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Some patients suffer from maculopathy or retinopathy. Patients noticed the loss of central vision or blurred eye occurring at various times after infection; e.g., from immediately after the disease onset to several weeks or months later. One or both eyes could be affected [33–35], and the affected eyes had macular edema with exudates containing a white mass covering the macular area with or without retinal hemorrhage, vasculitis, infarction or vitreous haze [34–39]. In addition, retinal detachment, uveitis, or arterial occlusion [35,36,39–41] was reported in some patients. In many cases, a complete recovery of vision does not occur, and chorioretinal scarring can remain in macular and paramacular areas, in spite of the resorption of exudates [34,35,37–41], while some patients show partial improvement in vision after several months of RVFV infection.
In the 1930s–40s, many RVFV laboratory infections occurred due to a lack of appropriate biosafety procedures [21–25]. However, the patients in most of these and later outbreaks suffered from self-limiting and nonfatal illness [4,6,22,23,25–28]. Typically, the incubation period for RVF is 4 to 6 days. Symptoms start abruptly with severe chills, malaise, dizziness, weakness, severe headache, nausea and/or sensation of fullness over the liver region. These symptoms are followed by an elevated body temperature (38.8 °C to 39.5 °C); decreased blood pressure; pain in the back, shoulders, neck or legs; rigor; shivering; flushed face; red eye with sore; constipation; insomnia and/or photophobia. Occasionally, other symptoms are seen which include epistaxis, abdominal pain, lack of gustatory discrimination, vomiting and/or diarrhea. Some lessening of symptoms can be observed on the 3rd day, and the body temperature often decreases to a normal level by the 4th day after the onset of symptoms. However, within 1 to 3 days after the recovery of body temperature, some patients again experience a temporal recurrence of high fever with a severe headache for a few days. Moreover, patients may have a long-lasting high fever for as much as 10 days. After body temperature becomes normal, some patients may develop a massive coronary thrombosis, persistent aching of legs for two weeks, or persistent abdominal discomfort for weeks. The palpable enlargement of the liver and spleen is not common. In the convalescence phase, patients often experience weakness, malaise, a tendency to sweat, frequent headaches, pain on motion of the eye, and a sense of imbalance. Virus has been demonstrated in the blood during the febrile period (3–4 days), whereas neutralizing antibody also starts appearing around the 4th day of the onset of symptoms.
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.
Powassan virus (POWV) is the only North American member of the tick-borne encephalitis complex (TBE-C) of viruses, which are transmitted by the bite of an infected tick. Other members of the TBE-C include the following flaviviruses: tick-borne encephalitis virus (TBEV) in Eastern Europe and Western Asia, Omsk hemorrhagic fever virus in Siberia, Kyasanur Forest disease virus in India, Alkhurma virus in Saudi Arabia, and Louping ill virus in the United Kingdom. TBE-C viruses can cause a wide range of disease in humans, from mild febrile illness with biphasic fever to encephalitis or hemorrhagic fever (1). POWV is composed of two genetically and ecologically distinct lineages (2). Prototype POWV (lineage I) is transmitted primarily by Ixodes cookei, a tick with a narrow vertebrate host range that rarely feeds on humans. Powassan virus lineage II, also known as deer tick virus (DTV), is transmitted by Ixodes scapularis, a tick with a broad host range that also transmits the infectious agents that cause Lyme disease, anaplasmosis, ehrlichiosis, and babesiosis (3). Since the late 1990s, POWV infections have been reported in the Northeastern and Midwestern parts of the United States as well as in Canada, and incidence is increasing (4). Because the territory of I. scapularis is expanding and the prevalence of POWV in ticks and mammals is increasing, POWV poses an increasing threat (5, 6). In a recent survey, I. scapularis ticks collected from the northwest quadrant of Wisconsin from 2011 to 2012 demonstrated a POWV infection frequency of 4.67% (7). This is similar in frequency to a survey conducted in that same area in 1998 (8). Although POWV is rarely diagnosed as a cause of encephalitis, infections can be severe, and neurologic sequelae are common (9). Additionally, the potential for concurrent transmission with other tick-borne pathogens, particularly Borrelia burgdorferi, the causative agent of Lyme disease, has not been previously studied in North America.
