Pathogenicity of genotype 2.1 classical swine fever virus isolated from Japan in 2019 in pigs.

Classical swine fever (CSF) re-emerged in Japan in 2018 for the first time in 26 years. The disease has been known to be caused by a moderately pathogenic virus, rather than the highly pathogenic virus that had occurred in the past. However, the underlying pathophysiology remains unknown. This study conducted an experimental challenge on specific pathogen-free (SPF) pigs in a naïve state for 2, 4, and 6 weeks and confirmed the disease state during each period by clinical observation, virus detection, and pathological necropsy. We revealed the pathological changes and distribution of pathogens and virus-specific antibodies at each period after virus challenge. These results were comprehensively analyzed and approximately 70% of the pigs recovered, especially at 4- and 6-week post-virus challenge. This study provides useful information for future countermeasures against CSF by clarifying the pathogenicity outcomes in unvaccinated pigs with moderately pathogenic genotype 2.1 virus.

Diverse Molecular Mechanisms Underlying Microbe-Inducing Male Killing in the Moth Homona magnanima.

Male killing (MK) is a type of reproductive manipulation induced by microbes, where sons of infected mothers are killed during development. MK is a strategy that enhances the fitness of the microbes, and the underlying mechanisms and the process of their evolution have attracted substantial attention. Homona magnanima, a moth, harbors two embryonic MK bacteria, namely, Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), and a larval MK virus, Osugoroshi virus (OGV; Partitiviridae). However, whether the three distantly related male killers employ similar or different mechanisms to accomplish MK remains unknown. Here, we clarified the differential effects of the three male killers on the sex-determination cascades and development of H. magnanima males. Reverse transcription-PCR demonstrated that Wolbachia and Spiroplasma, but not OGVs, disrupted the sex-determination cascade of males by inducing female-type splice variants of doublesex (dsx), a downstream regulator of the sex-determining gene cascade. We also found that MK microbes altered host transcriptomes in different manners; Wolbachia impaired the host dosage compensation system, whereas Spiroplasma and OGVs did not. Moreover, Wolbachia and Spiroplasma, but not OGVs, triggered abnormal apoptosis in male embryos. These findings suggest that distantly related microbes employ distinct machineries to kill males of the identical host species, which would be the outcome of the convergent evolution. IMPORTANCE Many microbes induce male killing (MK) in various insect species. However, it is not well understood whether microbes adopt similar or different MK mechanisms. This gap in our knowledge is partly because different insect models have been examined for each MK microbe. Here, we compared three taxonomically distinct male killers (i.e., Wolbachia, Spiroplasma, and a partiti-like virus) that infect the same host. We provided evidence that microbes can cause MK through distinct mechanisms that differ in the expression of genes involved in sex determination, dosage compensation, and apoptosis. These results imply independent evolutionary scenarios for the acquisition of their MK ability.

Establishment of Intestinal Organoid from Rousettus leschenaultii and the Susceptibility to Bat-Associated Viruses, SARS-CoV-2 and Pteropine Orthoreovirus.

Various pathogens, such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, are threatening human health worldwide. The natural hosts of these pathogens are thought to be bats. The rousette bat, a megabat, is thought to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Additionally, the rousette bat showed a transient infection in the experimental inoculation of SARS-CoV-2. In the current study, we established and characterized intestinal organoids from Leschenault's rousette, Rousettus leschenaultii. The established organoids successfully recapitulated the characteristics of intestinal epithelial structure and morphology, and the appropriate supplements necessary for long-term stable culture were identified. The organoid showed susceptibility to Pteropine orthoreovirus (PRV) but not to SARS-CoV-2 in experimental inoculation. This is the first report of the establishment of an expandable organoid culture system of the rousette bat intestinal organoid and its sensitivity to bat-associated viruses, PRV and SARS-CoV-2. This organoid is a useful tool for the elucidation of tolerance mechanisms of the emerging rousette bat-associated viruses such as Ebola and Marburg virus.

Genetic diversity of enterovirus G detected in faecal samples of wild boars in Japan: identification of novel genotypes carrying a papain-like cysteine protease sequence.

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.

Complete genome sequencing and genetic analysis of a Japanese porcine torovirus strain detected in swine feces.

