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Nipah is paramyxovirus of the genus Henipavirus (family Paramyxoviridae) with a high fatality rate (69). Infection in humans usually causes severe encephalitic and respiratory disease (70). After inoculation with Nipah virus (NiV), Syrian hamsters also develop characterisitic neurological disease (12). Similar to symptoms after human infection, pathological lesions are the most severe and extensive in the hamster brain and viral antigen and RNA can be detected in neurons (11), lung (71), kidney, and spleen (11). The Syrian hamsters in the majority of NiV infection studies are treated by intraperitoneal (IP) injection or intranasal (IN.) delivery and these models have revealed that different inoculation method can cause diverse pathological responses (11). In Wong's work, IP injection of NiV in Syrian hamsters caused primarily neurological disease, while IN delivery developed neurological symptoms as well as labored breathing due to lung infection in the final stages of disease (11). Disease progression is usually much rapid and the time to death post-infection is shorter following intraperitoneal rather than intranasal inoculation (72). Since the Syrian hamster has shown suitability for studying NiV infection, it was further used to study the viral transmission (73–75), demonstrating that Nipah virus is transmitted efficiently via direct contact and inefficiently via fomites, but not via aerosols. Regarding the use of these models for development of disease treatment and prophylaxis, recent studies have shown that pretreatment with Poly(I)-poly(C12U) can significantly decrease the mortality caused by NiV infection of Syrian hamster (76). In addition, the model was used as a platform for evaluation of vaccines for NiV (77–80). Walpita et al. discovered purified NiV-like particles (VLP) can protect the Syrian hamster using either multiple-dose or single-dose vaccination regimens followed by NiV challenge (81).
Coronaviruses (CoVs) prior to the SARS outbreak were only known to be the second cause of the common cold after rhinoviruses. At least four different species can cause mild, self-limiting upper respiratory tract infections in humans: alphacoronaviruses HCoV-229E and HCoV-NL63, and betacoronaviruses HCoV-HKU1 and HCoV-OC43. More recently, two more additional pathogenic human-CoV were identified: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV).23 SARS-CoV was first identified in China in February 2003, and 4 months later, >8000 cases had been reported with about 800 deaths in 27 different countries worldwide.24 SARS-CoV has a wide host range and it is associated with wildlife meat industry. The natural history of the virus involves bats as primary hosts that then transmitted it to the intermediate amplifying hosts – as mask palm civets and raccoon dogs – that then could spread it to humans.23,25 Human-to-human transmission follows and can lead to large numbers of infected patients and is considered the main route of transmission in large-scale epidemics.9
MERS-CoV is phylogenetically related to SARS-CoV and share with SARS-CoV the origin in bats.23,26,27 Several CoVs have been identified in insectivorous and frugivorous bat species in various countries, indicating that bats may represent an important reservoir of these viruses.23 MERS-CoV was first identified in Saudi Arabia in 2012 and then spread to other countries causing hundreds of deaths.26,28 Clinical features of MERS-CoV are similar to SARS-CoV, although this virus has also been associated with several extrapulmonary manifestations, such as severe renal complications. Recent studies have indicated that dromedary camels may be the intermediate hosts and potential source of the virus for humans.26,29 In addition, the first experimental infection of bats with MERS-CoV has been described. The virus maintains the ability to replicate in the host without clinical signs of disease, supporting the general hypothesis that bats are the ancestral reservoir for MERS-CoV.30 Human-to-human transmission has also been reported. Based on epidemiological data, both animal-to-human and human-to-human transmission are considered to be important elements in MERS outbreak.26
Members of the Paramyxoviridae family are pleomorphic enveloped viruses divided into two subfamilies, Paramyxovirinae and Pneumovirinae. Paramyxovirinae has recently been subdivided into seven genera: Aquaparamyxovirus, Avulavirus, Ferlavirus, Henipavirus, Morbillivirus, Respirovirus, and Rubulavirus (http://ictvonline.org/virusTaxonomy.asp?version=2012). Viruses of this family affect a wide range of animals, including primates, birds, carnivores, ungulates, snakes, cetaceans and humans, and cause a wide variety of infections, such as measles, mumps, pneumonia and encephalitis in humans, and distemper, peste des petits ruminants, Newcastle disease and respiratory tract infections in animals. However, several paramyxoviruses (PVs) have not been classified into any of these seven genera, including Nariva virus (NarPV), Mossman virus (MosPV), Beilong virus (BeiPV), J virus (JPV),, Tupaia paramyxovirus (TupPV) and Tailam virus
, all of which belong to a group of novel paramyxoviruses isolated from wild animals, as well as Salem virus isolated from horses. Among them, only JPV has been shown to be pathogenic, causing extensive haemorrhagic lesions in rodents. Horizontal transmission is the principal mode of intraspecies PV infection, suggesting that contaminated faeces, urine or saliva may be responsible for spillover to other species.
Bats have a close evolutionary relationship with several genera of mammalian paramyxoviruses. Otherwise, bat-borne paramyxoviruses are in close relationship to known paramyxoviruses of mammalian. These small mammals are known to harbour a broad diversity of PVs, including emergent henipaviruses (Nipah virus and Hendra virus) and rubulaviruses [Menangle virus, Tioman virus, Mapuera virus, and Tuhoko virus 1, 2 and 3 (ThkPV-1, ThkPV-2 and ThkPV-3)]. A very broad diversity of paramyxoviruses, including Henipa-, Rubula-, Pneumo- and Morbilli-related viruses, have been detected in six of ten tested bat families. Whereas most of the viruses identified in bats do not seem to cause clinical disease in these animals, there have been reports of rabid bats, and of unusually large numbers of animals succumbing to infection by rabies virus.
As part of a large-scale investigation of viral diversity in bats and of associated zoonotic risks, we have previously detected a bat paramyxovirus in one insectivorous African sheath-tailed bat (Coleura afra), exhibiting several hemorrhagic lesions at necropsy. We therefore examined occurrence of this bat paraymxovirus in other bats.
Emerging infectious diseases under this category were subcategorized into 1a, 1b and 1c. Subcategory 1a covers known pathogens that occur in new ecological niches/geographical areas. A few past examples belonging to this subcategory are the introduction and spread of West Nile virus in North America; chikungunya virus of the Central/East Africa genotype in Reunion Island, the Indian subcontinent and South East Asia; and dengue virus of different serotypes in the Pacific Islands and Central and South America.18,19,20,21,22,23 Factors that contributed to the occurrence of emerging infectious diseases in this subcategory include population growth; urbanization; environmental and anthropogenic driven ecological changes; increased volume and speed of international travel and commerce with rapid, massive movement of people, animals and commodities; and deterioration of public health infrastructure. Subcategory 1b includes known and unknown infectious agents that occur in new host ‘niches'. Infectious microbes/agents placed under this subcategory are better known as ‘opportunistic' pathogens that normally do not cause disease in immunocompetent human hosts but that can lead to serious diseases in immunocompromised individuals. The increased susceptibility of human hosts to infectious agents is largely due to the HIV/acquired immune deficiency syndrome pandemic, and to a lesser extent, due to immunosuppression resulting from cancer chemotherapy, anti-rejection treatments in transplant recipients, and drugs and monoclonal antibodies that are used to treat autoimmune and immune-mediated disorders. A notable example is the increased incidence of progressive multifocal leukoencephalopathy, a demyelinating disease of the central nervous system that is caused by the polyomavirus ‘JC' following the increased use of immunomodulatory therapies for anti-rejection regimens and for the treatment of autoimmune diseases.24,25,26 Subcategory 1c includes known and unknown infectious agents causing infections associated with iatrogenic modalities. Some examples of emerging infections under this subcategory include therapeutic epidural injection of steroids that are contaminated with Exserhilum rostratum and infectious agents transmitted from donor to recipients through organ transplantation, such as rabies virus, West Nile virus, Dandenong virus or Acanthamoeba.27,28,29,30,31
Paramyxoviridae constitute a wide viral family that includes human and animal pathogens. Several bat-borne paramyxoviruses have been recognized such as parainfluenza type 2 virus, Mapuera, Menangle and Tioman viruses and two infectious agents of emerging diseases, such as Nipah and Hendra viruses.20
Nipah and Hendra viruses, classified as the genus Henipavirus, are capable of causing severe, potentially fatal diseases in humans.20 Fruit bats of the Pteropus genus are the common reservoir hosts of the Nipah and Hendra viruses.20
Nipah virus (NiV) first emerged in 1998 in Malaysia, causing an outbreak of respiratory illness and encephalitis in pigs.21 Pig-to-human transmission of Nipah virus – associated with severe febrile encephalitis – was described and it was thought to occur through close contact with infected animals. Although uncommon, human-to-human transmission of virus was also described.21 In two other outbreaks in Bangladesh and India, an intermediate animal host was not identified, suggesting bat-to-human and human-to-human transmissions.
Hendra virus (HeV) causes a fatal respiratory disease in both humans and horses.20,22 Several outbreaks of HeV have occurred in Australia. Horse is the intermediate host and the virus is likely transmitted via ingestion of feed, pasture or water contaminated with urine, saliva and feces of infected bats. Horse-to-human transmission occurs when there is close contact with ill animals.20 To date, human-to-human transmission has not been observed.
The difficulty of estimating prevalence of emerging bat viruses may reflect true low prevalence, low test sensitivity, or sampling of tissues in which virus does not persist. Pathogens that cause acute infections may circulate at low prevalence or may be heterogeneously distributed in space and time and therefore require large sample sizes to detect.
