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The virus is responsible for causing severe and rapidly progressing illness in humans with the respiratory system as well as the central nervous system (CNS) mainly getting affected (Hossain et al. 2008). The signs and symptoms of the disease appear 3–14 days post NiV exposure. Initially, there is a high rise of temperature along with drowsiness and headache. This is followed by mental confusion as well as disorientation, ultimately progressing towards coma within 1–2 days. A critical complication of the NiV infection is encephalitis. During initial phase, the respiratory problems may become evident. There is development of atypical pneumonia. Coughing along with acute respiratory distress may be evident in certain patients (Hossain et al. 2008; Williamson and Torres-Velez 2010). There may be sore throat, vomiting, along with muscle aches (www.medicinenet.com). There may be development of septicemia along with impairment of the renal system and bleeding from the gastrointestinal tract. In severe cases within a period of 24–48 h, there may be development of encephalitis along with seizures that ultimately leads to coma (Giangaspero 2013). It is crucial to note that transmission of the virus is more common from patients having labored breathing than those having no respiratory problems (www.cdc.gov; Luby et al. 2009).
The central nervous system (CNS), a marvel of intricate cellular and molecular interactions, maintains life and orchestrates homeostasis. Unfortunately, the CNS is not immune to alterations that lead to neurological disease, some resulting from acute, persistent or latent viral infections. Several viruses have the ability to invade the CNS, where they can infect resident cells, including the neurons. Although rare, viral infections of the CNS do occur. However, their incidence in clinical practice is difficult to evaluate precisely. For instance, in cases of viral encephalitis involving the most prevalent viruses known to reach the CNS (mainly herpesviruses, arboviruses and enteroviruses), an actual viral presence can only be detected in 3 to 30 cases out of 100,000 persons. Considering all types of viral infections, between 6000 and 20,000 cases of encephalitis that require hospitalization occur every year in the United States, representing about 6 cases per 100,000 infected persons every year. As the estimated charge for each case lies between $58,000 and $89,600, an evaluation of the total annual health cost is of half a billion dollars. Due to the cost associated with patient care and treatment, CNS viral infections cause considerably more morbidity and disabilities in low-income/resource-poor countries.
Very common worldwide, viral infections of the respiratory tract represent a major problem for human and animal health, imposing a tremendous economic burden. These respiratory infections induce the most common illnesses and are a leading cause of morbidity and mortality in humans worldwide, causing critical problems in public health, especially in children, the elderly and immune-compromised individuals. Viruses represent the most prevalent pathogens present in the respiratory tract. Indeed, it is estimated that about 200 different viruses (including influenza viruses, coronaviruses, rhinoviruses, adenoviruses, metapneumoviruses, such as human metapneumovirus A1, as well as orthopneumoviruses, such as the human respiratory syncytial virus) can infect the human airway. Infants and children, as well as the elderly represent more vulnerable populations, in which viruses cause 95% and 40% of all respiratory diseases, respectively. Among the various respiratory viruses, some are constantly circulating every year in the human populations worldwide, where they can be associated with a plethora of symptoms, from common colds to more severe problems requiring hospitalization. Moreover, in addition to the many “regular” viruses that circulate and infect millions of people every year, new respiratory viral agents emerge from time to time, causing viral epidemics or pandemics associated with more serious symptoms, such as neurologic disorders. These peculiar events usually take place when RNA viruses like influenza A, human coronaviruses, such as MERS-CoV and SARS-CoV, or henipaviruses, present in an animal reservoir, cross the species barrier as an opportunistic strategy to adapt to new environments and/or new hosts. These zoonoses may have disastrous consequences in humans, and the burden is even higher if they have neurological consequences.
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.
Magnetic resonance imaging (MRI) studies in human patients have revealed that the cortex, pons as well as temporal lobes of brain get involved. There may be bilateral abnormalities in the white matter of the brain. In the cerebral cortex, there may be more than one hyperintensities (T1-weighted) which are very much similar to necrosis of the laminar cortex. Lesions may also become evident in corpus callosum, brain stem, as well as cortex of the cerebrum. It is crucial to note in this regard that diffusion-weighted (DW)-MRI is employed to detect such lesions (Goh et al. 2000; Lim et al. 2002; Ang et al. 2018). There may be presence of disseminated microinfarction in the brain due to thrombosis induced by vasculitis. The neurons may also get involved directly. Vasculitic lesions of similar nature may be found in the kidneys, heart, as well as respiratory tract (Ang et al. 2018). It is also interesting to note that blood vessels of medium and small size show most involvement in case of infection due to NiV, which results in development of syncytia (multinucleated) along with fibrinoid necrosis (Ang et al. 2018).
