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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.
Mumps typically begins with a prodrome of low-grade fever, myalgias, anorexia, malaise and headache. Over the next 1–3 days, the patient develops earache and tenderness over the parotid gland, which becomes noticeably enlarged and painful (Figure 1). Parotitis is typically seen in 31–65% (with some authoritative texts citing 60–70%) of cases of mumps infection. In about three quarters of patients, the other parotid gland becomes involved.12,13 The parotitis is nonsuppurative and typically progresses for about three days and lasts for approximately one week.13 Patients often have trismus and have difficulty chewing and speaking. In about 10% of cases, other salivary glands, especially the submandibular gland, can become involved and can mimic anterior cervical lymphadenopathy.
Vesicular stomatitis is a viral disease which primarily affects cattle, horses, and swine. It occurs in enzootic and epizootic forms in the tropical and subtropical areas. The disease is rarely life-threatening but can have a significant financial impact on the horse industry. Vesicular stomatitis virus (VSV) is the prototype of the genus Vesiculovirus in family Rhabdoviridae. The virus has two serologically distinct serotypes, VSV-New Jersey (NJ) and VSV-Indiana (IND). The neutralizing antibodies generated by these two serotypes are not cross-reactive. The IND serogroup has three subtypes IND-1 (classical IND) IND-2 (cocal virus) and IND-3 (alagoas virus) The virus is endemic in South America, Central America, Southern Mexico, Venezuela, Colombia, Ecuador and Peru but the disease has been reported in South Africa in 1886 and 1897 and France in years 1915 and 1917.
The disease has been reported across continents in Belize, Bolivia, Brazil, Colombia, Costa Rica, Ecuador, El Salvador, Guatemala, Honduras, Mexico, Nicaragua, Pakistan, Panama, Peru, USA and Venezuela [91, 92]. Outbreaks historically occurred in all regions of the USA but have been limited to western states in 1995, 1997, 1998, 2004, 2005, 2006, 2009, 2010, and 2012 [93, 94]. While VS has been reported in horses at about 800 premises in eight states. VSV spread to Europe during the First World War and periodically appears in South Africa. The Chandipura virus, a Vesiculovirus caused encephalitis outbreaks in different states of India leading to mortalities in children. Isfahan another virus in this genus is endemic in Iran [89, 97]. The countries with incidence/serological evidence of vesicular stomatitis are presented in Fig. (2).
Clinical disease has been observed in cattle, horses, pigs and camels whereas sheep, goats and llamas tend to be resistant. White-tailed deer and numerous species of small mammals in the tropics are considered as wild hosts. Many species, including cervids, nonhuman primates, rodents, birds, dogs, antelope, and bats have shown serological evidence of infection. Experimentally different animals like mice, rats, guinea-pig, deer, raccoons, bobcats, and monkeys can be infected.
The virus is zoonotic and causes flu-like symptoms characterized by fever, chills, nausea, vomiting, headache, retrobulbar pain, myalgia, sub-sternal pain, malaise, pharyngitis, conjunctivitis, and lymphadenitis in humans. Vesicular lesions may be present in the pharynx, buccal mucosa, or tongue. Encephalitis is rare but may occur in children [107, 108].
The transmission is more likely by trans-cutaneous or transmucosal route. The virus can be transmitted through direct contact with infected animals having lesions of the disease or by blood-feeding insects. In endemic areas, Lutzomyia sp. (sand fly) is proven biologic vectors. Black flies (Simulidae) are the most likely biologic insect vector in USA. Other insects may also act as mechanical vectors. Saliva, exudates and epithelium from open vesicles are sources of virus. Plants and soil are also suspected as the source of virus.
Horses of all ages appear equally susceptible but lesions do not appear in all susceptible horses. The lesions of the disease resemble foot-and-mouth disease in cattle and the other viral vesicular diseases in pigs. The horses are resistant to foot and mouth disease and susceptible to VS. VSV is the only viral vesicular disease of livestock that infects horses. VSV is also the most important of these four viruses as a zoonotic agent for humans. When vesicular stomatitis occurs in horses, blanched raised or broken vesicles or blister-like lesions develop on the tongue, mouth lining, nose and lips. In some cases, lesions also develop on the udder or sheath or the coronary bands of horses. Animals may become anorectic, lethargic and have pyrexia. One of the most obvious clinical signs is drooling of saliva or frothing at the mouth. The rupture of the blisters creates painful ulcers in the mouth. The surface of the tongue may slough. Excessive salivation is often mistaken as a dental problem or colic. There may be weight loss due to mouth ulcers as animal finds it too painful to eat. The lesions around the coronary band may cause lameness and laminitis. In severe cases, the lesions on the coronary band may cause the hoof to slough. Animals usually recover completely within two weeks. Morbidity rates vary between 5 and 70% but mortality is rare. Vesicular stomatitis like disease disabled 4000 horses during the Civil War in 1862. Major epidemics in the US occurred in 1889, 1906, 1916, 1926, 1937, 1949, 1963, 1982, and 1995, with minor outbreaks during many other years. No specific treatment is available for the disease. Anti-inflammatory medications as supportive care help to minimize swelling and pain. Dressing the lesions with mild antiseptics may help avoid secondary bacterial infections. If fever, swelling, inflammation or pus develops around the sores, treatment with antibiotics may be required. The animals should be quarantined at least for 21 days after recovery of the last case before moving to other places. Vaccines for livestock are available in some Latin American countries.
