[Show abstract][Hide abstract] ABSTRACT: A major reservoir of Nipah virus is believed to be the flying fox genus Pteropus, a fruit bat distributed across many of the world's tropical and sub-tropical areas. The emergence of the virus and its zoonotic transmission to livestock and humans have been linked to losses in the bat's habitat. Nipah has been identified in a number of indigenous flying fox populations in Thailand. While no evidence of infection in domestic pigs or people has been found to date, pig farming is an active agricultural sector in Thailand and therefore could be a potential pathway for zoonotic disease transmission from the bat reservoirs. The disease, then, represents a potential zoonotic risk. To characterize the spatial habitat of flying fox populations along Thailand's Central Plain, and to map potential contact zones between flying fox habitats, pig farms and human settlements, we conducted field observation, remote sensing, and ecological niche modeling to characterize flying fox colonies and their ecological neighborhoods. A Potential Surface Analysis was applied to map contact zones among local epizootic actors.
Flying fox colonies are found mainly on Thailand's Central Plain, particularly in locations surrounded by bodies of water, vegetation, and safe havens such as Buddhist temples. High-risk areas for Nipah zoonosis in pigs include the agricultural ring around the Bangkok metropolitan region where the density of pig farms is high.
Passive and active surveillance programs should be prioritized around Bangkok, particularly on farms with low biosecurity, close to water, and/or on which orchards are concomitantly grown. Integration of human and animal health surveillance should be pursued in these same areas. Such proactive planning would help conserve flying fox colonies and should help prevent zoonotic transmission of Nipah and other pathogens.
BMC Veterinary Research 12/2015; 11(1):81. DOI:10.1186/s12917-015-0390-0 · 1.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Influenza A viruses have the remarkable characteristic of sustainability in the environment. Mucus from snails
contains sialic acids as the one that may allow avian influenza virions to bind to the vertebrate host cell
membrane. Subsequently snails could potentially promote persistence and/or concentration of influenza Avirions
through mucus, in wetland environment when virus is released from bird infected feces.
This article describes experimental research on the potential outcome of apple snails regarding the persistence
and concentration of the H5N1 influenza virions in fresh water. The presence of virus was detected from water
and snails by hemagglutination test, and H5N1 viral genetic material determined by quantitative RT-PCR
Active virus in the water was demonstrated up to twelve days after water infestation without snails, and up to
fourteen days with snails. Also, up to eleven days, the virus and genetic material were detected and tittered from
snails. Although the presence of snails did not significantly change the persistence of H5N1 virus in the water,
number of positive snail sampled and quantitative RT-PCR data suggest that snails may have the ability to
concentrate and carry viral particles.
Ultimately snails could play a role in the virus ecology by concentrating viral particles from water and
facilitating virus contact with the bird hosts that feed on them.
[Show abstract][Hide abstract] ABSTRACT: bstract
Influenza A viruses have the remarkable characteristic of sustainability in the environment. Mucus from snails contains sialic acids as the one that may allow avian influenza virions to bind to the vertebrate host cell membrane. Subsequently snails could potentially promote persistence and/or concentration of influenza Avirions through mucus, in wetland environment when virus is released from bird infected feces.
This article describes experimental research on the potential outcome of apple snails regarding the persistence and concentration of the H5N1 influenza virions in fresh water. The presence of virus was detected from water and snails by hemagglutination test, and H5N1 viral genetic material determined by quantitative RT-PCR
Active virus in the water was demonstrated up to twelve days after water infestation without snails, and up to fourteen days with snails. Also, up to eleven days, the virus and genetic material were detected and tittered from snails. Although the presence of snails did not significantly change the persistence of H5N1 virus in the water, number of positive snail sampled and quantitative RT-PCR data suggest that snails may have the ability to concentrate and carry viral particles.
Ultimately snails could play a role in the virus ecology by concentrating viral particles from water and facilitating virus contact with the bird hosts that feed on them.
[Show abstract][Hide abstract] ABSTRACT: N-linked glycosylation of the influenza virus hemagglutinin (HA) protein plays crucial roles in HA structure and function, evasion of neutralizing antibodies, and susceptibility to innate soluble antiviral factors. The N-linked glycosylation site at position 158 of highly pathogenic H5N1 virus was previously shown to affect viral receptor-binding preference. H5N1 viruses show heterogeneity with respect to the presence of this glycosylation site. Clade 1 viruses that caused outbreaks in Southeast Asia in 2004 contained this glycosylation site, while the site is absent in the more recent clade 2 viruses. Here, we show that elimination of this glycosylation site increases viral virulence in mice. The mutant lacking the glycosylation site at position 158 showed unaltered growth kinetics in vitro and a comparable level of sensitivity to a major antiviral protein found in respiratory secretions, surfactant protein D (SP-D).
