Detection, sequencing, and tissue distribution of piscine orthoreovirus 2-like virus in diseased coho salmon in Alaska.

We performed a diagnostic disease investigation on a cohort of coho salmon (Oncorhynchus kisutch) fingerlings in Alaska exhibiting anorexia, gaping mouths, anemia, and increased mortality. Histologic examination revealed mild-to-severe myocardial degeneration and lymphohistiocytic and neutrophilic myocarditis, moderate splenic histiocytosis, and mild renal histiocytosis. Piscine orthoreoviruses 1 and 3 were not detected by molecular methods, and no other viruses could be cultured on 3 common diagnostic fish cell lines. De novo assembly produced a viral genome of 10 linear segments with >80% homology to piscine orthoreovirus 2 (PRV2) encoding all 11 PRV2 proteins. An in situ hybridization probe using RNAscope was developed against 697 viral nucleotides identified by sequencing, which revealed viral genome in heart, spleen, gill, kidney, liver, blood, and the lamina propria of the intestines. Our findings are supportive of a novel piscine orthoreovirus most closely related to PRV2 associated with morbidity and mortality of coho salmon in the northeastern Pacific.

IL-6/STAT3 axis is hijacked by GCRV to facilitate viral replication via suppressing type â IFN signaling.

Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type Ⅰ IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.

Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus), Spain, 2022.

Epizootic hemorrhagic disease (EHD) virus serotype 8 (EHDV-8) emerged in Spain in autumn 2022. In this study, we aimed to (1) characterize the clinical and lesional presentation of EHDV infection in European red deer (Cervus elaphus), and (2) study the spatial spread of the virus in wild ruminants in Spain after its introduction, in 2022/2023. We confirmed EHDV infection in two clinically compatible sick red deer by PCR and detection of anti-EHDV specific antibodies. EHDV infection occurred in red deer with hyperacute to acute clinical signs and lesions associated to vascular changes leading to death of the animals. Partial sequences of variable segment 2 (VP2) and segment 5 (NS1) genes of the detected viruses had >99% nucleotide identity with EHDV-8 sequences from Tunisia and Italy. In a cross-sectional serological study of EHDV in 592 wild ruminants, mainly red deer (n=578), in southwestern Spain, we detected anti-EHDV antibodies in 37 of 592 samples (6.3%; 95% confidence interval: 4.3-8.2), all from red deer and from the localities where clinical cases of EHD were confirmed in red deer. We conclude that EHDV-8 infection causes severe EHD in European red deer. The serosurvey revealed a limited spread of EHDV-8 in Spanish wild ruminant populations in the first year of virus detection in Spain.

Analyses and Insights into Genetic Reassortment and Natural Selection as Key Drivers of Piscine orthoreovirus Evolution.

Piscine orthoreovirus (PRV) is a pathogen that causes heart and skeletal muscle inflammation in Salmo salar and has also been linked to circulatory disorders in other farmed salmonids, such as Oncorhynchus kisutch and Oncorhynchus mykiss. The virus has a segmented, double-stranded RNA genome, which makes it possible to undergo genetic reassortment and increase its genomic diversity through point mutations. In this study, genetic reassortment in PRV was assessed using the full genome sequences available in public databases. This study used full genome sequences that were concatenated and genome-wide reassortment events, and phylogenetic analyses were performed using the recombination/reassortment detection program version 5 (RDP5 V 5.5) software. Additionally, each segment was aligned codon by codon, and overall mean distance and selection was tested using the Molecular Evolutionary Genetics Analysis X software, version 10.2 (MEGA X version 10.2). The results showed that there were 17 significant reassortment events in 12 reassortant sequences, involving genome exchange between low and highly virulent genotypes. PRV sequences from different salmonid host species did not appear to limit the reassortment. This study found that PRV frequently undergoes reassortment events to increase the diversity of its segmented genome, leading to antigenic variation and increased virulence. This study also noted that to date, no reassortment events have been described between PRV-1 and PRV-3 genotypes. However, the number of complete genomic sequences within each genotype is uneven. This is important because PRV-3 induces cross-protection against PRV-1, making it a potential vaccine candidate.

Deep sequencing identified miR-193b-3p as a positive regulator of autophagy targeting Akt3 in Ctenopharyngodon idella CIK cells during GCRV infection.