There may be consolidation of varying degree along with hemorrhages (either petechiae or ecchymosis) in the lungs of affected pigs at necropsy. Froth-filled bronchi along with trachea are commonly observed. In certain instances, there may be presence of blood stained fluids in the trachea and bronchi. Congestion along with generalized edema is present in kidneys and brain. Both the cortex as well as suface of kidneys may become congested (Nor et al. 2000). There may be pneumonia (moderate to high) along with formation of syncytial cells in the endothelial cell lining of the blood vasculatures as revealed histologically (Chua et al. 2000; Nor et al. 2000). In the CNS and other major organs like lungs and kidneys, there may be development of small vessel vasculopathy (disseminated) in case of acute infection (Wong et al. 2009). Generalized vasculitis along with fibrinoid necrosis and mononuclear cell infiltration may be noticed in the brain, kidneys and lungs. Viral antigens at greater concentration may be present in the blood vascular endothelial cells (especially in the lungs) as is revealed immunohistologically. In the upper respiratory tract of pigs in the lumen viral antigens are evident amidst the cellular debris which is suggestive of the possible transmission of NiV through exhalation (Nor et al. 2000; Kulkarni et al. 2013). In dogs, kidneys may show congestion with severe hemorrhage. Exudates may be present in the bronchi and trachea (Nor 1999; Kulkarni et al. 2013).
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.
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.
Pigs also suffered during the 1998/99 Malaysian outbreak, but this was only diagnosed as part of the investigation following the human cases. The severity of symptoms of NiV infection in pigs varied with age. In suckling pigs (<4 weeks old), mortality could be high (up to 40%) and labored breathing and muscle tremors were evident. In growing pigs (1 to 6 months), an acute febrile (>39.9°C) illness was observed with respiratory signs ranging from increased or forced respiration to a harsh, loud non-productive cough, open mouth breathing, and epistaxis (26). In some cases these respiratory signs were accompanied by one or more of the following neurological signs: trembles, neuralgic twitches, muscle fasciculation, tetanic spasms, incoordination, rear leg weakness, or partial paralysis. Pigs of this age had high morbidity and low mortality (<5%) (26–28). Some animals over 6 months of age died rapidly (within 24 h) without signs of clinical disease. Respiratory signs were reported in adult pigs, as with younger animals, although these were less obvious (labored breathing, bloody nasal discharge, increased salivation) and neurological signs included head pressing, bar biting, tetanic spasms and convulsions. First trimester abortions were also reported (26–28).
In an experimental infection study, pigs were inoculated subcutaneously with a NiV isolate from the central nervous system of a fatally infected human patient. Infection elicited respiratory and neurological symptoms consistent with those observed in naturally infected Malaysian pigs, which included febrile illness, incoordination, mucosal nasal discharge, and persistent cough (29). Pigs inoculated orally with the same dose did not show clinical signs although they still shed virus. In a second study, piglets were inoculated oronasally with a human NiV isolate (30). All infected animals showed a transient increase in body temperature between 4 and 12 days post-infection. Two of these animals developed transient respiratory signs, mild depression and a hunched stance. Both these studies concluded that NiV infection in pigs had no pathognomonic features i.e., the clinical signs observed were non-specific. This can make field diagnosis of NiV infection in pigs difficult, as observed in the outbreak in Malaysia (16, 28).
The name proposed for the disease caused by NiV infection of pigs was “porcine respiratory and neurological syndrome” (also known as “porcine respiratory and encephalitis syndrome”), or, in peninsular Malaysia, “barking pig syndrome” (28). NiV infection was included as the sixth pig disease notifiable to the OIE World Organization for Animal Health (31). The OIE approve diagnostics and recommends preventative and control measures for a range of transboundary livestock diseases.
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).
Hendra virus and Nipah virus are the only identified zoonotic members of the paramyxovirus family, and both are highly pathogenic in humans. Hendra virus infection has resulted in multiple horse and four human fatalities since its emergence in Australia in 1994, with outbreaks in 2008 and 2009 leading to rising concern in the Australian horse breeding industry. Nipah virus emerged in Malaysia in 1999, causing an outbreak of viral encephalitis that led to 105 human fatalities out of 265 reported cases. Containment of the 1999 Nipah virus epidemic required the sacrifice of more than 1 million swine. Continued Nipah outbreaks have occurred in Southeast Asia, with mortality rates of up to 70% and suspected human-to-human transmission. Numerous molecular features have led to the placement of Hendra and Nipah viruses within a new genus in the paramyxovirus family, the henipaviruses (Figure 1). The principal reservoir species for both viruses is thought to be Pteropus fruit bats, but a number of other species have been shown to be susceptible to infection.
Human metapneumovirus (HMPV) was first identified in 2001, but unlike Hendra and Nipah, HMPV is not a new human virus resulting from zoonotic transmission. Instead, HMPV is a long-term human pathogen that was only identified by careful analysis of samples from children with respiratory tract disease for which an etiological agent had not been identified. Subsequent studies indicate that HMPV is a major causative agent of respiratory tract infections worldwide, causing between 5% and 15% of lower respiratory tract infections in young children. HMPV has been circulating in the human population since at least 1958. Sequence analysis places HMPV in the Pneumovirinae subfamily, along with RSV.
