Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic virus that can cause severe viral encephalitis. Initial interactions between JEV and host cells are required for productive viral infection and initiation of the viral life cycle. The elucidation of these interactions is critical, not only to understand the pathogenesis of JEV infection, but also to design efficient antiviral strategies. In this review, we outline the known viral and cellular components involved in JEV entry into host cells, with a particular focus on the initial virus-host cell interaction on the cell surface and the downstream early events such as endocytosis, membrane fusion, and viral genome release.

Soil viral ecology is a growing research field; however, the state of knowledge still lags behind that of aquatic systems. Therefore, to facilitate progress, the first Soil Viral Workshop was held to encourage international scientific discussion and collaboration, suggest guidelines for future research, and establish soil viral research as a concrete research area. The workshop took place at Søminestationen, Denmark, between 15 and 17th of June 2022. The meeting was primarily held in person, but the sessions were also streamed online. The workshop was attended by 23 researchers from ten different countries and from a wide range of subfields and career stages. Eleven talks were presented, followed by discussions revolving around three major topics: viral genomics, virus-host interactions, and viruses in the soil food web. The main take-home messages and suggestions from the discussions are summarized in this report.

BACKGROUND: Gut microbiota is crucial for immune homeostasis and is associated with the prognosis of chronic hepatitis B infection. Peyer's patches (PPs), characterized by intestinal mucosa localization, are involved in the gut microbiota-mediated immune response. However, whether and how PPs orchestrate gut microbiota-modulated anti-hepatitis B virus (HBV) response remain elusive. This study aims to elucidate the role of PPs in gut microbiota-mediated anti-HBV adaptive immunity. METHODS: We investigated the effects of gut microbiota and PPs on adaptive immune responses by transcriptomic, phenotypic, and functional analyzes from an HBV mouse model with gut commensal microbiota and PP-depleting interventions. RESULTS: Depletion of gut microbiota impaired systemic adaptive immune responses, resulting in a delayed HBV antigen clearance. Differentially expressed genes analysis of PPs revealed that pathways related to adaptive immune responses were significantly downregulated in gut microbiota-deficient mice. Notably, the depletion of PPs could abolish gut microbiota-boosted intrahepatic HBV-specific T cell response, leading to a higher serum hepatitis B surface antigen level in mice. CONCLUSION: PPs orchestrate gut microbiota-mediated intrahepatic anti-HBV cellular immunity, underlining the significance of remote manipulating the "gut microbiota-PPs" axis for achieving optimum anti-HBV response.

The newly discovered phage vB_EcoP_E212 (also known as E212) was characterized and its genome was annotated in this study, which was conducted in Jilin, China. Transmission electron microscopy indicates that phage E212 belongs to the class Caudoviricetes. This phage exclusively infects enterotoxigenic E. coli K88. E212 was found to have a short latent period of 20 min, and a burst size of 125 PFU/cell. Additionally, E212 remained stable at all pH levels (3.0-12.0) and temperatures between -20 and 60 ºC. The genome of the phage E212 consists of 38,252 bp dsDNA molecule with a G + C content of 46.98%. The genome is projected to include 53 ORFs but no tRNAs. This phage lacks homologs of virulence factors or antimicrobial resistance genes, but it has lysogeny-related genes. Phage E212 was placed in the genus Lederbergvirus as a result of nucleotide sequence alignment and phylogenetic analysis.