Archives of Virology 12/2014; 160(2). DOI:10.1007/s00705-014-2306-x · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A questionnaire was used to collect data on small poultry farm management and wild bird observed in poultry keeping areas to identify putative risk factors for infection with HPAI H5N1. The study was conducted in 2008 in four subdistricts of central Thailand that had experienced outbreaks of HPAI H5N1 in poultry. Descriptive and inferential analyses including univariable analyses and multivariable logistic regression were used to identify putative risk factors. Risk factors included purchasing native chickens/fighting cocks from commercial hatcheries, replacing or restocking birds individually, and observing lesser whistling ducks (Dendrocygna javanica) on the farm daily. Selecting healthy animals when purchasing animals to ensure that they were disease free was a protective factor. To fully understand the epidemiology of infection of small poultry farms with HPAI H5N1, control of movement of domestic poultry and serological and virological testing of the poultry population should be applied.
Zoonoses and Public Health 05/2013; 60:209-214. DOI:10.1111/j.1863-2378.2012.01515.x · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brown-headed gulls (Larus brunnicephalus), winter visitors of Thailand, were tracked by satellite telemetry during 2008–2011 for investigating their roles in the highly pathogenic avian influenza (HPAI) H5N1 virus spread. Eight gulls negative for influenza virus infection were marked with solar-powered satellite platform transmitters at Bang Poo study site in Samut Prakarn province, Thailand; their movements were monitored by the Argos satellite tracking system, and locations were mapped. Five gulls completed their migratory cycles, which spanned 7 countries (China, Bangladesh, India, Myanmar, Thailand, Cambodia, and Vietnam) affected by the HPAI H5N1 virus. Gulls migrated from their breeding grounds in China to stay overwinter in Thailand and Cambodia; while Bangladesh, India, Myanmar, and Vietnam were the places of stopovers during migration. Gulls traveled an average distance of about 2400 km between Thailand and China and spent 1–2 weeks on migration. Although AI surveillance among gulls was conducted at the study site, no AI virus was isolated and no H5N1 viral genome or specific antibody was detected in the 75 gulls tested, but 6.6% of blood samples were positive for pan-influenza A antibody. No AI outbreaks were reported in areas along flyways of gulls in Thailand during the study period. Distance and duration of migration, tolerability of the captive gulls to survive the HPAI H5N1 virus challenge and days at viral shedding after the virus challenging suggested that the Brown-headed gull could be a potential species for AI spread, especially among Southeast Asian countries, the epicenter of H5N1 AI outbreak.
PLoS ONE 11/2012; 7(11). DOI:10.1371/journal.pone.0049939 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Elephant endotheliotropic herpesvirus 1 (EEHV1) can cause fatal hemorrhagic disease in Asian elephants (Elephas maximus). Several studies have described this virus as a major threat to young Asian elephants. A SYBR Green I-based real-time polymerase chain reaction (PCR) was developed to identify EEHV1 on trunk swabs and necropsied tissues. Two of 29 (6.9%) trunk swab samples from healthy Asian elephants were positive for EEHV1. The viruses were analyzed and classified as EEHV1A based on 231 nucleotides of the terminase gene. Necropsied spleen and heart tissue showed the highest level and second highest levels of DNA virus copy accumulation, respectively. The detection limit of the test was 276 copies/μl of DNA. There was no cross-reaction with other mammalian herpesviruses, such as herpes simplex virus 1 and equine herpesvirus 2. Inter- and intra-assay showed low coefficients of variation values indicating the reproducibility of the test. The results indicated that the test can be practically used for epidemiological study, clinical diagnosis, and management and control of EEHV1.
[Show abstract][Hide abstract] ABSTRACT: Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems.