The Paramyxoviridae family within the order of Mononegavirales includes a large number of human and animal viruses that are responsible for a wide spectrum of diseases. Measles virus (MV) is one of the most infectious human viruses known, and has been targeted by the World Health Organization for eradication through the use of vaccines. The paramyxovirus family includes several other viruses with high prevalence and public health impact in humans, like respiratory syncytial virus (RSV), human metapneumovirus (HMPV), mumps virus (MuV), and the parainfluenza viruses (PIV). In addition, newly emerging members of the Paramyxoviridae family – hendra and nipah virus – have caused fatal infections in humans upon zoonoses from animal reservoirs,,. In animals, Newcastle disease virus (NDV) is and Rinderpest virus (RPV) was among the viruses with the most devastating impact on animal husbandry. Members of the Paramyxoviridae family switch hosts at a higher rate than most other virus families and infect a wide range of host species, including humans, non-human primates, horses, dogs, sheep, pigs, cats, mice, rats, dolphins, porpoises, fish, seals, whales, birds, bats, and cattle. Thus, the impact of paramyxoviruses to general human and animal welfare is immense.
The Paramyxoviridae family consists of two subfamilies, the Paramyxovirinae and the Pneumovirinae. The subfamily Paramyxovirinae includes five genera: Rubulavirus, Avulavirus, Respirovirus, Henipavirus and Morbillivirus. The subfamily Pneumovirinae includes two genera: Pneumovirus and Metapneumovirus
. Classification of the Paramyxoviridae family is based on differences in the organization of the virus genome, the sequence relationship of the encoded proteins, the biological activity of the proteins, and morphological characteristics,. Virions from this family are enveloped, pleomorphic, and have a single-stranded, non-segmented, negative-sense RNA genome. Complete genomic RNA sequences for known members of the family range from 13–19 kilobases in length. The RNA consists of six to ten tandemly linked genes, of which three form the minimal polymerase complex; nucleoprotein (N or NP), phosphoprotein (P) and large polymerase protein (L). Paramyxoviruses further uniformly encode the matrix (M) and fusion (F) proteins, and – depending on virus genus – encode additional surface glycoproteins such as the attachment protein (G), hemagglutinin or hemagglutinin-neuraminidase (H, HN), short-hydrophic protein (SH) and regulatory proteins such as non-structural proteins 1 and 2 (NS1, NS2), matrix protein 2 (M2.1, M2.2), and C and V proteins,.
Routine diagnosis of paramyxovirus infections in humans and animals is generally performed by virus isolation in cell culture, molecular diagnostic tests such as reverse transcriptase polymerase chain reaction (RT-PCR) assays, and serological tests. Such tests are generally designed to be highly sensitive and specific for particular paramyxovirus species. However, to detect zoonotic, unknown, and newly emerging pathogens within the Paramyxoviridae family, these tests may be less suitable. Development of virus family-wide PCR assays has greatly facilitated the detection of previously unknown and emerging viruses. Examples of such PCR assays are available for the flaviviruses, coronaviruses, and adenoviruses. For the Paramyxoviridae, Tong et al. described semi-nested or nested PCR assays to detect members of the Paramyxovirinae or Pneumovirinae subfamily or groups of genera within the Paramyxovirinae subfamily. Although these tests are valuable for specific purposes, nesting of PCR assays and requirement for multiple primer-sets are sub-optimal for high-throughput diagnostic approaches, due to the higher risk of cross-contamination, higher cost, and being more laborious.” Here, a PCR assay is described that detects all genera of the Paramyxoviridae with a single set of primers without the requirement of nesting. This assay was shown to detect all known viruses within the Paramyxoviridae family tested. As the assay is implemented in a high-throughput format of fragment analysis, the test will be useful for the rapid identification of zoonotic and newly emerging paramyxoviruses.
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.
Bats are an important reservoirs of different pathogenic agents, and many of them have already caused disease outbreaks worldwide.More than 200 viruses have been associated with bats, and almost all are RNA viruses probably owing to their great ability to adapt to changing environmental conditions through a higher genetic variability.Bacteria in bats and their putative threat to humans remain poorly studied.
In order to characterize infection of NiV-M, NiV-B and HeV in the ferret model and to determine the optimal challenge dose for subsequent studies, groups of 4 animals were challenged with 10-fold dilutions (10, 102, 103, or 105 tissue culture infectious dose 50%, or TCID50) of these three henipavirus strains via the intranasal (IN) route. Clinical signs were first noticeable on day 5 post infection (pi), and included development of fever, labored breathing, mild paralysis, generalized tremors and subcutaneous edema of the head and neck, followed by rapidly progressing clinical signs on days 6 and 7 pi. This coincided with a small increase in body temperature of HeV and NiV-B infected animals on day 5 pi (S1 Fig). Moribund animals displayed clinical signs that included lack of grooming, hunched posture, ataxia, severe depression, labored breathing, occasional subcutaneous edema of the neck and head, vomiting; and neurological signs such as continuous licking, tremors, imbalance, myoclonus, head tilt, hind limb paralysis and seizures. The majority of animals succumbed of infection by day 6–8 pi with a lethal dose 50% (LD50) for HeV, NiV-B and NiV-M of 5, 32 and 22 TCID50, respectively (Fig 1A–1C). Several ferrets receiving low infective doses (10 or 102 TCID50) displayed no clinical signs throughout the period of observation and did not seroconvert, suggesting that the LD50 is also the minimal infectious dose at which 50% is infected.