BACKGROUND: Hypertension may increase the infection risk of multiple viruses. The evidence for the association between hypertension and Epstein-Barr virus (EBV) reactivation is still largely lacking. METHODS: The study was based on the baseline information of a population-based prospective cohort from high-risk areas of nasopharyngeal carcinoma (NPC). Using two EBV reactivation classification criteria, we explored the relationship between hypertension and EBV reactivation through logistic regression models. RESULTS: We included a total of 12,159 subjects. Among them, 3,945 (32.45%) were EBV arbitrary seropositive, and 1,547 (12.72%) were EBV comprehensive seropositive. Hypertension was associated with an increased risk of EBV reactivation, with odds ratios (ORs) of 1.17 (95% CI = 1.08-1.27) for EBV arbitrary seropositive subjects and 1.16 (95% CI = 1.03-1.30) for EBV comprehensive seropositive subjects. Two types of antihypertensive drugs were associated with decreased risk of EBV reactivation: β-adrenergic receptor-blocking agents (β-blockers) (OR = 0.51, 95% CI = 0.42-0.61 for EBV arbitrary seropositive subjects; OR = 0.62, 95% CI = 0.47-0.81 for EBV comprehensive seropositive subjects) and angiotensin converting enzyme inhibitors (ACEIs) (OR = 0.61, 95% CI = 0.41-0.88 for EBV arbitrary seropositive subjects; OR = 0.58, 95% CI = 0.32-0.98 for EBV comprehensive seropositive subjects). CONCLUSIONS: Hypertension was associated with an increased risk of EBV reactivation in high-incidence areas of NPC. β-blockers and ACEIs reduce this risk, and thus might be used for NPC prevention in endemic areas.

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication.

The emergence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a threat to public health. Polymyxin-B is generally considered a last-resort antibiotic. In this study, we isolated a carbapenem- and polymyxin-B resistant K. pneumoniae phage BL02 for the first time in Southwestern China and evaluated its biological characteristics and whole-genome sequence. Polymyxin-B resistant K. pneumoniae, (CK02), was isolated from the blood of a male with severe septic shock, and phage BL02 was screened and purified from the hospital sewage. BL02 could lyse 40 out of 46 CRKP isolates (86.96%) and has high activity in the pH range of 6-10 and the temperature range of 4-55 °C. The latency period of BL02 was about 10 min and the lysis period was about 50 min. The genome results showed that BL02 was a linear dsDNA with a total length of 175,595 bp and a GC content of 41.83%. A total of 275 ORFs were predicted and no tRNA, rRNA, drug resistance genes, or virulence genes were found in the genome. Phylogenetic analysis showed that BL02 belongs to the family Straboviridae. Treatment of infected mice with two antibiotics (tigecycline or ceftazidime/avibactam) resulted in 7-day survival rates of 28.57% and 42.86%, respectively. In contrast, the survival rate of mice in the single-dose BL02-treated group was 71.43%. In summary, this preclinical study isolated a phage capable of lysing polymyxin-B resistant K. pneumoniae and validated its safety and efficacy in an in vivo model, which provides a reference for further research on controlling MDR pathogens.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections.

Nipah virus (NiV) is a zoonotic pathogen with airborne transmission and high case fatality rates in humans. There is currently no treatment or vaccine against NiV infection approved for humans or animals, therefore early diagnosis is the key to control any potential outbreaks. In this study, we developed an optimized one-pot assay using recombinase polymerase amplification (RPA) coupled to CRISPR/Cas13a for the molecular detection of NiV. The one-pot RPA-CRISPR/Cas13a assay for NiV detection was specific and did not cross-react against other selected (re)-emerging pathogens. The sensitivity of the one-pot RPA-CRISPR/Cas13a assay for NiV detection can detect as little as 103 cp/μL of total synthetic NiV cDNA. The assay was then validated with simulated clinical samples. The results for the one-pot RPA-CRISPR/Cas13a assay could be visualized with either fluorescence or lateral flow strips for convenient clinical or field diagnostics, providing a useful supplement to the gold-standard qRT-PCR assay for detecting NiV detections.

Enterococcus gallinarum, a gut pathobiont, is an opportunistic pathogen that carries the risk of antibiotic resistance in the clinic and has been proven to drive autoimmunity in both mice and humans. Screening for novel bacteriophages targeting Enterococcus gallinarum is expected to provide a promising strategy for controlling such infections or regulating related chronic diseases. In the present study, we isolated a novel lytic Enterococcus gallinarum phage, Phi_Eg_SY1, which presents favourable thermostability and pH stability. Further assays indicated that Phi_Eg_SY1 can efficiently adsorb and lyse the host bacteria in vitro. Genomic and phylogenetic analyses suggested that Phi_Eg_SY1 does not contain virulence or lysogeny genes and presents a novel unassigned evolutionary lineage among the related dsDNA phages. Phi_Eg_SY1 is therefore considered to be suitable for further applications.