Adenoviruses (AdVs) are non-enveloped icosahedral double-stranded DNA viruses that infect a number of vertebrate hosts, including humans and nonhuman primates. The genus Mastadenovirus within the Adenoviridae family includes 7 human adenoviral species A-G (HAdV-A through HAdV-G) and 1 simian adenoviral species A (SAdV-A). In humans, infections by adenoviruses cause conjunctivitis, gastroenteritis, hepatitis, myocarditis, and acute respiratory illness, ranging from the “common cold” syndrome to fatal outbreaks of pneumonia,. However, existing animal models of adenovirus infection to date have been primarily confined to rodents,, and no nonhuman primate (NHP) model has been established to study adenoviruses that infect humans and/or NHPs.
We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV) in association with a fatal outbreak of pneumonia and hepatitis in a closed colony of captive New World titi monkeys (Callicebus cupreus), with evidence for potential cross-species transmission to a human researcher and family member with concurrent acute respiratory symptoms. The origin and natural host reservoir for TMAdV remained unknown, although neutralizing antibodies to TMAdV were found in 23 titi monkeys demonstrating clinical signs, 14 titi monkeys exposed to animals demonstrating clinical signs, 1 rhesus macaque (Macaca mulatta), and the 2 humans. Studies by other groups have also revealed the widespread presence of closely related adenoviruses in both human and nonhuman primates,,, and large-scale serological surveys have detected antibodies to monkey adenoviruses in humans living in endemic regions,. In addition, human adenovirus species E and G each contain only one member isolated from humans,, with the remaining members all isolated from monkeys or apes. Collectively, these data have raised concerns regarding the potential of adenoviruses as sources for emerging zoonotic disease in humans.
To further investigate the pathogenicity of the novel adenovirus TMAdV, we sought to develop an in vivo animal model of infection and disease for TMAdV. While TMAdV was originally discovered in a closed colony of captive titi monkeys (Callicebus cupreus) at the California National Primate Research Center (CNPRC), we recognized that in vivo testing in this monkey species was contraindicated due to the significant devastation to the colony caused by the virus. Since TMAdV was able to be successfully propagated in the marmoset lymphocyte cell line B95a in vitro (Table 1), we instead elected to pursue in vivo testing of TMAdV infection in the common marmoset (Callithrix jacchus).
The common marmoset is a New World primate that is small, easily handled, and highly susceptible to infectious agents, and thus a suitable nonhuman primate model for infectious disease,. Marmosets have been successfully used to characterize a number of emerging viral diseases, including filovirus- and arenavirus-induced hemorrhagic fevers, encephalitis, and severe acute respiratory syndrome (SARS). Since titi monkeys (Family Pithecidae, subfamily Callicebiniae) and marmoset monkeys (Family Cebidae, subfamily Callitrichinae) are classified into separate families, the use of the marmoset for in vivo testing of TMAdV infection also afforded the opportunity to directly test the capacity of TMAdV, and, by extension, adenoviruses in general, to cross the species barrier and cause productive infection in a related yet taxonomically distinct secondary host.
In post-pubertal males, the most common complication of mumps infection is orchitis. In the era prior to the advent of the MMR vaccine, orchitis occurred in between 12% to 66% of post-pubertal males with mumps. In the post-vaccination era, orchitis has been reported in 15% to 40% of post-pubertal males.12 Orchitis typically occurs about 10 days after the onset of parotitis, although it can be seen up to six weeks later. Orchitis is typically unilateral, but bilateral orchitis manifests in 15–30% of cases.13 Orchitis may be accompanied by epididymitis up to 85% of the time.17
Mumps orchitis can lead to a range of testicular complications. True infertility following mumps orchitis is rare but subfertility has been seen in up to 13% of patients. Subfertility can occur even without accompanying testicular atrophy.7, 18–20 Testicular atrophy (any reduction in testicular size) occurs in 30–50% of patients with orchitis.6 Abnormalities of spermatogenesis have been observed to occur in up to half of patients for up to three months after recovery from the acute illness.20 Mumps orchitis and subsequent testicular atrophy have been weakly associated with the development of testicular tumors, including cancer, with an incidence of 0.5%.7, 21, 22
Other complications of mumps infection include meningitis, which may occur in up to 10% of cases. When meningitis does occur, it is typically seen 3–4 days after the onset of parotitis.6 Acute encephalitis and encephalomyelitis are rare. When acute encephalitis due to mumps occurs, it is typically self-limiting. Acute encephalomyelitis, on the other hand, tends to be much more severe. Case fatality rates for acute encephalomyelitis due to mumps virus are up to 10%, while the overall case fatality rate due to CNS complications from mumps virus has been reported to be about 1%.23
Sensorineural hearing loss is another CNS complication of mumps infection. Permanent unilateral hearing loss has been reported to occur in 1 of every 20,000 cases. Bilateral hearing loss is much less frequent. Other rare CNS complications include Guillain Barre Syndrome, transverse myelitis, facial palsy, cerebellar ataxia and flaccid paralysis.7
