Genetic and antigenic dynamics of influenza A viruses of swine on pig farms in Thailand.

Surveillance studies of influenza A virus of swine (IAV-S) have accumulated information regarding IAVs-S circulating in Thailand, but how IAVs-S evolve within a farm remains unclear. In the present study, we isolated 82 A(H1N1)pdm09 and 87 H3N2 viruses from four farms from 2011 through 2017. We then phylogenetically and antigenically analyzed the isolates to elucidate their evolution within each farm. Phylogenetic analysis demonstrated multiple introductions of A(H1N1)pdm09 viruses that resembled epidemic A(H1N1)pdm09 strains in humans in Thailand, and they reassorted with H3N2 viruses as well as other A(H1N1)pdm09 viruses. Antigenic analysis revealed that the viruses had acquired antigenic diversity either by accumulating substitutions in the hemagglutinin protein or through the introduction of IAV-S strains with different antigenicity. Our results, obtained through continuous longitudinal surveillance, revealed that IAV-S can be maintained on a pig farm over several years through the generation of antigenic diversity due to the accumulation of mutations, introduction of new strains, and reassortment events.

Pathogenicity of Genetically Similar, H5N1 Highly Pathogenic Avian Influenza Virus Strains in Chicken and the Differences in Sensitivity among Different Chicken Breeds.

Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.

Co-infection of influenza A viruses of swine contributes to effective shuffling of gene segments in a naturally reared pig.

Following the 2009 H1N1 pandemic, surveillance activities have been accelerated globally to monitor the emergence of novel reassortant viruses. However, the mechanism by which influenza A viruses of swine (IAV-S) acquire novel gene constellations through reassortment events in natural settings remains poorly understood. To explore the mechanism, we collected 785 nasal swabs from pigs in a farm in Thailand from 2011 to 2014. H3N2, H3N1, H1N1 and H1N2 IAVs-S were isolated from a single co-infected sample by plaque purification and showed a high degree of diversity of the genome. In particular, the H1N1 isolates, possessing a novel gene constellation previously unreported in Thailand, exhibited greater variation in internal genes than H3N2 IAVs-S. A pair of isolates, designated H3N2-B and H1N1-D, was determined to have been initially introduced to the farm. These results demonstrate that numerous IAVs-S with various gene constellations can be created in a single co-infected pig via reassortment.

The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production.

The viral protein Npro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I IFN (IFN-α/ß) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-α/ß induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3-degrading activity. Restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This led to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-α/ß production.

Isolation of the pandemic (H1N1) 2009 virus and its reassortant with an H3N2 swine influenza virus from healthy weaning pigs in Thailand in 2011.

A total of 300 nasal swabs were collected from 5 pig farms in two provinces in the Eastern part of Thailand in February 2011 and were subjected to viral isolation of influenza A viruses. Two H3N2 and 6 H1N1 influenza A viruses were isolated from swabs collected from clinically healthy weaning pigs on farms in Chonburi and Chachoengsao provinces, respectively. The H3N2 isolates consisted of the hemagglutinin (HA) and neuraminidase (NA) genes closely related to Thai SIVs and derived from a cluster of human seasonal H3N2 strains circulating around 1996-1997. The remaining gene segments of the isolates originated from the Pandemic (H1N1) 2009 (A (H1N1) pdm09) virus. Antigenicity of the H3N2 isolates was distinguishable from a human seasonal vaccine strain in the 1996-1998 seasons that represented antigenicity of the seasonal strains around 1996-1998. Nasal swabs from a Chachoengsao farm yielded A (H1N1) pdm09 viruses in chicken embryonated eggs and MDCK cells. A (H1N1) pdm09 viruses isolated in this study grew poorly in MDCK cells. Deduced amino acid sequences of the HA1 region of the HA protein of egg isolated viruses were identical to the sequences directly amplified from original swab samples. Our result demonstrated that the A (H1N1) pdm09 virus has been established in the Thai pig population and this has resulted in genetic reassortment with Thai SIV that previously circulated among pigs.

Highly pathogenic avian influenza (H5N1) in Myanmar, 2006-2010.

Highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first reported in Myanmar in 2006. In this study, we have characterized 6 HPAI (H5N1) viruses recovered from 2007-2010 as well as three additional available nucleotide sequences representing Myanmar AI outbreaks. Phylogenetic analysis showed that the Myanmar viruses belong to HPAI (H5N1) clades 7, 2.3.2 and 2.3.4. The result suggested that the HPAI (H5N1) viruses recovered from Myanmar had been introduced into the country by multiple introductions. Genetic analysis of the viruses confirmed the HPAI characteristics of the viruses.

Swine influenza virus infection in different age groups of pigs in farrow-to-finish farms in Thailand.

