Molecular characterization of bovine ephemeral fever virus in Thailand between 2013 and 2017.

Bovine ephemeral fever (BEF) is an arthropod-borne disease caused by bovine ephemeral fever virus (BEFV), a negative sense, single-stranded RNA virus. BEFV is endemic in tropical and sub-tropical regions including Thailand, a country in mainland Southeast Asia. However, there are few studies on BEFV and no available information regarding molecular characteristics of BEFV in Thailand. Therefore, the aims of this study were to genetically characterize Thai BEFVs and reveal their evolutions by phylogenetic analysis of G gene ectodomain sequences. From 2013 to 2017, blood samples were collected from bovine that matched with BEF case definition from three regions of Thailand. Thai BEFV G genes and a whole genome of an isolate, East Asia/TH/LRI0045/2016, were sequenced and characterized. Additionally, their phylogenies were constructed. This is the first report on genetics of BEFV in Southeast Asia. G ectodomain encoding region of Thai BEFV found during 2013-2017 are closely related to the second and third sub-clades of East Asia lineage. In addition, we observed mutation in the putative P' ORF of all Thai BEFVs which generated a premature stop codon. Thai G gene sequences are closely related to those of mainland Chinese and Taiwanese isolates. The whole genomic sequences of Thai BEFV and East Asia/China/JT02 L/2002 possess common characteristics, suggesting shared evolutionary relationship between East and Southeast Asian strains. Further studies on relationship between animal translocation, circulation of BEFV in Greater Mekong subregion and acquisition of more G gene sequences may improve understanding of BEFV epidemiology in mainland Southeast Asia.

Interferon gamma induces cellular protein alteration and increases replication of porcine circovirus type 2 in PK-15 cells.

Porcine circovirus type 2 (PCV2) infections may lead to the development of subclinical signs or chronic systemic syndromes, collectively known as "porcine circovirus-associated disease" (PCVAD) in swine. Interferon gamma (IFN-γ) is known to enhance PCV2 replication in vitro, and immune mediators may act as pivotal factors in triggering PCV2 infection progression toward PCVAD. We determined the effects of IFN-γ on PCV2 replication in PK-15 cells. PCV2 was cultured in the presence or absence of exogenous swine IFN-γ (swIFNγ). Growth curve analysis in PK-15 cells revealed that PCV2 could replicate to a significantly higher titer in swIFNγ medium. To investigate the host cell response upon PVC2 infection, differential expression of proteins in PCV2-infected PK-15 cells with or without swIFNγ stimulation was analyzed by proteomics (LC-MS/MS) analysis. A large proportion of the differentially expressed proteins in swIFNγ-treated PCV2-infected cells were found to be involved in apoptosis, cellular stress responses, cell survival/proliferation pathways, and inflammatory responses. We further confirmed the expression of these differentially expressed proteins at the mRNA levels by qRT-PCR. PCV2 infection in PK-15 cells in the presence of IFN-γ resulted in upregulation of cellular proteins in responses to stress, cell survival, and cell proliferation (Hsp90, MAP3K7, RAS-GTPase, c-myc, and 14-3-3 epsilon) as well as in an increase in the levels of proteins (CASP9 and TRAF5) related to the apoptosis pathways. Thus, PCV2 exploits several cellular biological processes through IFN activation for enhancing viral replication. This is the first evidence of IFN-γ promoting PCV2 replication in vitro via a mechanism similar to that used by several human viruses.

Genetic diversity of porcine circovirus type 2 (PCV2) in Thailand during 2009-2015.

Porcine circovirus type 2 (PCV2), the essential cause of porcine circovirus associated disease (PCVAD), has evolved rapidly and it has been reported worldwide. However, genetic information of PCV2 in Thailand has not been available since 2011. Herein, we studied occurrence and genetic diversity of PCV2 in Thailand and their relationships to the global PCV2 based on ORF2 sequences. The results showed that 306 samples (44.09%) from 56 farms (80%) were PCV2 positive by PCR. Phylogenetic trees constructed by both neighbor-joining and Bayesian Inference yielded similar topology of the ORF2 sequences. Thai PCV2 comprise four clusters: PCV2a (5.5%), PCV2b (29.41%), intermediate clade 1 (IM1) PCV2b (11.03%) and PCV2d (54.41%). Genetic shift of PCV2 in Thailand has occurred similarly to the global situation. The shift from PCV2b to PCV2d was clearly observed during 2013-2014. The viruses with genetically similar to the first reported PCV2 in 2004 have still circulated in Thailand. The first Thai PCV2b and PCV2d were closely related to the neighboring countries. The haplotype network analysis revealed the relationship of PCV2 in Thailand and other countries. These results indicate that genetic diversity of PCV2 in Thailand is caused by genetic drift of the local strains and intermittent introduction of new strains or genotypes from other countries. Genetic evolution of PCV2 in Thailand is similar to that occurs globally.

Screening for lead compounds and herbal extracts with potential anti-influenza viral activity.

Nonstructural protein 1 (NS1) of the highly pathogenic avian influenza virus (H5N1) contains a conserved RNA binding domain (RBD) that inhibits antiviral functions of host-innate immune response. Dimerization of NS1 forms a central groove and binds to double stranded (ds) RNA. This region might serve as a potential drug target. In this study, three dimensional structure model of NS1 RBD protein was constructed and virtual screening was performed to identify lead compounds that bound within and around the central groove. The virtual screening showed that 5 compounds bound within the central groove with binding energy ranging between -16.05 and -17.36 Kcal/mol. Two commercially available compounds, estradiol and veratridine, were selected for using in an in vitro screening assay. The results showed that neither of the compounds could inhibit the association between dsRNA and NS1 RBD protein. In addition, 34 herbal extracts were examined for their inhibitory effects. Five of them were able to inhibit association between NS1 RBD and dsRNA in electrophoresis mobility shift assay. Four herbs, Terminalia belirica, Salacia chinensis, Zingiber montanum and Peltophorum pterocarpum, could reduce > 50% of infectivity of H5N1 in a cell-based assay, and it is worth further studying their potential use as source of antiviral drugs.

Genesis and genetic constellations of swine influenza viruses in Thailand.

Swine influenza virus (SIV) is one of the most important zoonotic agents and the origin of the most recent pandemic virus. Asia is considered to be the epicenter for genetic exchanging of influenza A viruses and Southeast Asia including Thailand serves as a reservoir to maintain the persistence of the viruses for seeding other regions. Therefore, searching for new reassortants in this area has been routinely required. Although SIVs in Thailand have been characterized, collective information regarding their genetic evolution and gene constellations is limited. In this study, whole genomes of 30 SIVs isolated during clinical target surveillance plus all available sequences of past and currently circulating Thai SIVs were genetically characterized based on their evolutionary relationships. All genetic pools of Thai SIVs are comprised of four lineages including classical swine (CS), Eurasian swine (EAs), Triple reassortants (TRIG) and Seasonal human (Shs). Out of 84 isolates, nine H1N1, six H3N2 and one H1N2 strains were identified. Gene constellations of SIVs in Thailand are highly complex resulting from multiple reassortments among concurrently circulating SIVs and temporally introduced foreign genes. Most strains contain gene segments from both EAs and CS lineages and appeared transiently. TRIG lineage has been recently introduced into Thai SIV gene pools. The existence of EAs and TRIG lineages in this region may increase rates of genetic exchange and diversity while Southeast Asia is a persistent reservoir for influenza A viruses. Continual monitoring of SIV evolution in this region is crucial in searching for the next potential pandemic viruses.

Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses.

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.