Oophoritis (ovarian inflammation) has been reported to occur in 5% of post-pubertal females. Symptoms of oophoritis may include lower abdominal pain, vomiting and fever. Long-term sequelae of oophoritis, while rare, may include infertility or premature menopause. Mastitis (breast inflammation) has also been reported as a complication of mumps infection in post-pubertal females.7 In some studies, mumps infection in early pregnancy has been linked with spontaneous abortion, with one study identifying a 27% rate of fetal death after first trimester mumps infection compared with 13% in a control group.7, 24 A second, more recent study has not shown the same association between spontaneous abortion and mumps infection in early pregnancy.25 As of early 2016, there is no reported association between perinatal mumps infection and significant congenital malformations.7
Other rare complications associated with mumps infection include pancreatitis (with rare reported cases of severe hemorrhagic pancreatitis), ECG abnormalities (depressed ST segments, prolonged PR intervals and inverted T waves), myocarditis, polyarthritis, abnormal renal function (with rare reports of severe or fatal nephritis), hepatitis, acalculous cholecystitis, kerato-uveitis, hemophagocytic syndrome and thrombocytopenia.7 (Table)
Throughout recorded history, infectious diseases have plagued human existence. One effective approach to limiting these diseases has been vaccination. For example, in a recent report by Roush and colleagues at the U.S. Centers for Disease Control and Prevention (CDC), ever since the introduction of vaccines the incidence of infectious diseases like diphtheria, mumps, pertussis, tetanus, hepatitis A and B, Haemophilus influenza and varicella zoster has declined by more than 80% in the U.S. Furthermore, after the introduction of vaccines, large scale transmission of measles, rubella, and polio has been eliminated in the U.S., while smallpox has been eradicated worldwide. However, new emerging infectious pathogens such as HIV (human immunodeficiency virus), SARS coronavirus (severe acute respiratory syndrome virus), and highly pathogenic avian influenza (H5N1) viruses have adapted strategies to rapidly change their genetic compositions. As the influenza pandemic of 1918 (H1N1) killed approximately 20 to 50 million people worldwide, massive disease and death is similarly feared from newly emerging pathogens. In addition, the current novel swine derived H1N1 pandemic further exemplifies the need for a rapid and effective vaccine against emerging pathogens. Thus a vaccination strategy to control emerging diseases will require a more effective and rapid response than available from conventional approaches such as live-attenuated vaccines, inactivated vaccines, or protein subunit vaccines. Plasmid DNA vaccines, as reviewed in this article, may be an option to effectively combat current emerging infectious diseases.
Human microbiologic infections, known as zoonoses, are acquired directly from animals or via arthropods bites and are an increasing public health problem. More than two thirds of emerging human pathogens are of zoonotic origin, and of these, more than 70% originate from wildlife. In novel environments, viruses, particularly RNA viruses, can easily cross the species barrier by mutations, recombinations or reassortments of their genetic material, resulting in the capacity to infect novel hosts. Because of their adaptive abilities, RNA viruses represent more than 70% of the viruses that infect humans. When socio-economic and ecologic changes affect their environment, humans may encounter increased contact with emerging viruses that originate in wild or domestic animals.
Wolfe et al. in 2007 and Karesh et al. in 2012 described different stages in the switch from an animal-specific infectious agent into a human-specific pathogen. The key stage is the transition of a strictly animal-specific infectious agent (originating from wildlife or domestic animals) to exposed human populations, resulting in sporadic human infections (Figure 1). If the pathogen is able to adapt to its human host and acquire the means to accomplish an inter-human transmission, horizontal human-to-human transmission occurs and maintains the viral cycle. Sometimes, an intermediate host, such as a domestic animal, is the link between sylvatic viral circulation and human viral circulation. For example, some human infections originating from bats, such as Nipah, Hendra, SARS and Ebola viral infections, may involve intermediate amplification in hosts such as pigs, horses, civets and primates, respectively (Figure 1). Genetic, biologic, social, political or economic factors may explain a switch in viral host targets. For example, climate changes may influence the geographical repartition of vector arthropods, leading to new areas of the distribution of infectious diseases, like Aedes albopictus and Chikungunya infections in the Mediterranean. Morens et al. listed different key factors that may contribute to the emergence or re-emergence of infectious diseases, such as microbial adaptation to a new environment, biodiversity loss, ecosystem changes that lead to more frequent contact between wildlife and domestic animals or human populations, human demographics and behavior, economic development and land use, international travel and commerce, etc.. These patterns of transmission allow identifying different animals to follow in order to monitor the appearance of new or re-emerging infectious agents before its first detection in the human populations. Therefore, hematophagous arthropods, wildlife and domestic animals may serve as targets for zoonotic and arboviral disease surveillance, particularly because sampling procedures and long-term follow-up studies are more easily performed in these hosts than in humans.