We sequenced the complete genome of a porcine torovirus (PToV) strain from Japan for the first time. Whole-genome analysis revealed that this strain (Iba/2018) has a mosaic sequence composed of at least three genome backgrounds, related to US, Chinese and German PToV strains. Clear recombination breakpoints were detected in the M and HE coding regions. A similarity plot and structural analysis demonstrated that the HE coding region exhibits the highest diversity, and the most sequence variation was found in the lectin domain. PToVs were divided into two lineages in the HE region, whereas clear lineages were not found in other regions.

Establishment of a novel experimental model for muscle-invasive bladder cancer using a dog bladder cancer organoid culture.

In human and dogs, bladder cancer (BC) is the most common neoplasm affecting the urinary tract. Dog BC resembles human muscle-invasive BC in histopathological characteristics and gene expression profiles, and could be an important research model for this disease. Cancer patient-derived organoid culture can recapitulate organ structures and maintains the gene expression profiles of original tumor tissues. In a previous study, we generated dog prostate cancer organoids using urine samples, however dog BC organoids had never been produced. Therefore we aimed to generate dog BC organoids using urine samples and check their histopathological characteristics, drug sensitivity, and gene expression profiles. Organoids from individual BC dogs were successfully generated, expressed urothelial cell markers (CK7, CK20, and UPK3A) and exhibited tumorigenesis in vivo. In a cell viability assay, the response to combined treatment with a range of anticancer drugs (cisplatin, vinblastine, gemcitabine or piroxicam) was markedly different in each BC organoid. In RNA-sequencing analysis, expression levels of basal cell markers (CK5 and DSG3) and several novel genes (MMP28, CTSE, CNN3, TFPI2, COL17A1, and AGPAT4) were upregulated in BC organoids compared with normal bladder tissues or two-dimensional (2D) BC cell lines. These established dog BC organoids might be a useful tool, not only to determine suitable chemotherapy for BC diseased dogs but also to identify novel biomarkers in human muscle-invasive BC. In the present study, for the 1st time, dog BC organoids were generated and several specifically upregulated organoid genes were identified. Our data suggest that dog BC organoids might become a new tool to provide fresh insights into both dog BC therapy and diagnostic biomarkers.

Phylogenetic analysis of novel posaviruses detected in feces of Japanese pigs with posaviruses and posa-like viruses of vertebrates and invertebrates.

Posaviruses and posa-like viruses are unclassified viruses with sequence similarity to viruses of the order Picornavirales. They have been reported in various vertebrates and invertebrates. We identified 11 posavirus-like sequences in porcine feces and performed phylogenic analysis. Previously reported Japanese posaviruses and those identified in this study clustered with posavirus 1, 4, and 7 and husavirus 1, while five viruses branched into three independent lineages, tentatively named posavirus 10, 11, and 12. Interestingly, posaviruses, except for posavirus 8 and 9, husaviruses, and rasaviruses, formed a cluster consisting of viruses only from pigs, humans, and rats, while posavirus 8 and 9, fisavirus, and basaviruses clustered with posa-like viruses from invertebrates.

Complete genomic analysis and molecular characterization of Japanese porcine sapeloviruses.

The Porcine Sapelovirus (PSV) is an enteric virus of pigs that can cause various disorders. However, there are few reports that describe the molecular characteristics of the PSV genome. In this study, almost the entire genomes of 23 PSVs detected in Japanese pigs were analyzed using bioinformatics. Analysis of the cis-active RNA elements showed that the predicted secondary structures of the internal ribosome entry site in the 5' untranslated region (UTR) and a cis-replication element in the 2C coding region were conserved among PSVs. In contrast, those at the 3' UTR were different for different PSVs; however, tertiary structures between domains were conserved across all PSVs. Phylogenetic analysis of nucleotide sequences of the complete VP1 region showed that PSVs exhibited sequence diversity; however, they could not be grouped into genotypes due to the low bootstrap support of clusters. The insertion and/or deletion patterns in the C-terminal VP1 region were not related to the topology of the VP1 tree. The 3CD phylogenetic tree was topologically different from the VP1 tree, and PSVs from the same country were clustered independently. Recombination analysis revealed that recombination events were found upstream of the P2 region and some recombination breakpoints involved insertions and/or deletions in the C-terminal VP1 region. These findings demonstrate that PSVs show genetic diversity and frequent recombination events, particularly in the region upstream of the P2 region; however, PSVs could currently not be classified into genotypes and conserved genetic structural features of the cis-active RNA elements are observed across all PSVs.