BACKGROUND: Understanding swine influenza virus (SIV) ecology has become more and more important from both the pig industry and public health points of views. However, the mechanism whereby SIV occurs in pig farms is not well understood. The purpose of this study was to develop a proper strategy for SIV surveillance. FINDINGS: We conducted longitudinal monitoring in 6 farrow-to-finish farms in the central region of Thailand from 2008 to 2009. Nasal swabs and serum samples were collected periodically from clinically healthy pigs consisting of sows, fattening pigs, weaned piglets and pigs transferred from other farms. A total of 731 nasal swabs were subjected to virus isolation and 641 serum samples were subjected to detection of SIV antibodies against H1 and H3 subtypes using the hemagglutination inhibition test and ELISA. Twelve SIVs were isolated in this study and eleven were from piglets aged 4 and 8 weeks. Phylogenetical analysis revealed that SIVs isolated from different farms shared a common ancestor. Antibodies against SIVs were detected in fattening pigs on farms with no SIV isolation in the respective periods studied. These observations suggested that piglets aged 8 weeks or younger could be a main target for SIV isolation. Farm-to-farm transmission was suggested for farms where pigs from other farms are introduced periodically. In addition, antibodies against SIVs detected in fattening pigs could be a marker for SIV infection in a farm. CONCLUSIONS: The present study provided important information on SIV surveillance that will enable better understanding of SIV ecology in farrow-to-finish farms.

Host cytokine responses of pigeons infected with highly pathogenic Thai avian influenza viruses of subtype H5N1 isolated from wild birds.

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.

Differential host gene responses in mice infected with two highly pathogenic avian influenza viruses of subtype H5N1 isolated from wild birds in Thailand.

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.

Genetic variations of nucleoprotein gene of influenza A viruses isolated from swine in Thailand.

BACKGROUND: Influenza A virus causes severe disease in both humans and animals and thus, has a considerably impact on economy and public health. In this study, the genetic variations of the nucleoprotein (NP) gene of influenza viruses recovered from swine in Thailand were determined. RESULTS: Twelve influenza A virus specimens were isolated from Thai swine. All samples were subjected to nucleotide sequencing of the complete NP gene. Phylogenetic analysis was conducted by comparing the NP gene of swine influenza viruses with that of seasonal and pandemic human viruses and highly pathogenic avian viruses from Thailand (n = 77). Phylogenetic analysis showed that the NP gene from different host species clustered in distinct host specific lineages. The NP gene of swine influenza viruses clustered in either Eurasian swine or Classical swine lineages. Genetic analysis of the NP gene suggested that swine influenza viruses circulating in Thailand display 4 amino acids unique to Eurasian and Classical swine lineages. In addition, the result showed 1 and 5 amino acids unique to avian and human lineages, respectively. Furthermore, nucleotide substitution rates showed that the NP gene is highly conserved especially in avian influenza viruses. CONCLUSION: The NP gene sequence of influenza A in Thailand is highly conserved within host-specific lineages and shows amino acids potentially unique to distinct NP lineages. This information can be used to investigate potential interspecies transmission of influenza A viruses. In addition, the genetic variations of the NP gene will be useful for monitoring the viruses and preparing effective prevention and control strategies for potentially pandemic influenza outbreaks.

Alterations in receptor-binding properties of swine influenza viruses of the H1 subtype after isolation in embryonated chicken eggs.

Alterations of the receptor-binding properties of swine influenza A viruses (SIVs) during their isolation in embryonated chicken eggs have not been well studied. In this study, the receptor-binding properties of classical H1 SIVs isolated solely in eggs or Madin-Darby canine kidney (MDCK) cells were examined. Sequencing analysis revealed substitutions of D190V/N or D225G in the haemagglutinin (HA) proteins in egg isolates, whereas MDCK isolates retained HA genes identical to those of the original viruses present in the clinical samples. Egg isolates with substitution of either D190V/N or D225G had increased haemagglutinating activity for mouse and sheep erythrocytes, but reduced activity for rabbit erythrocytes. Additionally, egg isolates with D225G had increased haemagglutination activity for chicken erythrocytes. A direct binding assay using a sialyl glycopolymer that possessed either a 5-N-acetylneuraminic acid (Neu5Ac) alpha2,6galactose (Gal) or a Neu5Acalpha2,3Gal linkage revealed that the egg isolates used in this study showed higher binding activity to the Neu5Acalpha2,3Gal receptor than MDCK isolates. Increased binding activity of the egg isolates to the Neu5Acalpha2,3Gal receptor was also confirmed by haemagglutination assay with resialylated chicken erythrocytes by Galbeta1,3/4GlcNAcalpha2,3-sialyltransferase. These observations were reinforced by flow-cytometric and N-glycan analyses of the erythrocytes. The alpha2,3-linked sialic acids were expressed predominantly on the surface of mouse and sheep erythrocytes. Chicken erythrocytes expressed Neu5Acalpha2,3Gal more abundantly than Neu5Acalpha2,6Gal, and rabbit erythrocytes expressed both 5-N-glycolylneuraminic acid (Neu5Gc) alpha2,6Gal and Neu5Acalpha2,6Gal. Our results demonstrate clearly that classical H1 SIVs undergo alterations in receptor-binding activity associated with an amino acid substitution in the HA protein during isolation and propagation in embryonated chicken eggs.

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.