Similarly to other arboviral infections, POWV diagnosis is complex, requiring review of clinical and travel history in addition to knowledge of and access to diagnostic testing (10). Serologic testing remains the primary method for diagnosis of POWV infection, with an emphasis on the detection of POWV-specific IgM antibodies in serum or plasma. Until recently, commercial laboratory testing has been unavailable for POWV in the United States. Prior to this, a positive POWV IgM enzyme immunoassay (EIA) result confirmed by plaque reduction neutralization test (PRNT), a 4-fold or greater increase in titers between acute- and convalescent-phase sera, or culture or direct identification of virus-specific nucleic acids at a state public health laboratory or the Centers for Disease Control and Prevention (CDC) (11) has been the mainstay of diagnostic testing.
We describe here a laboratory-developed, serologic test panel, commercially available at a reference laboratory, for the detection of IgG and IgM antibodies to POWV in serum and plasma samples. The first test in the panel is a highly sensitive, commercial TBE-C screen by EIA. Per the manufacturer, cross-reactivity with other flaviviruses is expected, particularly with West Nile virus (WNV) and dengue virus (DENV) antibody-positive samples. Samples that are screen positive are then confirmed for POWV by indirect immunofluorescence assay (IFA). Performance characteristics of the test panel were optimized, and validation studies were performed to assess the analytical sensitivity, reproducibility, and specificity/cross-reactivity of the serologic test panel for use in routine diagnostic testing.
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.
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.
Viral encephalomyelitis is an important cause of morbidity and mortality worldwide, and many encephalitic viruses are emerging and re-emerging due to changes in virulence, spread to new geographic regions, and adaptation to new hosts and vectors. The term encephalomyelitis refers to inflammation in the brain and spinal cord that results from the immune response to virus infection. In humans, the viruses most commonly identified as causes of viral encephalomyelitis are herpesviruses and RNA viruses in the enterovirus (e.g., polio, enterovirus 71), rhabdovirus (e.g., rabies), alphavirus (e.g., eastern equine, Venezuelan equine, and western equine encephalitis), flavivirus (e.g., West Nile, Japanese encephalitis, Murray Valley, and tick-borne encephalitis), and bunyavirus (e.g., La Crosse) families. Other virus families with members that can cause acute encephalitis are the paramyxoviruses (e.g., Nipah, Hendra) and arenaviruses (e.g., lymphocytic choriomeningitis, Junin). However, this is certainly not a complete list, because for most cases of human viral encephalitis the etiologic agent is not identified, even when heroic attempts are made.
The primary target cells for most encephalitic viruses are neurons, although a few viruses attack cerebrovascular endothelial cells to cause ischemia and stroke or glial cells to cause demyelination, encephalopathy, or dementia–. Widespread infection of neurons may occur or viruses may display preferences for particular types of neurons in specific locations in the central nervous system (CNS). For instance, herpes simplex virus (HSV) type 1 often infects neurons in the hippocampus to cause behavioral changes, while poliovirus preferentially infects motor neurons in the brainstem and spinal cord to cause paralysis and Japanese encephalitis virus infects basal ganglia neurons to cause symptoms similar to those of Parkinson’s disease.
Because infections with encephalitic viruses are initiated outside the CNS (e.g., with an insect bite, skin, respiratory, or gastrointestinal infection), innate and adaptive immune responses are usually mounted rapidly enough to prevent virus entry into the CNS. Therefore, most viruses that can cause encephalitis more often cause asymptomatic infection or a febrile illness without neurologic disease, and encephalomyelitis is an uncommon complication of infection.
Arboviruses are transmitted between arthropods (mosquitoes, ticks, sandflies, midges, bugs…) and vertebrates during the life cycle of the virus.8 Many arboviruses are zoonotic, i.e., transmissible from animals to humans.9,10 As far as we are aware, there are no confirmed examples of anthroponosis, i.e., transmission of arboviruses from humans to animals.9,10 The term arbovirus is not a taxonomic indicator; it describes their requirement for a vector in their transmission cycle.11,12 Humans and animals infected by arboviruses, may suffer diseases ranging from sub-clinical or mild through febrile to encephalitic or hemorrhagic with a significant proportion of fatalities. In contrast, arthropods infected by arboviruses do not show detectable signs of sickness, even though the virus may remain in the arthropod for life. As of 1992, 535 species belonging to 14 virus families were registered in the International Catalogue of Arboviruses.12 However, this estimate is continuously increasing as advances in virus isolation procedures and sequencing methods impact on virus studies. Whilst many current arboviruses do not appear to be human or animal pathogens, this large number of widely different and highly adaptable arboviruses provides an immense resource for the emergence of new pathogens in the future.
