, and generally do not develop pulmonary disease significant enough to require assisted ventilation or even supplemental oxygen [36] [37] [38] [39] 41] . As a result, the WHO diagnostic criteria were not reliable in identifying SARS in pediatric patients [38] . Some experts have recommended the term ''mild acute respiratory syndrome'' for SARS-CoV infection in children [35] .

Virus and Cells

SARS-CoV Urbani strain was obtained from the Centers for Disease Control and Prevention (Tom Ksiazek) in Atlanta, Georgia and had been passaged four times in Vero E6 cells (American Type Culture Collection, Manassas, Virginia, United States) before inoculation. Final preparations of SARS-CoV in PBS that were used in infection procedures (below) had a titer of 3 3 10 6 plaque-forming units (pfu)/ml. SARS-CoV-specific antibody titers were determined by plaque reduction neutralization test (PRNT) on the day of infection and at 8 wk postchallenge. PRNTs were performed on confluent Vero cells in 6-well plates as described [9] . Recombinant virus (icSARS-CoV) was used at passage 1 past the initial transfection, and the derivation of this molecular clone has already been reported [19] . The icSARS-CoV preparation titered at 1.25 3 10 6 pfu/ml.

Radiographs

Chest radiograph results were available for six of the animals. Three of them exhibited radiographic evidence of pulmonary disease. The two animals infected by the IV route (group III) had no significant change in chest radiographs, while three of four animals infected by a mucosal route (groups I and II) developed radiographic disease ( Figure 1 ). Two animals with radiographic disease were infected with the icSARS-CoV (both group I) and one animal (group II) was infected with wild-type virus.
Of the three animals with radiographic evidence of disease, two (292Q and 91-379) were infected by the IB/IN route (group I) and one (91-512) was infected by the CJ/IN route (group II). All developed significant airspace disease on PID 6. The group II animal had normal radiographs until PID 6, when it developed a perihilar left upper lobe infiltrate. By PID 8, there was perihilar disease in the right upper lobe as well. These findings were relatively subtle and began to resolve on PID 12. They were still evident, albeit continuing to resolve, by PID 18. The two group I animals had subtle changes on PID 2 (right mid-lower lobe for 292Q, right lower lobe for 91-379). Radiographic appearance was unchanged on PID 4 in 292Q, but 91-379 did have a slight increase in the right lower lobe opacity. On PID 6, both animals had dramatic radiographic changes, with 292Q developing increasing lower lobe disease and new right-upper lobe airspace disease and 91-379 showing a significant increase in the right lower lobe consolidation. Both 292Q and 91-379 had increasing disease on PID 8 and demonstrated slight improvement by PID 10. Changes completely resolved by PID 14 in 91-379, but mild residual and resolving changes were still evident in 292Q on PID 18.

Discussion

This study demonstrated that cynomolgus macaques infected with SARS-CoV develop clinical disease with pulmonary radiographic findings. The presence of radiographic disease appeared to depend upon the route of infection. Of four animals challenged by a mucosal route (groups I and II), three had radiographic evidence of pulmonary disease. Radiographic disease developed by PID 6, and it peaked and resolved rapidly. Neither animal in the intravenous challenge group (group III) developed radiographic disease.
Disease produced by wild-type SARS-CoV Urbani and by icSARS-CoV was clinically indistinguishable. Observable symptoms and laboratory parameters did not vary between the two viruses. Both icSARS-CoV-challenged animals developed radiographic disease. Of the two animals infected with wild-type virus by a mucosal route, only one developed radiographic disease, and radiographic findings were relatively subtle. We cannot exclude the possibility that icSARS-CoV produces more significant pulmonary disease, but the difference in findings is more likely related to route of infection and dose. Animals that received icSARS-CoV (both in group I) were infected in the bronchus with a higher dose of virus. Importantly, the fact that recombinant icSARS-CoV produced disease clinically similar to its Urbani parent supports the hypothesis that the molecular clone recapitulates the disease phenotype in the macaque. These findings suggest that SARS-CoV is the sole cause of SARS, without the involvement of coinfecting or passenger agents. Models using icSARS-CoV may pave the way for identification of genetic determinants of pathogenesis.
The radiographs in our study present a much clearer picture of pulmonary disease than have previous studies using NHPs. In Kuiken et al. [23] and Fouchier et al. [21] , pneumonia was evident in pathological specimens from necropsy at PID 6, but animals were not followed past this point. Only two of eight animals in the study by Rowe et al. [25] had pathological evidence of lung disease, but necropsies were performed 14 d postinfection. McAuliffe et al. [20] found histopathologic evidence of viral lung infection on PID 2 that was resolving by PID 4. None of these studies used any other objective method to measure pulmonary disease.
The radiographic appearance of pneumonia in our study may afford insight into NHP models of SARS. Radiography provides an objective but dynamic measure of pulmonary disease. Our radiographic findings are similar to radiographs of humans with less-severe cases of SARS [28, 31, 32] . There is growing evidence that SARS may present as mild or indolent disease in adults [29, 30, 48] . Such mild disease in adults does appear to be an exception, however. Overall, our experience confirms that clinical disease from SARS-CoV infection in NHPs does not closely reproduce SARS in adult humans.
The similarity between our findings and SARS in children deserves further examination. We observed unifocal or multifocal radiographic disease resolving quickly instead of progressing to severe diffuse disease, which is consistent with findings in children with SARS [37, 39, 42, 43] . Absence of fever may not be as significant as it would seem. In published reports, children with SARS did present with fever, but the data are biased, because fever was universally included in case definitions [36] [37] [38] [39] 41] . Fever in children less than ten years old who are diagnosed with SARS tends to be relatively mild and short in duration [36] [37] [38] [39] 41] . In these reports, it is possible that children with mild clinical illness from SARS who did not develop a significant fever may have remained undiagnosed. This premise is supported by at least one documented case of SARS in an adult without fever [49] . Hematologic findings, particularly lymphopenia, are more prevalent in children with SARS [36] [37] [38] [39] 41] than in the macaques of our study, but Rowe et al. [25] did find lymphopenia and thrombocytopenia in their animals.
We have described our experiments with SARS-CoV infection by a variety of routes in cynomolgus macaques. Wild-type SARS-CoV and recombinant icSARS-CoV produced similar disease in our macaque model. As in prior studies, we found overt clinical disease in animals infected by mucosal routes that was much less severe than adult human SARS. We found radiographic pulmonary disease in most of these animals that resembled radiographic findings in human SARS. Overall, the disease we observed had some similarities to SARS in young children. Further study is needed to determine the applicability of NHP models to the study of SARS.

