Supaporn Wacharapluesadee

Bat Nipah virus, Thailand

Surveillance for Nipah virus (NV) was conducted in Thailands bat population. Immunoglobulin G antibodies to NV were detected with enzyme immunoassay in 82 of 1,304 bats. NV RNA was found in bat saliva and urine. These data suggest the persistence of NV infection in Thai bats.

Human rabies: neuropathogenesis, diagnosis, and management

Rabies is an almost invariably fatal disease that can present as classic furious rabies or paralytic rabies. Recovery has been reported in only a few patients, most of whom were infected with bat rabies virus variants, and has been associated with promptness of host immune response and spontaneous (immune) virus clearance. Viral mechanisms that have evolved to minimise damage to the CNS but enable the virus to spread might explain why survivors have overall good functional recovery. The shorter survival of patients with furious rabies compared with those with paralytic rabies closely corresponds to the greater amount of virus and lower immune response in the CNS of patients with the furious form. Rabies virus is present in the CNS long before symptom onset: subclinical anterior horn cell dysfunction and abnormal brain MRI in patients with furious rabies are evident days before brain symptoms develop. How the virus produces its devastating effects and how it selectively impairs behaviour in patients with furious rabies and the peripheral nerves of patients with paralytic rabies is beginning to be understood. However, to develop a pragmatic treatment strategy, a thorough understanding of the neuropathogenetic mechanisms is needed.

Evidence for novel hepaciviruses in rodents.

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.

A Longitudinal Study of the Prevalence of Nipah Virus in Pteropus lylei Bats in Thailand: Evidence for Seasonal Preference in Disease Transmission

After 12 serial Nipah virus outbreaks in humans since 1998, it has been noted that all except the initial event in Malaysia occurred during the first 5 months of the year. Increasingly higher morbidity and mortality have been observed in subsequent outbreaks in India and Bangladesh. This may have been related to different virus strains and transmission capability from bat to human without the need for an amplifying host and direct human-to-human transmission. A survey of virus strains in Pteropus lylei and seasonal preference for spillover of these viruses was completed in seven provinces of Central Thailand between May 2005 and June 2007. Nipah virus RNA sequences, which belonged to those of the Malaysian and Bangladesh strains, were detected in the urine of these bats, with the Bangladesh strain being dominant. Highest recovery of Nipah virus RNA was observed in May. Of two provincial sites where monthly surveys were done, the Bangladesh strain was almost exclusively detected during April to June. The Malaysian strain was found dispersed during December to June. Although direct contact during breeding (in December to April) was believed to be an important transmission factor, our results may not entirely support the role of breeding activities in spillage of virus. Greater virus shedding over extended periods in the case of the Malaysian strain and the highest peak of virus detection in May in the case of the Bangladesh strain when offspring started to separate may suggest that there may be responsible mechanisms other than direct contact during breeding in the same roost. Knowledge of seasonal preferences of Nipah virus shedding in P. lylei will help us to better understand the dynamics of Nipah virus transmission and have implications for disease management.

Failure of therapeutic coma and ketamine for therapy of human rabies

The recent success in treating a human rabies patient in Milwaukee prompted the use of a similar therapeutic approach in a 33-year-old male Thai patient who was admitted in the early stages of furious rabies. He received therapeutic coma with intravenous diazepam and sodium thiopental to maintain an electroencephalographic burst suppression pattern, which was maintained for a period of 46 h, as well as intravenous ketamine (48 mg/kg/day) as a continuous infusion and ribavirin (48 to 128 mg/kg/day) via a nasogastric tube. He never developed rabies virus antibodies and he died on his 8th hospital day. At least three other patients have been treated unsuccessfully with a similar therapeutic approach. Because of the lack of a clear scientific rationale, high associated costs, and potential complications of therapeutic coma, the authors recommend caution in taking this approach for the therapy of rabies outside the setting of a clinical trial. More experimental work is also needed in cell culture systems and in animal models of rabies in order to develop effective therapy for human rabies.

Nucleic-acid sequence based amplification in the rapid diagnosis of rabies

Current serological tests do not reliably diagnose rabies. We describe a technique based on amplification of nucleic-acid sequences to detect rabies-specific RNA in the saliva and cerebrospinal fluid (CSF) of four living patients with rabies. Rabies RNA could be detected in either saliva or CSF, or both, in all patients and as early as day 2 after onset of symptoms. Both saliva and CSF should be serially tested because not every sample can be expected to be positive. The whole process, including automated extraction, isothermal amplification, and detection can be done within 4 h.