Historically, classic viral detection techniques were based on the intracerebral inoculation of suckling mice or viral isolation in culture and the subsequent observation of cytopathic effects on cell lines. Later, immunologic methods, e.g., seroneutralization or hemaglutination, were used to detect viral antigens in various complex samples. These techniques were based on the isolation of viral agents. With the progresses of molecular biology, polymerase chain reaction (PCR)-based methods became the main techniques for virus discovery and allowed the detection of uncultivable viruses, but these techniques required prior knowledge of closely related viral genomes. Next-Generation Sequencing (NGS) techniques make it possible to sequence all viral genomes in a given sample without previous knowledge about their nature. These techniques, known as viral metagenomics, have allowed the discovery of completely new viral species. Because of their low cost, the use of NGS techniques is exponentially increasing.
The transmission of infections between humans occurs after a pathogen from a wild or domestic animal contacts with exposed human populations. The human exposures may or may not be mediated by the bite of bloodsucking arthropods. Surveillance programs may target wildlife, domestic animals or arthropods for emerging viruses before their adaptation to human hosts.
HAstVs are a classic cause of viral diarrhea in children, along with rotavirus, norovirus, sapovirus and adenovirus. Seroprevalence studies indicate that most children in Europe encounter astrovirus before the age of two. Astrovirus-associated diarrhea is not reported in immunocompetent adults, as infection in childhood is considered to confer protective immunity. Additionally, humoral immunity is considered to play a major protective role, along with cellular adaptive immunity. Therefore, immunosuppressed patients and the elderly can also develop astrovirus-associated diarrhea.
In non-immunocompromised individuals, after an incubation period of 4–5 days, an astrovirus infection will induce a mild disease, characterized by mild and short watery diarrhea for two to three days, followed by nausea, vomiting, and abdominal pain, which usually resolves spontaneously. These symptoms are most often milder than a rotavirus infection. Recent seroprevalence studies have indicated that some infections can be asymptomatic as well. As reported for rotavirus and norovirus, astrovirus has also been associated with intussusception in infants. Although virological diagnosis of astrovirus-associated diarrhea is not routinely used in medical practice, it is sometimes used in epidemiological studies in the context of diarrheal outbreaks and surveillance of diarrheal diseases.
An astrovirus infection in immunocompromised individuals may induce gastroenteritis, but it can also lead to severe and sometimes fatal systemic and central nervous system (CNS) infections, as seen in multiple cases of astrovirus-associated encephalitis and meningitis. These reports are associated with newly identified HAstVs that belong to novel species (MAstV 6 and 9). Studies are under way to assess the actual disease burden associated with these novel neurotropic astroviruses in humans. These novel astroviruses are enteric viruses, associated with diarrhea and fecal carriage, but their pathogenicity in the non-immunosuppressed host has not yet been precisely determined, although a case of meningitis in an apparently healthy adult has recently been reported. Therefore, these newly-discovered viruses seem to share some clinical characteristics with enteroviruses, due to their association with diarrhea, but may also induce meningitis and encephalitis in the immunosuppressed patients. For this reason, their detection should now be part of the laboratory diagnostic work-up in patients, in particular those who are immunosuppressed and are diagnosed with meningitis or encephalitis of unknown cause.
In mammals, astroviruses have been reported in piglets, minks and dogs with preweaning diarrhea, but accurate diagnosis is complicated due to the prevalence of fecal shedding in healthy animals, which complicates the interpretation of the results. Therefore, etiological diagnostic is not a routine practice. In mink presenting with the so called “shaking mink syndrome”, and cattle with encephalitis, astroviruses can be tested in necropsy brain samples. Additionally, astroviruses have been associated with severe avian diseases (i.e., chicken diarrhea, duck hepatitis, turkey enteritis, and avian nephritis), and diagnosis can be made in severely affected flocks by reverse transcription polymerase chain reaction (RT-PCR), using necropsy samples in specialized laboratories.
Bats and the viruses they harbor have been of interest to the scientific community due to the unique association with some high consequence human pathogens in the absence of overt pathology. Virologic and serologic reports in the literature demonstrate the exposure of bats worldwide to arboviruses (arthropod-borne viruses) of medical and veterinary importance. However, the epidemiological significance of these observations is unclear as to whether or not bats are contributing to the circulation of arboviruses.
Historically, a zoonotic virus reservoir has been considered a vertebrate species which develops a persistent infection in the absence of pathology or loss of function, while maintaining the ability to shed the virus (e.g., urine, feces, saliva). Haydon et al. extended this definition of a reservoir to include epidemiologically-connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population. The significance of the relative pathogenicity of the infectious agent to the purported reservoir host has been debated. In the case of bats as a reservoir species, rigorous field and experimental evidence now exist to solidify the role of the Egyptian rousette bat (Rousettus aegyptiacus) as the reservoir for Marburg virus. Considering arboviruses, additional criteria must be met in order to consider a particular vertebrate species a reservoir. Reviewed by Kuno et al., these criteria include the periodic isolation of the infectious agent from the vertebrate species in the absence of seasonal vector activity, and the coincidence of transmission with vector activity. Further, the vertebrate reservoir must also develop viremia sufficient to allow the hematophagous arthropod to acquire an infectious bloodmeal in order for vector-borne transmission to occur. Bats have long been suspected as reservoirs for arboviruses, but experimental data that would support a role of bats as reservoir hosts for certain arboviruses remain difficult to collect. Here we synthesize what information is currently known regarding the exposure history and permissiveness of bats to arbovirus infections, and identify knowledge gaps regarding their designation as arbovirus reservoirs.