Despite the announcement of the successful eradication of smallpox in 1979, the last case of rinderpest in 2008 and the current campaigns to eradicate poliomyelitis and measles through mass-immunization programmes, we still face the prospect of emerging or reemerging viral pathogens that exploit changing anthropological behavioural patterns. These include intravenous drug abuse, unregulated marketing of domestic and wild animals, expanding human population densities, increasing human mobility, and dispersion of livestock, arthropods and commercial goods via expanding transportation systems. Consequently, the World Health Organization concluded that acquired immune deficiency syndrome, tuberculosis, malaria, and neglected tropical diseases will remain challenges for the foreseeable future.1 Understandably, the high human fatality rates reported during the recent epidemics of Ebola, severe acute respiratory syndrome and Middle East respiratory syndrome have attracted high levels of publicity. However, many other RNA viruses have emerged or reemerged and dispersed globally despite being considered to be neglected diseases.2,3 Chikungunya virus (CHIKV), West Nile virus (WNV) and dengue virus (DENV) are three of a large number of neglected human pathogenic arthropod-borne viruses (arboviruses) whose combined figures for morbidity and mortality far exceed those for Ebola, severe acute respiratory syndrome and Middle East respiratory syndrome viruses. For instance, for DENV, the number of cases of dengue fever/hemorrhagic fever is between 300–400 million annually, of which an estimated 22 000 humans die.4 Moreover, in the New World, within 12 months of its introduction, CHIKV caused more than a million cases of chikungunya fever according to Pan American Health Organization/World Health Organization, with sequelae that include persistent arthralgia, rheumatoid arthritis and lifelong chronic pain.5 Likewise, within two months of its introduction, to Polynesia, the number of reported cases exceeded 40 0006 and is currently believed to be approaching 200000 cases. Alarmingly, this rapid dispersion and epidemicity of CHIKV (and DENV or Zika virus in Oceania) is now threatening Europe and parts of Asia through infected individuals returning from these newly endemic regions. This is an increasingly worrying trend. For example, in France, from 1 May to 30 November, 2014, 1492 suspected cases of dengue or chikungunya fever were reported.7 Accordingly, this review focuses on the emergence or reemergence of arboviruses and their requirements and limitations for controlling these viruses in the future.
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).
We emphasize the importance of using molecular diagnostic tests which can provide rapid diagnosis and lead to appropriate therapeutic strategies and epidemiological measures.
Central nervous system (CNS) infections including meningitis and encephalitis are important causes of significant mortality and morbidity in the developing nations. Viruses are considered as important etiological agents of CNS infections, causing diseases ranging from febrile illness to myelitis to meningoencephalitis. However, in most cases, the etiology of CNS infection is not known due to lack of diagnostic capacity, standard clinical case definitions, or low levels of surveillance. Specific diagnosis for CNS infection is rarely made and usually categorized empirically as only “viral” or “bacterial”. There have been few reports on the viral etiologies of CNS infections in Indonesia except for Japanese encephalitis virus (JEV), a leading cause of acute encephalitis in children and young adults in the Southeast Asian region [2–5]. Still, JEV is significantly underreported in Indonesia. Furthermore, in endemic provinces like Bali where encephalitis is often suspected to be JEV, there is lack of laboratory capability to accurately determine the disease burden of JEV and other CNS viruses. The objective of this study was therefore to detect and identify the pathogens responsible for viral CNS infections amongst in-patients at a referral hospital in Manado, North Sulawesi, Indonesia.
Viral encephalitis is a relatively rare disease but with a potentially high morbidity and mortality. Advances in the diagnostic methods can lead to the identification of a large number of viruses that can affect the central nervous system.
Enteroviruses are non-enveloped single stranded RNA viruses of the genus Enterovirus within the family of Picornaviridae. Seven species of Enterovirus are associated with human disease: Enterovirus A-D and Rhinovirus A-C. While rhinoviruses commonly cause mild respiratory illness, enteroviruses A-D (EVs) are a significant cause of morbidity and mortality worldwide. Prior to being reclassified as EV A-D, EVs were originally classified as polioviruses (PV) 1–3, coxsackieviruses (CV) A1–24 and B1–6, echoviruses (E) 1–33 and numbered enteroviruses (68–121).
