Transmission and pathogenicity of canine H3N2 influenza virus in dog and guinea pig models.

BACKGROUND: Influenza A virus causes respiratory disease in many animal species as well as in humans. Due to the high human-animal interface, the monitoring of canine influenza in dogs and the study of the transmission and pathogenicity of canine influenza in animals are important. METHODS: Eight-week-old beagle dogs (Canis lupus familaris) (n = 13) were used for the intraspecies transmission model. The dogs were inoculated intranasally with 1 ml of 106 EID50 per ml of canine H3N2 influenza virus (A/canine/Thailand/CU-DC5299/2012) (CIV-H3N2). In addition, 4-week-old guinea pigs (Cavia porcellus) (n = 20) were used for the interspecies transmission model. The guinea pigs were inoculated intranasally with 300 µl of 106 EID50 per ml of CIV-H3N2. RESULTS: For the Thai CIV-H3N2 challenged in the dog model, the incoculated and direct contact dogs developed respiratory signs at 2 dpi. The dogs shed the virus in the respiratory tract at 1 dpi and developed an H3-specific antibody against the virus at 10 dpi. Lung congestion and histopathological changes in the lung were observed. For the Thai CIV-H3N2 challenge in the guinea pig model, the incoculated, direct contact and aerosol-exposed guinea pigs developed fever at 1-2 dpi. The guinea pigs shed virus in the respiratory tract at 2 dpi and developed an H3-specific antibody against the virus at 7 dpi. Mild histopathological changes in the lung were observed. CONCLUSION: The result of this study demonstrated evidence of intraspecies and interspecies transmission of CIV-H3N2 in a mammalian model.

Genetic characterization of hypervirulent Klebsiella pneumoniae responsible for acute death in captive marmosets.

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including bla SHV-67, bla SHV-11, oqxA, oqxB, and fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.

First cases of SARS-CoV-2 infection in dogs and cats in Thailand.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic in humans since late 2019. Here, we investigated SARS-CoV-2 infection in dogs and cats during COVID-19 quarantine at private veterinary hospitals in Thailand. From April to May 2021, we detected SARS-CoV-2 in three out of 35 dogs and one out of nine cats from four out of 17 households with confirmed COVID-19 patients. SARS-CoV-2 RNA was detected from one of the nasal, oral, rectal and environmental swabs of dog-A (15 years old, mixed breed, male dog), cat-B (1 year old, domestic shorthair, male cat), dog-C (2 years old, mixed breed, female dog) and dog-D (4 years old, Pomeranian, female dog). The animals tested positive for SARS-CoV-2 RNA from 4 to 30 days after pet owners were confirmed to be COVID-19 positive. The animals consecutively tested positive for SARS-CoV-2 RNA for 4 to 10 days. One dog (dog-A) showed mild clinical signs, while the other dogs and a cat remained asymptomatic during quarantine at the hospitals. SARS-CoV-2 specific neutralizing antibodies were detected in both the dogs and cat by surrogate virus neutralization tests. Phylogenetic and genomic mutation analyses of whole genome sequences of three SARS-CoV-2 strains from the dogs and cat revealed SARS-CoV-2 of the Alpha variant (B.1.1.7 lineage). Our findings are suggestive of human-to-animal transmission of SARS-CoV-2 in COVID-19-positive households and contamination of viral RNA in the environment. Public awareness of SARS-CoV-2 infection in pet dogs and cats in close contact with COVID-19 patients should be raised.

Draft Whole-Genome Sequence of a Klebsiella pneumoniae Strain Isolated from a Marmoset in Thailand.

Klebsiella pneumoniae is a Gram-negative bacterium that can cause infection in various kinds of animals and humans. Here, we report the genome sequence of K. pneumoniae isolated from a captive marmoset in Thailand.

Two decades of one health surveillance of Nipah virus in Thailand.