Pathogens that persist within their hosts may be sequestered in tissues that are difficult to sample non-lethally. For example, Ebola virus RNA has only been detected in the liver and spleen of wild bats. Viremia or shedding in excreta may be periodic, and, therefore, estimation of infection status from these samples may yield false negatives.
Prevalence data collected across space and time may allow identification of geographic patterns, such as travelling waves, that are likely to be caused by host-to-host transmission (SIR or SIRS dynamics) (Table 1). Age-structured prevalence data with an age-specific incidence skewed towards younger individuals suggests an endemic disease with SIR dynamics. If the duration of maternal-derived immunity is known, the mean age of infection is a direct reflection of the basic reproductive ratio of the disease in the population. Although prevalence data provides many insights into the dynamics of disease, there are few situations in which prevalence data alone can distinguish among our proposed epidemiological scenarios.
Respiratory syncytial virus (RSV), a member of the Orthopneumovirus genus, infects approximately 70% of infants before the age of 1 and almost 100% by the age of 2 years old, making it the most common pathogen to cause lower respiratory tract infection such as bronchiolitis and pneumonia in infants worldwide. Recent evidence also indicates that severe respiratory diseases related to RSV are also frequent in immunocompromised adult patients and that the virus can also present neuroinvasive properties. Over the last five decades, a number of clinical cases have potentially associated the virus with CNS pathologies. RSV has been detected in the cerebrospinal fluid (CSF) of patients (mainly infants) and was associated with convulsions, febrile seizures and different types of encephalopathy, including clinical signs of ataxia and hormonal problems. Furthermore, RSV is now known to be able to infect sensory neurons in the lungs and to spread from the airways to the CNS in mice after intranasal inoculation, and to induce long-term sequelae such as behavioral and cognitive impairments.
An additional highly prevalent human respiratory pathogen with neuroinvasive and neurovirulent potential is the human metapneumovirus (hMPV). Discovered at the beginning of the 21st century in the Netherlands, it mainly causes respiratory diseases in newborns, infants and immunocompromised individuals. During the last two decades, sporadic cases of febrile seizures, encephalitis and encephalopathies (associated with epileptic symptoms) have been described. Viral material was detected within the CNS in some clinical cases of encephalitis/encephalopathy but, at present, no experimental data from any animal model exist that would help to understand the underlying mechanism associated with hMPV neuroinvasion and potential neurovirulence.
Hendra virus (HeV) and Nipah virus (NiV) are both highly pathogenic zoonotic members of the Henipavirus genus and represent important emerging viruses discovered in the late 1990s in Australia and southern Asia. They are the etiological agents of acute and severe respiratory disease in humans, including pneumonia, pulmonary edema and necrotizing alveolitis with hemorrhage. Although very similar at the genomic level, both viruses infect different intermediate animal reservoirs: the horse for HeV and the pig for NiV as a first step before crossing the barrier species towards humans. In humans, it can lead to different types of encephalitis, as several types of CNS resident cells (including neurons) can be infected. The neurological signs can include confusion, motor deficits, seizures, febrile encephalitic syndrome and a reduced level of consciousness. Even neuropsychiatric sequelae have been reported but it remains unclear whether a post-infectious encephalo-myelitis occurs following infection. The use of animal models showed that the main route of entry into the CNS is the olfactory nerve and that the Nipah virus may persist in different regions of the brain of grivets/green monkeys, reminiscent of relapsing and late-onset encephalitis observed in humans.
Influenza viruses are classified in four types: A, B, C and D. All are endemic viruses with types A and B being the most prevalent and causing the flu syndrome, characterized by chills, fever, headache, sore throat and muscle pain. They are responsible for seasonal epidemics that affect 3 to 5 million humans, among which 500,000 to 1 million cases are lethal each year. Associated with all major pandemics since the beginning of the 20th century, circulating influenza A presents the greatest threat to human health. Most influenza virus infections remain confined to the upper respiratory tract, although some can lead to severe cases and may result in pneumonia, acute respiratory distress syndrome (ARDS) and complications involving the CNS. Several studies have shown that influenza A can be associated with encephalitis, Reye’s syndrome, febrile seizure, Guillain–Barré syndrome, acute necrotizing encephalopathy and possibly acute disseminated encephalomyelitis (ADEM). Animal models have shown that, using either the olfactory route or vagus nerve, influenza A virus may have access to the CNS and alter the hippocampus and the regulation of neurotransmission, while affecting cognition and behavior as long-term sequelae. The influenza A virus has also been associated with the risk of developing Parkinson’s disease (PD) and has recently been shown to exacerbate experimental autoimmune encephalomyelitis (EAE), which is reminiscent of the observation that multiple sclerosis (MS) relapses have been associated with viral infections (including influenza A) of the upper respiratory tract.
Another source of concern when considering human respiratory pathogens associated with potential neuroinvasion and neurovirulence is the Enterovirus genus, which comprises hundreds of different serotypes, including polioviruses (PV), coxsackieviruses (CV), echoviruses, human rhinoviruses (HRV) and enteroviruses (EV). This genus constitutes one of the most common cause of respiratory infections (going from common cold to more severe illnesses) and some members (PV, EV-A71 and -D68, and to a lesser extent HRV) can invade and infect the CNS, with detrimental consequences. Even though extremely rare, HRV-induced meningitis and cerebellitis have been described. Although EV infections are mostly asymptomatic, outbreaks of EV-A71 and D68 have also been reported in different parts of the world during the last decade. EV-A71 is an etiological agent of the hand–foot–mouth disease (HFMD) and has occasionally been associated with upper respiratory tract infections. EV-D68 causes different types of upper and lower respiratory tract infections, including severe respiratory syndromes. Both serotypes have been associated with neurological disorders like acute flaccid paralysis (AFP), myelitis (AFM), meningitis and encephalitis.
Last but not least, human coronaviruses (HCoV) are another group of respiratory viruses that can naturally reach the CNS in humans and could potentially be associated with neurological symptoms. These ubiquitous human pathogens are molecularly related in structure and mode of replication with neuroinvasive animal coronaviruses like PHEV (porcine hemagglutinating encephalitis virus), FCoV (feline coronavirus) and the MHV (mouse hepatitis virus) strains of MuCoV, which can all reach the CNS and induce different types of neuropathologies. MHV represents the best described coronavirus involved in short- and long-term neurological disorders (a model for demyelinating MS-like diseases). Taken together, all these data bring us to consider a plausible involvement of HCoV in neurological diseases.
YFV is an arthropod-borne virus of the genus Flavivirus (family Flaviviridae) and has high morbidity and mortality rates in regions of sub-Saharan Africa and South America (53). It was one of the first viruses of humans to be identified, isolated, propagated in vitro and studied by genomic sequencing (54). The study of infection mechanism of YFV has historically been hindered by the lack of appropriate small animal model and non-human primate (NHP) models have typically been used. More recently, several research groups have generated animal models using Syrian hamsters that can be successfully infected with YFV (55–58). McArthur et al. reported adapted viral strains (Asibi/hamster p7) allow the reproduction of yellow fever disease in hamsters with features similar to the human disease (59). Further, studies have also shown that infection of Syrian hamster results in immune responses that correspond to those observed in infected humans, with marked increases in IFN-γ, IL-2, TNF-α in the spleen, kidney, and heart, but reduced levels of these seen in the liver. In addition, these studies found increased levels of IL-10 and reduced levels of TGF-β in the liver, spleen, and heart in early and mid-stages of infection (60). Syrian hamster can be used both to study the pathogenesis of the YFV infection, and to validate antiviral drugs and antiviral therapies. Recent findings have shown that treatment with the anti-viral compounds 2′-C-methyl cytidine (61), T-1106 (62), IFN alfacon-1 (63), and BCX4430 (64) pre- and post-YFV exposure can significantly improve Syrian hamster survival. In a study by Julander et al. immunization with DEF201, an AdV type-5 vector expressing IFN alpha (IFN-α), can effectively reduce the viral titer in hamster's liver and serum post-YFV infection (65). Immunoprophylaxis with XRX-001, a vaccine containing inactivated yellow fever antigen with an alum adjuvant, can elicit high titers of neutralizing antibodies in vivo to protect Syrian hamsters from YFV infection (66, 67). Interestingly, Xiao et al. (67) and Tesh et al. (68) demonstrate that prior exposure of Syrian hamsters to heterologous flaviviruses reduces the risk of YFV infection.
Bats are considered a reservoir of severe emerging infectious diseases. Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), Nipah virus, Hendra virus, and Ebola virus are all thought to be bat-borne viruses1,2.
Notably, bats also host major mammalian paramyxoviruses from the family Paramyxoviridae, order Monone-gavirales3,4. While Henipaviruses (Nipah and Hendra viruses) in South East Asia and Australia are associated with fruit bats5, other paramyxoviruses have been detected not only in fruit bats but in insectivorous bats worldwide6–9. A potential pathway for Nipah virus transmission from bats to humans was found to be associated with a human-bat interface, specifically date palm sap shared by bats and humans10. In addition, serological evidence of possible human infection with a bat-originated paramyxovirus, Tioman virus11, reinforces the epidemiological role of bats in the emergence of pathogens such as paramyxoviruses in humans.