An ascovirus isolated from Spodoptera litura (Noctuidae: Lepidoptera) transmitted by the generalist endoparasitoid Meteorus pulchricornis (Braconidae: Hymenoptera).

The family Ascoviridae is a recently described virus family whose members are transmitted by parasitoids and cause chronic and lethal infections in lepidopteran insects. Little is known about the biology and ecology of ascoviruses, and few isolates have been found outside the United States. We report here the isolation of a new ascovirus variant from Spodoptera litura in Japan. Full genome sequence and phylogenetic analyses showed that this virus was closely related to variants in Heliothis virescens ascovirus-3a, and it was named HvAV-3j. HvAV-3j has a DNA genome of 191 718 bp, with 189 putative ORFs and a GC content of 45.6 %, and is highly similar to HvAV-3h, which was isolated in China. In a field survey, the endoparasitoid Meteorus pulchricornis caused a high percentage of parasitization in populations of S. litura larvae, and under laboratory conditions M. pulchricornis was able to transmit HvAV-3j from infected to uninfected larvae by oviposition. Meteorus pulchricornis is thus likely to be a major vector for HvAV-3j transmission in Japan. This species is recognized here for the first time as a vector of ascoviruses that parasitizes a range of host species that extends across families.

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system.

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples.

Involvement of the 3' Untranslated Region in Encapsidation of the Hepatitis C Virus.

Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3' end- to 5' end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3' untranslated region (UTR) with Core in vitro was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3' UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in trans. Mutations within the conserved stem-loops of the 3' UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3´ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3' UTR acts as a cis-acting element for encapsidation.

Whole genome analysis of a novel neurotropic bovine astrovirus detected in a Japanese black steer with non-suppurative encephalomyelitis in Japan.

While neurotropic bovine astroviruses (BoAstVs) have been identified in North America and Europe, their presence has never been reported in Asia. In this study, we detected BoAstV in the brain of a steer showing neurological signs. Phylogenetic analysis revealed that the identified virus belongs to the Virginia/Human-Mink-Ovine clade, which contains most of the neurotropic astroviruses including the neurotropic BoAstVs. Similarity plot analysis showed that the virus was closely related to the American BoAstV NeuroS1 strain with respect to the ORF regions and to the European BoAstV CH13 strain in the 3' untranslated region, suggesting the occurrence of intra-genotypic recombination events.

Application of the SureSelect target enrichment system for next-generation sequencing to obtain the complete genome sequence of bovine leukemia virus.

In this study, the SureSelect target enrichment system for Illumina Multiplexed Sequencing was applied to proviral DNA sequencing of bovine leukemia virus (BLV). The complete genomic DNA sequences of four Vietnamese BLV strains were successfully obtained with high read depth values and a genome coverage of 100% across all sequenced samples, in less than one week. This study provides the first complete Vietnamese BLV genome sequences. Their genetic variability and phylogenetic relationship were also analyzed and compared with those of 28 whole BLV genome sequences from different parts of the world. The results obtained provided new insights into the genetic diversity of the BLV tax gene, and further enabled us to identify nucleotide mutations in the gene that might not have been detected with the commercial detection kit that is currently available.

Correction to: Application of the SureSelect target enrichment system for next-generation sequencing to obtain the complete genome sequence of bovine leukemia virus.

Unfortunately, Figure 1 was incorrectly published in the original publication and the correct version is updated here.

Isolation of Pteropine orthoreovirus from Pteropus vampyrus in Garut, Indonesia.

Flying foxes belonging to the genus Pteropus are known to be reservoirs of zoonotic viruses. In this study, we describe the isolation of Pteropine orthoreovirus (PRV) from rectal swab samples of Pteropus vampyrus in Indonesia. PRV is an emerging zoonotic respiratory virus that can be transmitted from bats to humans. Rectal swabs (n = 91) were screened by PCR for PRV and 10 (11%) were positive. Phylogenetic analysis based on nucleotide sequences indicated that the S2, S3, S4, M3, L2, and L3 segments of one isolate (Garut-69) were closely related to previously isolated strains in Indonesia. The remaining gene segments showed both similarity and genetic divergence with other PRV strains, suggesting that re-assortment events had occurred. This is the first report of PRV infection to P. vampyrus in West Java, Indonesia.