In addition to the surveillance system for poliomyelitis, some countries have established comprehensive surveillance programs for circulating non-polio EVs [2, 3]. In recent years, surveillance activities have been enhanced in response to the emergence of hand, foot, and mouth disease (HFMD) causing enteroviruses in the Asia-Pacific region and the global spread of EV-D68 causing respiratory infections [5, 6].
EV infections are often asymptomatic, but may also result in a diverse spectrum of clinical illness, varying from mild febrile illnesses to severe disease of the cutaneous, gastrointestinal, respiratory, cardiovascular, and central nervous system (CNS) [7, 8]. Generally, EV A is associated with herpangina and hand, foot and mouth disease (HFMD), EV B with herpangina and sporadic and epidemic viral meningitis or encephalitis, EV C with poliomyelitis and EV D with respiratory infections [2, 9–11]. In Viet Nam, since 2005, various serotypes of EV A, most commonly enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), CV-A10, and CV-A6 have been associated with outbreaks of HFMD [12, 13] and EVs have also been frequently detected in aetiological studies of CNS and respiratory infections [14–18].
In the majority of aetiological studies only generic RT-PCR is used for detection of enteroviruses [14–16, 18, 19]. Information about specific enterovirus serotypes circulation and their associated clinical phenotypes therefore remains sparse.
Here we report the clinical associations and serotyping results of EVs that were previously detected in our studies of CNS and respiratory infections in southern and central Viet Nam between 1997 and 2010.
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.
This study was approved by the Medical Research Ethics Committee of R.D. Kandou General Hospital (Ethical Approval No. 066/EC-UPKT/III/2016) and Eijkman Institute Research Ethics Commission (Ethical Approval No. 78). Written informed consent for participating in the study was obtained from all of the patients, guardians, or accompanying close relatives.
Respiratory syncytial virus (RSV), a member of the Orthopneumovirus genus, infects approximately 70% of infants before the age of 1 and almost 100% by the age of 2 years old, making it the most common pathogen to cause lower respiratory tract infection such as bronchiolitis and pneumonia in infants worldwide. Recent evidence also indicates that severe respiratory diseases related to RSV are also frequent in immunocompromised adult patients and that the virus can also present neuroinvasive properties. Over the last five decades, a number of clinical cases have potentially associated the virus with CNS pathologies. RSV has been detected in the cerebrospinal fluid (CSF) of patients (mainly infants) and was associated with convulsions, febrile seizures and different types of encephalopathy, including clinical signs of ataxia and hormonal problems. Furthermore, RSV is now known to be able to infect sensory neurons in the lungs and to spread from the airways to the CNS in mice after intranasal inoculation, and to induce long-term sequelae such as behavioral and cognitive impairments.
An additional highly prevalent human respiratory pathogen with neuroinvasive and neurovirulent potential is the human metapneumovirus (hMPV). Discovered at the beginning of the 21st century in the Netherlands, it mainly causes respiratory diseases in newborns, infants and immunocompromised individuals. During the last two decades, sporadic cases of febrile seizures, encephalitis and encephalopathies (associated with epileptic symptoms) have been described. Viral material was detected within the CNS in some clinical cases of encephalitis/encephalopathy but, at present, no experimental data from any animal model exist that would help to understand the underlying mechanism associated with hMPV neuroinvasion and potential neurovirulence.
Hendra virus (HeV) and Nipah virus (NiV) are both highly pathogenic zoonotic members of the Henipavirus genus and represent important emerging viruses discovered in the late 1990s in Australia and southern Asia. They are the etiological agents of acute and severe respiratory disease in humans, including pneumonia, pulmonary edema and necrotizing alveolitis with hemorrhage. Although very similar at the genomic level, both viruses infect different intermediate animal reservoirs: the horse for HeV and the pig for NiV as a first step before crossing the barrier species towards humans. In humans, it can lead to different types of encephalitis, as several types of CNS resident cells (including neurons) can be infected. The neurological signs can include confusion, motor deficits, seizures, febrile encephalitic syndrome and a reduced level of consciousness. Even neuropsychiatric sequelae have been reported but it remains unclear whether a post-infectious encephalo-myelitis occurs following infection. The use of animal models showed that the main route of entry into the CNS is the olfactory nerve and that the Nipah virus may persist in different regions of the brain of grivets/green monkeys, reminiscent of relapsing and late-onset encephalitis observed in humans.
