Molecular identification and risk factor analysis of the first Lumpy skin disease outbreak in cattle in Mongolia.

Lumpy skin disease (LSD) is a transboundary viral infectious disease in cattle caused by a Capripoxvirus. LSD has been recently introduced in some Asian countries. However, in Mongolia, no report of LSD is publicly available. We clinically examined LSD symptoms in 1,034 cattle from 4 soum (district) in Dornod province in Mongolia. Sixty-one cattle of them were confirmed with symptoms of LSD and then viral P32 gene was detected by a PCR. The overall prevalence of LSD in cattle was 5.9%. Females odds ratios (OR)=2.27 than males, adults (>2.5-years-old, OR=3.68) than young (1-2.5-years-old) and calves (<1-year-old) were at higher risks for LSD cases in Mongolia, while locations near the tube well and pond water are major risk areas for viral transmission due to density of insects often is high. For virus isolation, skin nodule tissue samples of 4 cattle located in four distinct soums were used for viral propagation using the MDBK cell line. Internal terminal repeat region and RPO30 gene of 4 Mongolian isolates were amplified and sequenced. In the phylogenetic trees, Mongolian LSDVs (2021) were clustered together with the Chinese (2020) and Vietnamese isolates (2020). This is the first report alarming the LSD outbreak in Mongolia that was confirmed by our study. The newly isolated viruses would be a useful base for developing diagnostic tools and inactivated vaccine technology. A large-scale study of LSD is next priority for establishing successful control strategy of further disease outbreak.

Establishment of a mouse- and egg-adapted strain for the evaluation of vaccine potency against H3N2 variant influenza virus in mice.

Sporadic spreads of swine-origin influenza H3N2 variant (H3N2v) viruses were reported in humans, resulting in 437 human infections between 2011 and 2021 in the USA. Thus, an effective vaccine is needed to better control a potential pandemic for these antigenically distinct viruses from seasonal influenza. In this study, a candidate vaccine strain with efficient growth capacity in chicken embryos was established through serial blind passaging of A/Indiana/08/2011 (H3N2)v in mice and chicken embryos. Seven amino acid substitutions (M21I in PA; A138T, N165K, and V226A in HA; S312L in NP; T167I in M1; G62A in NS1 proteins) were found in the passaged viruses without a major change in the antigenicity. This mouse- and egg-adapted virus was used as a vaccine and challenge strain in mice to evaluate the efficacy of the H3N2v vaccine in different doses. Antibodies with high neutralizing titers were induced in mice immunized with 100 µg of inactivated whole-virus particles, and those mice were significantly protected from the challenge of homologous strain. The findings indicated that the established strain in the study was useful for vaccine study in mouse models.

Efficacy of Oral Vaccine against Classical Swine Fever in Wild Boar and Estimation of the Disease Dynamics in the Quantitative Approach.

Classical swine fever virus (CSFV) in the wild boar population has been spreading in Japan, alongside outbreaks on pigs, since classical swine fever (CSF) reemerged in September 2018. The vaccination using oral bait vaccine was initially implemented in Gifu prefecture in March 2019. In the present study, antibodies against CSFV in wild boar were assessed in 1443 captured and dead wild boars in Gifu prefecture. After the implementation of oral vaccination, the increase of the proportion of seropositive animals and their titer in wild boars were confirmed. Quantitative analysis of antigen and antibodies against CSFV in wild boar implies potential disease diversity in the wild boar population. Animals with status in high virus replication (Ct < 30) and non- or low-immune response were confirmed and were sustained at a certain level after initial oral vaccination. Through continuous vaccination periods, the increase of seroprevalence among wild boar and the decrease of CSFV-positive animals were observed. The epidemiological analysis based on the quantitative virological outcomes could provide more information on the efficacy of oral vaccination and dynamics of CSF in the wild boar population, which will help to improve the implementation of control measures for CSF in countries such as Japan and neighboring countries.

The clinically used serine protease inhibitor nafamostat reduces influenza virus replication and cytokine production in human airway epithelial cells and viral replication in mice.

The effects of the clinically used protease inhibitor nafamostat on influenza virus replication have not been well studied. Primary human tracheal (HTE) and nasal (HNE) epithelial cells were pretreated with nafamostat and infected with the 2009 pandemic [A/Sendai-H/108/2009/(H1N1) pdm09] or seasonal [A/New York/55/2004(H3N2)] influenza virus. Pretreatment with nafamostat reduced the titers of the pandemic and seasonal influenza viruses and the secretion of inflammatory cytokines, including interleukin-6 and tumor necrosis factor-α, in the supernatants of the cells infected with the pandemic influenza virus. HTE and HNE cells exhibited mRNA and/or protein expression of transmembrane protease serine 2 (TMPRSS2), TMPRSS4, and TMPRSS11D. Pretreatment with nafamostat reduced cleavage of the precursor protein HA0 of the pandemic influenza virus into subunit HA1 in HTE cells and reduced the number of acidic endosomes in HTE and HNE cells where influenza virus RNA enters the cytoplasm. Additionally, nafamostat (30 mg/kg/day, intraperitoneal administration) reduced the levels of the pandemic influenza virus [A/Hyogo/YS/2011 (H1N1) pdm09] in mouse lung washes. These findings suggest that nafamostat may inhibit influenza virus replication in human airway epithelial cells and mouse lungs and reduce infection-induced airway inflammation by modulating cytokine production.