BACKGROUND: Nipah virus (NiV) infection causes encephalitis and has > 75% mortality rate, making it a WHO priority pathogen due to its pandemic potential. There have been NiV outbreak(s) in Malaysia, India, Bangladesh, and southern Philippines. NiV naturally circulates among fruit bats of the genus Pteropus and has been detected widely across Southeast and South Asia. Both Malaysian and Bangladeshi NiV strains have been found in fruit bats in Thailand. This study summarizes 20 years of pre-emptive One Health surveillance of NiV in Thailand, including triangulated surveillance of bats, and humans and pigs in the vicinity of roosts inhabited by NiV-infected bats. METHODS: Samples were collected periodically and tested for NiV from bats, pigs and healthy human volunteers from Wat Luang village, Chonburi province, home to the biggest P. lylei roosts in Thailand, and other provinces since 2001. Archived cerebrospinal fluid specimens from encephalitis patients between 2001 and 2012 were also tested for NiV. NiV RNA was detected using nested reverse transcription polymerase chain reaction (RT-PCR). NiV antibodies were detected using enzyme-linked immunosorbent assay or multiplex microsphere immunoassay. RESULTS: NiV RNA (mainly Bangladesh strain) was detected every year in fruit bats by RT-PCR from 2002 to 2020. The whole genome sequence of NiV directly sequenced from bat urine in 2017 shared 99.17% identity to NiV from a Bangladeshi patient in 2004. No NiV-specific IgG antibodies or RNA have been found in healthy volunteers, encephalitis patients, or pigs to date. During the sample collection trips, 100 community members were trained on how to live safely with bats. CONCLUSIONS: High identity shared between the NiV genome from Thai bats and the Bangladeshi patient highlights the outbreak potential of NiV in Thailand. Results from NiV cross-sectoral surveillance were conveyed to national authorities and villagers which led to preventive control measures, increased surveillance of pigs and humans in vicinity of known NiV-infected roosts, and increased vigilance and reduced risk behaviors at the community level. This proactive One Health approach to NiV surveillance is a success story; that increased collaboration between the human, animal, and wildlife sectors is imperative to staying ahead of a zoonotic disease outbreak.

African Horse Sickness Virus Serotype 1 on Horse Farm, Thailand, 2020.

To investigate an outbreak of African horse sickness (AHS) on a horse farm in northeastern Thailand, we used whole-genome sequencing to detect and characterize the virus. The viruses belonged to serotype 1 and contained unique amino acids (95V,166S, 660I in virus capsid protein 2), suggesting a single virus introduction to Thailand.

Isolation and in vitro antimicrobial susceptibility of porcine Lawsonia intracellularis from Brazil and Thailand.

BACKGROUND: Lawsonia intracellularis is an obligate intracellular bacterium which cannot be cultured by conventional bacteriological methods. Furthermore, L. intracellularis needs enriched medium and a unique atmosphere for isolation, cultivation and propagation. Because of this,there are only a few isolates of L. intracellularis available and few studies in vitro demonstrating the susceptibility of this bacterium to antimicrobial agents. The objectives of this study were to isolate South American and Southeast Asia strains of L.intracellularis and to determine the in vitro antimicrobial activity against these isolates. Tested antimicrobials included: chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin(against both Brazilian and Thailand strains) and additionally, amoxicillin, zinc-bacitracin, carbadox, enrofloxacin, gentamicin, sulfamethazine, trimethoprim, spectinomycin and a combination (1:1) of spectinomycin and lincomycin were also tested against the Thai isolates. The minimum inhibitory concentration (MIC) was determined by the antimicrobial activity that inhibited 99% of L. intracellularis growth in a cell culture as compared to the control (antimicrobial-free). RESULTS: Two strains from Brazil and three strains from Thailand were successfully isolated and established in cell culture. Each antimicrobial was evaluated for intracellular and extracellular activity. Pleuromutilin group (valnemulin and tiamulin) and carbadox were the most active against L. intracellularis strains tested. Tylosin showed intermediate activity, chlortetracycline had variable results between low and intermediate activity, as well as spectinomycin, spectinomycin and lincomycin, amoxicillin, sulfamethazine and enrofloxacin. L. intracellularis was resistant to lincomycin, gentamicin, trimethoprim, colistin and bacitracin in in vitro conditions. CONCLUSIONS: This is the first report of isolation of L. intracellularis strains from South America and Southeast Asia and characterization of the antimicrobial susceptibility patterns of these new strains.