Pathophysiology of human paralytic rabies

Furious rabies is a well-recognized clinical disorder in humans but the paralytic form is not as easily identified. The mechanisms responsible for the weakness and longer survival periods are not clear. Several hypotheses have been proposed, including rabies virus variants associated with a particular vector, location of wounds, incubation period, influence of prior rabies vaccination, and virus localization in the central nervous system (CNS). However, none of these have been substantiated. Regarding molecular analyses of rabies viruses isolated from both furious and paralytic rabies patients, only minor genetic variations with no specific patterns in glyco-(G), phospho-(P), and nucleoprotein (N) sequences have been identified and arginine 333 in G protein was present in all samples. Regional distribution of rabies virus antigen in rabies patients whose survival periods were 7 days or less and magnetic resonance imaging (MRI) of the CNS indicated brainstem and spinal cord as predilection sites regardless of clinical presentations. There are clinical, electrophysiological, and pathological indications that peripheral nerve dysfunction is responsible for weakness in paralytic rabies whereas in furious rabies, even in the absence of clinical weakness, abundant denervation potentials with normal sensory nerve conduction studies and proximal motor latencies suggest anterior horn cell dysfunction. The lack of cellular immunity to rabies virus antigen accompanied by an absence of cerebrospinal fluid (CSF) rabies neutralizing antibody in most paralytic rabies patients may argue against role of an immune response against rabies virus—positive axons. Aberrant immune responses to peripheral nerve antigen, in particular those mediated by one or more cellular-dependent mechanisms, may be involved as is supported by the absence of putative anti-ganglioside antibodies commonly found in immune-mediated peripheral nerve diseases. Longer survival period in paralytic rabies may possibly be related to currently unidentified mechanism(s) on neuronal gene expression, required for virus transcription/replication and for maintaining neuronal survival.

Survey for Bat Lyssaviruses, Thailand

Surveillance for lyssaviruses was conducted among bat populations in 8 provinces in Thailand. In 2002 and 2003, a total of 932 bats of 11 species were captured and released after serum collection. Lyssavirus infection was determined by conducting virus neutralization assays on bat serum samples. Of collected samples, 538 were either hemolysed or insufficient in volume, which left 394 suitable for analysis. These samples included the following: Pteropus lylei (n = 335), Eonycteris spelaea (n = 45), Hipposideros armiger (n = 13), and Rousettus leschennaulti (n = 1). No serum samples had evidence of neutralizing antibodies when tested against rabies virus. However, 16 samples had detectable neutralizing antibodies against Aravan virus, Khujand virus, Irkut virus, or Australian bat lyssavirus; all were specifically associated with fruit bats P. lylei (n = 15) and E. spelaea (n = 1). These results are consistent with the presence of naturally occurring viruses related to new putative lyssavirus genotypes.

Transmission dynamics of rabies virus in Thailand: Implications for disease control

BackgroundIn Thailand, rabies remains a neglected disease with authorities continuing to rely on human death statistics while ignoring the financial burden resulting from an enormous increase in post-exposure prophylaxis. Past attempts to conduct a mass dog vaccination and sterilization program have been limited to Bangkok city and have not been successful. We have used molecular epidemiology to define geographic localization of rabies virus phylogroups and their pattern of spread in Thailand.MethodsWe analyzed 239 nucleoprotein gene sequences from animal and human brain samples collected from all over Thailand between 1998 and 2002. We then reconstructed a phylogenetic tree correlating these data with geographical information.ResultsAll sequences formed a monophyletic tree of 2 distinct phylogroups, TH1 and TH2. Three subgroups were identified in the TH1 subgroup and were distributed in the middle region of the country. Eight subgroups of TH2 viruses were identified widely distributed throughout the country overlapping the TH1 territory. There was a correlation between human-dependent transportation routes and the distribution of virus.ConclusionInter-regional migration paths of the viruses might be correlated with translocation of dogs associated with humans. Interconnecting factors between human socioeconomic and population density might determine the transmission dynamics of virus in a rural-to-urban polarity. The presence of 2 or more rabies virus groups in a location might be indicative of a gene flow, reflecting a translocation of dogs within such region and adjacent areas. Different approaches may be required for rabies control based on the homo- or heterogeneity of the virus. Areas containing homogeneous virus populations should be targeted first. Control of dog movement associated with humans is essential.

Furious and paralytic rabies of canine origin: neuroimaging with virological and cytokine studies.

Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors studied magnetic resonance imaging (MRI) and cytokine and virus distribution in rabies-infected dogs of both clinical types. MRI examination of the brain and upper spinal cord was performed in two furious and two paralytic dogs during the early clinical stage. Rabies viral nucleoprotein RNA and 18 cytokine mRNAs at 12 different brain regions were studied. Rabies viral RNA was examined in four furious and four paralytic dogs during the early stage, and in one each during the late stage. Cytokine mRNAs were examined in two furious and two paralytic dogs during the early stage and in one each during the late stage. Larger quantities of rabies viral RNA were found in the brains of furious than in paralytic dogs. Interleukin-1β and interferon-γ mRNAs were found exclusively in the brains of paralytic dogs during the early stage. Abnormal hypersignal T2 changes were found at hippocampus, hypothalamus, brainstem, and spinal cord of paralytic dogs. More widespread changes of less intensity were seen in furious dog brains. During the late stage of infection, brains from furious and paralytic rabid dogs were similarly infected and there were less detectable cytokine mRNAs. These results suggest that the early stage of furious dog rabies is characterized by a moderate inflammation (as indicated by MRI lesions and brain cytokine detection) and a severe virus neuroinvasiveness. Paralytic rabies is characterized by delayed viral neuroinvasion and a more intense inflammation than furious rabies. Dogs may be a good model for study of the host inflammatory responses that may modulate rabies virus neuroinvasiveness.