West Nile virus (WNV) is a zoonotic Flavivirus belonging to the family Flaviviridae. The virus is transmitted by mosquitoes and causes fatal encephalitis in human, equines and birds.
WNV was first isolated and identified in 1937 from a woman presented with mild febrile illness in the Nile district of Uganda. It has been described in Africa, Europe, South Asia, Oceania and North America. Countries with incidence/serological evidence are presented in Fig. (1). In North America, more than 1.8 million people have been infected, with over 12,852 reported cases of encephalitis or meningitis and 1,308 deaths from 1999 to 2010. The mortality rate in human varies from 3-15% and can reach up to 50% in clinically affected horses. WNV in India has been confirmed by seroprevalence and by virus isolation on different occasions from mosquitoes bat and man [42, 45-47]. WNV infection has also been reported in animals and birds.
Horses and human are the main hosts. Animals other than horses may be susceptible to WNV, but rarely become ill. Antibodies have been found in serum samples from bats, horse, dogs, cats, racoons, opossums, squirrels, domestic rabbits, eastern striped skunks, cows, sheep, deer and pigs. The virus is transmitted to humans by mosquitoes. About 20% of the infected people develop fever with other symptoms. Fatal, neurologic illness occurs in less than 1% of infected people.
WNV is amplified by continuous transmission cycles between mosquitoes and birds. Generally Culex mosquitoes are the vectors and passerine birds are the vertebrate reservoirs in enzootic transmission cycles. The virus is carried in the salivary glands of infected mosquitoes and transmitted to susceptible birds during blood-sucking. Competent bird reservoirs sustain an infectious viraemia for 1 to 4 days subsequent to exposure, and then develop life-long immunity. Horses, human and most other mammals rarely develop the infectious levels of viraemia and are dead-end hosts. Few cases in human have been spread through blood transfusions, organ transplants, breast feeding and during pregnancy. The ticks observed to be infected naturally include Ornithodoros maritimus, Argas hermanni and Hyalomma marginatum and the virus has also been isolated from other species of hard ticks in Africa, Europe and Asia [36, 63, 64]. Swallow bugs (Oeciacus hirundinis) have been implicated as vectors in Austria. In most of the horses bitten by carrier mosquitoes, there is no disease. Approximately 33% of the infected horses develop severe disease and die or are affected severely. The time between the bite of an infected mosquito and appearance of clinical signs ranges from 3 to 14 days. The symptoms in horses may vary from none to trembling, skin twitching and ataxia. There can be sleepiness, dullness, listlessness, facial paralysis, difficulty in urination and defecation, and inability to rise. In some horses, there can be mild fever, blindness, seizures, and other signs.
There is no effective treatment for clinical WNV infection in humans, horses or any other animal. Vaccines are available for control of WNV in horses in the USA. Vaccination of horses protects valuable animals from a potentially fatal disease, but trade and competition practices make this undesirable as some countries use positive antibody tests and impose import restrictions. WNV encephalitis is so rare in human that vaccine development may not be feasible economically.
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.
According to data released by the World Health Organization (WHO), infectious agents causing lower respiratory infections, diarrheal diseases, and tuberculosis were ranked in the top ten causes of death worldwide, resulting in 5.7 million deaths in 2016 (1). It is clear that we need to improve our understanding of these diseases and pathogenic agents in order to develop more effective drugs and vaccines. To this end, we need a suitable animal model that can most accurately mimic the pathogenesis of infection as infection usually induces a complex process of host immune responses that in vitro experiments are unable to simulate. Only in vivo models can accurately assess the complexity of host responses and allow the efficacy and adverse effects of drugs or vaccine to be evaluated.
The Syrian hamster (Mesocricetus auratus) has been used as an animal model to study human-associated diseases for over 60 years. A number of studies have documented that Syrian hamsters represent better models for analysis of viral infections compared to murine models as the similarity to humans with regard to disease symptoms, pathognesis and immune responses is greater (2–4). It has been demonstrated by us and others that human cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12), are fully functional in hamster models, but not in mouse models (5, 6). Together with other advantages, such as fast reproductive rate and ease of handling, Syrian hamsters are a superior choice compared with other small animals.