Although most of the attention was focused on the characterization of changes in the Spike protein among variants of SARS-CoV-2 virus, mutations outside the Spike region are likely to contribute to virus pathogenesis, virus adaptation and escape to the immune system. Phylogenetic analysis of SARS-CoV-2 Omicron strains reveals that several virus sub-lineages could be distinguished, from BA.1 up to BA.5. Regarding BA.1, BA.2 and BA.5, several mutations concern viral proteins with antagonistic activity to the innate immune system, such as NSP1 (S135R), which is involved in mRNAs translation, exhibiting a general shutdown in cellular protein synthesis. Additionally, mutations and/or deletions in the ORF6 protein (D61L) and in the nucleoprotein N (P13L, D31-33ERS, P151S, R203K, G204R and S413R) have been reported, although the impact of such mutations on protein function has not been further studied. The aim of this study was to better investigate the innate immunity modulation by different Omicron sub-lineages, in the attempt to identify viral proteins that may affect virus fitness and pathogenicity. Our data demonstrated that, in agreement with a reduced Omicron replication in Calu-3 human lung epithelial cells compared to the Wuhan-1 strain, a lower secretion of interferon beta (IFN-β) from cells was observed in all sub-lineages, except for BA.2. This evidence might be correlated with the presence of a mutation within the ORF6 protein (D61L), which is strikingly associated to the antagonistic function of the viral protein, since additional mutations in viral proteins acting as interferon antagonist were not detected or did not show significant influence. Indeed, the recombinant mutated ORF6 protein failed to inhibit IFN-β production in vitro. Furthermore, we found an induction of IFN-β transcription in BA.1 infected cells, that was not correlated with the cytokine release at 72 h post-infection, suggesting that post-transcriptional events can be involved in controlling the innate immunity.

A newly discovered tick-borne virus called the severe fever with thrombocytopenia syndrome virus (SFTSV) can cause the severe fever with thrombocytopenia syndrome (SFTS). The mortality and incidence rate of SFTS patients remain extremely high due to the fast global dissemination of its arthropod vectors, and the mechanism of viral pathogenesis remains largely unknown. In this study, high-throughput RNA sequencing (RNA-Seq) was used to sequence HEK 293 cells treated with SFTSV at four time points. 115, 191, 259, and 660 differentially expressed genes (DEGs) were identified at 6, 12, 24, and 48 h post-infection, respectively. We found that SFTSV infection induced the expression of genes responsible for numerous cytokine-related pathways, including TNF, CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, and CCL20. With the extension of infection time, the expression of most genes involved in these pathways increased significantly, indicating the host's inflammatory response to SFTSV. Moreover, the expression levels of GNA13, ARHGEF12, RHOA, ROCK1, and MYL12A, elements of the platelet activation signaling pathway, were downregulated during SFTSV infection, suggesting that the SFTSV infection may cause thrombocytopenia by inhibiting platelet activation. Our results contribute to further understanding the interaction between SFTSV and the host.

The emergence and rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (BA.1.1) has attracted global attention. The numerous mutations in the spike protein suggest that it may have altered susceptibility to immune protection elicited by the existing coronavirus disease 2019 (COVID-19) infection. We used a live virus neutralization test and SARS-CoV-2 pseudotype vesicular stomatitis virus vector-based neutralization assay to assess the degree of immune escape efficiency of the original, Delta (B1.617.2), and Omicron strains against the serum antibodies from 64 unvaccinated patients who had recovered from COVID-19 and the results were strongly correlated. The convalescent serum neutralization was more markedly reduced against the Omicron variant (9.4-57.9-fold) than the Delta variant (2.0-4.5-fold) as compared with the original strain. Our results demonstrate the reduced fusion and notable immune evasion capabilities of the Omicron variants, highlighting the importance of accelerating the development of vaccines targeting them.