In addition to these bat paramyxoviruses with zoonotic potential, other new paramyxoviruses have been reported. These include several new mammalian paramyxoviruses such as Beilong virus and J virus, which remain unassigned under the family Paramyxoviridae12. Recent bat-associated paramyxoviruses were proposed to be grouped in a separate phylogenetic clade within a potentially separate genus such as Shaanvirus13 which was distantly related to Jeilongvirus14. In addition, novel strains of bat paramyxoviruses in diverse genera have been reported continuously15–17. Based on the recent papers, bat paramyxoviruses found worldwide to date have belonged to the genera Rubulavirus, Morbillivirus, Henipavirus and the unclassified proposed genera Shaanvirus. Expanded classifications for grouping newly identified viruses in bats can be accomplished by further studying the biological characteristics of novel paramyxoviruses as well as genome characterization18.
In this study, active surveillance was performed to reveal paramyxoviruses circulating in Korean bats. A total of 232 bat samples were collected at 48 sites in natural bat habitats and tested for the possible existence of paramyxoviruses.
Western equine encephalitis (WEE) is an uncommon viral illness of horses and human. WEE virus (WEEV) is an Alphavirus of the family Togaviridae which is maintained between birds and mosquitoes, occasionally causing disease in humans and equids [135, 136]. This is an arbovirus transmitted by mosquitoes of the genera Culex and Culiseta. It is a recombinant between Sindbis and Eastern equine encephalitis like viruses. It has also been reported to cause disease in poultry, game birds and ratites. WEEV is normally maintained between Culex tarsalis mosquitoes and birds. WEE has several subtypes consisting Sindbis, Aura, Ft. Morgan and Y 62–33. WEEV previously isolated in the south and eastern USA has been shown to belong to the HJ virus serogroup.
Horses and humans are often referred to as “dead-end” hosts as the virus does not build to high enough levels in blood to infect other mosquitoes. Most people infected with WEE virus will have either no symptoms or a very mild illness. A small percentage of people, especially infants and elderly people to a lesser extent, may develop encephalitis. Approximately 5-15% of these encephalitis cases are fatal, and about 50% of surviving infants will have permanent brain damage.
Geographically, WEEV exists throughout uine deaths were estimated in central America and northern portions of South America, Mexico and Canada. In the US, WEEV exist in the western two third of the country. Outbreaks of the disease have been recorded since 1847. In 1930 about 6000 horses and mules were infected leading to about 50% mortalities in California. The largest epidemic was recorded in 1937 and 1938 in USA and Canada. In 1938 outbreak an estimated 264000 equids were infected with a morbidity of 21.4%. In the USA, WEE is seen primarily in provinces west of the Mississippi River. During 1941, there was an outbreak of WEE in several states of US and Canada causing 300,000 cases of encephalitis in mules and horses and 3336 cases in humans. The 1970s saw 209 human cases; 87 were reported during the 1980s, only 4 cases during the 1990s, and no cases have been reported in the USA or Canada since 1998. The last documented human case in North America occurred in 1994, and the virus has not been detected in mosquito pools since 2008. In human, WEEV infections tend to be asymptomatic or cause mild disease after a short incubation period of 2–7 days with nonspecific symptoms, e.g., sudden onset of fever, headache, nausea, vomiting, anorexia and malaise. In some cases, additional symptoms of altered mental status, weakness and signs of meningeal irritation may be observed. In a minority of infected individuals, encephalitis or encephalomyelitis occurs and may lead to neck stiffness, confusion, tonic-clonic seizures, somnolence, coma and death. WEEV is considered as agent that the US researched as potential biological weapons before the nation suspended its biological weapons program.
In horses, infections with WEEV begin with fever, inappetence and lethargy, progressing to various degrees of excitability and then drowsiness, ultimately leading to paresis, seizures and coma in 5-10 day course of the disease. The WEEV mortality rate in horses is higher than humans. Mortality of horses showing clinical signs of WEE is 20–50%. These symptomatic horses either progress to recumbency or die from WEE infections.
There is no treatment for WEE other than supportive care. Formalin-inactivated whole viral vaccines for EEE, WEE, and VEE are commercially available in mono-, bi-, or trivalent form. Previously non vaccinated adult horses require booster. For adult horses in temperate climates, an annual vaccine within 4 wk of the start of the arbovirus season is recommended. However, for horses that travel between areas affected by the virus, 2 or even 3 times vaccination in a year is recommended. Mares should be vaccinated 3–4 wk before foaling to induce colostral antibody.
Venezuelan equine encephalitis (VEE) is an arbovirus infection transmitted by mosquitoes. VEE viruses (VEEV) are classified in the genus Alphavirus, family Togaviridae. The VEE virus complex is composed of six subtypes (I–VI); Subtype I includes five antigenic variants (AB–F), of which variants 1-AB and 1-C are associated with epizootics in equines and concurrent epidemics in humans. The epizootic variants 1-AB and 1-C are thought to originate from mutations of the enzootic 1-D serotype. The enzootic strains are 1-D, 1-E and 1-F of subtype I, subtype II, four antigenic variants (A–D) of subtype III, and subtypes IV–VI. The enzootic viruses do not produce clinical encephalomyelitis in the equines normally. Enzootic VEE strains have been identified as Everglades (subtype II) in the Florida, variant I-E in Central American countries and Mexico, variants I-D and I-E in Panama, variant I-D in Venezuela, Colombia, variants 1-D, III-C, and III-D in Peru, variant III-B and subtype V in French Guiana, variant I-D in Ecuador, variant III-A in Suriname and Trinidad, variants I-F and III-A and subtype IV in Brazil and subtype VI in Argentina. In an atypical ecological niche, variant III-B has been isolated in the USA (Colorado and South Dakota) in an unusual association with birds. Countries with incidence/serological evidence are presented in Fig. (4).
The primary vectors for the bird or rodent-mosquito life cycle are members of the Melanoconion subgenus (Culex cedecci). Epizootic VEEV strains (I-AB and I- C) are transmitted by several mosquito vectors (e.g., Aedes and Psorophora spp.) to equids.
Infections with VEE virus (VEEV) may present, in both humans and horses, as either encephalitic disease or as simply a febrile disease without profound neurologic signs. Horses may die after a very acute course, even without any neurologic signs, but mortality in humans is generally low. Horses are not dead-end hosts for VEEV epizootic strains like they are for EEEV and WEEV. Horses are, in fact, the key reservoir species for the epizootic strains of VEEV that cause clinical disease in both horses and humans.
Epizootic subtypes highly pathogenic to equines, can spread rapidly through large populations. Equines are the primary animal species and serve as amplifying hosts for epizootic VEE virus strains. Blood-sucking insects feed on infected horses, pick up this virus and transmit to other animals or humans. Animal like cattle, swine, and dogs, can become infected, but they neither show the signs of the disease nor contribute to spread. Aerosol transmission has been reported in human from laboratory accidents [128, 132]. Infections with both epizootic and enzootic variants are infectious to human beings and can occur in laboratory workers. The workers handling infectious VEE viruses or their antigens should take preventive measures including use of containment facilities and vaccination.
VEE can cause disease in equines including horses, mules, donkeys and zebras. Cattle, swine, chickens and dogs have been shown to seroconvert after epizootics; mortality has been observed in domesticated rabbits, dogs, goats and sheep. Humans also can contract this disease. Epidemics of VEE involving tens of thousands of humans have been reported. The mortality rates in equines during epizootics have been 19-83% while 4-14% in human beings associated with neurological disease
It usually causes influenza like symptoms in adults, but in children and horses it can cause severe encephalitis. Equines may suddenly die or exhibit progressive central nervous system disorders. Infections with VEEV may present, in both humans and horses, as either encephalitic disease or as simply a febrile disease without profound neurologic signs. Horses may die after a very acute course, even without any neurologic signs, but mortality in humans is generally low. Young and immune compromised horses are most likely to develop clinical signs. It causes only low morbidity and mortality in man but high morbidity and mortality in animals.
The epizootic VEE was initially limited to northern and western South America in Venezuela, Colombia, Ecuador, Peru and Trinidad, but the epizootics have been reported in years from 1969 to 1972 in Guatemala, Nicaragua, El Salvador, Honduras, Costa Rica, Belize, Mexico, and the United States of America due to variant 1-AB. Epizootics caused by I-AB or I-C virus have not occurred in North America and Mexico after 1972. Equine and human epizootic VEE viruses were subtype 1-C from Venezuela in 1993, 1995 and 1996 and Colombia in 1995. In 1960 over 200,000 human cases and more than 100,000 equine deaths were estimated in Central Colombia. Countless cases in horses and 75,000-100,000 human cases with more than 300 fatal encephalitis cases occurred in Venezuela and Colombia in 1995. Equine disease associated with VEEV-IE occurred in Mexico and human cases of VEEV ID-associated disease occurred in Peru from 1993 to 95. Subtype II has been isolated from humans and mosquitoes from Florida; subtype III has been isolated from the Rocky Mountains and northern plains states. Sylvatic VEE viruses are endemic in North, Central, and South America in swampy environments with persistent fresh or brackish water. Epizootics have been associated with a mutation to a subtype I (A, B, C, and possibly E), a change in mammalian pathogenesis, and change to several bridge vectors.
Treatment of viral encephalitis is supportive, as there are no specific antiviral therapies. The two VEE vaccines, a modified-live vaccine (TC-83) and an inactivated adjuvant vaccine, have been used in field. Horses were vaccinated with TC-83 vaccine during outbreak in Mexico and Texas in 1971 as equine vaccine was not available but it is still in use for humans working with VEE. Formalin-inactivated virulent VEE virus vaccines are not recommended for use in equids due to risk of residual virulence.