Tropism and Induction of Cytokines in Human Embryonic-Stem Cells-Derived Neural Progenitors upon Inoculation with Highly- Pathogenic Avian H5N1 Influenza Virus.

Central nervous system (CNS) dysfunction caused by neurovirulent influenza viruses is a dreaded complication of infection, and may play a role in some neurodegenerative conditions, such as Parkinson-like diseases and encephalitis lethargica. Although CNS infection by highly pathogenic H5N1 virus has been demonstrated, it is unknown whether H5N1 infects neural progenitor cells, nor whether such infection plays a role in the neuroinflammation and neurodegeneration. To pursue this question, we infected human neural progenitor cells (hNPCs) differentiated from human embryonic stem cells in vitro with H5N1 virus, and studied the resulting cytopathology, cytokine expression, and genes involved in the differentiation. Human embryonic stem cells (BG01) were maintained and differentiated into the neural progenitors, and then infected by H5N1 virus (A/Chicken/Thailand/CUK2/04) at a multiplicity of infection of 1. At 6, 24, 48, and 72 hours post-infection (hpi), cytopathic effects were observed. Then cells were characterized by immunofluorescence and electron microscopy, supernatants quantified for virus titers, and sampled cells studied for candidate genes.The hNPCs were susceptible to H5N1 virus infection as determined by morphological observation and immunofluorescence. The infection was characterized by a significant up-regulation of TNF-α gene expression, while expressions of IFN-α2, IFN-ß1, IFN-γ and IL-6 remained unchanged compared to mock-infected controls. Moreover, H5N1 infection did not appear to alter expression of neuronal and astrocytic markers of hNPCs, such as ß-III tubulin and GFAP, respectively. The results indicate that hNPCs support H5N1 virus infection and may play a role in the neuroinflammation during acute viral encephalitis.

Prevalence of porcine circovirus-2 DNA-positive ovarian and uterine tissues in gilts culled due to reproductive disturbance in Thailand.

The present study aimed to determine the prevalence of porcine circovirus-2 (PCV-2) DNA-positive ovarian and uterine tissues in gilts culled due to reproductive disturbance in Thailand. Tissues (70 ovaries and 102 uteri) and serum (n = 102) samples from 102 gilts were included. PCV-2 DNA was detected by using polymerase chain reactions. The localisation of PCV-2 antigen was determined by immunohistochemistry, and PCV-2 antibody was evaluated by ELISA. PCV-2 DNA was detected in 30.0 % (21/70) of the ovaries and in 45.1 % (46/102) of the uteri. Age did not influence the frequency of PCV-2 DNA detection in these reproductive organs of gilts (P > 0.05). The prevalence of PCV-2 DNA-positive uterine tissue in gilts culled due to non-reproductive problems (20.0 %) was lower than gilts culled due to abortion (85.0 %), abnormal vaginal discharge (47.5 %) and anoestrus (53.5 %) (P < 0.05). The prevalence of PCV-2 DNA-positive uterine tissue in the gilts with high antibody titres (23.0 %) was lower than in gilts with low antibody titres (57.6 %) and seronegative gilts (64.5 %) (P < 0.05). PCV-2 immunostaining was detected in the endometrial cells, lymphocytes and macrophages of the uteri and in oocytes and granulosa cells of the ovaries. In conclusion, the detection of PCV-2 in the reproductive organs reveals an important potential impact of this virus on the reproductive apparatus in gilts.

