Whole-genome analysis of two bovine rotavirus C strains: Shintoku and Toyama.

Rotavirus C (RVC) has been detected frequently in epidemic cases and/or outbreaks of diarrhoea in humans and animals worldwide. Because it is difficult to cultivate RVCs serially in cell culture, the sequence data available for RVCs are limited, despite their potential economical and epidemiological impact. Although whole-genome sequences of one porcine RVC and seven human RVC strains have been analysed, this has not yet been done for a bovine RVC strain. In the present study, we first determined the nucleotide sequences for five as-yet under-researched genes, including the NSP4 gene, from a cultivable bovine RVC, the Shintoku strain, identified in Hokkaido Prefecture, Japan, in 1991. In addition, we elucidated the ORF sequences of all segments from another bovine RVC, the Toyama strain, detected in Toyama Prefecture, Japan, in 2010, in order to investigate genetic divergence among bovine RVCs. Comparison of segmental nucleotide and deduced amino acid sequences among RVCs indicates high identity among bovine RVCs and low identity between human and porcine RVCs. Phylogenetic analysis of each gene showed that the two bovine RVCs belong to a cluster distinct from human and porcine RVCs. These data demonstrate that RVCs can be classified into different genotypes according to host species. Moreover, RVC NSP1, NSP2 and VP1 amino acid sequences contain a unique motif that is highly conserved among rotavirus A (RVA) strains and, hence, several proteins from bovine RVCs are suggested to play important roles that are similar to those of RVAs.

Efficacy of commercial live vaccines against QX-like infectious bronchitis virus in Japan.

Infectious bronchitis, an acute and highly contagious disease that affects chickens, is caused by the infectious bronchitis virus (IBV). The antigenic variant QX-like IBV was first reported in China in 1996 and is now endemic in many countries. Our previous study reported the first detection and isolation of QX-like IBVs in Japan and that they were genetically related to the recently detected strains in China and South Korea. The pathogenicity of 2 Japanese QX-like IBV strains (JP/ZK-B7/2020 and JP/ZK-B22/2020) was evaluated by inoculating specific pathogen-free (SPF) chickens with 102 to 106 median embryo infectious dose. Both strains caused clinical signs of respiratory symptoms, gross tracheal lesions, and moderate-to-severe suppression of tracheal ciliostasis. To evaluate the efficacy of commercial IBV live vaccines against the JP/ZK-B7/2020 strain, vaccinated SPF chickens were challenged with the JP/ZK-B7/2020 strain at 104 EID50 (median embryo infectious dose). Only the JP-Ⅲ vaccine provided high levels of protection (reduced suppression of tracheal ciliostasis and reduced viral loads in organs), whereas the Mass vaccine showed little protective effect. Virus neutralization test results and comparisons between IBV genotypes based on the S1 gene suggested that QX-like and JP-III genotypes were closely related. These results suggest that the JP-III IBV vaccine, which has relatively high S1 gene homology with QX-like IBVs, is effective against Japanese QX-like IBV strain.

Molecular Prevalence Study of QX-like Infectious Bronchitis Virus in Japan from 2016 to 2023.

Infectious bronchitis is an acute and highly contagious disease in chickens caused by the infectious bronchitis virus (IBV), which has caused significant economic losses to the poultry industry worldwide. The antigenic variant, the QX-like genotype (GI-19 lineage), has been currently reported in epidemics in East Asia, Southeast Asia, the Middle East, Europe, and Africa. We first reported an epidemic of Japanese QX-like IBVs genetically related to Chinese and South Korean strains in the Kyushu region of Japan in 2020. However, because their nationwide prevalence was unknown, we performed a nationwide survey. The testing of 419 reverse transcription (RT)-PCR-positive samples (376 layers and 43 broilers) of IBV field strains between April 2016 and March 2022 detected two QX-like IBVs in 2019 and 2021 broiler samples from one region. A survey of fecal samples collected from 122-layer farms nationwide between November 2022 and January 2023 detected QX-like IBV genes from seven farms in various regions. Phylogenetic tree analysis on the basis of the S1 gene showed that all QX-like IBVs detected in Japan were genetically related to recent Chinese and South Korean strains. A new RT-PCR assay was developed to distinguish between QX-like IBV and other IBV variants prevalent in Japan, whose results were consistent with those of previously reported identification methods. These results suggest that QX-like IBV is rapidly spreading in Japan and that countermeasures are necessary.