The MAstV-1 species is comprised of HAstV-1–8, and surveillance has revealed that HAstV-1 is the most commonly detected type in children, followed by HAstV-2–5, whereas HAstV-6–8 have been rarely detected. HAstV-4 and HAstV-8 have been associated with infection of older children and longer duration of diarrhea (>7 days). A HAstV-4 strain was also isolated from an infant with fatal meningoencephalitis. Based upon the phylogenetic analysis of the ORF2 region, different lineages within each HAstV type have been proposed; HAstV-1 (HAstV-1a–d) and HAstV-2 (HAstV-2a–d) have been divided into four lineages, whereas HAstV-3 (HAstV-3a–b) and HAstV-4 (HAstV-4a–c) have been classified into two and three lineages, respectively.
Avian influenza A (H5N1) was first reported to infect a human in 1997 in Hong Kong; 6 additional confirmed and 2 possible cases were reported in Hong Kong during the subsequent 7 months (9) and ultimately resulted in a total of 18 cases with 6 deaths (10). Since its emergence, this virus has been associated with continuing sporadic cases and small clusters and a high case-fatality proportion of 59% in humans. While the virus has not yet developed the capacity to spread easily from humans to humans, if it were to do so, the combination of greater transmissibility between humans, the lack of pre-existing immunity in the population, and high case-fatality proportion has the potential to cause substantial global mortality (10). Significant progress has been made worldwide over the past decade in the ability to rapidly detect and respond to the emergence of such a pathogen. While the response to the 2009 H1N1 pandemic demonstrated this growing global capacity, the potential for greater severity associated with an influenza H5N1 pandemic would be a much greater challenge. During 2012, outbreaks of highly pathogenic avian influenza H5N1 have continued to be reported in poultry, most recently confirmed in Bangladesh, Bhutan, Cambodia, Chinese Taipei, Egypt, Hong Kong, India, Japan, Republic of Korea, Myanmar, Nepal, and Vietnam (11). During 2012, 32 human infections with H5N1 influenza were reported from Bangladesh, Cambodia, China, Egypt, Indonesia, and Vietnam; most were associated with exposure to poultry, and 20 (62.5%) of these cases were fatal (12). Although influenza H5N1 remains poorly transmissible among humans, recently published research highlights the potential for mutations that would yield greater transmissibility among mammals (13–15). In addition to influenza H5N1, we continue to watch for reports of other novel influenza subtypes being reported. For example, the GDD Operations Center closely monitored pandemic A (H1N1) 2009 virus infection (2009 H1N1), which was first detected in April 2009 and spread rapidly across the world. Additionally, during 2012 we began monitoring an outbreak of highly pathogenic influenza H7N3 among poultry in Mexico first reported in June, which was subsequently associated with two non-fatal influenza infections in humans (16). Because the GDD Operations Center’s surveillance activities are solely international, we did not monitor, for example, cases of influenza A (H3N2) associated with swine in the United States; however, with our surveillance techniques we would be able to identify cases of novel influenza that occur outside the United States, such as the above example of H7N3 in Mexico. Figure 1 depicts CDC international responses to countries’ requests for assistance to cases or outbreaks of influenza H5N1 and 2009 H1N1 which occurred from January 2007 to August 2012.
We examined TMAdV infectivity by inoculating marmosets intranasally with 0.1 mL of 105 TCID50/mL of P10 virus for which sufficient viral stocks were available. The inoculation dose (104 TCID50) was chosen to be similar to the physiologic doses used in experimental nasal inoculation of nonhuman primates at the TBRI with acute respiratory viruses such as RSV (respiratory syncytial virus). Three marmosets, CJ29012, CJ29019, and CJ29020, were inoculated with culture-adapted TMAdV (passage 13), while one marmoset, CJ29021, was inoculated with cell culture media as a control (Fig. 2A). All animals appeared normal until approximately 5 to 10 days after infection, when clinical signs presented in 3/3 inoculated monkeys (clinical scores >4) (Fig. 2C; Table S2). Animals exhibited reduced activity, had decreased stool production, became anorexic, and developed low-grade fevers. Marmoset CJ29012 demonstrated the most severe clinical signs and was in guarded physical condition with nasal congestion and sneezing by day 10. After day 11, all 3 experimentally infected animals recovered quickly, with marmosets CJ29019 and CJ29020 fully recovered at the time of euthanasia on day 15. To assess the effect of re-infection with TMAdV, marmoset CJ29012 was re-inoculated on day 15 and, along with control marmoset CJ29021, was observed for an additional 21 days prior to euthanasia at day 36. No clinical signs of infection were observed in marmoset CJ29012 (experimental TMAdV animal) or marmoset CJ29021 (experimental control animal) after re-inoculation (clinical scores < = 4).