HeV and NiV are currently classified as BSL-4 agents and consequently human efficacy studies for testing potential therapeutic products are not easily achievable. In 2002, the U.S. Food and Drug Administration (FDA) implemented the Animal Efficacy Rule for the development of therapeutic products under these circumstances. Specifically, FDA can rely on evidence derived from animal studies in the evaluation of product effectiveness when particular criteria are met, such as a well-understood mechanism for both the pathogenicity of the agent and the underlying mode of action of the product. Importantly, the therapeutic effect must also be demonstrated in more than one animal species. Once these criteria have been met, human clinical trials could commence, most likely in populations at high risk of natural infection by HeV and NiV.
A strong epidemiological association existed in Malaysia between human NiV infection and close direct contact with pigs, especially sick and dying animals. No direct association with flying foxes was made. An epidemiological link has been noted between HeV infection in people and horses dying from HeV disease. Again, neither HeV disease nor seroconversion has ever been identified in wildlife carers who came into close and regular contact with sick and injured bats. While both horses and pigs have been experimentally infected with HeV and NiV, respectively [42, 44], neither represents a practical option for multiple experimental efficacy studies. Large animals such as horses are difficult to manage in sufficient numbers under BSL-4 conditions and in pigs NiV infection is mostly inconsequential from a clinical perspective. For the purposes of this review we will focus on the smaller animal model systems that have been explored (Table 1). There was no serological evidence of NiV infection in rodents in Malaysia [9, 45]. However, attempts were made to infect mice experimentally. NiV and HeV do not cause disease in mice after subcutaneous administration (5,000 TCID50 HeV) (Crameri, G and Eaton, B.T., unpublished observations) or with either an intranasal (6x105 pfu NiV) or intraperitoneal (107 pfu NiV) challenge of NiV, although the HeV is lethal if administered intracranially. Rabbits are not susceptible to HeV associated disease when challenged subcutaneously (5,000 TCID50 HeV). Guinea pigs were experimentally infected with HeV soon after its discovery (5,000-50,000 TCID50 HeV) and developed generalized vasculitis affecting lung, kidney, spleen, lymph nodes, gastrointestinal tract, and skeletal and intercostal muscles [36, 38]. However, in spite of vascular involvement of the lung, there was little or no pulmonary edema. NiV infection in the guinea pig (50,000 TCID50 NiV) clinically manifested as ruffled fur and abnormal (less fearful) behavior with gross pathology limited to edema of the mesentery, broad ligament, and retroperitoneal tissues. Significant microscopic pathology included vasculitis with fibrinoid necrosis and endothelial syncytial cell formation in the myocardium, kidney, lymph node, spleen, myometrium, retroperitoneal tissues, and submucosal vessels of the bladder. Oophoritis and the presence of hemorrhagic corpora lutea were also noted together with endometrial degeneration and necrosis accompanied by multinucleated cells.
A golden hamster animal model for acute NiV virus infection has been established where, importantly, encephalitis and neuron infection were demonstrated similar to those seen in NiV-infected humans. The LD50 values for hamsters inoculated by intraperitoneal and intranasal routes were 270 pfu and 47,000 pfu, respectively. Notably, the brain was the most severely affected organ in terms of vascular and parenchymal lesions. Neurons in the vicinity of vascultis showed numerous cytoplasmic eosinophilic inclusion bodies and both viral antigen and RNA were found extensively throughout neurons. Ultrastructural analysis revealed cytoplasmic inclusions composed of defined herringbone nucleocapsids typical of paramyxoviruses. However, although HeV and NiV clearly cause systemic disease in humans, NiV was not detected in serum samples from infected hamsters. Moreover, the pathology seen in the lungs and kidneys of NiV-infected hamsters differed from that seen in HeV-infected horses and NiV-infected humans. HeV infection of hamsters has not been reported.
Experimental HeV infection of cats has been performed and findings from those studies were similar to those of horses and humans, namely generalized vascular disease with the most severe effects seen in the lung [36, 44]. NiV infection in cats was comparable to that observed with HeV except that with NiV there was also extensive inflammation of the upper and lower respiratory tract epithelium, associated with the presence of viral antigen, similar to the severe respiratory disease observed in humans in the recent NiV outbreaks in Bangladesh. Cats succumb to infection 6 to 10 days following parenteral inoculation of as low as 500 TCID50 NiV, or oronasal administration of 50,000, TCID50 of low passage, plaque purified HeV [41, 42, 46] or NiV (Bossart, K., Bingham, J. and Middleton, D., unpublished data). Gross pathology common to both HeV and NiV infection of cats consisted of hydrothorax, dense purple-red consolidation in the lung with fluid accumulation and froth in the bronchi (reviewed in. Histologically cats infected with NiV develop a necrotizing alveolitis, with necrotic foci developing within a range of other organs, particularly kidney, spleen, lymph nodes, bladder, ovaries, adrenal and meninges. Studies on cats that were euthanized early in the course of the infection (1 day after the onset of fever, as determined by radiotelemetry measurement of body temperature) indicated that alveolar infection by NiV preceded vascular infection suggesting an increased tropism for alveolar epithelium as compared to vascular tissue (Bingham, J., unpublished observation). Thus evidence to date indicates that the cat represents an animal model in which henipavirus induced pathology closely resembles the lethal respiratory disease caused by HeV and NiV in humans.
Experimental NiV infection in Pteropid bats has been attempted. All bats that were challenged with 50,000 TCID50 NiV remained clinically well throughout the study period and no febrile responses were recorded following NiV inoculation via a parenteral route. Challenged bats developed a sub-clinical infection characterized by episodic viral shedding in urine, limited presence of virus within selected viscera and seroconversion. No gross abnormalities were identified on post-mortem examination of animals at various times post exposure; all bat tissues were negative upon immunohistochemical labeling for NiV antigen.
Recently, we have been evaluating the potential of the ferret as an improved animal model for NiV infection. Ferrets have emerged as important animal models for several major respiratory diseases including highly pathogenic avian influenza, severe acute respiratory syndrome (SARS), and also morbilliviruses, the closest relatives to HeV and NiV. Significant similarity exists between ferret and human lung physiology and morphology and ferrets have been used previously for toxicology and biological safety assessment studies. We have confirmed that ferrets are susceptible to NiV infection with disease developing 6 to 10 days following oronasal administration of 500 to 50,000 TCID50 (Bossart, K., Bingham, J. and Middleton, D., unpublished data). A lower dose of 50 TCID50 failed to produce infection as defined by fever, illness, detection of virus, viral antigen or viral genome, histopathological lesions or seroconversion. Infection first manifested as fever and this was followed by inappetance and severe depression. A mild increase in respiratory rate in some animals was attributable to fever with one ferret showing tremors and hindlimb weakness. Gross pathology included subcutaneous edema of the head, hemorrhagic lymphadenopathy of submandibular, retropharyngeal and sometimes visceral lymph nodes, numerous diffuse pin-point hemorrhagic nodules scattered throughout the pulmonary parenchyma and petechial hemorrhages of the renal cortex. Histopathological lesions included focal necrotizing alveolitis, vasculitis, degeneration of glomerular tufts, and focal necrosis in a range of other tissues, including lymph nodes, spleen, adrenal cortex, bladder and ovary. Lesions in each case were associated with significant quantities of viral antigen, as determined by immunohistochemical staining (Fig. 1). Syncytial cells were also frequently present in lesions. NiV genome was detected in blood, brain, liver, testes, adrenal, kidney, lung, lymph node and spleen. These preliminary results are encouraging and indicate that the ferret may be another suitable model for human infection.
The golden hamster, cat and ferret are practical laboratory animal models that can be used to evaluate aspects of disease caused by either HeV or NiV, and each is more amenable to therapeutic efficacy testing than large domestic animals particularly for these BSL-4 pathogens. In hamsters NiV-associated encephalitis was unique among the small animal models; however, it is unclear if this was due to the length of clinical course, where animals were kept until death occurred naturally. In all other studies, most animals were euthanized before advanced disease onset (Table 1). In summary, exploration and validation of such animal models is critical for the future exploration of therapeutic intervention strategies as is the development of non-human primate models for both HeV and NiV infection which has yet to be attempted in earnest.
NiV is the most recently emerging zoonotic and highly deadly virus having pandemic threat. As an emerging and recognized zoonotic pathogen discovered in modern times, NiV causes severe febrile illness and high fatality rates in affected persons and is posing an ongoing high risk to the health of humans worldwide (Clayton 2017; Mukherjee 2017; Thibault et al. 2017). NiV is an uncommon but has become a deadly virus responsible for causing high fatality rates of 40–75%. Fruit bats (Pteropus) serve as natural hosts (wildlife reservoir) and pigs are the intermediate hosts for NiV zoonotic cycle (Paul 2018). During a large outbreak of acute encephalitis in Malaysia in 1998, the virus was discovered in affected patients having contact with sick pigs. The pigs got infection from bats, and then NiV spread proficiently among pig-to-pig, and thereafter from pig-to-man. Moreover, it has been revealed that Pteropus vampyrus and Pteropus hypomelanus (flying foxes in the Malysian Islands) bear the virus in saliva as well as urine, indicating their potential to act as natural reservoir of the virus (Looi and Chua 2007). It is interesting to note that there is always risk of spill over associated with NiV infection. Interaction of the molecular as well as ecological factors collectively that govern the susceptible nature of populations of animals (domestic) as well as humans are not understood yet well (Thibault et al. 2017).