Estudio de la prevalencia molecular del virus de la bronquitis infecciosa similar a la cepa QX en Japón entre los años 2016 al 2023. La bronquitis infecciosa es una enfermedad aguda y altamente contagiosa del pollo causada por el virus de la bronquitis infecciosa (IBV), que ha causado importantes pérdidas económicas a la industria avícola en todo el mundo. Actualmente se ha reportado la variante antigénica, el genotipo similar a la cepa QX (cepa GI-19), en brotes en el este de Asia, el sudeste de Asia, el Medio Oriente, Europa y África. Primeramente, se reportó un brote con virus japoneses similares a QX que eran genéticamente relacionados con cepas chinas y surcoreanas en la región de Kyushu en Japón en el 2020. Sin embargo, debido a que se desconocía su prevalencia a nivel nacional, se realizó una encuesta a nivel nacional. Mediante el análisis de 419 muestras positivas a la presencia de cepas de campo por transcripción reversa (RT) y PCR (376 de ponedoras y 43 de pollos de engorde) entre abril del 2016 y marzo del 2022 se detectaron dos virus de bronquitis infecciosa similares a la cepa QX en el 2019 y en el 2021 en muestras de pollos de engorde de una región. Una encuesta de muestras fecales recolectadas de granjas de 122 ponedoras en todo el país entre noviembre del 2022 y enero del 2023 detectó genes de bronquitis infecciosa similares a la cepa QX de siete granjas en varias regiones. El análisis del árbol filogenético basado en el gene S1 mostró que todos los virus similares a la cepa QX detectados en Japón estaban relacionados genéticamente con cepas recientes de China y Corea del Sur. Se desarrolló un nuevo ensayo de RT-PCR para distinguir entre los virus de bronquitis infecciosa similares a la cepa QX y otras variantes del IBV prevalentes en Japón, cuyos resultados fueron consistentes con los de los métodos de identificación reportados anteriormente. Estos resultados sugieren que virus de bronquitis infecciosa similares a la cepa QX se están extendiendo rápidamente en Japón y que se necesitan medidas de control.

Detection and isolation of QX-like infectious bronchitis virus in Japan.

The antigenic variant QX-like infectious bronchitis virus (IBV) is endemic in several countries. In Japan, the QX-like genotype is classified as the JP-III genotype based on the partial S1 gene and as the GI-19 genotype based on the complete S1 gene. This study showed that QX-like IBVs and JP-III IBVs can be identified based on the amino acid polymorphism of the S1 glycoprotein. Furthermore, genetic analysis of several IBV field strains detected in commercial broiler farms across the Kyushu area in 2020 revealed Japanese QX-like IBVs, which are highly homologous to the QX-like IBVs recently detected in China and South Korea. Herein, QX-like IBV field strains were isolated for evaluating commercial vaccine efficacy in our future studies.

Immunobiotic Bifidobacteria Strains Modulate Rotavirus Immune Response in Porcine Intestinal Epitheliocytes via Pattern Recognition Receptor Signaling.

In this work, we aimed to characterize the antiviral response of an originally established porcine intestinal epithelial cell line (PIE cells) by evaluating the molecular innate immune response to rotavirus (RVs). In addition, we aimed to select immunomodulatory bacteria with antiviral capabilities. PIE cells were inoculated with RVs isolated from different host species and the infective titers and the molecular innate immune response were evaluated. In addition, the protection against RVs infection and the modulation of immune response by different lactic acid bacteria (LAB) strains was studied. The RVs strains OSU (porcine) and UK (bovine) effectively infected PIE cells. Our results also showed that RVs infection in PIE cells triggered TLR3-, RIG-I- and MDA-5-mediated immune responses with activation of IRF3 and NF-κB, induction of IFN-ß and up-regulation of the interferon stimulated genes MxA and RNase L. Among the LAB strains tested, Bifidobacterium infantis MCC12 and B. breve MCC1274 significantly reduced RVs titers in infected PIE cells. The beneficial effects of both bifidobacteria were associated with reduction of A20 expression, and improvements of IRF-3 activation, IFN-ß production, and MxA and RNase L expressions. These results indicate the value of PIE cells for studying RVs molecular innate immune response in pigs and for the selection of beneficial bacteria with antiviral capabilities.

