Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant.

In middle to late 2023, a sublineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. We performed multiscale investigations, including phylogenetic analysis, epidemic dynamics modeling, infection experiments using pseudoviruses, clinical isolates, and recombinant viruses in cell cultures and experimental animals, and the use of human sera and antiviral compounds, to reveal the virological features of the newly emerging EG.5.1 variant. Our phylogenetic analysis and epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T are critical to its increased viral fitness. Experimental investigations on the growth kinetics, sensitivity to clinically available antivirals, fusogenicity, and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 are comparable to those of XBB.1.5. However, cryo-electron microscopy revealed structural differences between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible in our experimental setup. Our multiscale investigations provide knowledge for understanding the evolutionary traits of newly emerging pathogenic viruses, including EG.5.1, in the human population.

The Amino Acid at Position 95 in the Matrix Protein of Rabies Virus Is Involved in Antiviral Stress Granule Formation in Infected Cells.

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.

Definition of the immune evasion-replication interface of rabies virus P protein.

Rabies virus phosphoprotein (P protein) is a multifunctional protein that plays key roles in replication as the polymerase cofactor that binds to the complex of viral genomic RNA and the nucleoprotein (N protein), and in evading the innate immune response by binding to STAT transcription factors. These interactions are mediated by the C-terminal domain of P (PCTD). The colocation of these binding sites in the small globular PCTD raises the question of how these interactions underlying replication and immune evasion, central to viral infection, are coordinated and, potentially, coregulated. While direct data on the binding interface of the PCTD for STAT1 is available, the lack of direct structural data on the sites that bind N protein limits our understanding of this interaction hub. The PCTD was proposed to bind via two sites to a flexible loop of N protein (Npep) that is not visible in crystal structures, but no direct analysis of this interaction has been reported. Here we use Nuclear Magnetic Resonance, and molecular modelling to show N protein residues, Leu381, Asp383, Asp384 and phosphor-Ser389, are likely to bind to a 'positive patch' of the PCTD formed by Lys211, Lys214 and Arg260. Furthermore, in contrast to previous predictions we identify a single site of interaction on the PCTD by this Npep. Intriguingly, this site is proximal to the defined STAT1 binding site that includes Ile201 to Phe209. However, cell-based assays indicate that STAT1 and N protein do not compete for P protein. Thus, it appears that interactions critical to replication and immune evasion can occur simultaneously with the same molecules of P protein so that the binding of P protein to activated STAT1 can potentially occur without interrupting interactions involved in replication. These data suggest that replication complexes might be directly involved in STAT1 antagonism.

Differential role of NSs genes in the neurovirulence of two genogroups of Akabane virus causing postnatal encephalomyelitis.

Akabane virus (AKAV) is a member of the genus Orthobunyavirus, family Peribunyaviridae. In addition to AKAV strains that cause fetal Akabane disease, which is characterized by abortion in ruminants, some AKAV strains cause postnatal infection characterized by nonsuppurative encephalomyelitis in ruminants. Here, we focused on the NSs protein, a virulence factor for most viruses belonging to the genus Orthobunyavirus, and we hypothesized that this protein would act as a neurovirulence factor in AKAV strains causing postnatal encephalomyelitis. We generated AKAV strains that were unable to produce the NSs protein, derived from two different genogroups, genogroups I and II, and then examined the role of their NSs proteins by inoculating mice intracerebrally with these modified viruses. Our results revealed that the neurovirulence of genogroup II strains is dependent on the NSs protein, whereas that of genogroup I strains is independent of this protein. Notably, infection of primary cultured bovine cells with these viruses suggested that the NSs proteins of both genogroups suppress innate immune-related gene expression with equal efficiency. These results indicate differences in the determinants of virulence of orthobunyaviruses.

Avian paramyxovirus serotype-1 isolation from migratory birds and environmental water in southern Japan: An epidemiological survey during the 2018/19-2021/2022 winter seasons.