Recent research has highlighted complex and close interaction between miRNAs, autophagy, and viral infection. In this study, we observed the autophagy status in CIK cells infected with GCRV at various time points. We found that GCRV consistently induced cellar autophagy from 0 h to 12 h post infection. Subsequently, we performed deep sequencing on CIK cells infected with GCRV at 0 h and 12 h respectively, identifying 38 DEMs and predicting 9581 target genes. With the functional enrichment analyses of GO and KEGG, we identified 35 autophagy-related target genes of these DEMs, among which akt3 was pinpointed as the most central hub gene using module assay of the PPI network. Then employing the miRanda and Targetscan programs for prediction, and verification through a double fluorescent enzyme system and qPCR method, we confirmed that miR-193 b-3p could target the 3'-UTR of grass carp akt3, reducing its gene expression. Ultimately, we illustrated that grass carp miR-193 b-3p could promote autophagy in CIK cells. Above results collectively indicated that miRNAs might play a critical role in autophagy of grass carp during GCRV infection and contributed significantly to antiviral immunity by targeting autophagy-related genes. This study may provide new insights into the intricate mechanisms involved in virus, autophagy, and miRNAs.

Genome and evolution of Tibet orbivirus, TIBOV (genus Orbivirus, family Reoviridae).

Tibet orbivirus (TIBOV) was first isolated from Anopheles maculatus mosquitoes in Xizang, China, in 2009. In recent years, more TIBOV strains have been isolated in several provinces across China, Japan, East Asia, and Nepal, South Asia. Furthermore, TIBOVs have also been isolated from Culex mosquitoes, and several midge species. Additionally, TIBOV neutralizing antibodies have been detected in serum specimens from several mammals, including cattle, sheep, and pigs. All of the evidence suggests that the geographical distribution of TIBOVs has significantly expanded in recent years, with an increased number of vector species involved in its transmission. Moreover, the virus demonstrated infectivity towards a variety of animals. Although TIBOV is considered an emerging orbivirus, detailed reports on its genome and molecular evolution are currently lacking. Thus, this study performed the whole-genome nucleotide sequencing of three TIBOV isolates from mosquitoes and midges collected in China in 2009, 2011, and 2019. Furthermore, the genome and molecular genetic evolution of TIBOVs isolated from different countries, periods, and hosts (mosquitoes, midges, and cattle) was systematically analyzed. The results revealed no molecular specificity among TIBOVs isolated from different countries, periods, and vectors. Meanwhile, the time-scaled phylogenetic analysis demonstrated that the most recent common ancestor (TMRCA) of TIBOV appeared approximately 797 years ago (95% HPD: 16-2347) and subsequently differentiated at least three times, resulting in three distinct genotypes. The evolutionary rate of TIBOVs was about 2.12 × 10-3 nucleotide substitutions per site per year (s/s/y) (95% HPD: 3.07 × 10-5, 9.63 × 10-3), which is similar to that of the bluetongue virus (BTV), also in the Orbivirus genus. Structural analyses of the viral proteins revealed that the three-dimensional structures of the outer capsid proteins of TIBOV and BTV were similar. These results suggest that TIBOV is a newly discovered and rapidly evolving virus transmitted by various blood-sucking insects. Given the potential public health burden of this virus and its high infectious rate in a wide range of animals, it is significant to strengthen research on the genetic variation of TIBOVs in blood-feeding insects and mammals in the natural environment and the infection status in animals.

Epidemiological Analyses of the First Incursion of the Epizootic Hemorrhagic Disease Virus Serotype 8 in Tunisia, 2021-2022.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-transmitted viral disease and a World Organization for Animal Health (WOAH)-listed disease of domestic and wild ruminants since 2008. EHDV is transmitted among susceptible animals by a few species of midges of genus Culicoides. During the fall of 2021, a large outbreak caused by the epizootic hemorrhagic disease virus (EHDV), identified as serotype 8, was reported in Tunisian dairy and beef farms with Bluetongue virus (BTV)-like clinical signs. The disease was detected later in the south of Italy, in Spain, in Portugal and, more recently, in France, where it caused severe infections in cattle. This was the first evidence of EHDV-8 circulation outside Australia since 1982. In this study, we analyzed the epidemiological situation of the 2021-2022 EHDV outbreaks reported in Tunisia, providing a detailed description of the spatiotemporal evolution of the disease. We attempted to identify the eco-climatic factors associated with infected areas using generalized linear models (GLMs). Our results demonstrated that environmental factors mostly associated with the presence of C. imicola, such as digital elevation model (DEM), slope, normalized difference vegetation index (NDVI), and night-time land surface temperature (NLST)) were by far the most explanatory variables for EHD repartition cases in Tunisia that may have consequences in neighboring countries, both in Africa and Europe through the spread of infected vectors. The risk maps elaborated could be useful for disease control and prevention strategies.

