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The H9 and H6 subtypes of low pathogenicity avian influenza viruses (LPAIVs) cause substantial economic losses in poultry worldwide, including Vietnam. Herein, we characterized Vietnamese H9 and H6 LPAIVs to facilitate the control of avian influenza. The space-time representative viruses of each subtype were selected based on active surveillance from 2014 to 2018 in Vietnam. Phylogenetic analysis using hemagglutinin genes revealed that 54 H9 and 48 H6 Vietnamese LPAIVs were classified into the sublineages Y280/BJ94 and Group II, respectively. Gene constellation analysis indicated that 6 and 19 genotypes of the H9 and H6 subtypes, respectively, belonged to the representative viruses. The Vietnamese viruses are genetically related to the previous isolates and those in neighboring countries, indicating their circulation in poultry after being introduced into Vietnam. The antigenicity of these subtypes was different from that of viruses isolated from wild birds. Antigenicity was more conserved in the H9 viruses than in the H6 viruses. Furthermore, a representative H9 LPAIV exhibited systemic replication in chickens, which was enhanced by coinfection with avian pathogenic Escherichia coli O2. Although H9 and H6 were classified as LPAIVs, their characterization indicated that their silent spread might significantly affect the poultry industry.

The inhibitory effects of 5-aminolevulinic acid phosphate (5-ALA), an important amino acid for energy production in the host, against viral infections were previously reported. Here, the antiviral effects of 5-ALA against classical swine fever virus (CSFV) belonging to the genus Pestivirus in the Flaviviridae family and its possible mechanisms were investigated. CSFV replication was suppressed in swine cells supplemented with 5-ALA or its metabolite, protoporphyrin IX (PPIX). The infectivity titer of CSFV was decreased after mixing with PPIX extracellularly. In addition, the activities of the replication cycle were decreased in the presence of PPIX based on the CSFV replicon assay. These results showed that PPIX exerted antiviral effects by inactivating virus particles and inhibiting the replication cycle. To evaluate the in vivo efficacy of 5-ALA, pigs were supplemented daily with 5-ALA for 1 week before virus inoculation and then inoculated with a virulent CSFV strain at the 10(7.0) 50% tissue culture infectious dose. The clinical scores of the supplemented group were significantly lower than those of the nonsupplemented group, whereas the virus growth was not. Taken together, 5-ALA showed antiviral effects against CSFV in vitro, and PPIX played a key role by inactivating virus particles extracellularly and inhibiting the replication cycle intracellularly.

Many high pathogenicity avian influenza (HPAI) cases in wild birds due to H5N1 HPAI virus (HPAIV) infection were reported in northern Japan in the winter of 2021-2022. To investigate the epidemiology of HPAIVs brought to Japan from surrounding areas, a genetic analysis of H5 HPAIVs isolated in northern Japan was performed, and the pathogenicity of the HPAIV in chickens was assessed by experimental infection. Based on the genetic analysis of the hemagglutinin gene, pathogenic viruses detected in northern Japan as well as one in Sakhalin, the eastern part of Russia, were classified into the same subgroup as viruses prevalent in Europe in the same season but distinct from those circulating in Asia in winter 2020-2021. High identities of all eight segment sequences of A/crow/Hokkaido/0103B065/2022 (H5N1) (Crow/Hok), the representative isolates in northern Japan in 2022, to European isolates in the same season could also certify the unlikeliness of causing gene reassortment between H5 HPAIVs and viruses locally circulating in Asia. According to intranasal challenge results in six-week-old chickens, 50% of the chicken-lethal dose of Crow/Hok was calculated as 10(4.5) times of the 50% egg-infectious dose. These results demonstrated that the currently prevalent H5 HPAIVs could spread widely from certain origins throughout the Eurasian continent, including Europe and the Far East, and implied a possibility that contagious viruses are gathered in lakes in the northern territory via bird migration. Active monitoring of wild birds at the global level is essential to estimate the geographical source and spread dynamics of HPAIVs.

Many high pathogenicity avian influenza (HPAI) cases in wild birds due to H5N1 HPAI virus (HPAIV) infection were reported in northern Japan in the winter of 2021-2022. To investigate the epidemiology of HPAIVs brought to Japan from surrounding areas, a genetic analysis of H5 HPAIVs isolated in northern Japan was performed, and the pathogenicity of the HPAIV in chickens was assessed by experimental infection. Based on the genetic analysis of the hemagglutinin gene, pathogenic viruses detected in northern Japan as well as one in Sakhalin, the eastern part of Russia, were classified into the same subgroup as viruses prevalent in Europe in the same season but distinct from those circulating in Asia in winter 2020-2021. High identities of all eight segment sequences of A/crow/Hokkaido/0103B065/2022 (H5N1) (Crow/Hok), the representative isolates in northern Japan in 2022, to European isolates in the same season could also certify the unlikeliness of causing gene reassortment between H5 HPAIVs and viruses locally circulating in Asia. According to intranasal challenge results in six-week-old chickens, 50% of the chicken-lethal dose of Crow/Hok was calculated as 10(4.5) times of the 50% egg-infectious dose. These results demonstrated that the currently prevalent H5 HPAIVs could spread widely from certain origins throughout the Eurasian continent, including Europe and the Far East, and implied a possibility that contagious viruses are gathered in lakes in the northern territory via bird migration. Active monitoring of wild birds at the global level is essential to estimate the geographical source and spread dynamics of HPAIVs.

The GPE(-) strain is a live attenuated vaccine for classical swine fever (CSF) developed in Japan. In the context of increasing attention for the differentiating infected from vaccinated animals (DIVA) concept, the achievement of CSF eradication with the GPE(-) proposes it as a preferable backbone for a recombinant CSF marker vaccine. While its infectious cDNA clone, vGPE(-), is well characterized, 10 amino acid substitutions were recognized in the genome, compared to the original GPE(-) vaccine seed. To clarify the GPE(-) seed availability, this study aimed to generate and characterize a clone possessing the identical amino acid sequence to the GPE(-) seed. The attempt resulted in the loss of the infectious GPE(-) seed clone production due to the impaired replication by an amino acid substitution in the viral polymerase NS5B. Accordingly, replication-competent GPE(-) seed variant clones were produced. Although they were mostly restricted to propagate in the tonsils of pigs, similarly to vGPE(-), their type I interferon-inducing capacity was significantly lower than that of vGPE(-). Taken together, vGPE(-) mainly retains ideal properties for the CSF vaccine, compared with the seed variants, and is probably useful in the development of a CSF marker vaccine.