Molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in Central Region, Thailand.

Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak in dairy cows in the Central Region, Thailand. BCoV was molecularly detected from bloody diarrheic cattle feces by using nested PCR. Agarose gel electrophoresis of three diarrheic fecal samples yielded from the 25 samples desired amplicons that were 488 base pairs and sequencing substantiated that have BCoV. The sequence alignment indicated that nucleotide and amino acid sequences, the three TWD isolated in Thailand, were more quite homologous to each other (amino acid at position 39 of TWD1, TWD3 was proline, but TWD2 was serine) and closely related to OK-0514-3strain (virulent respiratory strain; RBCoV).The amino acid sequencing identities among TWD1, TWD2,TWD3, and OK-0514-3 strain were 96.0 to 96.6%, those at which T3I, H65N, D87G, H127Y, andQ136R were changed. In addition, the phylogenetic tree of the hypervariable region S1subunit spike glycoprotein BCoV gene was composed of three major clades by using the 54 sequences generated and showed that the evolutionally distance, TWD1, TWD2, and TWD3 were the isolated group together and most similar to OK-0514-3 strain (98.2 to 98.5% similarity). Further study will develop ELISA assay for serologic detection of winter dysentery disease.

Economic and value chain analysis to support an investigation and risk mitigation efforts on Marek's disease in layers in the southern part of Thailand.

Background and Aim: Marek's disease (MD) is a common lymphoproliferative disease affecting chickens and causing economic losses in commercial poultry. The MD outbreak was noticed in the southern part of Thailand in 2019. The suspected cases were found with an abnormal number of cases of layers dying with clinical signs, for example, weakness and emaciation, with evidence of MD gross lesions. This study aimed to raise awareness of the MD outbreak through value chain analysis (VCA), identifying associated possible risk factors, and estimating the associated economic impact. Materials and Methods: Value chain analysis, including seasonal calendar, value chain diagram, and layer movement mapping of the layer industry, was conducted. High-risk stakeholders were identified on the basis of risk practices and interactions between stakeholders. A case-control study was conducted to determine risk factors associated with the MD outbreak on layer farms, and partial budget analysis was used to estimate economic losses associated with MD. Results: The value chain diagram showed the linkages between stakeholders, including estimation of the percentage of products moved from one stakeholder group to another and the negotiated price. Fourteen out of 35 layer farms were case farms. Farm size and source of birds were significantly associated with the MD outbreak. The MD outbreak caused total economic losses of 295,823 USD. Farms that slaughtered infected birds with additional revenues incurred losses of 140,930 USD, whereas farms that culled infected birds without additional revenue returned incurred losses of 1995 USD. Conclusion: The VCA provided a better understanding of the layer and egg businesses in South Thailand and guided the development of questionnaires for outbreak investigation. The potential risk factor findings suggested the need for further exploration of the source of the MD outbreak.

Induction of TNF-alpha in human macrophages by avian and human influenza viruses.

The highly pathogenic avian influenza virus H5N1 is known to induce high level of tumor necrosis factor alpha (TNF-alpha) from primary macrophages. However, it is still unclear whether current H5N1 strains also induce high TNF-alpha production, as most of the data were derived from extinct clade 0 H5N1 strain. Here, we show that current clade 1 and 2 H5N1 strains induce variable levels of TNF-alpha that are not necessarily higher than those induced by seasonal influenza viruses. The result suggests that hyper-induction of TNF-alpha in human macrophages is not always associated with a highly pathogenic phenotype. We further tested the contribution of the NS gene segment from H5N1 isolates to TNF-alpha induction by using reverse genetics. While NS conferred some variation in TNF-alpha induction when incorporated into an H1N1 virus genetic background, it did not affect TNF-alpha induction in an H5N1 virus genetic background, suggesting that other viral genes are involved.

A simple screening assay for receptor switching of avian influenza viruses.

BACKGROUND: Adaptation of the receptor-binding preference from alpha2,3- to alpha2,6-linked sialic acid is an essential step for an avian influenza virus to transmit efficiently in human population and become a pandemic virus. The currently available assays for receptor-binding preference are complex and not widely available. OBJECTIVES: A simple high-throughput screening assay will facilitate early detection of a potential pandemic virus, which is crucial for the prevention and control of the possible pandemic. We wanted to develop a simple assay to differentiate influenza viruses with alpha2,3- or alpha2,6-linked receptor-binding preference. STUDY DESIGN: The assay employs a specific sialidase (from Salmonella thyphimurium) that can eliminate alpha2,3-linked sialic acid from red blood cells. A reduction of hemagglutination titer indicates alpha2,3-linked receptor preference in this assay. RESULTS: Using a panel of H5N1 avian influenza isolates and H1/H3 human influenza isolates, as well as mutated H5 reverse genetics virus, the assay could accurately differentiate the viruses according to their receptor-binding preference. Furthermore, the assay was sufficiently sensitive to detect a minor variant with alpha2,6-linkage-specificity in a background of alpha2,3-linkage-specific virus. CONCLUSIONS: We have developed a simple screening assay capable of detecting avian influenza viruses that have switched their receptor-binding preference.

Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.

A double-antigen sandwich ELISA was developed for the detection of antibodies to influenza A viruses. A recombinant nucleoprotein (rNP) of influenza A virus was used as a capture antigen and an HRP-conjugate for detecting the antibodies. A total of 125 serum samples from birds of different species including chickens, geese, open-billed storks, Khaki Campbell ducks, lesser whistling ducks, and pigeons with known antibodies were tested by ELISA. The sensitivity and the specificity of ELISA were found to be 98% and 97.3%, respectively. The assay was able to detect the presence of influenza A antibodies as early as the fourth day post-inoculation in ducks infected experimentally with influenza A (H5N1) virus. Excellent agreement (97.6%) was obtained between this sandwich ELISA and the hemagglutination inhibition (HI) tests (kappa=0.95). The double-antigen sandwich ELISA correlated well with a commercial avian influenza (AI) multispecies ELISA and was slightly more sensitive than the AI multispecies ELISA. These findings indicate that the double-antigen sandwich ELISA based on rNP may offer an effective screening method for serodiagnosis of influenza A virus. The double-antigen sandwich ELISA also enables the detection of antibodies to influenza A viruses in different species without the need for species-specific secondary antibodies.

Erythrocyte binding preference of avian influenza H5N1 viruses.

Five erythrocyte species (horse, goose, chicken, guinea pig, and human) were used to agglutinate avian influenza H5N1 viruses by hemagglutination assay and to detect specific antibody by hemagglutination inhibition test. We found that goose erythrocytes confer a greater advantage over other erythrocyte species in both assays.

Surveillance for reassortant virus by multiplex reverse transcription-PCR specific for eight genomic segments of avian influenza A H5N1 viruses.

Avian influenza H5N1 virus is a global threat. An emergence of a reassortant virus with a pandemic potential is a major concern. Here we describe a multiplex reverse transcription-PCR assay that is specific for the eight genomic segments of the currently circulating H5N1 viruses to facilitate surveillance for a virus resulting from reassortment between human influenza virus and the H5N1 virus.