Besides Malaysia, the fruit bats of Pteropus genus serve as the main reservoir of NiV in Thailand and Cambodia. Apart from drinking raw date palm sap contaminated by bats as a cause of initial outbreak, man-to-man and animal-to-man transmission is also a major mode of spread of the infection during an ongoing outbreak. Further, it has been found that direct contact of the susceptible population with the respiratory and body secretions of the infected patients increases the risk of acquiring the infection. During the NiV outbreak in Thakurgaon district, northwest Bangladesh, anti-NiV antibodies were detected in half of the Pteropus bats tested (Chadha et al. 2006; Gurley et al. 2007; Homaira et al. 2010a,b; Clayton 2017; Thibault et al. 2017). Other major public health threats appear to be acquiring NiV infection from the susceptible food and domestic animals. Many domesticated mammals seem to be susceptible to Nipah virus. This virus can be maintained in pig populations, but other domesticated animals such as sheep, goats, dogs, cats and horses appear to be incidental hosts acquiring the infection during outbreaks. Fruits punctured by the bat and contaminated with their saliva forma common source of transmission of NiV infection from bats to domestic animals. Consumption of fruits eaten partially by fruit bats may cause infection in pigs which may then transmit it to humans. Contact with sick cow was reported to have caused a case of human infection in Bangladesh (Chua 2003; Luby et al. 2012; Siddique et al. 2016; http://www.cfsph.iastate.edu/Factsheets/pdfs/nipah.pdf).
Consumption of fruits, vegetables or water contaminated with saliva, urine or fecal matter of infected bats could also be a possible mode of transmission to man and animals (Luby et al. 2009). Date palm sap can be used to prepare alcoholic beverages and such beverages when consumed can lead to human infection (Harit et al. 2006; Simons et al. 2014). Evidence from several NiV outbreaks indicate that consumption of undercooked meat from infected animals or handling of infected animals in the home, farm or slaughter houses may also pose risk of animal-to-man transmission (Chanchal et al. 2018).
Close contact with symptomatic patients or their infectious secretions has been implicated for human transmission of NiV in Bangladesh. Specific exposures can pose a high risk of person-to-person transmission, though sustained transmission do not occur in humans. Studies conducted in animal models further support this fact (Clayton 2017). The recent NiV outbreak in Kerala, India, which caused encephalitis in humans, raised global health concerns (Paul 2018).
The potential for a global pandemic due to NiV appears to stem from several features: availability of susceptible human population, several viral strains withpotential for person-to-person transmission, and error-prone nature of RNA virus replication. Outbreaks of NiV disease in densely populated regions like South Asia can lead to pandemics, due to extensive global travel and trade connectivity (Luby 2013). Many ecological and molecular factors underlie NiV spillover into humans and human and animal susceptibility to it, though the intricate interaction between these is unclear (Thibault et al. 2017). Research studies need to be undertaken to elaborate the molecular mechanisms of the respiratory transmission of NiV in order to reduce the risk of human-to-human transmission. Improved surveillance and vaccination strategies must also be adopted (Luby 2013).
Epithelial cells that line the respiratory tract are the first cells that can be infected by respiratory viruses. Most of these infections are self-limited and the virus is cleared by immunity with minimal clinical consequences. On the other hand, in more vulnerable individuals, viruses can also reach the lower respiratory tract where they cause more severe illnesses, such as bronchitis, pneumonia, exacerbations of asthma, chronic obstructive pulmonary disease (COPD) and different types of severe respiratory distress syndromes. Besides all these respiratory issues, accumulating evidence from the clinical/medical world strongly suggest that, being opportunistic pathogens, these viruses are able to escape the immune response and cause more severe respiratory diseases or even spread to extra-respiratory organs, including the central nervous system where they could infect resident cells and potentially induce other types of pathologies.
Like all types of viral agents, respiratory viruses may enter the CNS through the hematogenous or neuronal retrograde route. In the first, the CNS is being invaded by a viral agent which utilizes the bloodstream and in the latter, a given virus infects neurons in the periphery and uses the axonal transport machinery to gain access to the CNS. In the hematogenous route, a virus will either infect endothelial cells of the blood-brain-barrier (BBB) or epithelial cells of the blood-cerebrospinal fluid barrier (BCSFB) in the choroid plexus (CP) located in the ventricles of the brain, or leukocytes that will serve as a vector for dissemination towards the CNS. Viruses such as HIV, HSV, HCMV, enteroviruses such as coxsackievirus B3, flaviviruses, chikungunya virus (CHIKV) and echovirus 30 have all been shown to disseminate towards the CNS through the hematogenous route. Respiratory viruses such as RSV, henipaviruses, influenza A and B and enterovirus D68 are also sometimes found in the blood and, being neuroinvasive, they may therefore use the hematogenous route to reach the CNS. As they invade the human host through the airway, the same respiratory viruses may use the olfactory nerve to get access to the brain through the olfactory bulb. On the other hand, these viruses may also use other peripheral nerves like the trigeminal nerve, which possesses nociceptive neuronal cells present in the nasal cavity, or alternatively, the sensory fibers of the vagus nerve, which stems from the brainstem and innervates different organs of the respiratory tract, including the larynx, the trachea and the lungs.
Although the CNS seems difficult for viruses to penetrate, those pathogens that are able to do so may disseminate and replicate very actively and will possibly induce an overreacting innate immune response, which may be devastating. This situation may lead to severe meningitis and encephalitis that can be fatal, depending on several viral and host factors (including immunosuppression due to disease or medications) that may influence the severity of the disease.
Recently, a very interesting manuscript produced by Bookstaver and collaborators underlined the difficulties of precisely deciphering the epidemiology and identifying the causal agent of CNS infections. These difficulties are mainly due to the tremendous variation in the symptoms throughout the disease process and to the myriad of viruses that can cause CNS infections. As stated in their report, these authors underlined that the clinical portrait of viral infections is often nonspecific and requires the clinician to consider a range of differential diagnoses. Meningitis (infection/inflammation in meninges and the spinal cord) produces characteristic symptoms: fever, neck stiffness, photophobia and/or phonophobia. Encephalitis (infection/inflammation in the brain and surrounding tissues) may remain undiagnosed since the symptoms may be mild or non-existent. Symptoms may include altered brain function (altered mental status, personality change, abnormal behavior or speech), movement disorders and focal neurologic signs, such as hemiparesis, flaccid paralysis or paresthesia. Seizures can occur during both viral meningitis and encephalitis. Furthermore, viral encephalitis may also be difficult to distinguish from a non-viral encephalopathy or from an encephalopathy associated with a systemic viral infection occurring outside the CNS. Considering all these observations, it is therefore mandatory to insist on the importance of investigating the patient’s history before trying to identify a specific viral cause of a given neurological disorder.
In humans, a long list of viruses may invade the CNS, where they can infect the different resident cells (neuronal as well as glial cells) and possibly induce or contribute to neurological diseases, such as acute encephalitis, which can be from benign to fatal, depending on virus tropism, pathogenicity as well as other viral and patient characteristics. For instance, 30 years ago, the incidence of children encephalitis was as high as 16/100,000 in the second year of life, while progressively reducing to 1/100,000 by the age of 15. More recent data indicate that, in the USA, the herpes simplex virus (HSV) accounts for 50–75% of identified viral encephalitis cases, whereas the varicella zoster virus (VZV), enteroviruses and arboviruses are responsible for the majority of the other cases in the general population. Several other viruses can induce short-term neurological problems. For example, the rabies virus, herpes simplex and other herpes viruses (HHV), arthropod-borne flaviviruses such as the West Nile virus (WNV), Japanese encephalitis virus (JEV), chikungunya virus (CHIKV), Zika virus (ZIKV), alphaviruses such as the Venezuelan, Western and Eastern equine encephalitis viruses and enteroviruses affect millions of individuals worldwide and are sometimes associated with encephalitis, meningitis and other neurological disorders. The presence of viruses in the CNS may also result in long-term neurological diseases and/or sequelae. Human immunodeficiency virus (HIV) induces neurodegeneration, which lead to motor dysfunctions and cognitive impairments. Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease associated with reactivation of latent polyoma JC virus (JCV). Progressive tropical spastic paraparesis/HTLV-1-associated myelopathy (PTSP/HAM) is caused by human T-lymphotropic virus (HTLV-1) in 1–2% of infected individuals. Measles virus (MV), a highly contagious common virus, is associated with febrile illness, fever, cough and congestion, as well as a characteristic rash and Koplik’s spots. In rare circumstances, significant long-term CNS diseases, such as post-infectious encephalomyelitis (PIE) or acute disseminated encephalomyelitis (ADEM), occur in children and adolescents. Other examples of rare but devastating neurological disorders are measles inclusion body encephalitis (MIBE), mostly observed in immune-compromised patients, and subacute sclerosing panencephalitis (SSPE) that appears 6–10 years after infection.
Yet, with the exception of HIV, no specific virus has been constantly associated with specific human neurodegenerative disease. On the other hand, different human herpes viruses have been associated with Alzheimer’s disease (AD), multiple sclerosis (MS) and other types of long-term CNS disorders. As accurately stated by Majde, long-term neurodegenerative disorders may represent a “hit-and-run” type of pathology, since some symptoms are triggered by innate immunity associated with glial cell activation. Different forms of long-term sequelae (cognitive deficits and behavior changes, decreased memory/learning, hearing loss, neuromuscular outcomes/muscular weakness) were also observed following arboviral infections.