Although Syrian hamsters have historically been used in diseases research, their value as an animal model in the study of infectious diseases has only recently been realized. With advancements in gene editing technologies, their popularity has increased significantly (Figure 1). The use of genetically engineered Syrian hamster (GESH) models is critical for understanding disease progression and for developing prophylactic and therapeutic treatment regimens. The first STAT2 gene knockout (KO) Syrian hamster was developed in 2014, using the CRISPR/Cas9 system to target the hamster germline (7). STAT2 is a crucial element of the type I interferon (IFN) signal transduction pathway and the hamster model has emerged as the only small animal model permissive for Adenovirus (AdV) infection, thus, the STAT2 KO model has been critical for the characterization of Adenovirus pathogenesis (8).
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.
Although bat bites may be the main transmission route coming to mind, pathogen transmission involving bat bites has been documented mostly for rabies virus (Rhabdoviridae). The common vampire bat (Desmodus rotundus) can, for instance, naturally transmit rabies to other species when biting to feed on blood, particularly to livestock and sometimes to humans. Mycoplasma has also been detected in common vampire bat blood and saliva and might be transmitted between bats, for instance, during aggressive behaviors. Obligate blood-feeding bats are, however, restricted to Central and South America and represent only a very small proportion of the bat species diversity (<0.005%; 3/1,200). Most bat species do not naturally bite humans unless intentional contacts occur (e.g., veterinarian and field biologists involved in bat capture and handling, people trying to remove bats from houses).
Contact with bat body fluids (saliva, urine, and feces) is increasingly recognized as an important mechanism of pathogen spillover to humans. Human encroachment into bat habitats as well as increasing urbanization, which facilitates bat roosting in artificial structures, are likely to increase contact with bat body fluids. For example, Nipah virus (Paramyxoviridae) human infection cases reported in Bangladesh were associated with the consumption of raw sap from date palm trees contaminated with fruit bat saliva and urine. In the case of Marburg virus (Filoviridae), experimental studies indicate that bat-to-bat transmission may occur via saliva and aerosols, suggesting that the virus may be transmitted to other hosts by a similar mechanism. This hypothesis is supported by investigations revealing that most humans infected with Marburg virus had entered bat (Rousettus aegyptiacus) caves before becoming sick and reported regular contacts with bats or their secretions.
Hunting, preparation, and consumption of bats as bushmeat have also been pointed out as a potential source of infection, especially for Ebola virus. For instance, the putative first human case of the 2007 Ebola outbreak in the Democratic Republic of Congo would have bought freshly killed bats for consumption. The fruit bat Eidolon helvum, which is the most frequently hunted and traded bat species in many African countries (e.g., more than 120,000 E. helvum are sold yearly in markets in Ghana), has been shown to be infected with Henipa-related viruses. This highlights the substantial exposure of local hunters and consumers to viruses of potential zoonotic importance.
Nipah virus (NiV) together with Hendra virus (HeV) are closely related highly pathogenic zoonoses and are the type species within the paramyxovirus genus Henipavirus. Both viruses can cause significant morbidity and mortality in a variety of vertebrate species including humans. The henipaviruses are categorized as zoonotic biosafety level 4 (BSL-4) agents which has limited an extensive examination of their in vivo pathogenic features and the development and evaluation of therapeutics or vaccines. NiV and HeV are select agents of biodefense concern that are classified as priority pathogens in category C by the National Institute of Allergy and Infectious Diseases and the Centers for Disease Control and Prevention, with the potential to cause significant morbidity and mortality in humans and major economic and public health impacts (reviewed). Pteropid bats (family Pteropodidae), commonly known as flying foxes, are the predominate natural reservoirs for both HeV and NiV (reviewed) although evidence of henipavirus infection has now been reported in a wider range of both frugivorous and insectivorous bats,.
Since their initial recognition, both viruses have repeatedly re-emerged. In total, 13 HeV outbreaks have occurred in Australia in 1994, 1999, 2004, and 2006–2009, and have always involved horses as an intermediate host with some human infections including four fatalities, the most recent in September 2009 (reviewed),. NiV has also repeatedly caused spill-over events involving hundreds of human cases since 1998 with at least nine recognized occurrences primarily in Bangladesh and India since 2001 (reviewed) with the most recent in March 2008. Several of the more recent NiV outbreaks have had higher rates of acute respiratory distress syndrome in conjunction with encephalitis, epidemiological findings consistent with multiple rounds of person-to-person transmission, higher case fatality rates (∼75%), and direct transmission of virus from flying foxes to humans via contaminated food has been demonstrated,.