Malaria knowledge, preventive actions, and treatment-seeking behavior among ethnic minorities in Ratanakiri Province, Cambodia: a community-based cross-sectional survey.

BACKGROUND: Malaria incidence has been steadily declining in Cambodia, where the government is aiming to eliminate malaria by 2025. Successful malaria elimination requires active engagement and participation of communities to recognize malaria symptoms and the development of prompt treatment-seeking behavior for early diagnosis and appropriate treatment. This study examined malaria knowledge, preventive actions, and treatment-seeking behavior among different groups of ethnic minorities and Khmer in Ratanakiri Province, Cambodia. METHODS: Face-to-face interviews were conducted in December 2015, targeting 388 mothers with children under 2 years old, who belonged to ten ethnic minority groups or the Khmer group living in 62 rural villages in Ratanakiri. In addition to describing mothers' knowledge and actions for malaria prevention, logistic regression analysis was performed to identify determinants of fever during the most recent pregnancy and among children under two. RESULTS: Overall 388 mothers were identified for enrollment into the study of which 377 (97.2%) were included in analyses. The majority of mothers slept under bed nets at home (95.8%) and wore long-sleeved clothes (83.8%) for malaria prevention. However, knowledge of malaria was limited: 44.6% were aware of malaria symptoms, 40.6% knew the malaria transmission route precisely, and 29.2% knew of mosquito breeding places. Staying overnight at a farm hut was significantly associated with having fever during the most recent pregnancy (adjusted odds ratio [AOR] 2.008, 95% confidence interval [CI]: 1.215-3.321) and a child having fever (AOR 3.681, 95% CI 1.943-6.972). Mothers' partaking in a variety of malaria preventive actions was protective against fever in children (AOR 0.292, 95% CI: 0.136-0.650). Among those who had fever during pregnancy, 39.4% did not seek treatment. CONCLUSION: Although the majority of mothers took malaria preventive actions, knowledge of malaria epidemiology and vector ecology and treatment-seeking behavior for fever were limited. Staying overnight at farm huts, regardless of the differences in socio-demographic and socio-cultural characteristics, was strongly associated with fever episodes during pregnancy and childhood. This study indicates the necessity of spreading accurate malaria knowledge, raising awareness of health risks related to agricultural practices, and promoting treatment-seeking behavior among ethnic minorities to strengthen their engagement in malaria elimination.

Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein.

BACKGROUND: Bovine enterovirus (BEV) belongs to the species Enterovirus E or F, genus Enterovirus and family Picornaviridae. Although numerous studies have identified BEVs in the feces of cattle with diarrhea, the pathogenicity of BEVs remains unclear. Previously, we reported the detection of novel kobu-like virus in calf feces, by metagenomics analysis. In the present study, we identified a novel BEV in diarrheal feces collected for that survey. Complete genome sequences were determined by deep sequencing in feces. Secondary RNA structure analysis of the 5' untranslated region (UTR), phylogenetic tree construction and pairwise identity analysis were conducted. RESULTS: The complete genome sequences of BEV were genetically distant from other EVs and the VP1 coding region contained novel and unique amino acid sequences. We named this strain as BEV AN12/Bos taurus/JPN/2014 (referred to as BEV-AN12). According to genome analysis, the genome length of this virus is 7414 nucleotides excluding the poly (A) tail and its genome consists of a 5'UTR, open reading frame encoding a single polyprotein, and 3'UTR. The results of secondary RNA structure analysis showed that in the 5'UTR, BEV-AN12 had an additional clover leaf structure and small stem loop structure, similarly to other BEVs. In pairwise identity analysis, BEV-AN12 showed high amino acid (aa) identities to Enterovirus F in the polyprotein, P2 and P3 regions (aa identity ≥82.4%). Therefore, BEV-AN12 is closely related to Enterovirus F. However, aa sequences in the capsid protein regions, particularly the VP1 encoding region, showed significantly low aa identity to other viruses in genus Enterovirus (VP1 aa identity ≤58.6%). In addition, BEV-AN12 branched separately from Enterovirus E and F in phylogenetic trees based on the aa sequences of P1 and VP1, although it clustered with Enterovirus F in trees based on sequences in the P2 and P3 genome region. CONCLUSIONS: We identified novel BEV possessing highly divergent aa sequences in the VP1 coding region in Japan. According to species definition, we proposed naming this strain as "Enterovirus K", which is a novel species within genus Enterovirus. Further genomic studies are needed to understand the pathogenicity of BEVs.