Including the few examples listed above, more than one hundred infectious agents (much of them being viruses) have been described as potentially encephalitogenic and an increasing number of positive viral identifications are now made with the help of modern molecular diagnostic methods. However, even after almost two decades into the 21st century and despite tremendous advances in clinical microbiology, the precise cause of CNS viral infections often remains unknown. Indeed, even though very important technical improvements were made in the capacity to detect the etiological agent, identification is still not possible in at least half of the cases. Among all the reported cases of encephalitis and other encephalopathies and even neurodegenerative processes, respiratory viruses could represent an underestimated part of etiological agents.
Despite the importance of NiV as an emerging disease with the potential for pandemic, no therapeutics or vaccines are approved for use in humans or livestock species. Due to the lethal nature of NiV infection, producing a safe, live attenuated vaccine with no potential for reversion is difficult. However, recombinant NiV mutants, attenuated in hamster and ferret models, have been shown to generate strong neutralizing antibody responses (32, 33). More commonly, NiV vaccine approaches have focused individual candidate antigens delivered as subunit vaccines or using viral vectors. The most studied vaccine candidate is the soluble form of the G protein (sG) from the related Hendra virus (HeV). HeV and the NiV Malaysia strain share between 68 and 92% amino acid homology between their proteins; with F and G proteins sharing 88 and 83% homology, respectively (34). Both F and G envelope glycoproteins are regarded as vaccine candidate antigens since they are the targets of NiV neutralizing antibodies (35).
An adjuvanted HeV sG protein subunit-based vaccine (Equivac® HeV, Zoetis) has been licensed in Australia to protect horses against HeV and to reduce the zoonotic risk to humans (36). Equivac® HeV protects ferrets and African green monkeys (AGMs) after experimental challenge with NiV, as well as HeV (37, 38). Surprisingly, this vaccine failed to protect pigs from experimental NiV challenge (39). Since the vaccine induced cross-neutralizing antibodies but not measurable T cell responses, the authors concluded that both arms of the adaptive immune response may be required for protection against NiV and HeV. These studies also potentially highlight that adjuvants can have species specific effects and tailoring of adjuvants to the target species may be required or considered in the context of preclinical models. The experimental viral vectored vaccine candidates for NiV include vesicular stomatitis virus, rabies virus, canarypox virus (ALVAC strain), adeno-associated virus (AAV), measles virus, Newcastle disease virus (NDV) and Venezuelan equine encephalitis virus (40). ALVAC expressing NiV G or F (ALVAC-G and ALVAC-F) was found to protect pigs against NiV challenge 2 weeks after the second immunization (41). High titres of NiV neutralizing antibodies were induced with the ALVAC-G vaccine, while despite the low levels of neutralizing antibodies induced by the ALVAC-F; all vaccinated pigs were protected against virulent NiV challenge. Recombinant attenuated NDV expressing NiV glycoproteins have been shown to induce long lasting NiV-specific nAbs in pigs, with the vector expressing NiV G performing better than NiV F (42). However, no challenge was performed in this study and it remains to be determined whether these paramyxovirus-based vaccine candidates are efficacious. Compared to canarypox vectors, NDV-based vectors have a number of advantages including their high titer propagation in chicken eggs removing the requirement for cell culture (41, 42). Despite these encouraging results and the continued threat posed by NiV, no vaccine candidate has progressed toward market for either pigs or humans.
Hendra virus (HeV) and Nipah virus (NiV) are closely related highly pathogenic paramyxoviruses that have emerged independently in the past 15 years and continue to emerge in new locations. Flying foxes in the genus Pteropus are considered to be the natural reservoir for both viruses as demonstrated by seroconversion and isolation of HeV- and NiV-like viruses from bat tissues and secretions [1, 2]. Additionally, extensive serological surveys have not demonstrated the presence of HeV- or NiV-specific antibodies in other species. Indeed the flying fox geographic range encompasses all locations where HeV and NiV have been found. Paramyxoviruses are large, enveloped, negative-sense ssRNA viruses, and include such well-known members as measles virus (MeV), simian virus 5 (SV5), and respiratory syncytial virus (RSV). It is a diverse virus family, with various members causing common upper and lower respiratory tract infections to less common manifestations of neurological disease. In contrast, NiV and HeV are distinguished from all other paramyxoviruses most notably by their broad species tropism and high case fatality, and they have been classified into the new Henipavirus genus within the family Paramyxoviridae. HeV has appeared sporadically in Australia since 1994 where infection has been transmitted from horses to humans (reviewed in). Presumably horses become infected through spillover events from flying foxes, although no virus has been isolated from flying foxes during outbreaks. In horses, the disease presented as a severe respiratory infection, while one of the two reported human mortalities had severe respiratory disease, and the other succumbed to encephalitis 13 months following the presumed time of exposure. Recent outbreaks where horse fatalities were documented include 1999, 2004 and 2006 and although no human mortalities have occurred, one mildly ill, seroconverting, human case has been reported. NiV first appeared in peninsular Malaysia and Singapore in 1998-9 and the majority of infections occurred in pigs with subsequent transmission to humans (reviewed in [9, 10]). In pigs, infection was largely subclinical; however, where clinical disease was observed, it manifested as respiratory and encephalitic disease with low fatality ratios for respiratory cases. In contrast, humans developed severe febrile encephalitis with high case fatality and up to 25% of NiV cases also exhibited respiratory signs including non-productive cough. Interestingly, both relapsing and late-onset encephalitis syndromes with significant fatality (∼18%) have been recognized following either acute NiV encephalitic episodes or non-encephalitic/ asymptomatic infection. NiV has re-emerged numerous times since 1998: twice in 2001 in Bangladesh and West Bengal India, again in 2003 in Bangladesh, three times in Bangladesh in 2004 and 2005, and most recently in 2007 in Nadia, India. Significant observations in all of the Bangladesh and Indian NiV outbreaks have included a higher incidence of acute respiratory distress syndrome in conjunction with encephalitis, epidemiological findings consistent with person-to-person transmission, and higher case fatality ratios (∼75%). Furthermore, no intermediate or amplification host has been identified and direct transmission of NiV from the reservoir host to humans has been suggested. In West Bengal in 2001 there was no concurrent illness in animals and in Bangladesh in 2004 the common epidemiological link among cases was drinking fresh date palm sap [12, 17]. In general, it is believed that date palm sap is regularly contaminated by flying foxes and their excretions.
NiV and HeV are classified as zoonotic biosafety level 4 (BSL-4) viruses and infectious virus can only be studied at a handful of laboratories worldwide. Both viruses have also been included among the various pathogenic agents of biodefense concern and each are classified as priority pathogens in category C by the Centers for Disease Control and Prevention (CDC) and the National Institute of Allergy and Infectious Diseases (NIAID). The category C agents include high emerging pathogens with the potential for causing morbidity and mortality with major economic and health impacts. Henipaviruses in particular, could be engineered for mass dissemination because of their availability from natural sources and their relative ease of propagation and dissemination. Currently there are no specific antiviral therapies or vaccines available for treating or preventing NiV or HeV infection resulting from a natural outbreak, laboratory accident or deliberate misuse.
Over one-half of all known human pathogens originated from animals, and over 75% of emerging infectious diseases identified in the last three decades were zoonotic.1 The threat of veterinary pathogens to human health continues to grow because of increasing population density and urbanization, global movement of people and animals, and deforestation accompanied by increased proximity of human and wildlife habitats. Recent emerging infectious diseases have been concentrated in tropical Africa, Latin America, and Asia, with outbreaks usually occurring within populations living near wild animals.1 Identification of animal reservoirs from which zoonosis may emerge and detection and characterization of pathogens in these reservoirs will facilitate timely implementation of control strategies for new zoonotic infections.2 Therefore, pathogen discovery studies in animal reservoirs represent an integral part of public health surveillance.
Bats have long been known as natural hosts for lyssaviruses, and more recently, they have been recognized as potential reservoirs for emerging human pathogens, including henipaviruses, filoviruses, and severe acute respiratory syndrome (SARS) related coronaviruses.3,4 Novel viruses are documented in bats every year, which has drawn increasing attention to these mammalian reservoirs that are uniquely associated with a variety of known and potential zoonotic pathogens. In this study, we report the detection of nucleic acids of adenoviruses, rhabdoviruses, and paramyxoviruses in bats from Kenya.
Emerging viruses belonging to the Paramyxoviridae family (Henipaviruses) can cause severe respiratory illness and/or encephalitis in humans. Ferrets infected with henipaviruses exhibit similar symptoms as humans including respiratory signs such as cough and nasal discharge, neural signs such as depression,32 and high mortality rates with most experimentally infected ferrets succumbing within 1 week.31 While the virus is detected in pharyngeal and rectal secretions, it is currently unclear if ferrets could serve as a transmission model for the disease.31, 32 Ferrets infected intranasally with henipaviruses similarly display clinical illness.31, 34 Assessment of immune gene expression by Leon et al31 in both lungs and brain tissues of the infected ferrets revealed upregulation of macrophage markers such as CD40 and CD80 in both lung and brain tissues, whereas lymphocytic markers were unchanged in the lungs.
Longitudinal sampling of the infection status of individuals can help distinguish between scenarios if four conditions are met: (1) Prevalence of the infection must be high enough to detect pathogen within a small number of sampled individuals but must not approach 100% so that variations can be observed. (2) The period over which individuals are resampled must exceed that of the infectious period (and therefore the infectious period must be known). (3) The host’s lifetime must far exceed the infectious period. (4) Pathogen must be detectable without lethal sampling. It is our contention that, if these conditions are met, it should be possible to distinguish among scenarios. In a population with waning immunity and cyclical reinfection, the probability that a given individual becomes infected is expected to be equal to the prevalence of infection among other individuals of similar age and the same sex. Therefore, past infection status is not expected to predict present infection status. In contrast, if individuals are persistently infected but shed episodically, the prevalence of infection among previously positive individuals will be higher than among the population, and past infection increases the likelihood of positive infection status (Table 1). However, large sample sizes and intermediate levels of prevalence probably are required to distinguish cyclical reinfection from persistent infections, and alternative explanations for patterns of prevalence may be difficult to reject. For example, the individuals most likely to be exposed may appear to be persistently infected. In this case, complementing longitudinal sampling with sequence data may improve inferences.