[Show abstract][Hide abstract] ABSTRACT: Highly pathogenic avian influenza virus (HPAIV) of the H5N1 subtype has been reported to infect pigeons asymptomatically or induce mild symptoms. However, host immune responses of pigeons inoculated with HPAIVs have not been well documented. To assess host responses of pigeons against HPAIV infection, we compared lethality, viral distribution and mRNA expression of immune related genes of pigeons infected with two HPAIVs (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) isolated from wild birds in Thailand. The survival experiment showed that 25% of pigeons died within 2 weeks after the inoculation of two HPAIVs or medium only, suggesting that these viruses did not cause lethal infection in pigeons. Pigeon04 replicated in the lungs more efficiently than T.sparrow05 and spread to multiple extrapulmonary organs such as the brain, spleen, liver, kidney and rectum on days 2, 5 and 9 post infection. No severe lesion was observed in the lungs infected with Pigeon04 as well as T.sparrow05 throughout the collection periods. Encephalitis was occasionally observed in Pigeon04- or T.sparrow05-infected brain, the severity, however was mostly mild. To analyze the expression of immune-related genes in the infected pigeons, we established a quantitative real-time PCR analysis for 14 genes of pigeons. On day 2 post infection, Pigeon04 induced mRNA expression of Mx1, PKR and OAS to a greater extent than T.sparrow05 in the lungs, however their expressions were not up-regulated concomitantly on day 5 post infection when the peak viral replication was observed. Expressions of TLR3, IFNα, IL6, IL8 and CCL5 in the lungs following infection with the two HPAIVs were low. In sum, Pigeon04 exhibited efficient replication in the lungs compared to T.sparrow05, but did not induce excessive host cytokine expressions. Our study has provided the first insight into host immune responses of pigeons against HPAIV infection.
PLoS ONE 08/2011; 6(8):e23103. DOI:10.1371/journal.pone.0023103 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Thailand, highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 had been isolated from various wild birds during the HPAI outbreak in poultries. In this study, we examined the pathogenicity of two wild bird isolates (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) in mice. They showed similar replication in several organs and lethal outcome. However, on day 3 post-infection, Pigeon04 induced mRNA expression of proinflammatory cytokines (IL6 and TNFα) and MIP-2, neutrophil chemoattractant, in the lungs, resulting in severe pneumonia that was accompanied by neutrophil infiltration. In contrast, on day 7 post-infection, T.sparrow05 induced the expression of several cytokines to a greater extent than Pigeon04; it also potently induced mRNA expression of several cytokines in brains of the infected mice that triggered frequent inflammatory events. In sum, our study demonstrated that two HPAI viruses induced different host responses, despite having similar replications, resulting in lethal outcome in mice.
[Show abstract][Hide abstract] ABSTRACT: A serological and virological surveillance program to investigate the HPAI H5N1 virus in wild bird populations was undertaken from February 2007 to October 2008. The purpose of the survey was to investigate the infection status in free ranging wild birds in Banglane district, Nakhon Pathom province, central Thailand. Samples from wild birds were collected every two months. Choanal and cloacal swabs, serum and tissue samples were collected from 421 birds comprising 44 species. Sero-prevalence of the virus tested by H5N1 serum neutralization test (using a H5N1 virus clade 1; A/chicken/Thailand/vsmu-3-BKK/2004) was 2.1% (8 out of 385 samples; 95% CI 0.7, 3.5). Species that were antibody positive included rock pigeons (Columba livia), Asian pied starling (Gracupica contra), spotted dove (Streptopelia chinensis), oriental magpie robin (Copsychus saularis), blue-tailed bee-eater (Merops philippinus), myna (Acridotheres spp.), and pond heron (Ardeola spp.). Prevalence by H5N1 virus isolation was 0.5% (2 out of 421 samples; 95% CI 0.0, 1.1); the two H5N1 virus-positive samples were from Asian pied starling (Gracupica contra) and white vented myna (Acridotheres grandis). Positive virological samples were collected in June 2007 while all positive serology samples were collected between May and August except for one sample collected in December 2007. No positive samples were collected in 2008. Molecular studies revealed that the wild bird H5N1 viruses were closely related to poultry viruses isolated in other parts of Thailand. However, there was no poultry H5N1 prevalence study performed in the study site during the time of this wild bird survey. Interpretation of source of virus isolates would include spill-over of H5N1 viruses from contaminated sources due to movement of domestic poultry and/or fomites from other areas; or infection of wild birds within the outbreak locations and then translocation by wild bird movement and interaction with wild birds inhabiting distant locations.
[Show abstract][Hide abstract] ABSTRACT: Individuals infected with the 2009 pandemic virus A(H1N1) developed serological response which can be measured by hemagglutination-inhibition (HI) and microneutralization (microNT) assays.