Re-Invasion of H5N8 High Pathogenicity Avian Influenza Virus Clade 2.3.4.4b in Hokkaido, Japan, 2020.

Global dispersion of high pathogenicity avian influenza (HPAI), especially that caused by H5 clade 2.3.4.4, has threatened poultry industries and, potentially, human health. An HPAI virus, A/northern pintail/Hokkaido/M13/2020 (H5N8) (NP/Hok/20) belonging to clade 2.3.4.4b, was isolated from a fecal sample collected at a lake in Hokkaido, Japan where migratory birds rested, October 2020. In the phylogenetic trees of all eight gene segments, NP/Hok/20 fell into in the cluster of European isolates in 2020, but was distinct from the isolates in eastern Asia and Europe during the winter season of 2017-2018. The antigenic cartography indicates that the antigenicity of NP/Hok/20 was almost the same as that of previous isolates of H5 clade 2.3.4.4b, whereas the antigenic distances from NP/Hok/20 to the representative strains in clade 2.3.4.4e and to a strain in 2.3.4 were apparently distant. These data imply that HPAI virus clade 2.3.4.4b should have been delivered by bird migration despite the intercontinental distance, although it was not defined whether NP/Hok/20 was transported from Europe via Siberia where migratory birds nest in the summer season. Given the probability of perpetuation of transmission in the northern territory, periodic updates of intensive surveys on avian influenza at the global level are essential to prepare for future outbreaks of the HPAI virus.

Potency of an Inactivated Influenza Vaccine against a Challenge with A/Swine/Missouri/A01727926/2015 (H4N6) in Mice for Pandemic Preparedness.

H4 influenza viruses have been isolated from birds across the world. In recent years, an H4 influenza virus infection has been confirmed in pigs. Pigs play an important role in the transmission of influenza viruses to human hosts. Therefore, it is important to develop a new vaccine in the case of an H4 influenza virus infection in humans, considering that this virus has a different antigenicity from seasonal human influenza viruses. In this study, after selecting vaccine candidate strains based on their antigenic relation to one of the pig isolates, A/swine/Missouri/A01727926/2015 (H4N6) (MO/15), an inactivated whole-particle vaccine was prepared from A/swan/Hokkaido/481102/2017 (H4N6). This vaccine showed high immunogenicity in mice, and the antibody induced by the vaccine showed high cross-reactivity to the MO/15 virus. This vaccine induced sufficient neutralizing antibodies and mitigated the effects of an MO/15 infection in a mouse model. This study is the first to suggest that an inactivated whole-particle vaccine prepared from an influenza virus isolated from wild birds is an effective countermeasure in case of a future influenza pandemic caused by the H4 influenza virus.

Genetic and antigenic characterization of H5 and H7 avian influenza viruses isolated from migratory waterfowl in Mongolia from 2017 to 2019.

The circulation of highly pathogenic avian influenza viruses (HPAIVs) of various subtypes (e.g., H5N1, H5N6, H5N8, and H7N9) in poultry remains a global concern for animal and public health. Migratory waterfowls play important roles in the transmission of these viruses across countries. To monitor virus spread by wild birds, active surveillance for avian influenza in migratory waterfowl was conducted in Mongolia from 2015 to 2019. In total, 5000 fecal samples were collected from lakesides in central Mongolia, and 167 influenza A viruses were isolated. Two H5N3, four H7N3, and two H7N7 viruses were characterized in this study. The amino acid sequence at hemagglutinin (HA) cleavage site of those isolates suggested low pathogenicity in chickens. Phylogenetic analysis revealed that all H5 and H7 viruses were closely related to recent H5 and H7 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds in Asia and Europe. Antigenicity of H7Nx was similar to those of typical non-pathogenic avian influenza viruses (AIVs). While HPAIVs or A/Anhui/1/2013 (H7N9)-related LPAIVs were not detected in migratory waterfowl in Mongolia, sporadic introductions of AIVs including H5 and H7 viruses into Mongolia through the wild bird migration were identified. Thus, continued monitoring of H5 and H7 AIVs in both domestic and wild birds is needed for the early detection of HPAIVs spread into the country.

Evaluation of a rapid isothermal nucleic acid amplification kit, Alere™ i Influenza A&B, for the detection of avian influenza viruses.

Rapid and accurate diagnosis of influenza virus infection is essential for quick responses for both human and animal health. The Alere™ i Influenza A&B is a novel isothermal nucleic acid amplification kit that can detect and differentiate between influenza A and B viruses in human specimens in approximately 15 min. In the present study, the performance of the Alere™ i Influenza A&B kit was evaluated for its ability to detect avian influenza virus in chickens. The kit was able to detect representative avian influenza virus strains (hemagglutinin subtypes H1-H16, including the recently isolated H5 and H7 highly pathogenic avian influenza viruses), and the detection limit of the kit for these viruses varied between 10-1.4-102.1 50% egg-infective dose per test, which is higher than the analytical sensitivity of the antigen detection immunochromatography kit ESPLINE® A INFLUENZA. In experimentally infected chickens inoculated with a highly pathogenic avian influenza virus strain A/chicken/Hokkaido/002/2016 (H5N6), viral RNA was detected in the tracheal and cloacal swabs. These results indicate that this kit has the potential to be used as a rapid screening test of influenza A virus infection in chickens.