Influenza viruses are classified in four types: A, B, C and D. All are endemic viruses with types A and B being the most prevalent and causing the flu syndrome, characterized by chills, fever, headache, sore throat and muscle pain. They are responsible for seasonal epidemics that affect 3 to 5 million humans, among which 500,000 to 1 million cases are lethal each year. Associated with all major pandemics since the beginning of the 20th century, circulating influenza A presents the greatest threat to human health. Most influenza virus infections remain confined to the upper respiratory tract, although some can lead to severe cases and may result in pneumonia, acute respiratory distress syndrome (ARDS) and complications involving the CNS. Several studies have shown that influenza A can be associated with encephalitis, Reye’s syndrome, febrile seizure, Guillain–Barré syndrome, acute necrotizing encephalopathy and possibly acute disseminated encephalomyelitis (ADEM). Animal models have shown that, using either the olfactory route or vagus nerve, influenza A virus may have access to the CNS and alter the hippocampus and the regulation of neurotransmission, while affecting cognition and behavior as long-term sequelae. The influenza A virus has also been associated with the risk of developing Parkinson’s disease (PD) and has recently been shown to exacerbate experimental autoimmune encephalomyelitis (EAE), which is reminiscent of the observation that multiple sclerosis (MS) relapses have been associated with viral infections (including influenza A) of the upper respiratory tract.
Another source of concern when considering human respiratory pathogens associated with potential neuroinvasion and neurovirulence is the Enterovirus genus, which comprises hundreds of different serotypes, including polioviruses (PV), coxsackieviruses (CV), echoviruses, human rhinoviruses (HRV) and enteroviruses (EV). This genus constitutes one of the most common cause of respiratory infections (going from common cold to more severe illnesses) and some members (PV, EV-A71 and -D68, and to a lesser extent HRV) can invade and infect the CNS, with detrimental consequences. Even though extremely rare, HRV-induced meningitis and cerebellitis have been described. Although EV infections are mostly asymptomatic, outbreaks of EV-A71 and D68 have also been reported in different parts of the world during the last decade. EV-A71 is an etiological agent of the hand–foot–mouth disease (HFMD) and has occasionally been associated with upper respiratory tract infections. EV-D68 causes different types of upper and lower respiratory tract infections, including severe respiratory syndromes. Both serotypes have been associated with neurological disorders like acute flaccid paralysis (AFP), myelitis (AFM), meningitis and encephalitis.
Last but not least, human coronaviruses (HCoV) are another group of respiratory viruses that can naturally reach the CNS in humans and could potentially be associated with neurological symptoms. These ubiquitous human pathogens are molecularly related in structure and mode of replication with neuroinvasive animal coronaviruses like PHEV (porcine hemagglutinating encephalitis virus), FCoV (feline coronavirus) and the MHV (mouse hepatitis virus) strains of MuCoV, which can all reach the CNS and induce different types of neuropathologies. MHV represents the best described coronavirus involved in short- and long-term neurological disorders (a model for demyelinating MS-like diseases). Taken together, all these data bring us to consider a plausible involvement of HCoV in neurological diseases.
The Flaviviridae is a large family of positive-strand RNA viruses, that comprises four genera: Flavivirus, Pegivirus, Pestivirus, and Hepacivirus. The Flavivirus genus consists of more than 70 viruses, many of which are arthropod-borne human pathogens that cause a variety of clinical diseases, ranging from asymptomatic to mild fever to more severe diseases including encephalitis and hemorrhagic fever [1, 2] Most flaviviruses are transmitted through the bite of an infected arthropod vector, mainly Aedes genus (Aedes aegypti and to a lesser extent, Aedes albopictus) and Cluex mosquitos, and most were once maintained by animal reservoirs in sylvatic transmission cycles. Many flaviviruses, however, such as dengue virus, yellow fever and Zika virus, are now principally maintained by mosquito-borne transmission with a possible human-to-human transmission through transfusion of infected blood or transplantation of infected tissue.
Some flaviviruses can cause globally significant vector-borne diseases with a substantial public health impact such as dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV) and yellow fever virus (YFV). Other members of the Flaviviridae family that have a more regional impact include Murray Valley encephalitis virus (MVEV) in Oceania, St. Louis encephalitis virus (SLEV) in North America, and tick-borne encephalitis virus (TBEV) in Europe. Over the past few decades, many of these flaviviruses have re-emerged for a range of reasons including decreases in mosquito control efforts, rapid changes in climate and vector's demography, dense urbanization, population growth and globalization with increased transportation and trade activities. Examples include the geographic spread of DENV throughout the tropical world; JEV throughout south Asia, Australasia and the Pacific; ZIKV into South and Central America; YFV into the Americas and the invasion of WNV into much of North America [1, 5].