MicroNT and HI assays for specific antibody to the 2009 pandemic virus were conducted in serum samples collected at the end of the first epidemic wave from various groups of Thai people: laboratory confirmed cases, blood donors and health care workers (HCW) in Bangkok and neighboring province, general population in the North and the South, as well as archival sera collected at pre- and post-vaccination from vaccinees who received influenza vaccine of the 2006 season. This study demonstrated that goose erythrocytes yielded comparable HI antibody titer as compared to turkey erythrocytes. In contrast to the standard protocol, our investigation found out the necessity to eliminate nonspecific inhibitor present in the test sera by receptor destroying enzyme (RDE) prior to performing microNT assay. The investigation in pre-pandemic serum samples showed that HI antibody was more specific to the 2009 pandemic virus than NT antibody. Based on data from pre-pandemic sera together with those from the laboratory confirmed cases, HI antibody titers ≥ 40 for adults and ≥ 20 for children could be used as the cut-off level to differentiate between the individuals with or without past infection by the 2009 pandemic virus.
Based on the cut-off criteria, the infection rates of 7 and 12.8% were estimated in blood donors and HCW, respectively after the first wave of the 2009 influenza pandemic. Among general population, the infection rate of 58.6% was found in children versus 3.1% in adults.
PLoS ONE 01/2011; 6(1):e16164. DOI:10.1371/journal.pone.0016164 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: All 16 subtypes of avian influenza viruses of low pathogenicity (LPAIV) as well as their hemagglutinin (H) antigens, and four 2009 pandemic influenza A (H1N1) virus isolates were assayed for hemagglutinating activity against 5 erythrocyte species: goose, guinea pig, human group O, chicken and horse. Of all viruses and antigens assayed, the highest hemagglutination (HA) titers were obtained with goose and guinea pig erythrocytes. Hemagglutinating activity of replicating LPAIV and LPAIV antigens decreased, in order, with chicken and human group O; meanwhile, horse erythrocytes yielded lowest or no HA titer. Moreover, the 2009 pandemic viruses did not agglutinate both horse and chicken erythrocytes. Our study concluded that goose and guinea pig erythrocytes are the best in HA assay for all subtypes of influenza viruses.
[Show abstract][Hide abstract] ABSTRACT: Background: It had been reported 40 years ago that Asian openbill storks migrated from Bangladesh to Thailand for breeding. During the first outbreak of HPAI H5N1 virus in Thailand in 2004, storks were suspected to bring in the virus along with their migration from Bangladesh to Thailand. However, there was no report of H5N1 outbreak in Bangladesh at that time. Storks feed on apple snails living in freshwater marshes, rice paddy fields and ditches. They share the same habitats as free ranging ducks. This study aimed to determine migratory route of storks and its role in the spread of AI during their movement in Thailand. Methods: Four birds from 2 colonies of approximately 10,000 storks in Nakhon Pathom and Bueng Boraphet, Nakhon Sawan provinces, were tracked since April 2007 with satellite transmitters (Microwave) and monitored by Argos satellite with positioning by Google Earth Programme version 4.3. Surveillance for H5N1 spread by storks was conducted by searching for AI outbreak in domestic poultry and other wild birds living along their flyways in Thailand. Results: For longer than 2 years of monitoring, all 4 storks have never migrated out of Thailand. Birds in Nakhon Pathom moved out from one area to the other areas, probably related to crop rotation and snail population, but birds in Nakhon Sawan did not move far. During breeding season, birds came back to their old nested areas or nearby to form nests. The areas of home range of their habitats vary from 45.0 to 2476.8 km 2 per month (95% utility distribution) as calculated by Fixed Kernel Home Range programme. No AI outbreak was found in animals living along the flyway. Viral isolation assay in 500 storks yielded negative result. H5N1 antibody at titers ≥80 was detected in 0.67% (4/397) of storks by microneutralization assay. Conclusion: Asian openbill storks now become resident birds of Thailand. Their flyways did not correlate with H5N1 outbreaks in both domestic poultry and wild birds; and AI infection rate in storks was low. Our study suggested that storks do not play important role on AI spread in Thailand.