A caveat to this method is that longitudinal, systematic sampling of wild animals, particularly highly mobile or migratory species, requires major effort. For example, large populations of Eidolon helvum (the reservoir hosts of henipaviruses and Lagos bat virus) migrate across continents. Even in resident populations, such as urban populations of Pteropus alecto (the reservoir host of Hendra virus), recapture of individuals within populations that include tens of thousands of individuals is difficult if not impossible. In contrast to studies in migratory canopy-dwelling species, capture-mark-recapture methods have been implemented in cave-dwelling species. For example, Frick et al. reported recapture rates of 0.10 to 0.35 during longitudinal studies of Myotis lucifugusi, and Smith et al. reported recapture rates of up to 0.81 during longitudinal studies of Myotis macropus.
If recapture is possible, generating a consistent time-series of recaptures may be challenging, affecting the ability to make inferences on the temporal resolution of the underlying processes. Nevertheless, some mechanisms may be ruled out even with incomplete time-series data. For example, SIR dynamics are unlikely in an animal in which virus is detected, not detected, and then detected again. However, genomics still is necessary to differentiate SIRS from SILI dynamics.
SARS‐CoV infection causes acute respiratory distress in humans with mortality rates of up to 10%.135 While worldwide outbreaks have not been reported since 2004, there is still a lack of vaccines and effective treatment measures. Ferrets display clinical signs of infection such as elevated body temperatures, sneezing, increase in lymphocyte counts and lesions in the respiratory tract and alveolar oedema7 and are therefore a good mammalian model to study the pathogenesis of SARS‐CoV (reviewed in7, 136) and evaluate vaccines (reviewed in14, 137, 138, 139). In terms of immunity, ferrets exhibit strong antiviral interferon responses after infection and vaccination as measured by interferon response gene expression levels.5, 13 However, leucocyte counts and interferon‐related gene expression were decreased upon re‐infection,5 suggesting that innate immune dysregulation is a possible mechanism of pathogenesis, though a protective antibody response was also evident during attempts to re‐infect ferrets.8
To determine efficacy of ChAdOx1 NiVB against NiV Malaysia and HeV, groups of 10 hamsters were vaccinated with a single dose of ChAdOx1 NiVB or a single dose of ChAdOx1 GFP at D-28 (Fig 3A). As before, virus neutralizing antibodies could be detected after vaccination with ChAdOx1 NiVB but not upon injection with ChAdOx1 GFP (Average VN titer ± SEM = 68.6 ± 13.6) (Fig 3B). Subsequently, hamsters were challenged with either NiV Malaysia or HeV (1000 LD50) via intraperitoneal inoculation on D0 (Fig 3A). All vaccinated animals challenged with NiV Malaysia survived with no signs of disease such as weight loss at any stage throughout the experiment. In contrast, animals challenged with NiV Malaysia that received ChAdOx1 FGP all succumbed to infection between D5 and D6. These animals experienced weight loss and respiratory and neurological signs (Fig 3C and 3D). Statistical analysis demonstrated that survival in the vaccinated group was significantly different from the control group (P = 0.0012).
Oropharyngeal swabs were taken daily and assessed for infectious virus. None of the vaccinated animals challenged with NiV Malaysia shed virus at any timepoint. In contrast, control animals challenged with NiV Malaysia were found to shed virus at D5 and D6 (Fig 3E).
Four animals from both groups were euthanized at D5 and lung and brain tissue were harvested. Infectious virus could only be detected in lung and brain tissue of animals from the control group (average virus titer lung ± SEM = 1.5 x 105 ± 5.2 x 104 TCID50/g, brain ± SEM = 6.8 x 101 ± 4.4 x 101 TCID50/g) and was not detected in any tissue of the vaccinated animals (Fig 3F).
Four out of six vaccinated animals challenged with HeV succumbed to disease between D5 and D7. The two survivors showed minimal weight loss (<2%) and no signs of disease. Animals that received ChAdOx1 FGP all succumbed to HeV infection between D4 and D6. These animals showed weight loss as well as respiratory and neurological signs (Fig 3C and 3D). Log-rank (Mantel-Cox) test demonstrated that survival in the vaccinated group was significant (P = 0.0476) compared to the control group.
Oropharyngeal swabs were taken daily and assessed for infectious virus. None of the vaccinated animals challenged with HeV shed virus at any timepoint. In contrast, control animals challenged with HeV were found to shed virus at D4, D5 and D6 (Fig 3E).
Four animals from both groups were euthanized at D4 and lung and brain tissue were harvested. Infectious virus was detected in three out of four lungs of the vaccinated animals and all lungs of the control animals (average virus titer ± SEM = 5.2 x 105 ± 3.6 x 105 and 4.4 x 106 ± 2.2 x 106 TCID50/g tissue for vaccinated and control animals, respectively). No statistical difference in infectious virus titer was found between the two groups using an unpaired one-tailed Student’s t-test (P = 0.0674). Infectious virus was only detected in brain tissue of animals from the control group (average titer ± SEM = 4.6 x 102 ± 2.0 x 102 TCID50/g) and not in vaccinated animals (Fig 3F).
Harvested lung tissue was then evaluated for pathological changes. All four groups of hamsters developed pulmonary lesions. All animals challenged with HeV and control animals challenged with NiV Malaysia developed bronchointerstitial pneumonia which was indistinguishable from the lesions described for the control animals in the homologous challenge study. Vaccinated hamsters challenged with NiV Malaysia developed mild to moderate bronchointerstitial pneumonia and did not display any evidence of pulmonary edema, fibrin or hemorrhage. ISH demonstrated viral RNA predominantly in type I pneumocytes and rarely in vascular and bronchiolar smooth muscle and endothelial cells in animals challenged with HeV and control animals challenged with NiV Malaysia. In vaccinated animals challenged with NiV Malaysia, however; there was very little RNA present and only in type I pneumocytes in areas of inflammation (Fig 4).
Nipah virus (NiV) is an enveloped, single stranded, negative sense RNA paramyxovirus, genus Henipavirus. The natural hosts and wildlife reservoirs of NiV are Old World fruit bats of the genus Pteropus (1). Both Nipah and the related Hendra virus possess a number of features that distinguish them from other paramyxoviruses. Of particular note is their broad host range which is facilitated by the use of the evolutionary conserved ephrin-B2 and –B3 as cellular receptors (2). The NiV attachment glycoprotein (G) is responsible for binding to ephrin-B2/-B3 (3). Following receptor binding, the G protein dissociates from the fusion (F) protein. Subsequently, the F protein undergoes a series of conformational changes which in turn initiates fusion of the viral and host membrane allowing entry (4). During viral replication, the F protein is synthesized and cleaved into fusion active F1 and F2 subunits. These subunits are subsequently transported back to the cell surface to be incorporated into budding virions, or facilitate fusion between infected and adjacent uninfected cells (5). This cell-to-cell fusion results in the formation of multinucleated cells called syncytia, and greatly influences the cyopathogenicity of NiV as it allows spread of the virus, even in the absence of viral budding (5, 6).
NiV infection is currently classed as a stage III zoonotic disease, meaning it can spill over to humans and cause limited outbreaks of person-to-person transmission (7, 8). NiV outbreaks have been recognized yearly in Bangladesh since 2001 as well as occasional outbreaks in neighboring India (Figure 1). These outbreaks have been characterized by person-to-person transmission and the death of over 70% of infected people (10, 11). In May 2018, the first ever outbreak in southern India was reported. A total of 19 NiV cases, of which 17 resulted in death, were reported in the state of Kerala. Pteropus giganteus bats from areas around the index case in Kozhikode, Kerala, were tested at the National High Security Animal Diseases Laboratory at Bhopal. Of these, 19% were found to be NiV positive by RT-PCR (12). Characteristics of NiV that increase the risk of it becoming a global pandemic include: humans are already susceptible; many NiV strains are capable of person-to-person transmission; and as an RNA virus, NiV has a high mutation rate (13). NiV has been found to survive for up to 4 days when subjected to various environmental conditions, including fruit bat urine and mango flesh (14). Whilst survival time was influenced by fluctuations in both temperature and pH, the ability for NiV to be spread by fomites could play a role in outbreak situations.
The first and still most devastating NiV outbreak occurred in peninsular Malaysia from September 1998 to May 1999 (15, 16). The link to pigs in this outbreak was obvious as 93% of the infected patients had contact with pigs (17). If a NiV strain were to become human-adapted and infect communities in Southeast Asia where there are high human and pig densities and pigs are a primary export commodity, infection could rapidly spread and humanity could face its most devastating pandemic (8, 11, 18).