A B S T R A C T Background

The emergence of severe acute respiratory syndrome (SARS) in 2002 and 2003 affected global health and caused major economic disruption. Adequate animal models are required to study the underlying pathogenesis of SARS-associated coronavirus (SARS-CoV) infection and to develop effective vaccines and therapeutics. We report the first findings of measurable clinical disease in nonhuman primates (NHPs) infected with SARS-CoV.

Methods and Findings

In order to characterize clinically relevant parameters of SARS-CoV infection in NHPs, we infected cynomolgus macaques with SARS-CoV in three groups: Group I was infected in the nares and bronchus, group II in the nares and conjunctiva, and group III intravenously. Nonhuman primates in groups I and II developed mild to moderate symptomatic illness. All NHPs demonstrated evidence of viral replication and developed neutralizing antibodies. Chest radiographs from several animals in groups I and II revealed unifocal or multifocal pneumonia that peaked between days 8 and 10 postinfection. Clinical laboratory tests were not significantly changed. Overall, inoculation by a mucosal route produced more prominent disease than did intravenous inoculation. Half of the group I animals were infected with a recombinant infectious clone SARS-CoV derived from the SARS-CoV Urbani strain. This infectious clone produced disease indistinguishable from wild-type Urbani strain.

Introduction

In late 2002, Chinese health officials reported an unusual number of atypical pneumonia cases in Guangdong Province, and within 2 months, the World Health Organization (WHO) was alerted of an outbreak widespread throughout the province [1] . By March 2003, the illness designated severe acute respiratory syndrome (SARS) had spread to Hong Kong, Singapore, Vietnam, and Toronto, Canada [2] . The global SARS outbreak ended that July, but during its existence the disease caused 774 fatalities and had a significant economic impact on Southeast Asia [3] [4] [5] . A special concern was its predilection for nosocomial spread, as 21% of SARS cases occurred in healthcare workers . In certain local outbreaks, hospital staff accounted for over 50% of cases, and nosocomial spread to other patients or family members accounted for a significant proportion of SARS cases [6] .
Radiographic findings in children with SARS are also less significant than in adults, in both presentation and progression [40] . Up to 50% of children have normal initial chest radiographs [35] . In children with abnormal radiographs, unilateral, focal airspace disease predominates [36, 37, 39] . Most children have worsening of radiographic disease as illness progresses, with multifocal or bilateral lung involvement developing in 20-50% of cases [39, 42] . Radiographic abnormalities in children generally resolve quickly, within 6-14 d [37, 42, 43] .
In this report, we document the results of observational studies of SARS-CoV and icSARS-CoV infection in nonhuman primates. These studies focused on clinical and virologic parameters associated with infection in cynomolgus macaques in an attempt to examine the underlying mechanism of disease and to study a potential animal model for SARS.

Discussion

In our study, frequency and source of SARS-CoV genome detection correlated with route of inoculation. Animals infected intravenously (group III) had a paucity of positive specimens. Viruria appearing after day 10 was the only persistent finding in these animals (Table 3 ). Animals infected mucosally (groups I and II) had a greater number of positive specimens from nasal swabs and throat swabs. The two group II animals had the mos