Clinical symptoms and histopathological changes in coho salmon affected by the erythrocytic inclusion body syndrome (EIBS) are caused by the infection of piscine orthoreovirus 2 (PRV-2).

The relationship of histopathological changes and the infection of Piscine orthoreovirus 2 (PRV-2) was investigated in coho salmon that were suffering from the erythrocytic inclusion body syndrome (EIBS). Immunohistochemical observations revealed abundant σ1 protein of PRV-2 in the spongy layer of the ventricle of the heart, where severe myocarditis was observed. In the spleen, the virus protein was detected in many erythrocytes, some of which were spherical-shaped and apparently dead. The number of erythrocytes was decreased in the spleen compared to the apparently healthy fish. The virus protein was also detected in some erythrocytes in blood vessels. The viral protein was often detected in many macrophages ingesting erythrocytes or dead cell debris in the spleen or in the kidney sinusoids. Large amounts of the viral genomic segment L2 were also detected in these organs by RT-qPCR. Many necrotic foci were found in the liver, although the virus protein was not detected in the hepatocytes. These results suggest that the primary targets of PRV-2 are myocardial cells and erythrocytes and that clinical symptoms such as anaemia or jaundice and histopathological changes such as myocarditis in EIBS-affected coho salmon are caused by PRV-2 infection.

Isolation of Epizootic Hemorrhagic Disease Virus Serotype 10 from Culicoides tainanus and Associated Infections in Livestock in Yunnan, China.

Two strains of viruses, JC13C644 and JC13C673, were isolated from Culicoides tainanus collected in Jiangcheng County, Yunnan Province, situated along the border area shared by China, Laos, and Vietnam. JC13C644 and JC13C673 viruses can cause cytopathic effect (CPE) in mammalian cells BHK21 and Vero cells, and cause morbidity and mortality in suckling mice 48 h after intracerebral inoculation. Whole-genome sequencing was performed, yielding complete sequences for all 10 segments from Seg-1 (3942nt) to Seg-10 (810nt). Phylogenetic analysis of the sub-core-shell (T2) showed that the JC13C644 and JC13C673 viruses clustered with the Epizootic Hemorrhagic Disease Virus (EHDV) isolated from Japan and Australia, with nucleotide and amino acid homology of 93.1% to 98.3% and 99.2% to 99.6%, respectively, suggesting that they were Eastern group EHDV. The phylogenetic analysis of outer capsid protein (OC1) and outer capsid protein (OC2) showed that the JC13C644 and JC13C673 viruses were clustered with the EHDV-10 isolated from Japan in 1998, with the nucleotide homology of 98.3% and 98.5%, and the amino acid homology of 99.6% and 99.6-99.8%, respectively, indicating that they belong to the EHDV-10. Seroepidemiological survey results demonstrated that JC13C644 virus-neutralizing antibodies were present in 29.02% (177/610) of locally collected cattle serum and 11.32% (89/786) of goat serum, implying the virus's presence in Jiangcheng, Yunnan Province. This finding suggests that EHDV-10 circulates not only among blood-sucking insects in nature but also infects local domestic animals in China. Notably, this marks the first-ever isolation of the virus in China and its discovery outside of Japan since its initial isolation from Japanese cattle. In light of these results, it is evident that EHDV Serotype 10 exists beyond Japan, notably in the natural vectors of southern Eurasia, with the capacity to infect local cattle and goats. Therefore, it is imperative to intensify the surveillance of EHDV infection in domestic animals, particularly focusing on the detection and monitoring of new virus serotypes that may emerge in the region and pose risks to animal health.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

Identification, pathological, and genomic characterization of novel goose reovirus associated with liver necrosis in geese, China.