It is estimated that there are over 390 million DENV infections per year, of which 96 million manifests clinically with varying degrees of severity and 3.9 billion people in 128 countries are at risk of infection. Similarly, high incidence rates for symptomatic cases of JEV were reported over the past three decades to reach 2.4 per year per 100,000 population. Epidemic waves of YFV are projected to result in 30,000 to 200,000 clinical cases per year with case-fatality rates ranging from 2 to 15% [9–12]. WNV, first appeared in the northeastern USA in 1999, are spread presently across much of the USA and southern Canada. For example, in 2015, the CDC reported 2,175 cases of WNV, of which 1,616 (74%) were hospitalized and 146 (7%) died. In the developing world, WNV incidence is likely to be underestimated due to political, psychological, and economic barriers to reporting [12, 14].
Although most human flavivirus infections are asymptomatic or have an undifferentiated febrile illness, a small percentage of affected individuals develop acute fever that can progress to severe clinical manifestations such as hemorrhage, vascular leakage and encephalitis. Currently, our knowledge of the host-related factors that influence the pathogenesis of severe disease is inadequate to allow prediction of who will develop severe clinical illness. However, some mechanisms and etiological factors underlying inter-individual variations in response to flavivirus infections have been identified. Interactions of virus-encoded proteins with human innate immune pathways; the effect of host-cell surface molecules in virus binding and entry; the role of viral protein nuclear localization in the host cell response; and the flavivirus replication dynamics within multiple immune systems have all been considered as host-pathogen interaction events that may regulate viral virulence or attenuation and the subsequent disease severity. Over the past few years, however, host-related factors such as preexisting chronic conditions, e.g., cardiovascular diseases, diabetes, obesity, and asthma have received attention as predictors for increased risk of progression to severe flavivirus infection [19–21]. Recent studies have raised the proposition that cardiovascular disease, stroke, diabetes, respiratory diseases and renal disorders may contribute, together with old age, to severe clinical manifestations of dengue [19, 20]. A few studies of WNV and JEV infections, and responses to YFV vaccination, have also explored the role of chronic comorbidities in the prognosis of infections. Given the lack of specific medical treatment for flavivirus diseases, effective public health surveillance for vector-borne infections together with continuing vector control efforts will be critical to preventing infection. However, elucidating the impact of comorbidities to the severity of disease when infection occurs will be critical to identifying vulnerable populations, to whom effective interventions protocols and individually-tailored clinical monitoring practices should be particularly targeted.
The objective of this study is to systematically review the existing literature on the prevalence of the most common non-communicable comorbidities related to the cluster of metabolic syndromes-associated diseases, such as diabetes mellitus, heart diseases, hypertension, asthma, stroke and obesity in flavivirus infections and to evaluate the difference of their prevalence in severe vs. non-severe clinical outcomes to infection. Identifying and characterizing associations between comorbidities and severity of flavivirus infections will be significant factor in designing public health measures that aim to prevent the severe outcomes of infection.
Japanese encephalitis (JE) is caused by the Japanese encephalitis virus (JEV), and is one of the most important mosquito-borne diseases with a mortality rate as high as 20% to 50%, and is widely distributed in most of East and South-east Asia and parts of Oceania. Up to 50,000 human cases of JE are reported annually in Asian countries, of which 10,000–15,000 result in fatality. A high proportion (nearly 50%) of survivors, especially young children and those greater than 65 years of age, exhibit permanent neurologic and psychiatric sequelae. A wide range of animals including swine, equines and birds can also be infected. Pigs, as well as birds, serve as amplifying and reservoir hosts,. Further, JEV infection has accounted for significant economic losses in the pig industry due to fetal encephalitis and reproductive failure in pregnant sows and hypospermia in boars. There is no specific treatment available for JE, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. JEV non-structural protein 1 (NS1) has been shown to induce both humoral and cell-mediated immunity against JE,. Further, like other flaviviruses, NS1 is able to elicit protective immunity without the risk of antibody-dependent enhancement. These characteristics make NS1 an attractive alternative immunogen. As such, much research is currently being devoted to NS1-based vaccine development,,. Although NS1 is not present in the virion, NS1-induced antibodies can protect against infection in vivo by an undetermined mechanism, which presumably depends on the Fc portion of the antibody since they kill their target cells through a complement-dependent pathway,.