In 1975, the first observation by EM of 28–30 nm particles was reported in the stool of babies with gastroenteritis. The star-shaped surface configuration of the viruses rapidly led the author to propose the name of “astrovirus” (derived from the Greek “astron” which means “star”) and, since then, this morphological characteristic has been widely used for the detection of astrovirus infection in both humans and animals. Direct EM is complicated by the fact that only a minority of virions exhibit a complete star-shaped structure, and careful searching may be necessary to distinguish between, for example, astrovirus and calicivirus, which are similar in size. Sensitivity of EM is also dependent on elevated concentrations of particles, usually around 107 per gram of stool. The use of immune electron microscopy (IEM) techniques using specific antibodies or convalescent sera can improve the sensitivity of the detection and help with the typing or the detection of new viral agents. Due to the limitations described above, the use of EM for the diagnosis of viral infections has been superseded by molecular methods, and therefore, it is rarely available or used in clinical laboratories anymore.
In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis. Aichi virus infection has been shown to elicit an immune response. Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus (0.5% and 1.8%) was too low to find a significant association with diarrhea. In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea. Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis. Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals. Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected. However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis. Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature. This high sequence diversity has also been observed within particular outbreaks of gastroenteritis, limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified.
Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis (norovirus) was identified in an outbreak of diarrhea in Norwalk (California, United States). Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis, astrovirus in infantile diarrhea cases, enteric adenoviruses in the feces of children with acute diarrhea, and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan. All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below.
Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases (diarrhea cases where an enteropathogen could be found), reviewed in the literature. The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea. Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro. Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%–12.7% of all gastroenteritis cases around the globe. Sapovirus outbreaks occur throughout the year and can be foodborne. For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host.
Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults. Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection. Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases. Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools. The virus can replicate in human embryonic kidney cells and was detected by electron microscopy (EM). Adenoviruses are responsible for around 1.5%–5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature. Of the 57 identified adenovirus types, only adenoviruses type 40 and 41 are associated with diarrhea. Next to these two types, adenovirus type 52 can also cause gastroenteritis, although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41. Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature.
Human enterovirus A71 (EV-71) belongs to the Enterovirus genus within the family of Picornaviridae. The EV-71 genome is a single-stranded, positive sense RNA with approximately 7411 nucleotides, and consists of an open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). Internal ribosome entry site (IRES)-dependent translation initiates synthesis of the viral polyprotein, which is subsequently cleaved into structural proteins (VP1-VP4) and non-structural proteins (2A-2C and 3A-3D). The RNA genome is enclosed in an icosahedral capsid assembled from 60 copies of each of the four structural proteins.
EV-71 was first described in 1969, after its isolation from a two-month-old infant with aseptic meningitis in California, USA. Several EV-71 epidemics with high mortality rates occurred in Bulgaria and Hungary in 1975 and 1978, respectively. Since then, many EV-71 outbreaks have been reported in Taiwan, Australia, Singapore, Malaysia, China, Vietnam and Cambodia.
EV-71 infections usually manifest as mild hand, foot and mouth disease (HFMD), characterized by fever, mouth ulcers, and vesicles on the palms and feet. Unlike other HFMD-related enteroviruses, EV-71 also causes severe neurological manifestations, such as poliomyelitis-like acute flaccid paralysis and brainstem encephalitis in infants and children below 6 years old. The fatal brainstem encephalitis is characterized by rapid progression of cardiopulmonary failure. Patients with neurological involvement who survive often have permanent neurological sequelae, with delayed neurodevelopment and reduced cognitive function.
Similar to the global poliovirus (PV) eradication initiative, an EV-71 vaccine is likely to be the most effective way to control, and hopefully eradicate disease. Several promising EV-71 vaccine candidates are currently under clinical trial. Nevertheless, effective antivirals are still needed for treatment of infected patients with severe disease. This review will highlight the potential targets for EV-71 antivirals as well as recent developments and future prospects of antivirals against EV-71 infections.
Astroviruses are classified within the unassigned Astroviridae family and are non-enveloped viruses characterized by a positive sense, single-stranded RNA (ssRNA) genome 6.4–7.9 kb long comprised of a 5′-untranslated region (UTR), three open reading frames (ORFs)—ORF1a, ORF1b, and ORF2, a 3′-UTR, and a poly A tail. The ORF1a region encodes a non-structural polyprotein (serine protease), ORF1b encodes a polyprotein including the RNA-dependent RNA polymerase (RdRp), and ORF2 encodes the viral capsid protein. A further ORF, termed ORFX, has been observed in classic HAstVs and some mammalian astroviurses, overlapping the 5′ end of ORF2 which may be translated through a leaking scanning mechanism. Astroviruses exhibit several distinctive features in addition to a distinctive morphology. The viruses lack a RNA-helicase domain encoded within the genome and utilize a ribosomal frameshifting mechanism to translate the RdRp, which distinguishes the Astroviridae family from other non-enveloped ssRNA virus families such as Picornaviridae and Caliciviridae. The greatest diversity in the genome is within the ORF2 region, which is characterized by a highly-conserved N-terminal domain (amino acids (aa) 1–424), a hypervariable domain (aa 425–688) which is believed to form the capsid spike and contribute to receptor binding, and a highly acidic C-terminal domain.
Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates. Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay.
