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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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

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

The viral protein N-pro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, N-pro suppresses type I IFN (IFN-alpha/beta) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the N-pro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of N-pro coordinating zinc by means of the amino acid residues 0112, 0134, 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-alpha/beta 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 N-pro 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 N-pro contributed to higher stability of the reconstructed N-pro compared with the N-pro from the Thai isolate. This led to enhanced interaction of N-pro 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 N-pro are involved in the stability of N-pro, in interaction of N-pro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-alpha/beta production

RT-PCR Survey of Emerging Paramyxoviruses in Cave-dwelling Bats

Bats are the reservoir hosts for several Paramyxoviruses including two serious zoonotic viruses, Hendra virus and Nipah virus which are responsible for fatal infections in animals and humans. These two viruses are sufficiently different from previously described Paramyxoviruses and are included in a new genus, Henipavirus. We report here a survey of cave-dwelling, insectivorous bats in Thailand for the presence of henipaviruses. Pooled urine samples were collected in nine caves inhabited by six different bat species in the northern (Chiangmai and Nakornsawan) and southern (Songkla and Satoon) provinces of Thailand. A reverse transcription PCR (RT-PCR) assay using henipavirus-specific primers derived from the conserved region of the RNA polymerase (L) gene was used to detect known and unknown viruses in this genus. Samples from seven out of nine caves surveyed tested positive by RT-PCR. Nucleotide sequences of the PCR bands revealed the presence of diverse strains (three clusters and seven divergent genotypes) of previously uncharacterised paramyxovirus(es). Phylogenetic analysis based on the deduced L protein sequence revealed close correlations between the positive samples and the recently described but unclassified paramyxoviruses: Beilong virus and J-virus. This is the first report on the prevalence of paramyxovirus variants in cave-dwelling bats and highlights the importance of further epidemiological surveillance in bats

Summary of the experimental infections at 10⁶EID₅₀.

<p>Summary of the experimental infections at 10⁶EID₅₀.</p

Viral distribution in chickens infected with three HPAIVs at a dose of 10⁶ EID₅₀.

<p>Three chickens in each group were sacrificed at 24 h post-inoculation. Virus titers are presented as the mean values ± standard deviation. ⁑<i>P</i> < 0.01 vs. pigeon-04 and crane-05. ‡ <i>P</i> < 0.01 vs. crane-05.</p

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

<div><p>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 <i>Mx</i> and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.</p></div