Pathogenicity of highly pathogenic avian influenza viruses of H5N1 subtype isolated in Thailand for different poultry species.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused several rounds of outbreaks in Thailand. In this study, we used 3 HPAI viruses isolated in Thailand in January 2004 from chicken, quail, and duck for genetic and pathogenetic studies. Sequence analysis of the entire genomes of these isolates revealed that they were genetically similar to each other. Chickens, quails, domestic ducks, and cross-bred ducks were inoculated with these isolates to evaluate their pathogenicity to different host species. A/chicken/Yamaguchi/7/04 (H5N1), an HPAI virus isolated in Japan, was also used in the chicken and quail studies for comparison. All four isolates were shown to be highly pathogenic to chickens and quails, with 100% mortality by 10(6) EID50 inoculants of the viruses. They caused sudden death in chickens and quails within 2-4 days after inoculation. The mean death times (MDT) of quails infected with the Thai isolates were shorter than those of chickens infected with the same isolates. Mortality against domestic and cross-bred ducks ranged from 50 to 75% by intranasal inoculation with the 10(6) EID50 viruses. Neurological symptoms were observed in most of the inoculated domestic ducks and appeared less severe in the cross-bred ducks. The MDTs of the ducks infected with the Thai isolates were 4.8-6 days post-inoculation. Most of the surviving ducks infected with the Thai isolates had sero-converted until 14 dpi. Our study illustrated the pathobiology of the Thai isolates against different poultry species and would provide useful information for improving control strategies against HPAI.

Molecular epidemiological analysis of highly pathogenic avian influenza H5N1 subtype isolated from poultry and wild bird in Thailand.

A comprehensive molecular epidemiological analysis was performed on highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype derived from poultry and wild bird during 2004-2007 in Thailand. Sequence analysis followed by phylogenetic analysis was applied to all eight segments of the viruses. Viruses belonging to clades 1 and 2.3.4 in the HA phylogenetic tree have been shown to circulate in Thailand. Our analysis revealed differential evolution of the HPAI viruses among clade 1 strains. Isolates from Phichit province in 2006 resided in two distinct branches, designated 1.p1 and 1.p2. A hemagglutination inhibition test with a panel of monoclonal antibodies demonstrated a possible antigenic drift between the Phichit isolates. Involvement of free-grazing duck practice in the area was discussed as a cause of the differential evolution among the Phichit isolates. A branch, designated 1-TGWB and consisting exclusively of isolates from zoological tigers and wild birds, was evident in all phylogenetic trees constructed in the study. The branch's existence indicated that the HPAI viruses could have been maintained in the wild bird population for a certain period, although no involvement of wild birds in HPAI transmission to poultry was evident in this study.

Genetic characterization of H1N1, H1N2 and H3N2 swine influenza virus in Thailand.

Swine have been known to be a suitable host for influenza A virus. In Thailand, phylogenetic analysis on swine influenza virus (SIV) has as yet not been attempted. The present report presents molecular and phylogenetic analysis performed on SIV in Thailand. In this study, 12 SIV isolates from the central and eastern part of Thailand were subtyped and the molecular genetics of hemagglutinin and neuraminidase were elucidated. Three subtypes, H1N1, H1N2 and H3N2, are described. Phylogenetic analysis of the SIV hemagglutinin and neuraminidase genes shows individual clusters with swine, human or avian influenza virus at various global locations. Furthermore, amino acid substitutions were detected either at the receptor binding site or the antigenic sites of the hemagglutinin gene.

Genetic diversity of swine influenza viruses isolated from pigs during 2000 to 2005 in Thailand.

BACKGROUND: Recent studies have revealed the existence of genetic diversity in swine influenza viruses (SIVs) in the world. In Thailand, there has been a little information on the molecular characteristics of the SIVs since the first isolation of viruses of H1N1 and H3N2 subtypes in the late 1970s. Our previous study demonstrated that Thai H1N1 SIVs possessed the classical swine H1 and avian-like swine N1 genes (Takemae et al., Proceedings of the Options for the Control of Influenza VI.2007;350-353). OBJECTIVES: In the present study, we genetically characterized 12 SIVs including those of H1N1, H1N2 and H3N2 subtypes isolated between 2000 and 2005. METHODS: We determined the entire nucleotide sequences of the eight gene segments of those isolates. RESULTS: Phylogenetic analysis revealed the existence of nine distinct genotypes amongst the Thai SIVs. These genotypes arose from multiple introductions of classical swine, avian-like swine and human viruses. The existence of two distinct sublineages within classical swine H1 and NS, avian-like swine PA and M and human H3 and N2 genes of the Thai SIVs suggested that introduction of viruses of classical swine, avian-like swine and human origins occurred twice respectively into the Thai pig population. The predominance of avian-like swine genes amongst the Thai SIVs was evident. In particular, three polymerase (PB1, PB2 and PA) and matrix genes of avian-like swine origin were retained in all the Thai SIVs examined. CONCLUSIONS: These observations may suggest that genes of avian-like swine lineages have some advantages to be maintained in pigs as seen in the SIVs established through multiple introductions in other regions.