Since 2021, a novel strain of goose reovirus (GRV) has emerged within the goose farming industry in Guangdong province, China. This particular viral variant is distinguished by the presence of white necrotic foci primarily localized in the liver and spleen, leading to substantial economic losses for the poultry industry. However, the etiology, prevalence and genomic characteristics of the causative agent have not been thoroughly investigated. In this study, we conducted an epidemiological inquiry employing suspected GRV samples collected from May 2021 to September 2022. The macroscopic pathological and histopathological lesions associated with GRV-infected clinical specimens were examined. Moreover, we successfully isolated the GRV strain and elucidated the complete genome sequence of the isolate GD21/88. Through phylogenetic and recombination analysis, we unveiled that the GRV strains represent a novel variant resulting from multiple reassortment events. Specifically, the µNS, λC, and σNS genes of GRV were found to have originated from chicken reovirus, while the σA gene of GRV exhibited a higher degree of similarity with a novel duck reovirus. The remaining genes of GRV were traced back to Muscovy duck reovirus. Collectively, our findings underscore the significance of GRV as a pathogenic agent impacting the goose farming industry. The insights gleaned from this study contribute to a more comprehensive understanding of the epidemiology of GRV in Southern China and shed light on the genetic reassortment events exhibited by the virus.

Grass carp superoxide dismutases exert antioxidant function and inhibit autophagy to promote grass carp reovirus (GCRV) replication.

Superoxide dismutases (SODs) are potent antioxidants crucial for neutralizing reactive oxygen species (ROS) and protecting organisms from oxidative damage. In this study, we successfully cloned and analyzed two SOD genes, CiSOD1 and CiSOD2, from grass carp (Ctenopharyngodon idellus). CiSOD1 consists of two CuZn signature motifs and two conserved cysteine residues, while CiSOD2 contains a single Mn signature motif. The expression of CiSODs was found to be ubiquitous across all examined tissues, with their expression levels significantly altered after stimulation by grass carp reovirus (GCRV) or pathogen-associated molecular patterns (PAMPs). CiSOD1 was observed to be uniformly distributed in the cytoplasm, whereas CiSOD2 localized in the mitochondria. Escherichia coli transformed with both CiSODs demonstrated enhanced host resistance to H2O2 and heavy metals. Additionally, purified recombinant CiSOD proteins effectively protected DNA against oxidative damage. Furthermore, overexpression of CiSODs in fish cells reduced intracellular ROS, inhibited autophagy, and then resulted in the promotion of GCRV replication. Knockdown of CiSODs showed opposite trends. Notably, these roles of CiSODs in autophagy and GCRV replication were reversed upon treatment with an autophagy inducer. In summary, our findings suggest that grass carp SODs play an important role in decreasing intracellular ROS levels, inhibiting autophagy, and subsequently promoting GCRV replication.

First in vitro and in vivo evaluation of recombinant IL-1ß protein as a potential immunomodulator against viral infection in fish.

IL-1ß is an important proinflammatory cytokine with multifaceted modulatory roles in immune responses. In fish, recombinant IL-1ß has been employed in the control of bacterial diseases, while the antiviral mechanisms of IL-1ß remain largely unknown, and the efficacy of recombinant IL-1ß as an immunomodulator to prevent viral diseases is still not determined. This study evaluated the immunomodulatory effects of recombinant grass carp IL-1ß against grass carp reovirus (GCRV) in vitro and in vivo. Firstly, the mature form (Ser111-Lys270) of grass carp IL-1ß was identified, and its recombinant protein (designated as rgcIL-1ß) was prepared through prokaryotic expression. Then, an in vitro evaluation model for rgcIL-1ß activity was established in the CIK cells, with the appropriate concentration (600 ng/mL) and effect time (1 h). In vitro, rgcIL-1ß could not only induce the production of proinflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNF-α but also a series of antiviral factors including IFN-1, IFN-2, IFN-γ, and ISG15. Mechanistically, transcriptome analysis and western blotting confirmed that rgcIL-1ß activated multiple transcriptional factors, including NF-κB, IRF1, IRF3, and IRF8, and the signal pathways associated with inflammatory cytokines and antiviral factors expression. Expectedly, rgcIL-1ß treatment significantly inhibited GCRV replication in vitro. In vivo administration of rgcIL-1ß via intraperitoneal pre-injection significantly aroused an antiviral response to restrict GCRV replication and intense tissue inflammation in grass carp, demonstrating the immunomodulatory effects of rgcIL-1ß. More importantly, rgcIL-1ß administrated with 10 ng/g and 1 ng/g could improve the survival rate of grass carp during GCRV infection. This study represents the first time to comprehensively reveal the immunomodulatory and antiviral mechanisms of IL-1ß in fish and may also pave the way for further developing recombinant IL-1ß as an immunotherapy for the prevention and control of fish viral diseases.

Occurrence of salmonid alphavirus and piscine orthoreovirus-1 infections in migrating salmon (Salmo salar L.) post-smolt in western Norway.