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis (2-DE), protein chip, mass spectrometry, and suspension array technology. However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods. Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been one of the most important and widely used techniques for the identification and characterization of new viruses. Two TEM techniques are usually used for this purpose: negative staining on an electron microscopic grid coated with a support film and (ultra) thin section TEM of infected cells, fixed, pelleted, dehydrated, and embedded in epoxy plastic. Negative staining can be conducted on highly concentrated suspensions of purified virus or cell culture supernatants. For some viruses, TEM can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or concentrated stool material (rotaviruses and noroviruses). For successful detection of viruses in ultrathin sections of infected cells, at least 70% of cells must be infected, and so either high multiplicity of infection (MOI) or rapid virus multiplication is required.
Viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location or, for some viruses, from the ultrastructural cytopathology and specific structures forming in the host cell during virus replication. Usually, ultrastructural characteristics are sufficient for the identification of a virus at the level of a family. In certain cases, confirmation can be obtained by immuno-EM performed either on virus suspension before negative staining or on ultrathin sections. This requires virus-specific primary antibodies, which might be not available in the case of a novel virus. For on-section immuno-EM, OsO4 post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic (usually LR White). The ultrastructure of most common viruses is well documented in good atlases and book chapters and many classical publications of the 1960s, 1970s, and 1980s. Several excellent reviews were recently published on the use of TEM in the detection and identification of viruses.
Encephalitis is the most serious complication of herpes simplex virus (HSV) infection.6 While rare, HSV is commonly included among causes of viral encephalitis. Since HSV infection can be treated effectively with acyclovir, it is critical to rapidly and accurately establish the diagnosis and initiate treatment to reduce morbidity and mortality. Culture of cerebrospinal fluid (CSF) is insensitive for the diagnosis of HSV encephalitis, so brain biopsy, a very invasive procedure with significant morbidity, was historically required to make a diagnosis, with results not available for days afterward. Several studies demonstrated that detection of viral DNA by polymerase chain reaction (PCR) using CSF samples performed equivalently to brain biopsy, including a landmark study in 1995.7-9 Given the ease of collecting CSF samples, the lower risk of complication, the lower cost, and speed of results, PCR became the standard of care for diagnosing HSV CNS infections in the mid-1990s, with LDPs used for nearly 20 years. It was not until 2014 that the first FDA-cleared PCR test for the diagnosis of HSV encephalitis became available, with a second test cleared in 2015. Laboratory-developed testing procedures dramatically improved the quality of care for many patients and continue to be used successfully today, as the 2 cleared tests have limitations, requiring specific instrumentation not standard in many laboratories. Additionally, one of the tests is highly multiplexed, which may not be appropriate in all clinical situations.
Herpes simplex virus can also cause infections in neonates, with a frequency of 1:3500 to 1:5000 deliveries in the United States. The infection is acquired by exposure to maternal genital secretions. The presentation of neonatal HSV varies and includes skin, eye, and mouth infection, with encephalitis and disseminated disease in over 50% of cases. Again, rapid diagnosis is needed to prevent morbidity and sequelae from encephalitis. The diagnosis of encephalitis is made via PCR of CSF samples, while testing of plasma or serum is critical to diagnose disseminated disease. The ability to use plasma or serum is very important as it may be difficult to obtain enough CSF from a newborn to assess all diagnoses in the differential. Although there are 2 FDA-cleared assays to test CSF for HSV, there are no assays cleared for testing serum and plasma. Laboratory-developed testing procedures continue to play a critical role in the diagnosis and management of disseminated HSV infection in newborns.
Additionally, LDPs have performed with a high degree of accuracy and interlaboratory agreement. Blinded PT samples were distributed to 383 laboratories as part of a CAP PT survey for analysis of HSV; 91.6% correctly identified HSV-2, and 7.8% identified HSV, but not noting the subtype; an overall accuracy of 99.4% was obtained. Previous proficiency surveys showed similar results for samples containing HSV1. Over 90% of the laboratories used LDPs.10
Poliovirus isolation techniques also led to discovery and characterization of a large, ubiquitous group of picornaviruses termed “enteroviruses,” containing not only the three polioviruses but also at least 110 “nonpolio enteroviruses” (NPEVs) (12). Unlike classical fecal-oral transmission of polioviruses, some NPEVs are more commonly transmitted by the respiratory route. NPEVs can cause a wide array of disease syndromes, including respiratory infections, conjunctivitis, myositis, pleurodynia, myocarditis, maculopapular and vesicular rashes, hand-foot-and-mouth disease, herpangina, meningitis, encephalitis, so-called “neonatal viral sepsis,” possibly type 1 diabetes, and—occasionally—sporadic AFM (9, 10). Indeed, the very first NPEV discovered (coxsackievirus A1, in 1947) was isolated from a child with AFM (13). In temperate climates, NPEVs circulate together endemically and epidemically every late summer/fall, causing localized outbreaks of aseptic meningitis and other conditions. Although immunity to NPEVs is near-universal by early childhood (10), infections continure to occur because there are many different NPEV types, some evolving rapidly (10).