Newcastle disease caused by highly pathogenic viruses of avian paramyxovirus serotype-1 (APMV-1) is a highly contagious poultry disease. Although a large-scale epidemic of Newcastle disease had occurred in Japan between the 1950s and the 2000s, there have been no outbreaks anywhere since 2010. In addition, there are no reports of epidemiological surveys of APMV-1 in wild birds in Japan in the last 10 years. We conducted the first epidemiological survey of APMV-1 in the Izumi plain, Kagoshima prefecture of southern Japan from the winter of 2018 to 2022. A total of 15 APMV-1 strains were isolated, and isolation rates from roosting water and duck fecal samples were 2.51% and 0.10%, respectively. These results indicate that the isolation method from environmental water may be useful for efficient surveillance of APMV-1 in wild birds. Furthermore, this is the first report on the success of APMV-1 isolation from environmental water samples. Genetic analysis of the Fusion (F) gene showed that all APMV-1 isolates were closely related to virus strains circulating among waterfowl in Far East Asian countries. All isolates have avirulent motifs in their cleavage site of F genes, all of which were presumed to be low pathogenic viruses in poultry. However, pathogenicity test using embryonated chicken eggs demonstrated that some isolates killed all chicken embryos regardless of viral doses inoculated (102 -106 50% egg infectious dose). These results indicated that APMV-1 strains, which are potentially pathogenic to chickens, are continuously brought into the Izumi plain by migrating wild birds.

Genetically Diverse Highly Pathogenic Avian Influenza A(H5N1/H5N8) Viruses among Wild Waterfowl and Domestic Poultry, Japan, 2021.

Genetic analyses of highly pathogenic avian influenza H5 subtype viruses isolated from the Izumi Plain, Japan, revealed cocirculation of 2 genetic groups of clade 2.3.4.4b viruses among migratory waterfowl. Our findings demonstrate that both continuous surveillance and timely information sharing of avian influenza viruses are valuable for rapid risk assessment.

Analyses of cell death mechanisms related to amino acid substitution at position 95 in the rabies virus matrix protein.

We previously reported that the avirulent fixed rabies virus strain Ni-CE induces a clear cytopathic effect in mouse neuroblastoma cells, whereas its virulent progenitor, the Nishigahara strain, does not. Infection with Nishigahara and Ni-CE mutants containing a single amino acid substitution in the matrix protein (M) demonstrated that the amino acid at position 95 of M (M95) is a cytopathic determinant. The characteristics of cell death induced by Ni-CE infection resemble those of apoptosis (rounded and shrunken cells, DNA fragmentation), but the intracellular signalling pathway for this process has not been fully investigated. In this study, we aimed to elucidate the mechanism by which M95 affects cell death induced by human neuroblastoma cell infection with the Nishigahara, Ni-CE and M95-mutated strains. We demonstrated that the Ni-CE strain induced DNA fragmentation, cell membrane disruption, exposure of phosphatidylserine (PS), activation of caspase-3/7 and anti-poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, an early apoptosis indicator, whereas the Nishigahara strain did not induce DNA fragmentation, caspase-3/7 activation, cell membrane disruption, or PARP-1 cleavage, but did induce PS exposure. We also demonstrated that these characteristics were associated with M95 using M95-mutated strains. However, we found that Ni-CE induced cell death despite the presence of a caspase inhibitor, Z-VAD-FMK. In conclusion, our data suggest that M95 mutation-related cell death is caused by both the caspase-dependent and -independent pathways.

Transition in genetic constellations of H3N8 and H4N6 low-pathogenic avian influenza viruses isolated from an overwintering site in Japan throughout different winter seasons.

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site for migratory ducks and endangered cranes. We have surveyed avian influenza viruses (AIVs) in this area since 2012 and isolated low-pathogenic AIVs (LPAIVs) of various subtypes every winter season. H3N8 LPAIVs were isolated during the 2012/13 and 2016/17 seasons, and H4N6 LPAIVs were isolated during the 2012/13 and 2013/14 seasons. In the 2017/18 season, one H3N8 and two H4N6 LPAIV strains were isolated from environmental water samples. Genetic and phylogenetic analysis for each gene segment from these H3N8 and H4N6 LPAIVs suggested that our isolates were genetic reassortants generated by intermixing between AIVs circulating not only in Eurasia but also in Africa and/or North America. Comparison of the genetic constellations of our three isolates with their counterparts isolated during previous seasons from the Izumi plain revealed a drastic transition in the genetic constellations of both subtypes. These findings emphasize the importance of continuous surveillance of AIVs on the Izumi plain.

Virological characteristics of the SARS-CoV-2 BA.2.86 variant.

In late 2023, several SARS-CoV-2 XBB descendants, notably EG.5.1, were predominant worldwide. However, a distinct SARS-CoV-2 lineage, the BA.2.86 variant, also emerged. BA.2.86 is phylogenetically distinct from other Omicron sublineages, accumulating over 30 amino acid mutations in its spike protein. Here, we examined the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Additionally, four clinically available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 are significantly lower than those of BA.2 both in vitro and in vivo, the attenuated pathogenicity of BA.2.86 is likely due to its decreased replication capacity. These findings uncover the features of BA.2.86, providing insights for control and treatment.