JEV is a member of the family Flaviviridae, and the genus Flavivirus, and is primarily transmitted by Culex mosquitoes. Besides JEV, the Japanese encephalitis virus serocomplex of Flaviviridae includes the West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEM). JEV serogroup viruses and Dengue virus (DENV) have a similar ecology; it is very common that two or more of these flaviviruses co-circulate in some regions of the world–, and cross-reactivity can be demonstrated among these flaviviruses in serological tests. These cross-reactive responses could confound the interpretation of results during serological testing, including neutralization tests and enzyme-linked immunosorbent assays (ELISA). This serves to emphasize the utility of virus-specific epitopes for the differential diagnosis of disease and epidemiological surveys. The serological cross-reactivity is primarily caused by cross-reactive epitopes on the structural protein E,. In contrast, NS1 is more specific in serological testing of flavivirus infections, and it has been reported that NS1 can induce antibodies without cross-reactivity among flaviviruses, and even among different serotypes of DENV,, therefore the development of an NS1-based specific serological diagnosis is of great interest,,. First, it is necessary to precisely identify the B-cell epitopes on NS1. In this study we have identified and characterized five JEV NS1-specific epitopes with monoclonal antibodies. This work demonstrates progress toward the development of a specific serological diagnostic test for JEV infection, extends our understanding of the antigenic structure of JEV NS1, and could help inform vaccine design.
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.
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.
Samples from a total of 203 patients were included in this study, including from 79 patients with CNS infection and 124 with respiratory infection. When analyzing for the monthly distribution of cases, there was no clear peak among CNS cases, whereas two peaks of respiratory cases were found in April and November (Fig. 1).
Among patients with CNS infection (n = 79), 51 were children (median age 2 years, interquartile range [IRQ]: 1–5) and 28 were adults (20 years, IRQ: 17–29.5). All adults had EV detected in CSF and 93% (26/28) had meningitis as discharge diagnosis. Of the 51 children, 10 had EV detected in CSF, and the remaining (41/51) had EV detected in respiratory and/or rectal swabs. Encephalitis was the most common discharge diagnosis (30/41). Fifteen deaths were noted, and all were children with EV detected in swabs only (Table 1).
Among patients with respiratory infections, the median age was 12.7 months (IQR: 5.7–24.3). Inpatients (n = 65) were younger than outpatients (n = 59), due to study enrolment criteria (Table 2). Bronchiolitis (as assessed at the discretion of treating physicians) was the most common clinical diagnosis (57% in outpatients and 61% in inpatients). Thirty-five percent of inpatients had a clinical diagnosis of pneumonia (Table 2).
A previously well 69-year-old Australian man traveled to Thailand in early May 2017. The planned duration of travel was 12 days, and he did not attend a travel clinic prior to departure. The patient did not take malaria chemoprophylaxis, nor did he have a prior history of Japanese encephalitis virus (JEV) vaccination. He flew to Phuket, before traveling north to the popular tourist destination of Khao Lak, where he stayed in a beachside holiday resort. Heavy rainfall occurred during the trip, which limited holiday activities. He did not travel to rural or remote areas, but did receive numerous mosquito bites. On the eighth day of travel, he became unwell with lethargy and generalized muscle aches. He flew to Bangkok on the ninth day of the trip, and over the following 3 days his symptoms included ongoing lethargy, poor appetite, and drenching sweats, but no headache, meningism, or confusion. He returned to Australia on day 12 of travel and was admitted to a regional hospital the following day, now the fifth day after symptoms commenced (Figure 1).
Shortly after admission, on day 7 of his illness, he became confused. Cerebrospinal fluid (CSF) obtained by lumbar puncture demonstrated a glucose of 3.5 mmol/L (reference interval, 2.2–3.9 mmol/L), protein of 1.3 g/L (reference interval < 0.45), polymorphs of 280 × 10^6/L, lymphocytes of 90 × 10^6/L, and red blood cells of 54 × 10^6/L. He was commenced on empiric broad-spectrum antibiotics with vancomycin, meropenem, benzyl penicillin, and acyclovir. Due to a deteriorating conscious state, he was intubated the following day and transferred to a tertiary center. Neurological examination upon arrival revealed a generalized flaccid paralysis. A magnetic resonance image of the brain demonstrated no abnormalities. Seizure activity developed on day 10 of his illness, for which anticonvulsant medication was commenced.