In addition to their highly pathogenic nature, the henipaviruses are also distinguished from all other paramyxoviruses by their unusually broad host tropism. Host cell infection by NiV and HeV requires two membrane-anchored envelope glycoproteins; the attachment (G) glycoprotein which binds the viral receptor, and the fusion (F) glycoprotein which drives virus-host cell membrane merger. The henipavirus G glycoprotein lacks hemagglutinin and neuraminidase activities and the F glycoprotein is a typical class I fusion glycoprotein (reviewed in). The host cell membrane anchored proteins, ephrin-B2 and ephrin-B3 ligands, have been shown to be the receptors employed by the henipaviruses,,,. There are presently no licensed therapeutics available to treat infection caused by the henipaviruses. Recently, we isolated and extensively characterized a neutralizing human monoclonal antibody (hmAb), m102.4, which recognizes the receptor binding domain of the HeV and NiV G glycoproteins. This hmAb potently neutralized both viruses in vitro and maintained its biological activity in vivo suggesting its possible utility as a passive therapeutic modality following henipavirus infection. Here we report the development and characterization of a novel ferret model of acute NiV infection and associated disease as well as conduct the first henipavirus therapeutic antibody trial using the hmAb m102.4. Together, our data demonstrate that NiV-mediated disease in the ferret closely resembles that seen in humans with the presence of both respiratory and neurological disease. We further demonstrate that m102.4 is an effective post-exposure therapeutic representing the first antiviral drug candidate showing in vivo efficacy in treating lethal NiV-mediated disease, and it is the first human mAb therapeutic developed and tested for the treatment of henipavirus infection.
In humans, disease resulting from NiV infection can vary in intensity from an acute febrile illness or one progressing to severe central nervous and respiratory disease. Pathological findings show systemic vasculitis, necrotizing alveolitis and meningoencephalitis,. The disease in experimentally infected cats and hamsters is similar,; but in hamsters meninoencephalitis is more prominent, while cats develop an acute respiratory disease. Here, we sought to assess a new ferret model of NiV pathogenesis where our preliminary observations had confirmed susceptibility to NiV infection, with development of systemic vasculitis and involvement of the central nervous and respiratory systems. Ferrets have emerged as a model for several viral respiratory diseases including avian influenza, severe acute respiratory syndrome), and morbilliviruses, close relatives of henipaviruses. They offer the combined advantages over either of the aforementioned laboratory animal species of being relatively small mammals, while displaying complex behaviors especially in relation to their handlers that may be used to advantage in clinical assessments. They are however also sufficiently large to enable repeated collection of a wide range of clinical samples throughout the course of an experimental infection, as well as administration of potential therapies in a manner similar and consistent with human medicine.
We initiated a NiV minimal infectious dose study (MID50) for the purpose of determining an appropriate challenge dose for subsequent work that would reliably productively infect naïve ferrets. Doses of 50, 500, 5,000 or 50,000 TCID50 were each administered to groups of two ferrets oral-nasally; the most likely route of natural infection. Based on prior experience with NiV infection in cats,, and using similar parameters, we defined infection endpoints in ferrets for the purpose of humane euthanasia; these were used as surrogates for lethality. Endpoints were fever plus signs of rapidly progressing clinical illness including both generalized (e.g. inappetance, depression) and localizing (e.g.dyspnea, neurological signs) disease signs. Ferrets that received 50 TCID50 (1–50 and 2–50), and one animal that received 500 TCID50 (3–500) remained well throughout the period of observation, did not shed detectable virus or viral RNA, did not seroconvert, and their tissues were normal at post mortem and histological examination. The remaining ferrets developed fever (4–7 days post-infection (dpi)) and rapidly progressive clinical illness (6–8 dpi). Ferrets inoculated with 50,000 TCID50 (7–50000 and 8–50000) were euthanized 6 and 7 dpi, respectively; those inoculated with 5,000 TCID50 (5–5000 and 6–5000) were euthanized 8 and 10 dpi, respectively, and one ferret inoculated with 500 TCID50 (4–500) was euthanized 9 dpi.
Clinical signs in affected ferrets included severe depression, cough, serous nasal discharge, dyspnea and subcutaneous edema of the head (8–50000); severe depression, orthopnea, and expiratory dyspnea (7–50000); severe depression, orthopnea and cutaneous ecchymoses (6–5000); severe depression, vomiting and hypothermia (5–5000); and obtunded with tremor and hind limb paresis (4–500). Gross pathological findings comprised varying degrees of subcutaneous hemorrhagic edema of the head and neck, centered upon local lymph nodes in all diseased ferrets; focal raised pin-head hemorrhagic nodules scattered throughout the pulmonary parenchyma; and scattered intra-abdominal petechial hemorrhages. Histopathology of diseased ferrets revealed acute focal necrotizing alveolitis and pulmonary vasculitis, acute glomerular necrosis, focal necrosis of the spleen, and severe diffuse subacute inflammation of the organs and connective tissues of the head and neck. Less commonly, there was mild focal nonsuppurative meningitis (4–500, 5–5000 and 8–50000), focal cystitis (4–500), severe acute necrotizing salpingitis (4–500), acute focal coagulative necrosis of the adrenal cortex (4–500 and 5–5000), and severe acute thyroiditis (6–5000). Two ferrets demonstrated neurological signs (4–500, 5–5000) and had nonsuppurative meningitis. Syncytia were usually present in lesions and often contained abundant viral antigen (
). In mildly affected lymph nodes there was focal mononuclear cell and neutrophilic inflammation of the capsule, accompanied by a zone of subcapsular lymphocyte depletion. In more severely affected lymph nodes, there was severe extensive hemorrhagic and coagulative necrosis, often resulting in the destruction of the entire node with antigen staining mainly in syncytia at the living margin of the necrotic focus (
). Clinically healthy ferrets had no gross pathological changes, and no viral antigen or significant histological lesions were found in any tissues.