Improved method for avian influenza virus isolation from environmental water samples.

Environmental water-targeted surveillance of migratory aquatic birds at overwintering sites is potentially one of the most effective approaches for understanding the ecology of avian influenza viruses (AIVs). In this study, we improved the method for AIV isolation from environmental water samples by making a minor modification to our previously reported process. We experimentally demonstrated that the AIV recovery efficiency of the modified method was 10-100-fold higher than that of the original method. This improved isolation method allowed us to isolate a considerably larger number of AIV isolates from environmental water samples collected at an overwintering site for tens of thousands of migratory aquatic birds in Japan during the 2018/2019 winter season, compared with those during previous winter seasons. Genetic and phylogenetic analyses revealed that AIVs of the same subtypes with multiple genetic constellations were circulating in a single overwintering site during a single winter season. These findings indicate that our improved isolation method contributes to enhance environmental water-targeted surveillance and to a better understanding of AIV ecology in migratory aquatic bird populations by monitoring ongoing AIV circulation.

Genetic Characterization of Avian Influenza Viruses Isolated from the Izumi Plain, Japan in 2019/20 Winter Season.

The Izumi plain in the Kagoshima Prefecture, Japan, is known as an overwintering site for more than 30,000 migratory waterfowl, including endangered crane species. We previously reported that environmental water samples, from artificial wet paddies created as crane roost sites on the Izumi plain, are useful for avian influenza virus (AIV) surveillance. During the 2019/20 winter season, we collected 238 water samples from the crane roost sites and isolated 22 AIVs of six subtypes: one H1N1, one H3N2, seven H3N8, four H4N6, nine H6N6, and one H11N2 subtypes. Genetic analyses revealed that AIVs of the same subtype isolated from the Izumi plain during a single winter season exhibited multiple genetic constellations. Furthermore, phylogenetic analyses suggested that our H3N2 isolate may be a genetic reassortant between close relatives to our H3N8 and H11N2 isolates. Our study highlighted the importance of monitoring AIV circulation to better understand AIV ecology in migratory waterfowl populations.

Molecular detection of filarial nematode parasites in Japanese black bears (Ursus thibetanus japonicus) from Iwate Prefecture, Japan.

This study aimed to detect filarial parasites in blood samples of Japanese black bears (Ursus thibetanus japonicus) collected from Iwate Prefecture, Japan. Positive amplicons were obtained from 26 out of 30 samples by nested PCR targeting 18S ribosomal RNA gene and first internal transcribed spacer regions. DNA sequences of Mansonella sp. close to M. ozzardi and Dirofilaria sp. were detected for eight and 11 positive amplicons, respectively. Co-infection was detected for the remaining seven amplicons. Dirofilaria sp. was identified as D. ursi by further genetic analysis of 5S ribosomal RNA gene sequence. The results of this study will contribute to further investigations of Japanese black bears for monitoring their risk as a reservoir of possible zoonotic filarial parasites.

Newly-designed primer pairs for the detection of type 2 porcine reproductive and respiratory syndrome virus genes.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, caused by PRRS virus (PRRSV), that critically affects the swine industry. While the detection of PRRSV genes plays a key role in PRRS control, the PRRSV genome is known to undergo frequent mutation. Nevertheless, primer pairs widely used for the detection of PRRSV genes were designed between 1995 and 2010. The reliability of these primer pairs for the detection of currently circulating PRRSVs is therefore questionable. Here, we investigated the sensitivity of the previously reported primer pairs to detect PRRSV genes that have been recently isolated or detected in Japan. In addition, based on nucleotide sequences from the recent Japanese PRRSVs, we designed four new primer pairs for the detection of PRRSV genes. The sensitivity and specificity of the new primer pairs were evaluated by quantitative reverse transcription PCR using RNA extracted from PRRSV isolates, swine serum, and oral fluid specimens collected from PRRS-affected pigs, and swine sera collected from a PRRSV-free pig farm in Japan. One of novel primer pairs used in our study exhibited greater sensitivity than the previously reported primer pairs, and is thus more reliable for the detection of PRRSV genes.

Genetic Characterization of H5N8 Highly Pathogenic Avian Influenza Viruses Isolated from Falcated Ducks and Environmental Water in Japan in November 2020.