Porcine reproductive and respiratory syndrome virus detection in Thailand during 2005-2010 in relation to clinical problems, pig types, regions, and seasons.

The objectives of the present study were to determine the prevalence of porcine reproductive and respiratory syndrome virus (PRRSV) in Thailand between 2005 and 2010. The study was conducted by retrospectively investigating the detection of PRRSV from different pig types including boars, sows, piglets, nursery pigs, and fattening pigs from six regions of Thailand, i.e., the northern, eastern, northeastern, central, western, and southern parts. The data were obtained from cases submitted to the Chulalongkorn University Veterinary Diagnostic Laboratory for PRRSV detection between 2005 and 2010. Frequency analyses and generalized linear models were used to evaluate the prevalence of PRRSV in relation to various factors. In total, 2,273 tissues (n = 636), semen (n = 210) and serum (n = 1,427) samples were included. PRRSV was detected in 32.6 % (740/2,273) of the pigs. The virus was found in 43.1 %, 15.7 %, and 30.3 % in the tissues, semen, and serum samples, respectively (P < 0.001). The prevalence of PRRSV was highest in 2005 (43.6 %) and lowest in 2009 (23.6 %) (P < 0.001). The prevalence of PRRSV was highest in nursery pigs (43.7 %) and lowest in boars (15.4 %) (P < 0.001). The prevalence of PRRSV in the hot season (34.9 %) was higher than that found in the cool season (28.1 %, P = 0.018) but did not differ significantly compared to rainy season (34.0 %, P = 0.486). The strain of PRRSV isolated in the present study was genotype 2 (54.5 %), genotype 1 (31.0 %), and mixed genotypes (14.5 %). It can be concluded that PRRSV was detected in the tissue samples more frequently than the semen and serum samples. The prevalence of PRRSV was high in the nursery pigs. A high prevalence of PRRSV was found in the hot season, indicating that climatic factors may also contribute to the prevalence of PRRSV in Thailand. Of all the PRRSV detected, 31.0 %, 54.5 %, and 14.5 % belonged to genotype 1, genotype 2, and mixed genotypes, respectively.

Comparative study of pandemic (H1N1) 2009, swine H1N1, and avian H3N2 influenza viral infections in quails.

Quail has been proposed to be an intermediate host of influenza A viruses. However, information on the susceptibility and pathogenicity of pandemic H1N1 2009 (pH1N1) and swine influenza viruses in quails is limited. In this study, the pathogenicity, virus shedding, and transmission characteristics of pH1N1, swine H1N1 (swH1N1), and avian H3N2 (dkH3N2) influenza viruses in quails was examined. Three groups of 15 quails were inoculated with each virus and evaluated for clinical signs, virus shedding and transmission, pathological changes, and serological responses. None of the 75 inoculated (n = 45), contact exposed (n = 15), or negative control (n = 15) quails developed any clinical signs. In contrast to the low virus shedding titers observed from the swH1N1-inoculated quails, birds inoculated with dkH3N2 and pH1N1 shed relatively high titers of virus predominantly from the respiratory tract until 5 and 7 DPI, respectively, that were rarely transmitted to the contact quails. Gross and histopathological lesions were observed in the respiratory and intestinal tracts of quail inoculated with either pH1N1 or dkH3N2, indicating that these viruses were more pathogenic than swH1N1. Sero-conversions were detected 7 DPI in two out of five pH1N1-inoculated quails, three out of five quails inoculated with swH1N1, and four out of five swH1N1-infected contact birds. Taken together, this study demonstrated that quails were more susceptible to infection with pH1N1 and dkH3N2 than swH1N1.

Quail as a potential mixing vessel for the generation of new reassortant influenza A viruses.