Diagnostic assays performed on the initial and a repeat CSF (on day 10) were negative using conventional gel-based and real-time multiplex polymerase chain reactions (PCRs) for herpes viruses (herpes simplex 1 and 2 and varicella zoster), enterovirus, pan-flavivirus (Murray Valley encephalitis [MVE], Kunjin, dengue, West Nile, Zika, yellow fever, JEV), respiratory viruses (influenza, respiratory syncytial virus, parainfluenza, human metapneumovirus, picornavirus, adenovirus, coronavirus), meningococcus, and pneumococcus. The conventional pan-flavivirus PCR and JEV real-time PCR performed on plasma were also negative (see the Supplementary Data for detailed methods). HIV and syphilis serology were negative, as were bacterial cultures and screening swabs for Burkholderia pseudomallel.
On the fourteenth day of illness, JEV immunofluorescence serology (Euroimmun) performed in parallel on plasma from day 7, day 12, and day 13 of illness, was positive. The JEV IgG titre increased across the serial bleeds from 160 to 1280 to >2560 (confirmed by neutralization), suggesting recent infection, and JEV IgM was detected in all 3 specimens. Serology performed on CSF from day 10 was also positive for JEV IgM and IgG. The presence of measles IgG in plasma but not CSF was consistent with local production of JEV antibodies in CSF rather than contamination. Serology for MVE (EIA—total antibody) was weakly positive on day 7 but negative on days 12 and 13 and thought to represent nonspecific cross-reactivity. Dengue virus IgM, IgG, and NS1 antigen were not detected.
Given the results of serological testing, serial samples of plasma, whole blood, and urine were tested using the conventional gel based pan-flavivirus PCR and JEV real-time PCR. Notably, both urine and whole blood specimens were found to be persistently positive for JEV (confirmed by sequencing of the NS5 region), while plasma was found to be persistently negative (Figure 1). Reproducibility of these findings was confirmed by re-extracting and retesting the 4 most recent urine and whole blood specimens directly from the primary samples, and repeat testing was performed on separate PCR runs. Whole-genome sequencing and subsequent phylogenetic analysis (Supplementary Figure 1) identified the isolated JEV strain (VIDRL_JEV aligned) as a member of Genotype I. Our patient’s isolate localized within a subclade of viruses isolated from Thailand, geographically aligning with the travel history.
Aliquots of urine and whole blood were inoculated onto cultured cells to assess viral infectivity (see the Supplementary Data for detailed methods). Cytopathic effects were observed 7 days postinoculation from the day 14 urine specimen. The cell culture supernatant was tested by JEV real-time PCR and was found to be positive with a high level of detection, suggesting efficient viral replication.
Japanese encephalitis viral RNA was detected in urine samples out to day 26 after the onset of symptoms and in whole blood up until the final specimen was tested on day 28.
Electromyogram and nerve conduction studies performed on day 22 were consistent with an acute motor-axonal neuropathy or anterior horn cell pathology. No clinical improvement was evident after 4 days of intravenous immunoglobulin (IVIG) administered at a dose of 1 g/kg daily, and the patient died 30 days after the onset of symptoms.
No difference in sensitivity was observed between the viral strains tested or between lots of slides. A sample dilution of 1:20 for IgM and 1:40 for POWV IgG IFAs demonstrated the optimal balance between sensitivity and nonspecific background staining (Fig. 1). Tick-borne disease (TBD) samples with titers of 1:320 and 1:160 in the plaque reduction neutralization test using a 90% reduction cutoff (PRNT90) were assayed at optimized screening dilutions to confirm. All but one of the POWV encephalitis samples obtained from the New York State Department of Health (NYSDOH) with PRNT90 titers of 1:20 were detected by the TBE-C EIA screen and confirmatory POWV IFA. A PRNT90 titer of 1:20 was determined to be the limit of detection (LOD) for the serologic test panel and was confirmed as such using known PRNT90-positive samples (Table 1). At these screening dilutions, the serologic panel showed an analytical sensitivity of 89% (Table 1). Reproducibility studies showed 100% accordance (k = 1.0).
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.