Isolation and experimental inoculation of an S INDEL strain of porcine epidemic diarrhea virus in Japan.

In 2013, porcine epidemic diarrhea (PED) was reported in Japan for the first time in 7years and caused significant economic losses. In the present study, we isolated PED virus (PEDV) circulating in Japan using Vero cell cultures and analyzed sequences of S1 genes of these PEDV isolates. Sequence analysis revealed that one of these strains contained distinct insertion and deletions in the S gene (i.e., S INDEL). Furthermore, inoculation of PEDV into 1-week-old pigs demonstrated that the S INDEL strain had a lower pathogenicity than the North American (NA) prototype strain. This is the first report comparing pathogenicity of an S INDEL strain with the NA prototype strain following experimental inoculation. Excretion of PEDV in the feces of S INDEL strain-inoculated pigs occurred later than in NA prototype strain-inoculated pigs. Thus, our findings suggested that the S INDEL strain had different viral dynamics than the NA prototype strain.

Sequence and phylogenetic analyses of nonstructural protein 2 genes of species B porcine rotaviruses detected in Japan during 2001-2009.

Porcine rotavirus B (RVB) has been often detected in diarrhea of suckling and weaned pigs. Because it is difficult to serially cultivate RVBs in cell culture, the number of available sequence data for RNA segments other than VP7 and NSP1 in especially porcine RVBs is still limited. We performed genetic analysis focusing on nonstructural protein 2 (NSP2) using several porcine RVB strains, which were detected in diarrheic feces collected around Japan during 2001-2009. Comparison of NSP2 nucleotide and deduced amino acid sequences from porcine RVB strains exhibited low identities (64.0-99.9% in nt and 66.7-100.0% in aa) to those of other RVB strains. Phylogenetic analysis of RVB NSP2 revealed the presence of four clusters (N1-N4) including human plus murine, bovine and porcine clusters with cut-off values of 75% at the nt and 85% at the aa level. Furthermore, the NSP2 genes of porcine RVBs were divided into three genotypes, of which some porcine RVBs belonged into bovine-cluster. PB-70-H5 and PB-70-H3, which belonged to same pig farm, might be identical in NSP2 gene as shown sequence identity of 99.9%, nevertheless both had different VP7 genes each other. Thus, this data demonstrates the occurrence of gene reassortment among porcine RVBs derived from same pig farm. Our findings presented here would provide more valuable information to elucidate evolution of RVBs.

Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains.

Porcine rotavirus B (RVB) has frequently been detected in the diarrhea of suckling and weaned pigs. Because it is difficult to propagate RVBs serially in cell culture, little genetic information about RNA segments other than VP7, NSP1 and NSP2 is available for porcine RVBs. We conducted a phylogenetic analysis focusing on nonstructural protein 5 (NSP5) using 22 porcine RVB strains, which were identified in fecal samples collected around Japan. Sequence analysis showed that NSP5 genes of RVBs contain one ORF, in contrast to the corresponding gene of RVAs that have two ORFs. Comparison of NSP5 amino acid sequences from porcine RVBs with other RVBs revealed that eight serine and serine/threonine residues at the N- and C-terminal regions are highly conserved among RVBs. Phylogenetic analysis also indicated the presence of six clusters (H1-H6) including rat, human, porcine and bovine plus ovine clusters with cut-off values of 78% at the nucleotide level. Moreover, the NSP5 genes of porcine RVBs were divided to three clusters. The data presented here demonstrates that several porcine RVBs with distinctive genotypes are circulating among farms throughout Japan. Our findings provide important new insights into the evolutionary dynamics of RVBs.