Quail has been proposed as one of the intermediate hosts supporting the generation of newly reassortant influenza A viruses (IAVs) with the potential to infect humans. To evaluate the role of quail as an intermediate host of IAVs, co-infections of quail with swine-origin pandemic H1N1 2009 (pH1N1) and low pathogenic avian influenza (LPAI) duck H3N2 (dkH3N2) viruses (n=10) or endemic Thai swine H1N1 (swH1N1) and dkH3N2 viruses (n=10) were conducted. Three additional groups of five quail were each inoculated with pH1N1, swH1N1 and dkH3N2 as control groups to verify that each virus can infect quail. Our result showed that co-infected quail shed higher viral titers from the respiratory tract than single virus infected quail. This study confirmed that reassortant viruses could be readily generated in the respiratory tract of quail from both the pH1N1/dkH3N2 co-infected group (100% of quail generating reassortant viruses) and the swH1N1/dkH3N2 (33% of quail generating reassortant viruses) co-infected group without discernible clinical signs. The reassortment efficacy between the two combination of viruses was different in that the frequency of reassortant viruses was significantly higher in pH1N1/dkH3N2 co-infected quail (21.4%) compared to swH1N1/dkH3N2 co-infected quail (0.8%), indicating that gene combinations in pH1N1 have a higher potential to reassort with dkH3N2 compared to swH1N1. In summary, our result confirmed that quail could be an intermediate host of IAVs for generating new reassortant viruses. Our finding highlights the importance of monitoring IAVs especially pH1N1 in quail.

Seroprevalence of porcine reproductive and respiratory syndrome, Aujeszky's disease, and porcine parvovirus in replacement gilts in Thailand.

The present study investigated the seroprevalence of porcine reproductive and respiratory syndrome virus, Aujeszky's disease virus (ADV), and porcine parvovirus (PPV) in replacement gilts from selected five swine herds in Thailand. The study consisted of three parts. First, a retrospective data analysis on the seroprevalence of porcine reproductive and respiratory syndrome virus (PRRSV) and ADV glycoprotein I (gI) in gilts, sows, boars, nursery, and fattening pigs in five herds (n = 7,030). Second, a cross-sectional study on seroprevalence of PRRSV, ADV, and PPV (n = 200) in replacement gilts. Last, the seroprevalence of PRRSV, ADV, and PPV in gilts culled due to reproductive failure (n = 166). Across the herds, the seroprevalence of PRRSV and ADV was 79.3% and 5.3%, respectively. The cross-sectional study revealed that 87.5%, 4.0%, and 99.0% of the replacement gilts were infected with PRRSV, ADV, and PPV, respectively. In the gilts culled due to reproductive failure, the seroprevalence of PRRSV, ADV, and PPV was 73.5%, 28.3%, and 86.0%, respectively. Of these culled gilts, 75.5% had been infected with at least two viruses and 18.9% had been infected with all three viruses. It could be concluded that most of the replacement gilts were exposed to PRRSV (84%), PPV (97%), and ADV (4%) before entering the breeding house. PPV was an enzootic disease among the selected herds. The prevalence of ADV was higher in gilts culled due to reproductive disturbance than in the healthy gilts.

Single-step multiplex reverse transcription polymerase chain reaction assay for detection and differentiation of the 2009 H1N1 Influenza A virus pandemic in Thai swine populations.

A recently emerged H1N1 Influenza A virus (pandemic H1N1 (pH1N1)) with a Swine influenza virus (SIV) genetic background spread globally from human-to-human causing the first influenza virus pandemic of the 21st century. In a short period, reverse zoonotic cases in pigs followed by a widespread of the virus in the pig population were documented. The implementation of effective control strategies, rapid diagnosis, and differentiation of such virus from endemically circulating SIV in the various swine populations of the world is needed. To address the problem, a multiplex reverse transcription polymerase chain reaction assay utilizing a combination of the PB1, H1, and N1 primers that can rapidly and simultaneously subtype and screen for the presence of pH1N1 virus infection in Thai pigs was developed. The assay had 100% specificity and did not amplify genetic material from other subtypes of SIV, seasonal H1N1 human influenza (huH1N1) virus, highly pathogenic influenza H5N1 virus, and other important swine respiratory viral pathogens. The assay was able to both detect and subtype pH1N1 virus as low as 0.1-50% tissue culture infective doses/ml (TCID(50)/ml). The assay was used to screen 175 clinical samples obtained from SIV suspected cases, of which 6 samples were pH1N1 positive and were confirmed through virus isolation and whole genome sequencing. The results of the study suggested that the assay would be useful for the rapid diagnosis of pH1N1 in suspected Thai swineherds, where genetics of the endemically circulating SIV differ from the strains circulating in North American and European herds.