Hendra (HeV) and Nipah (NiV) viruses (genus Henipavirus, family Paramyxoviridae) are zoonotic pathogens that can cause severe acute respiratory distress and neurological disease in humans. HeV was initially identified during an outbreak of acute respiratory disease in horses in Australia in 1994. To date, 7 human cases of HeV infection have been identified, all associated with direct contact with infected horses and with a case fatality rate (CFR) of 57%. NiV was first identified as the cause of an outbreak of acute respiratory and neurological disease in pigs in 1998–99 that led to infections in humans in Malaysia (283 cases) and Singapore (11 cases) with a CFR of 38%. Subsequent outbreaks of NiV have occurred almost yearly in humans in Bangladesh and India with increased CFR (75%) and higher prevalence of respiratory disease (from 40% to 75%) [2, 4]. In addition, human-to-human transmission has only been observed during outbreaks of NiV in Bangladesh. The NiV strains isolated during outbreaks in Malaysia and Bangladesh are genetically distinct and have been designated as the Malaysia strain (NiV-M) and Bangladesh strain (NiV-B), respectively [5, 6].
In hamsters and other animal models, infection with HeV is generally more severe than infection with NiV-M and NiV-B [7, 8]. These possible differences in virulence between HeV and NiV-M have also been observed in the recently developed African green monkey model. Interestingly, in that same model, a recent study showed that NiV-B is more pathogenic, similar to what is seen during outbreaks of human disease. The differences in the pathogenic mechanisms of these strains, which ultimately drive the disparity of clinical outcomes and transmission profiles, remain largely unknown.
Ferrets are commonly used as experimental models of infection for a variety of respiratory viruses due to their susceptibility to these viruses and the close resemblance of the pathological features to those found in human infections [11, 12], including the development of severe respiratory and neurological disease during henipavirus infection [13–15]. Despite the limitations in ferret-specific reagents, the use of ferrets as experimental models of infection has gained interest since the publication of the ferret transcriptome and draft genome [16–18], which opened the doors to performing whole-genome gene expression analysis in this animal model.
In this study, we have performed a systematic characterization of HeV, NiV-B and NiV-M infection in a well-established lethal challenge model in ferrets and used next-generation mRNA sequencing to characterize the evolution of the host responses in key target organs, the lung and the brain. This analysis provides an integrated view of the functional components of the host responses that participate in the fight against the virus.
Long identified as potential sources of zoonotic pathogens, bats (Order Chiroptera) are now associated with several deadly emerging infectious viruses, including Hendra, Nipah, Marburg, Ebola, and Severe Acute Respiratory Syndrome coronavirus (SARS CoV). Although spillover from bats to humans or domestic animals remains rare, the case fatality rate from these diseases is high, and onward transmission can occur. Public health preparedness would benefit from understanding bat virus dynamics to allow predictions of viral spillover in space and time.
Spillover of bat viruses is often associated with discrete temporal and spatial pulses of virus excretion from the bats that function as reservoir hosts [3–6]. Outbreaks in livestock or humans occur seasonally with high annual variability. For example, spillover of Hendra virus in Australia, Nipah virus in Bangladesh, Marburg virus in Uganda, and Ebola virus in Central Africa is seasonal, but incidence and location of spillover infections vary among years [5–8]. Longitudinal surveys of bat colonies also have detected seasonal variation in the prevalence or seroprevalence of zoonotic viruses, including Nipah virus in Pteropus lylei in Thailand, Hendra virus in Pteropus sp. in Australia, and Marburg virus in Rousettus aegyptiacus in Uganda [6,9–12].
Markedly different underlying mechanisms can yield similar spatial and temporal patterns in prevalence and seroprevalence data. We outline three distinct scenarios that could generate pulses of viral excretion in bats (Figs 1 and 2). Although the scenarios fall along a continuum, each one leads to a different set of hypotheses that can be tested in the field or laboratory. Within- and between-host processes drive the first and second scenarios: pulses of transmission among bats with clearance of infection and either long-term (Fig 1A) or waning immunity (Fig 1B). Within-host processes drive the third scenario, with pulses triggered by viral reactivation in persistently infected bats (Fig 1C). A common driver among the three scenarios is seasonal forcing, which occurs through birth pulses, seasonal transmission, waning maternal immunity in young, and periods of environmental or physiological stress (Fig 2). Research efforts often reflect the working hypothesis that pulses are driven by between-host transmission or that pulses are driven by within-host processes of reactivation, but often without using data collection methods that would allow them to be distinguished [5,13–16]. In reality, the evidence for either hypothesis has not been fully assessed for the emerging viruses discussed here.