Circulating NPEVs usually are replaced, in part or in whole, by other NPEVs in subsequent seasons (9, 10); however, some NPEVs may reappear at intervals of 2 or more years (8–10, 14, 15). For example, in Southeast Asia (but not in the rest of the world), EV-A71 has occurred in 2- to 3-year cycles (8); in previous decades, 5-year cycles were noted for EVA9 and EVB5; and various other cyclic patterns have been noted for different NPEVs (9, 14). The cycles presumably reflect factors such as viral transmissibility, population immunity, and possibly NPEV elicitation of cross-reactive immunity to shared epitopes. Short-interval cyclicity is consistent with viral hypertransmissibility, which leads to high population herd immunity that prevents further spread until such time as new annual birth cohorts of susceptible persons can sufficiently dilute it. Such patterns were well described for measles and other childhood diseases in the prevaccine era.
There is an obvious paradox in temporal-geographic association between AFM and EV-D68, on the one hand, and difficulty detecting EV-D68 in AFM cases, on the other. In this regard, a precipitating EV-D68 infection, often with low-level viral replication (20), may well have run its course by the time of onset and diagnosis of AFM, several days to a week or more later. Early transient viremia during the respiratory prodrome might also have resolved by the time of AFM onset. Alternatively, local virus may cross the blood-brain barrier to extend proximally up nerve axons to the cord; this is believed to be the mechanism of ipsilateral trauma-associated “provocation poliomyelitis” (23). In addition, some NPEVs that spread by the respiratory route, including EV-D68, have low gastrointestinal tropism, hindering stool isolation (the standard poliovirus diagnostic technique).
It is noteworthy that while enterotropic polioviruses, and some other NPEVs that cause AFM, often can be isolated from stool for weeks, they, too, like nonenterotropic EV-D68, are uncommonly isolated from the CSF of paralytic cases (24). Similarly to epidemic polio, the AFM epidemic has been associated with cases of cranial nerve paralysis, bulbar paralysis, and meningoencephalitis (3, 20). Once viral damage to gray matter has occurred—via viral cytopathicity or a pathogenic immune response—intracellular virus may not be released into the (anatomically distant) spinal fluid and thus detected by lumbar puncture. Furthermore, although easily visible on MRI (3, 11), involved cord and bulbar gray matter cannot safely be biopsied to allow for direct virus isolation. For these reasons, EV-D68-induced pathogenic processes associated with early brief low-level viral replication and early transient viremia, or with direct axonal extension to internal cord gray matter, might well lead to AFM without providing good opportunity for viral detection.
Although EV-D68 appears to be good at covering its tracks, the epidemiologic evidence that EV-D68 is a major cause of epidemic AFM, while circumstantial, is nonetheless strong. Since historically many or even most cases of nonpolio AFP/AFM have been caused by circulating NPEVs (18, 24, 25), it is logical to suspect that during explosive EV-D68 epidemicity, many or most AFM cases would be caused by EV-D68 as well, even as AFM cases associated with other NPEVs continue to occur at lower background rates.
Members of the genus Orthonairovirus of medical and veterinary significance include Crimean Congo hemorrhagic fever virus (CCHFV) and Nairobi sheep disease virus (NSDV). CCHFV is transmitted by ticks in genera Rhipicephalus and Hyalomma. While neither live virus nor nucleic acid of CCHFV has been detected from bats, serologic evidence suggests past infection of populations of bats across a diverse geographic range. Further, bats are often parasitized by both soft and hard ticks, which occupy a diverse range of ecological niches in endemic countries. A 2016 seroprevalance study by Müller and colleagues examining 16 African bat species (n = 1,135) found that the prevalence of antibodies against CCHFV was much higher in cave-dwelling bats (3.6%–42.9%, depending on species) than foliage-living bats (0.6%–7.1%). They also screened 1,067 serum samples by RT-PCR, but all were negative for CCHFV nucleic acid. Experimental studies to assess the ability of bats to support replication of CCHFV have not been published.
Picornaviridae family is an extensive group of viruses causing enteric, neurological, and respiratory diseases. Members of this family including rhinoviruses A, B and C, coxsackievirus, echovirus, and enterovirus are responsible for important pediatric enteric and respiratory diseases. Several reports have shown that rhinovirus C is associated with asthma exacerbations.1, 2, 3, 4 Non‐polio enteroviruses such as coxsackievirus A16 and enterovirus 71 have been associated with hand, foot and mouth disease in the United States and Asia,5 whereas coxsackievirus A24 and enterovirus 70 have been related to conjunctivitis outbreaks and echoviruses 13, 18, and 30 have caused outbreaks of viral meningitis in the United States.6
In the late summer of 2014, clusters of severe respiratory infections due to enterovirus D68 had been reported in children throughout the United States, Canada, and the Netherlands.7 These children presented with asthma exacerbation and were generally characterized by low‐grade or absent fever, wheezing, dyspnea, and hypoxia.8, 9 Severe cases of the disease needed mechanical ventilation, and on rare occasions, children developed acute focal limb weakness with non‐enhancing spinal cord lesions following the respiratory illness.8, 9
The purposes of this report were to describe the clinical findings of an EV‐D68 infection outbreak in Mexico City and identify genetic relationship with the strains reported in United States, Europe, and Asia. During September, we detected an increase in enterovirus/rhinovirus identification on respiratory samples from hospitalized children with pneumonia or asthma exacerbation (none with paralysis), of which 24 were EV‐D68‐positive.
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.