Molecular characteristics and prevalence of small ruminant lentiviruses in goats in Japan.

Small ruminant lentiviruses (SRLVs), which comprise caprine arthritis-encephalitis virus (CAEV) and maedi-visna virus (MVV), are prevalent in goats and sheep worldwide, including in Japan. However, little is known about the molecular characteristics of goat lentiviruses in Japan. In this study, a molecular and phylogenetic analysis of the long gag region was performed. The phylogenic tree demonstrated that all samples belonged to SRLV subtype B1. Two clusters were identified, with one cluster distinct from previously reported strains of subtype B1. In addition, several alterations in the amino acid sequence were detected in immunodominant epitopes of the gag region. To gain a deeper understanding of the genetic diversity of SRLVs in Japan, it will be necessary to increase the sample size and conduct a broader survey. The present report is important for establishing baseline information on the prevalence of SRLV in Japan and providing data to develop a new, more sensitive diagnostic test for effective control of SRLV.

Characterization of cross-clade monoclonal antibodies against H5N1 highly pathogenic avian influenza virus and their application to the antigenic analysis of diverse H5 subtype viruses.

H5N1 highly pathogenic avian influenza viruses (HPAIVs) are a threat to both animal and public health and require specific and rapid detection for prompt disease control. We produced three neutralizing anti-hemagglutinin (HA) monoclonal antibodies (mAbs) using two clades (2.2 and 2.5) of the H5N1 HPAIV isolated in Japan. Blocking immunofluorescence tests showed that each mAb recognized different epitopes; 3B5.1 and 3B5.2 mAbs against the clade 2.5 virus showed cross-clade reactivity to all 26 strains from clades 1, 2.2, 2.3.2.1, 2.3.2.1a, b, c and 2.3.4, suggesting that the epitope(s) recognized are conserved. Conversely, the 1G5 mAb against the clade 2.2 virus showed reactivity to only clades 1, 2.3.4 and 2.5 strains. An analysis of escape mutants, and some clades of the H5N1 viruses recognized by 3B5.1 and 3B5.2 mAbs, suggested that the mAbs bind to an epitope, including amino acid residues at position 162 in the HA1 protein (R162 and K162). Unexpectedly, however, when five Eurasian-origin H5 low-pathogenic AIV (LPAIV) strains with R162 were examined (EA-nonGsGD clade) as well as two American-origin strains (Am-nonGsGD clade), the mAb recognized only EA-nonGsGD clade strains. The R162 and K162 residues in the HA1 protein were highly conserved among 36 of the 43 H5N1 clades reported, including clades 2.3.2.1a and 2.3.2.1c that are currently circulating in Asia, Africa and Europe. The amino acid residues (158-PTIKRSYNNTNQE-170) in the HA1 protein are probably an epitope responsible for the cross-clade reactivity of the mAbs, considering the epitopes reported elsewhere. The 3B5.1 and 3B5.2 mAbs may be useful for the specific detection of H5N1 HPAIVs circulating in the field.

First isolation and characterization of pteropine orthoreoviruses in fruit bats in the Philippines.

Pteropine orthoreovirus (PRV) causes respiratory tract illness (RTI) in humans. PRVs were isolated from throat swabs collected from 9 of 91 wild bats captured on the Mindanao Islands, The Philippines, in 2013. The nucleic acid sequence of the whole genome of each of these isolates was determined. Phylogenetic analysis based on predicted amino acid sequences indicated that the isolated PRVs were novel strains in which re-assortment events had occurred in the viral genome. Serum specimens collected from 76 of 84 bats were positive for PRV-neutralizing antibodies suggesting a high prevalence of PRV in wild bats in the Philippines. The bat-borne PRVs isolated in the Philippines were characterized in comparison to an Indonesian PRV isolate, Miyazaki-Bali/2007 strain, recovered from a human patient, revealing that the Philippine bat-borne PRVs had similar characteristics in terms of antigenicity to those of the Miyazaki-Bali/2007 strain, but with a slight difference (e.g., growth capacity in vitro). The impact of the Philippine bat-borne PRVs should be studied in human RTI cases in the Philippines.