Finally, we wanted to assess the protective effect of antibodies elicited after ChAdOx1 NiVB vaccination. Two groups of 15 hamsters were either vaccinated with ChAdOx1 NiVB or injected with ChAdOx1 FGP at D-56 and D-28. All animals were bled at D0 and we collected 13 and 15 mL respectively. IgG was purified from 10 mL pooled serum. Ten animals per group were then injected peritoneally with purified IgG. Animals were challenged with a lethal dose of NiV Bangladesh (1000 LD50) one day post passive transfer (Fig 5A). We were unable to detect neutralizing antibodies in serum obtained at D5 from four hamsters from each group. However, serum from animals treated with NiV antibodies was positive by ELISA against NiV G protein, albeit with a lower reciprocal titer than antibodies in serum obtained from single-dose vaccinated animals (Fig 5B). One out of six animals treated with NiV antibodies succumbed to disease on D11. No weight loss was observed, however the animal showed severe neurological signs. None of the other NiV antibody-treated animals experienced weight loss or signs of disease. Four out of six animals treated with GFP antibodies succumbed to disease between D6 and D8. These animals showed weight loss and respiratory or neurological signs. The two surviving animals did not show any signs of disease throughout the experiment. One of these animals did not seroconvert as measured by ELISA against NiV F and G protein, and it was suspected this animal was not infected. Therefore, this animal was excluded from the survival curve. The log-rank (Mantel-Cox) test demonstrated that survival in the treated group was significant (P = 0.0168) compared to the control group (Fig 5C and 5D).
Oropharyngeal swabs were taken daily and assessed for infectious virus. Shedding was minimal and found in one animal treated with NiV antibodies on D5, and five animals treated with GFP antibodies between D4 and D6 (Fig 5E).
Four animals from both groups were euthanized at D5 and lung and brain tissue were harvested. Infectious virus could only be detected in lung tissue of animals treated with GFP antibodies and was not detected in any tissue of the animals treated with NiV antibodies (Fig 5F).
Lung tissue harvested at D5 was then evaluated for pathological changes. Both groups of hamsters developed pulmonary lesions similar to those described in the homologous challenge study, however; the NiV antibody-treated hamsters developed mild to moderate pulmonary lesions whereas the control animals developed severe lesions. Additionally, none of the NiV antibody-treated hamsters displayed any pulmonary fibrin, edema or hemorrhage. ISH demonstrated viral RNA in type I pneumocytes in areas of inflammation. Abundance of viral RNA was notably less in animals treated with NiV antibodies (Fig 6).
Paramyxoviridae is a large and diverse family whose members have been isolated from many species of avian, terrestrial, and aquatic animal species around the world. Paramyxoviruses are pleomorphic, enveloped, cytoplasmic viruses that have a non-segmented, negative-sense RNA genome. The family is divided into two subfamilies, Paramyxovirinae and Pneumovirinae, based on their structure, genome organization, and sequence relatedness. The subfamily Paramyxovirinae contains five genera: Respirovirus, Rubulavirus, Morbillivirus, Henipavirus, and Avulavirus, while the subfamily Pneumovirinae contains two genera, Pneumovirus and Metapneumovirus. All paramyxoviruses that have been isolated to date from avian species can be segregated into two genera based on the taxonomic criteria mentioned above: genus Avulavirus, whose members are called the avian paramyxoviruses (APMV), and genus Metapneumovirus, whose members are called avian metapneumoviruses. The APMV of genus Avulavirus are separated into nine serotypes (APMV-1 through -9) based on Hemagglutination Inhibition (HI) and Neuraminidase Inhibition (NI) assays. Various strains of APMV-1, which is also called Newcastle disease virus (NDV), have been analyzed in detail by biochemical analysis, genome sequencing, and pathogenesis studies, and important molecular determinants of virulence have been identified. As a first step in characterizing the other APMV serotypes, complete genome sequences of one or more representative strains of APMV serotypes 2 to 9 were recently determined, expanding our knowledge about these viruses.
APMV-1 comprises all strains of NDV and is the best characterized serotype because of the severity of disease caused by virulent NDV strains in chickens. NDV strains vary greatly in their pathogenicity to chickens and are grouped into three pathotypes: highly virulent (velogenic) strains, which cause severe respiratory and neurological disease in chickens; moderately virulent (mesogenic) strains, which cause mild disease; and non-pathogenic (lentogenic) strains, which cause inapparent infections. In contrast, very little is known about the comparative disease potential of APMV-2 to APMV-9 in domestic and wild birds. APMV-2 strains have been isolated from chickens, turkeys and wild birds across the globe. APMV-2 infections in turkeys have been found to cause mild respiratory disease, decreases in egg production, and infertility. APMV-3 strains have been isolated from wild and domestic birds. APMV-3 infections have been associated with encephalitis and high mortality in caged birds. APMV-4 strains have been isolated from chickens, ducks and geese. Experimental infection of chickens with APMV-4 resulted in mild interstitial pneumonia and catarrhal tracheitis. APMV-5 strains have only been isolated from budgerigars (Melopsittacus undulatus) and cause depression, dyspnoea, diarrhea, torticollis, and acute fatal enteritis in immature budgerigars, leading to very high mortality. APMV-6 was first isolated from a domestic duck and was found to cause mild respiratory disease and drop in egg production in turkeys, but was avirulent in chickens. APMV-7 was first isolated from a hunter-killed dove and has also been isolated from a natural outbreak of respiratory disease in turkeys. APMV-7 infection in turkeys caused respiratory disease, mild multifocal nodular lymphocytic airsacculitis, and decreased egg production. APMV-8 was isolated from a goose and a feral pintail duck. APMV-9 strains have been isolated from ducks around the world. APMV types -2, -3, and -7 have been associated with mild respiratory disease and egg production problems in domestic chickens. There are no reports of isolation of APMV-5, -8 and -9 from poultry. But recent serosurveillance of commercial poultry farms in USA indicated the possible prevalence of all APMV serotypes excluding APMV-5 in chickens.
APMV-1 (NDV) is known to replicate in non-avian species including humans, although its only natural hosts are birds. APMV-1 infections in non-avian species are usually asymptomatic or mild. Clinical signs in human infections commonly involve conjunctivitis, which usually is transient and self-limiting. Presently, APMV-1 is being evaluated as a vaccine vector against human pathogens. When administered to the respiratory tract of non-human primates, NDV is highly restricted in replication, but foreign antigens expressed by recombinant NDV vectors are moderately to highly immunogenic. One of the major advantages of this approach is that most humans do not have pre-existing immunity to APMV-1. Pre-existing immunity is a potential drawback to using vectors derived from common human pathogens, and also can be a concern for any vector if two or more doses are necessary to elicit protective immunity. Therefore, we are investigating APMV types 2 to 9, which are antigenically distinct from APMV-1, as alternative human vaccine vectors. Also, some of these additional APMV types likely will have differences in replication, attenuation, and immunogenicity compared to APMV-1 that may be advantageous. However, the replication and pathogenicity of APMV-2 to -9 in non-avian species has not been studied. As a first step, we have evaluated the replication and pathogenicity of APMV-2 to -9 in hamsters. In this study, groups of hamsters were infected with a prototype strain of each APMV serotype by the intranasal route and monitored for virus replication, clinical symptoms, histopathology, and seroconversion. Our results showed that each of the APMV serotypes replicated in hamsters without causing adverse clinical signs of illness, although histopathologic evidence of disease was observed in some cases, and also induced high neutralizing antibody titers.
Nipah Virus (NiV) is a recently-recognised and highly pathogenic zoonotic paramyxovirus that can cause severe disease in man with high associated fatality rates (up to 100%).54 Outbreaks have occurred in Malaysia, Singapore and India with almost annual occurrence in Bangladesh. Human-to-human transmission is common in Bangladesh and has also been documented in India.55 Several species of pteropid fruit bats are known to be host reservoirs of NiV, with accumulating evidence that both NiV and other paramyxoviruses can circulate worldwide in bats.54-56 The high fatality rate, direct infection from natural reservoirs, infection following amplification in susceptible domestic livestock such as pigs, documented human-to-human transmission, and the potential ability to transverse the globe, all emphasise the pandemic potential of NiV.56
There are no clinically approved vaccines against NiV, however, one therapeutic approach (monoclonal antibody therapy) has recently completed a phase I clinical trial with results still to be reported.57 While monoclonal antibody treatment may be efficacious in a short window post-exposure, this treatment option is not suitable for large-scale use, and as such, vaccine development is a key research focus for the prevention of NiV-mediated disease. Advantageously, there are a number of animal models of NiV infection which are used in vaccine development programs and are considered to sufficiently mirror NiV-induced pathogenesis observed in humans, e.g. the hamster, ferret and African Green Monkey (AGM) models.58-60
While vaccine-mediated cellular immunity has been demonstrated to play a role in protection in preclinical models of NiV infection,61 the most advanced vaccine modalities demonstrating clear efficacy across multiple animal models have primarily induced humoral immunity. A soluble glycoprotein (sG) subunit vaccine from the related henipavirus Hendra virus (HeV) is an extensively studied vaccine that can protect ferrets and AGM from experimental challenge with NiV or HeV. Prime-boost regimens with adjuvanted HeV-sG subunit proteins are efficacious in stringent NiV challenge models, across a range of doses (4–100ug), and with pre-challenge neutralising antibody titres as low as 1:28.62,63 The HeV sG vaccine (Equivac® HeV) has been licensed to vaccinate horses in Australia against HeV.64 A number of viral vectored vaccines have also been tested and show promising immunogenicity and/or efficacy against NiV-mediated disease. These include poxvirus (canarypoxvirus ALVAC strain), vesicular stomatitis virus (VSV), rabies virus (RABV), adeno-associated virus (AAV), Newcastle disease virus (NDV) and Venezuelan equine encephalitis virus (VEEV); this topic has recently been comprehensively reviewed.56,65