The International Conference on Emerging Infectious diseases, Atlanta, Georgia. 11-14 July 2010, Atlanta, Georgia, USA.; 06/2010
[Show abstract][Hide abstract] ABSTRACT: Background: No new human case with H5N1 HPAI occurs in Thailand since July 2006. Nevertheless, the events of poultry die-off do not disappear. It happens sporadically during rainy to cold seasons, and mainly confines to the Yom–Nan River basin areas. How viruses persist in nature is not documented? It is suspected that healthy looking backyard chickens may be asymptomatically infected and contaminate the environment by their excreta. On the other hand, some virus isolates might have adapted themselves to live together with their hosts by decreasing their virulence. The present study, therefore, aimed to compare virulence of three H5N1 isolates in the backyard, Thai native-as well as in hybrid chickens by determining for their intravenous pathogenicity indices (IVPI). Methods: Three virus isolates used in this study were: A/Chicken/Thailand/VSMU-3/2004, isolated from a dead chicken; A/duck/Thailand/ICRC-V629/2008, isolated from an asymptomatic duck; and A/chicken/Thailand/ICRC-V1069/2008, isolated from an asymptomatic Thai native chicken. A group of ten 6-8 weeks old chickens (Gullus gallus) (Thai native or hybrid chicken) were inoculated intravenously with each virus at the inoculum dose of 16 HA units/100 μl/head and kept in the animal BSL3 facilities. The inoculated chickens were scored based on clinical signs and death for 10 days before the experiment was terminated. IVPI were calculated according to the OIE manual. Thai-native chickens, meanwhile, it was highly virulent in hybrids chickens (IVPI 2.06 and 0.95). Conclusions: Thai native chickens may be a special breed that can tolerate some H5N1 HPAI isolate as compared to hybrid chickens. Those asymptomatically infected backyard chickens might serve as potent virus spreaders. We need more data before concluding that the viruses have now become less virulent in order to adapt themselves to persist in the poultry population.
The International Conference on Emerging Infectious diseases. 11-14 July 2010, Atlanta, Georgia, USA; 06/2010
[Show abstract][Hide abstract] ABSTRACT: Wild bird surveillance for highly pathogenic avian influenza (HPAI) H5N1 virus from 2004 to 2007 in Thailand indicated that the prevalence of infection with avian influenza H5N1 virus in wild birds was low (1.0%, 95% confidence interval [CI]: 0.7-1.2, 60/6,263 pooled samples). However, the annual prevalence varied considerably over this period, with a peak of 2.7% (95% CI: 1.4, 4.1) in 2004. Prevalence dropped to 0.5% (95% CI: 0.3, 0.8]) and 0.6% (95% CI: 0.3, 1.0) in 2005 and 2006, respectively, and then increased to 1.8% (95% CI: 1.0, 2.6) in 2007. During this period, 16 species from 12 families of wild birds tested positive for H5N1 virus infection. All samples from juvenile birds were negative for H5N1 virus, whereas 0.6% (95% CI: 0.4, 0.9) of pooled samples from adult birds were positive. Most positive samples originated from peridomestic resident species. Infected wild bird samples were only found in provinces where poultry outbreaks had occurred. Detection of H5N1 virus infection in wild birds was reported up to 3 yr after eradication of the poultry outbreaks in those provinces. As observed with outbreaks in poultry, the frequencies of H5N1 outbreaks in wild birds were significantly higher in winter. Further understanding of the mechanisms of persistence and ongoing HPAI H5N1 transmission between wild birds and domestic poultry is needed.
[Show abstract][Hide abstract] ABSTRACT: Outbreaks of H5N1 avian influenza show strong seasonality. It is not clear where the source of virus originates from in each new outbreak season. This study sought to understand the nature of viral resurgence in recent outbreak seasons in Thailand, where the epidemic is relatively well controlled. In such a situation, indigenous viruses surviving the inter-outbreak season would have to pass through a bottleneck. In order to look for evidence of the bottleneck effect, viral genome sequences from recent outbreaks in the country were analysed. H5N1 avian influenza viruses were isolated from six outbreaks in the rainy season and winter of 2007 through to early 2008. Most of the outbreaks were in the Yom-Nan River basin in the southern part of the northern region of the country. Sequences of these viral isolates were identified as clade 1, genotype Z, similar to viruses from previous years in the central region of the country. The sequences clustered into two groups, one of which was closely related to viruses isolated from the same area in July 2006. These analyses indicated that there was a strong bottleneck effect on the virus population and that only a few lineages remained in the area. In addition, evidence of reassortment among these viruses was found. These indicated re-emergence of viruses from a small pool of indigenous sources that had been silently perpetuated over the dry summer months. Therefore, an approach to eradicate H5N1 avian influenza from the area by eliminating these local reservoirs may be feasible and should be seriously considered.
Journal of General Virology 02/2009; 90(Pt 1):216-22. DOI:10.1099/vir.0.005660-0 · 3.18 Impact Factor