Genetic characterization of 2008 reassortant influenza A virus (H5N1), Thailand.

In January and November 2008, outbreaks of avian influenza have been reported in 4 provinces of Thailand. Eight Influenza A H5N1 viruses were recovered from these 2008 AI outbreaks and comprehensively characterized and analyzed for nucleotide identity, genetic relatedness, virulence determinants, and possible sites of reassortment. The results show that the 2008 H5N1 viruses displayed genetic drift characteristics (less than 3% genetic differences), as commonly found in influenza A viruses. Based on phylogenetic analysis, clade 1 viruses in Thailand were divided into 3 distinct branches (subclades 1, 1.1 and 1.2). Six out of 8 H5N1 isolates have been identified as reassorted H5N1 viruses, while other isolates belong to an original H5N1 clade. These viruses have undergone inter-lineage reassortment between subclades 1.1 and 1.2 and thus represent new reassorted 2008 H5N1 viruses. The reassorted viruses have acquired gene segments from H5N1, subclade 1.1 (PA, HA, NP and M) and subclade 1.2 (PB2, PB1, NA and NS) in Thailand. Bootscan analysis of concatenated whole genome sequences of the 2008 H5N1 viruses supported the reassortment sites between subclade 1.1 and 1.2 viruses. Based on estimating of the time of the most recent common ancestors of the 2008 H5N1 viruses, the potential point of genetic reassortment of the viruses could be traced back to 2006. Genetic analysis of the 2008 H5N1 viruses has shown that most virulence determinants in all 8 genes of the viruses have remained unchanged. In summary, two predominant H5N1 lineages were circulating in 2008. The original CUK2-like lineage mainly circulated in central Thailand and the reassorted lineage (subclades 1.1 and 1.2) predominantly circulated in lower-north Thailand. To prevent new reassortment, emphasis should be put on prevention of H5N1 viruses circulating in high risk areas. In addition, surveillance and whole genome sequencing of H5N1 viruses should be routinely performed for monitoring the genetic drift of the virus and new reassorted strains, especially in light of potential reassortment between avian and mammalian H5N1 viruses.

Determination of antibody response to the human pandemic influenza H1N1 2009 among patients with influenza-like illness and high risk groups.

The global population has been exposed to the novel pandemic H1N1 influenza virus since mid March 2009, causing the expansion of respiratory illness around the world, including Thailand. To evaluate the antibody titers against human pandemic influenza (H1N1) in Thai people with influenza-like illness (ILI), 45 paired serum samples (acute and convalescent) were subjected to hemagglutination inhibition (HI) test and real-time RT-PCR. Most serum samples of ILI patients positive by real-time RT-PCR displayed an at least four-fold antibody increase of HI titers against pandemic influenza (H1N1). In addition, to determine cross-reactivity with human seasonal H1N1 influenza, viral antigen from the seasonal H1N1 was used to detect antibody against seasonal H1N1 influenza and all sera showed negative results. We also studied the single sera samples from the high risk medical personals collected before and after the pandemic influenza (HIN1) outbreaks for antibodies against seasonal H3 influenza virus infection. The results showed lack of cross-reactivity to the human pandemic H1N1 influenza virus. HI antibody testing to pandemic influenza (H1N1) can be used for the diagnosis, preventive and control measures of potential outbreaks.