Viral diseases are a serious problem in Atlantic salmon (Salmo salar L.) farming in Norway, often leading to reduced fish welfare and increased mortality. Disease outbreaks in salmon farms may lead to spread of viruses to the surrounding environment. There is a public concern that viral diseases may negatively affect the wild salmon populations. Pancreas disease (PD) caused by salmonid alphavirus (SAV) and heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus-1 (PRV-1) are common viral diseases in salmon farms in western Norway. In the current study, we investigated the occurrence of SAV and PRV-1 infections in 651 migrating salmon post-smolt collected from three fjord systems (Sognefjorden, Osterfjorden and Hardangerfjorden) located in western Norway in 2013 and 2014 by real-time RT-PCR. Of the collected post-smolts, 303 were of wild origin and 348 were hatchery-released. SAV was not detected in any of the tested post-smolt, but PRV-1 was detected in 4.6% of them. The Ct values of PRV-1 positive fish were usually high (mean 32.0; range: 20.1-36.8). PRV-1 prevalence in post-smolts from the three fjords was 6.1% in Sognefjorden followed by 4.8% in Osterfjorden and 2.3% in Hardangerfjorden. The prevalence PRV-1 was significantly higher in wild (6.9%) compared to hatchery-released post-smolt (2.6%). The occurrence of PRV-1 infection in the fish was lowest in the Hardangerfjorden which has the highest fish farming intensity. Our results suggest that SAV infection are uncommon in migrating smolt while PRV-1 infection can be detected at low level. These findings suggest that migrating smolts were at low risk from SAV or PRV-1 released from salmon farms located in their migration routes in 2013 and 2014.

Research Note: Genetic characterization and pathogenicity of an epidemic variant strain of avian reovirus.

The past few years have witnessed a rapid increase in cases of viral arthritis caused by avian reovirus (ARV) in chicken farms in China, attributed to the emergence of variant strains that render traditional vaccines ineffective, leading to substantial economic losses. In this study, we successfully isolated a novel ARV strain, designated as 2023ARV-GS-SDAU-1, from chickens in a broiler flock vaccinated with an ARV vaccine in Gansu province. We performed whole-genome sequencing and assessed its pathogenicity through 2 infection routes: oral administration and intraperitoneal injection. Our analysis revealed significant variations in the σA gene, associated with the inhibition of interferon secretion, compared to known ARV strains. The highest nucleotide identity observed was below 80%. Additionally, the σC gene exhibited notable variations compared to its homologous strains within the same group. Multiple alignment of the amino acid sequences classified the 2023ARV-GS-SDAU-1 strain under genotype I. Furthermore, our pathogenicity experiments indicated that the isolated strain exhibited more severe pathogenicity when administered via intraperitoneal injection in SPF chickens. In summary, our data suggest that the 2023ARV-GS-SDAU-1 strain represents a novel variant circulating in broiler flocks in China. These findings enrich currently available genetic information on ARV strains and provide a new complete genome sequence.

Genetic characterization of newly emerging avian reovirus variants in chickens with viral arthritis/tenosynovitis in Israel.

In recent years, new avian reovirus (ARV) variants caused a variety of symptoms in chickens worldwide, the most important of which was Viral arthritis/tenosynovitis which caused substantial economic losses and has become a concern to the worldwide chicken industry. In this study, we characterized emerging ARV variants in Israel and analyzed their genetic relationship with reference strains. One hundred thirty-four ARV variants were isolated from tendons and synovial fluids of commercial broiler chickens with signs of arthritis/tenosynovitis. Phylogenetic analysis of the partial segment of the sigma C (σC) gene confirmed that these field isolates from Israel could be clustered into all six known clusters. The majority of ARV isolates in Israel belonged to the genotypic cluster 5 (GC5). The strains in this study had a low sequence identity when compared to the commercial vaccine (strain S1133). The findings of this study demonstrated the genetic diversity of ARV strains in Israel from 2015 to 2022. It is reasonable to conclude from the preliminary results of this investigation that Israel has not been subject to selection pressure or the emergence of new ARV variants since the introduction of the live vaccine (ISR-7585). Due to the ongoing emergence of ARV variants, a robust epidemiological monitoring program supported by molecular biology techniques is required to track ARV strains in Israeli poultry flocks.

Experimental infection of cattle, sheep, and goats with the newly emerged epizootic hemorrhagic disease virus serotype 8.

Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) emerged in Europe for the first time in late 2022. In this study, we investigated the kinetics of EHDV-8 infection in cattle, sheep, and goats.  Following experimental infection with EHDV-8, four out of five calves displayed fever, while another calf exhibited ulcerative and crusty lesions of the muzzle. RNAemia peaked at day 7 post infection in all calves and remained relatively stable till the end of the study, at 78 days post infection. Infectious virus was isolated up to 21 days post infection in one calf. As far as small ruminants are concerned, one sheep experienced fever and two out of five had consistent RNAemia that lasted until the end of the study. Remarkably, infectious virus was evidenced at day 7 post infection in one sheep. In goats, no RNA was observed. All infected animals seroconverted, and a neutralizing immune response was observed in all species, with calves exhibiting a more robust response than sheep and goats. Our study provides insights into the kinetics of EHDV-8 infection and the host immune responses. We also highlight that sheep may also play a role in EHDV-8 epidemiology. Altogether, the data gathered in this study could have important implications for disease control and prevention strategies, providing crucial information to policy makers to mitigate the impact of this viral disease on livestock.

Comparative Transcriptomic Analysis Revealed Potential Differential Mechanisms of Grass Carp Reovirus Pathogenicity.

Grass carp reovirus (GCRV), one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella), can lead to grass carp hemorrhagic disease (GCHD). Currently, GCRV can be divided into three genotypes, but the comparison of their pathogenic mechanisms and the host responses remain unclear. In this study, we utilized the Ctenopharyngodon idella kidney (CIK) model infected with GCRV to conduct comparative studies on the three genotypes. We observed a cytopathic effect (CPE) in the GCRV-I and GCRV-III groups, whereas the GCRV-II group did not show any CPE. Moreover, a consistent trend in the mRNA expression levels of antiviral-related genes across all experimental groups of CIK cells was detected via qPCR and further explored through RNA-seq analysis. Importantly, GO/KEGG enrichment analysis showed that GCRV-I, -II, and -III could all activate the immune response in CIK cells, but GCRV-II induced more intense immune responses. Intriguingly, transcriptomic analysis revealed a widespread down-regulation of metabolism processes such as steroid biosynthesis, butanoate metabolism, and N-Glycan biosynthesis in infected CIK cells. Overall, our results reveal the CIK cells showed unique responses in immunity and metabolism in the three genotypes of GCRV infection. These results provide a theoretical basis for understanding the pathogenesis and prevention and control methods of GCRV.

Recombinant surface display vaccine enhances the immersion immune effect against grass carp reovirus in grass carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is subject to a hemorrhagic disease caused by grass carp reovirus (GCRV), which can lead to mass mortality in grass carp culture, causing significant economic loss. Vaccination is the most promising strategy for the prevention of infectious diseases. Immersion vaccination is considered the most effective disease prevention method for juvenile fish because it can be implemented on many fish at once and administered without causing stress. However, immune responses by immersion vaccination are markedly less robust due to the skin barrier and insufficient antigen uptake. The display of heterologous proteins on the cell surface has been explored as a delivery system for viral antigens in veterinary and human vaccine studies. To improve the efficacy of the immersion vaccine, the major capsid protein (VP7) of GCRV was co-displayed with Aeromonas hydrophila outer membrane protein a (OmpA) and major adhesion protein (Mah) on the outer membrane surface of nonpathogenic Escherichia coli BL21 using the anchoring motif of ice-nucleation protein (Inp). The immune responses and protection efficiency against GCRV infection via both the injection and immersion routes were evaluated. The results indicated that the activities of anti-oxidant enzymes (ACP, AKP, SOD and T-AOC), as well as the expression of immune-related genes (TNF-α, IL-1ß, MHCI and IgM) and specific VP7 antibody levels, were strongly increased in the grass carp from 7 to 21 days post-injection inoculation in a dose dependent manner. The cumulative mortality rates of injection-vaccinated groups were much lower than those of the control group after the GCRV challenge, and the relative percent survival (RPS) was greater than 80 %. Vitally, the surface co-display of vp7-Mah protein conferred marked protection to grass carp against GCRV infection after immersion administration (RPS >50 %); this was consistent with the production of high level of specific serum antibodies, non-specific immune responses, and the expression of immune-related genes. Moreover, the invasion analysis further showed that surface co-display of the vp7-Mah protein indeed significantly improved the invasion of E. coli BL21 (DE3) in vitro. Altogether, this study demonstrated that surface display GCRV core antigen vaccine system accompanied by invasion component from aquatic pathogenic microorganism is an effective prophylactic against GCRV viral diseases via the immersion administration approach.