Even though the different scenarios can lead to similar observed dynamics, strategies to predict or manage spillover risk will be different for the different underlying mechanisms (and mechanisms would be expected to vary between virus types). Strategies that confront dynamics driven by transmission between hosts are likely to focus on population density, connectivity among populations, or herd immunity. By contrast, strategies to address dynamics driven by within-host reactivation are likely to target pathogen biology or processes that produce stress.
As part of the international focus on surveillance of zoonoses and bat-borne viruses such as Ebola virus, understanding the dynamics of pathogens within animal reservoir hosts should be a research priority. However, research on wildlife diseases often focuses on cross-sectional surveillance methods within a limited geographic area and does not capture information that would allow inference to spatial and temporal dynamics. Many studies do not examine the drivers of disease dynamics and therefore cannot draw predictive inferences about the occurrence of zoonotic spillover. Identifying the drivers of bats’ excretion of virus would allow prediction of locations and times when the likelihood of spillover is high, potentially leading to better management or even prevention of spillover.
Here, we establish a framework to assess the evidence for the different mechanisms that could drive the observed epidemiological patterns of emerging bat viruses. We highlight the strengths and limitations of potential investigation methods and recommend ways to distinguish between scenarios, which require ambitious, interdisciplinary research (Table 1). We explain some distinct challenges associated with pathogen studies in bats. Although rabies is an important zoonosis of bat origin, we restrict discussion to the less well-understood emerging bat pathogens. Our concepts are transferable to numerous diseases that affect wildlife, domestic animals, and humans.
There have been two fatal cases of HeV infection in humans, the first presented with severe respiratory disease and upon autopsy the patient’s lungs had gross lesions of congestion, hemorrhage and edema associated with chronic alveolitis and syncytial cell formation. The second fatal case presented thirteen months post-exposure and exhibited leptomeningitis with lymphocytes and plasma cells and foci of necrosis in various parts of the brain parenchyma (reviewed in). Multinucleate endothelial cells were also present in the viscera as well as in the brain. During the first NiV outbreak in Malaysia encephalitis killed 105 of 265 infected individuals. Immuno- and histological features were described as a systemic endothelial infection accompanied by vasculitis, thrombosis, ischaemia and necrosis. These changes were especially noted in the central nervous system (CNS). Immunohistochemical analyses demonstrated the widespread presence of NiV antigens both in neurons and other parenchymal cells in necrotic foci in the CNS and in endothelial cells of affected blood vessels. Evidence of vasculitis and endothelial infection was also seen in most organs examined. Disseminated endothelial cell infection, vasculitis, thrombosis and CNS parenchymal cell infection all appear to play essential roles in the fatal outcome of human NiV infection [18, 20]. In the more recent NiV outbreaks in India and Bangladesh, similar clinical signs were noted. However, a higher incidence of acute respiratory distress syndrome in conjunction with encephalitis was also described [11, 12].
To determine whether vaccination with the rVSV-ΔG-NiVB vectors could prevent NiVM disease course in ferrets, we challenged ferrets intranasally with a lethal challenge dose of NiVM on day 0 (Figure 2A, *). The animals were closely monitored over the course of 22 days post challenge (p.c.) for clinical signs of illness. The specifically vaccinated animals in Groups 2-4 did not lose weight over the course of the study (Figure 3A) and were 100% protected against NiVM (Figure 3B), while the animals in the non-specifically vaccinated Group 1 succumbed to infection on days 7 or 8 (Figure 2A, ^), respectively (Figure 3B, Table 1). Clinical scores were recorded each day after challenge for each animal using a scoring system based on coat grooming, social behavior, and provoked behavior. The clinical scores for each animal correlated with the survival data as seen with the mean clinical score for each animal in the vaccinated groups having no score on any day p.c. versus the animals in Group 1 having clinical scores on days 5 to 8 p.c. Clinical signs in response to NiVB infection were more dramatic for the animals in Groups 1 when compared to the animals in the other three groups (Table 1). In all animals that succumbed to NiVM infection, the gross pathologic findings included varying severity of dehydration, ventral cervical subcutaneous hemorrhage with edema (Figure 4A) and crusting serous nasal discharge (Figure 4B). The internal gross pathologic findings from animals in Group 1 included varying severity of heavy, wet, diffusely mottled with dark pin point foci throughout the pulmonary parenchyma, multifocal pin point raised foci throughout the renal parenchyma, mottling of the spleen with diffuse splenomegaly, and diffuse reticulation of the liver. Each animal had diffuse hemorrhagic interstitial pneumonia (Figure 4C) and splenomegaly with multifocal necrosis (pic 4D, *). Additionally, diffuse reticulation of the liver (Figure 4D, +) and multifocal renal hemorrhage (Figure 4D, arrow) were noted. There were no external or internal gross pathologic findings of note in any of the Group 2-4 animals at the study endpoint (Figure 2A, day 22 ^).