We isolated two highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N8 clade 2.3.4.4b from falcated duck (Anas falcata) feces and environmental water collected at an overwintering site in Japan. Our isolates were almost genetically identical to each other and showed high genetic similarity with H5N8 HPAIVs recently isolated in South Korea, a distant part of Japan, and European countries. These results suggest the potential role of falcated ducks in the dissemination of HPAIVs.

Detection and molecular characterization of Babesia sp. in wild boar (Sus scrofa) from western Japan.

Wild animals often act as reservoirs of tick-borne Babesia and Theileria spp., which cause piroplasmosis. Therefore, epidemiological investigations about the distribution of these parasites in wild animals are important for evaluating the transmission risk to humans and livestock. In this study, we surveyed Babesia and Theileria spp. infecting wild boar (Sus scrofa) in Kagoshima and Yamaguchi prefectures and Tsushima island, which are all in western Japan, and performed molecular genetic analyses on the samples. DNA was extracted from either blood or liver samples of wild boar captured in Kagoshima prefecture in 2015, 2016, and 2018 and from blood samples from wild boar captured in Yamaguchi prefecture in 2013-2015 and Tsushima island in 2018. PCR screening for the partial 18S ribosomal RNA gene (18S rRNA) of both Babesia and Theileria spp. in wild boar revealed that 63.9 % (140 of 219 samples) were positive. Sequencing of all positive samples revealed that they were all the same Babesia species. Subsequent phylogenetic analyses showed that the parasite is closely related to Babesia sp. previously detected in the hard tick, Amblyomma testudinarium in Kagoshima, and further analyses suggested that this species is genetically related to Babesia gibsoni. On the other hand, no Theileria were detected in any of the samples. In summary, we observed a high prevalence of B. gibsoni-like Babesia sp. in wild boar in western regions of Japan. The host range, distribution, pathogenicity, and life cycle of this protozoan should be further evaluated.

Complete Genome Sequences of Two Akabane Virus Strains Causing Bovine Postnatal Encephalomyelitis in Japan.

Akabane virus (AKAV) (genus Orthobunyavirus, family Peribunyaviridae) is an arthropod-borne virus that causes congenital abnormalities in ruminants. Here, we report the complete genome sequences of two AKAV strains causing nonsuppurative encephalomyelitis in cattle by postnatal infection in Japan.

Molecular detection of tick-borne protozoan parasites in sika deer (Cervus nippon) from western regions of Japan.

The sika deer (Cervus nippon) is one of the most common species of wildlife in Japan. This study aimed to reveal the prevalence of tick-borne protozoan parasites in wild sika deer living in western Japan. We used nested polymerase chain reaction (PCR) to detect the 18S rRNA gene of tick-borne apicomplexan parasites (Babesia, Theileria, and Hepatozoon spp.) from 276 blood and liver samples from sika deer captured in the Yamaguchi, Oita, Kagoshima, Okayama, Ehime, Kochi, and Tokushima Prefectures. In total, 259 samples (259/276; 93.8%) tested positive in the nested PCR screening. Gene sequencing revealed that 99.6% (258/259) of positive samples contained Theileria sp. (sika 1), while Theileria sp. (sika 2), another Theileria species, was detected in only 3 samples. We also found that one sample from a sika deer captured in Kagoshima contained the gene of an unidentified Babesia sp. related to Babesia sp. Kh-Hj42, which was previously collected from tick in western Siberia. In conclusion, we found a high prevalence of piroplasms in sika deer from western Japan, and DNA analysis revealed that Theileria sp. (sika 1) had the highest infection rate.

Seroprevalence of Cryptosporidium parvum and Neospora caninum in cattle in the southern Kyushu region of Japan.

Cryptosporidium parvum and Neospora caninum are common parasites in domesticated cattle worldwide, including in Japan. We carried out a serological survey to detect C. parvum and N. caninum infection among cattle in the southern Kyushu region of Japan-including the small islands-by indirect enzyme-linked immunosorbent assay based on recombinant antigens. We found that total seropositivity in 570 Japanese black cattle was 96.3% for C. parvum and 18.4% for N. caninum. Although seroprevalence was correlated with cattle age, differences in the seroprevalence of C. parvum among age groups were not statistically significant. On the other hand, N. caninum seroprevalence increased with age, suggesting horizontal transmission through ingestion of food or water contaminated with oocysts. These findings underscore the importance of monitoring C. parvum and N. caninum in cattle and implementing measures to prevent the spread of infection to other livestock and to humans.