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Chimeric marker vaccine candidates, vGPE - /PAPeV E rns and vGPE - /PhoPeV E rns , have been generated and their efficacy and capability to differentiate infected from vaccinated animals were confirmed in previous studies. The safety profile of the two chimeric marker vaccine candidates, particularly in the potential reversion to virulence, was evaluated. Each virus was administered to pigs with a dose equivalent to the vaccination dose, and pooled tonsil homogenates were subsequently inoculated into further pigs. Chimeric virus vGPE - /PAPeV E rns displayed the most substantial attenuation, achieving this within only two passages, whereas vGPE - /PhoPeV E rns was detectable until the third passage and disappeared entirely by the fourth passage. The vGPE - strain, assessed alongside, consistently exhibited stable virus recovery across each passage without any signs of increased virulence in pigs. In vitro assays revealed that the type I interferon-inducing capacity of vGPE - /PAPeV E rns was significantly higher than that of vGPE - /PhoPeV E rns and vGPE - . In conclusion, the safety profile of the two chimeric marker vaccine candidates was affirmed. Further research is essential to ensure the stability of their attenuation and safety in diverse pig populations.

In the fall of 2022, high pathogenicity avian influenza viruses (HPAIVs) were detected from raptors and geese in Japan, a month earlier than in past years, indicating a shift in detection patterns. In this study, we conducted a phylogenetic analysis on H5N1 HPAIVs detected from six wild birds during the 2022/2023 season to determine their genetic origins. Our findings revealed that these HPAIVs belong to the G2 group within clade 2.3.4.4b, with all isolates classified into three subgroups: G2b, G2d, and G2c. The genetic background of the G2b virus (a peregrine falcon-derived strain) and G2d viruses (two raptors and two geese-derived strains) were the same as those detected in Japan in the 2021/2022 season. Since no HPAI cases were reported in Japan during the summer of 2022, it is probable that migratory birds reintroduced the G2b and G2d viruses. Conversely, the G2c virus (a raptor-derived strain) was first recognized in Japan in the fall of 2022. This strain might share a common ancestor with HPAIVs from Asia and West Siberia observed in the 2021/2022 season. The early migration of waterfowl to Japan in the fall of 2022 could have facilitated the early invasion of HPAIVs.

A previous study proved that vGPE - mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE - vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE - vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein E rns of the GPE - vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE - /PAPeV E rns and vGPE - /PhoPeV E rns , were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE - vaccine strain. Vaccination at a dose of 10 4.0 TCID 50 with either vGPE - /PAPeV E rns or vGPE - /PhoPeV E rns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE - /PAPeV E rns and vGPE - /PhoPeV E rns affirmed their properties, as the vGPE - vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.

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., Competing Interests: The funders did not have any influence on the analysis or interpretation of the results in this study. Authors declare no conflict of interest.

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.