The inactivation of avian influenza virus subtype H5N1 isolated from chickens in Thailand by chemical and physical treatments.

The objectives of this study were to determine the survival of avian influenza virus (AIV) subtype H5N1 under various physical and chemical treatments, including disinfectants, temperature and pH. The highly pathogenic AIVs subtype H5N1 were isolated from internal organs of suspected chickens and were characterized by the inoculation into chicken embryonated eggs (CEEs), hemagglutination (HA) test, hemagglutination inhibition (HI) test, reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing of hemagglutinin (H) and neuraminidase (N) genes. Three H5N1 isolates, at the concentration of 10(9) 50% embryo lethal dose (ELD(50))/ml, were used for the determination of the survival of the virus under different chemical and physical treatments. The chemical treatments were performed by incubating the viruses with various types of disinfectants including glutaraldehyde (Glu), hydrogen peroxide, quaternary ammonium compounds (QAC), Glu+QAC, iodine, chlorine, formalin and phenol, at 25 and 37 degrees C, for 0, 5, 7, and 14 days. The physical treatments included incubation of the viruses at 55, 60, 65, 70 and 75 degrees C for 10, 15, 30, 45 and 60 min or pH 3, 5, 7, 9 and 12. The results revealed that AIV H5N1 reference viruses, 2004.1, CUK-2/04 and 2004.2, showed low or no resistance against Glu+QAC, chlorine and phenol at both tested temperatures. Incubations at 70 degrees C for 60 min or at least 75 degrees C for at least 45 min could effectively inactivate all of the isolates, whereas all ranges of pH could not inactivate any of them. In this study, CUK-2/04 was more resistant to the disinfectants, temperatures, and pH compared to the other isolates.

Molecular evolution of H5N1 in Thailand between 2004 and 2008.

Highly pathogenic avian influenza (HPAI) H5N1 viruses have seriously affected the Asian poultry industry since their occurrence in 2004. Thailand has been one of those countries exposed to HPAI H5N1 outbreaks. This project was designed to compare the molecular evolution of HPAI H5N1 in Thailand between 2004 and 2008. Viruses with clade 1 hemagglutinin (HA) were first observed in early 2004 and persisted until 2008. Viruses with clade 2.3.4 HA were first observed in the northeastern region of Thailand between 2006 and 2007. Phylogenetic analysis among Thai isolates indicated that clade 1 viruses in Thailand consist of three distinct lineages: CUK2-like, PC168-like, and PC170-like viruses. The CUK2-like virus represents the predominant lineage and has been circulating throughout the course of the 4-year outbreaks. Analysis of recently isolated viruses has shown that the genetic distance was slightly different from viruses of the early outbreak and that CUK2-like viruses comprise the native strain. Between 2005 and 2007, PC168-like and PC170-like viruses were first observed in several areas around central and lower northern Thailand. In 2008, viruses reassorted from these two lineages, PC168-like and PC170-like viruses, were initially isolated in the lower northern provinces of Thailand and subsequently spread to the upper central part of Thailand. On the other hand, CUK2-like viruses were still detected around the lower northern and the upper central part of Thailand. Furthermore, upon emergence of the reassorted viruses, the PC168-like and PC170-like lineages could not be detected, suggesting that the only predominant strains still circulating in Thailand were CUK2-like and reassorted viruses. The substitution rate among clade 1 viruses in Thailand was lower. The virus being limited to the same area might explain the lower nucleotide substitution rate. This study has demonstrated that nationwide attempts to monitor the virus may help curb access and propagation of new HPAI viral genes.

Influenza virus (H5N1) in live bird markets and food markets, Thailand.

A surveillance program for influenza A viruses (H5N1) was conducted in live bird and food markets in central Thailand during July 2006-August 2007. Twelve subtype H5N1 viruses were isolated. The subtype H5N1 viruses circulating in the markets were genetically related to those that circulated in Thailand during 2004-2005.