Pseudostellaria heterophylla (P. heterophylla) is a popular Chinese medicinal herb that is cultivated widely in China. Viral infection is commonly encountered during the production of P. heterophylla. To identify viruses causing P. heterophylla disease, sRNA and mRNA libraries were built for 2 sets of P. heterophylla plants, one set that was planted only once (FGP) and one that was planted three consecutive three times (TGP) in a field, using virus-free tuberous roots as reproductive materials. A comprehensive procedure, including assembling virus-derived sRNA (vsRNA), assessing and cloning the full-length viral genome, building an infectious cloning vector and constructing a virus-based expression vector, was performed to identify viruses infecting P. heterophylla. Ultimately, 48 contig-related viruses were mined from 6 sRNA and 6 mRNA P. heterophylla libraries. A 9762-bp fragment was predicted to be the complete genome of TuMV virus. This sequence was cloned from P. heterophylla, and its infectivity was evaluated using the virus-infection model plant Nicotiana benthamiana (N. benthamiana) and host plant P. heterophylla. The resulting 9839-bp viral genome was successfully obtained from P. heterophylla and identified as a new P. heterophylla TuMV-ZR isolate. Simultaneously, TuMV-ZR infectious clones were shown to effectively infect P. heterophylla. Furthermore, TuMV-ZR expression vectors were developed, and the ability of a TuMV-ZR-based vector to express foreign genes was determined by analysis with the reporter gene EGFP. TuMV-ZR-based vectors were found to continuously express foreign genes in different organs of P. heterophylla throughout the whole vegetative period. In addition, TuMV-ZR vectors carrying EGFP accumulated in the tuberous roots of P. heterophylla, confirming that tuberous roots are key targets for viral infection and transmission. This study revealed the core pathogenicity of P. heterophylla mosaic virus and developed a new TuMV-ZR-based expression tool that led to long-term protein expression in P. heterophylla, laying the foundation for the identification of the mechanisms of P. heterophylla infection with mosaic viruses and developing tools to express value proteins in the tuberous roots of the medicinal plant P. heterophylla.

In May 2015, a high mortality event in farmed rainbow trout occurred in Jeollabuk-do province in Korea. Histopathological analysis revealed necrosis in the kidney, liver, branchial arch, and gills of moribund fish, and infectious hematopoietic necrosis virus (IHNV) was detected in the lesions by immunohistochemistry. Cytopathic effects were observed in EPC, FHM, and RTG-2 cell lines after inoculation with kidney and spleen tissues and IHNV was detected by reverse transcription polymerase chain reaction (PCR). The amplified PCR product was sequenced, and phylogenetic analysis placed IHNV in the JRt Nagano group. Both in vivo and in vitro trials were performed to compare the virulence properties between RtWanju15 isolate, which causes 100% mortality in imported fry, and a previous isolate RtWanju09 of the JRt Shizuoka group isolated from eggs of healthy broodfish. In vivo challenge with high dose on specific pathogen free (SPF) rainbow trout fry performed in Denmark with isolates RtWanju09, RtWanju15 and DF04/99 isolates showed a survival rates of 60%, 37.5% and 52.5% (average), respectively without statistical difference. The replication efficiency of the two isolates in the in vitro challenge was similar.

Avian reovirus (ARV) can commonly infect a flock and cause immunosuppressive diseases in poultry. The nonstructural protein p17 is involved in viral replication, and significant progress has been made in showing its ability to regulate cellular signaling pathways. In our previous study, to further investigate the effect of ARV p17 protein on viral replication, the host protein polyglu-tamine binding protein 1 (PQBP1) was identified to interact with p17 by a yeast two-hybrid system. In the current study, the interaction between PQBP1 and p17 protein was further confirmed by laser confocal microscopy and coimmunoprecipitation assays. In addition, the N-terminal WWD of PQBP1 was found to mediate the process of binding to the p17 protein. Interestingly, we found that ARV infection significantly inhibited PQBP1 expression. While the quantity of ARV replication was largely influenced by PQBP1, PQBP1 overexpression decreased ARV replication. In contrast, upon PQBP1 knockdown, the quantity of ARV was notably increased. ARV infection and p17 protein expression were both proven to induce PQBP1 to mediate cellular inflammation. In the current study, we revealed through qRT‒PCR, ELISA and Western blotting methods that PQBP1 plays a positive role in ARV-induced inflammation. Furthermore, the mechanism of this process was shown to involve the NFκB-dependent transcription of inflammatory genes. In addition, PQBP1 was shown to regulate the phosphorylation of p65 protein. In conclusion, this research provides clues to elucidating the function of the p17 protein and the pathogenic mechanism of ARV, especially the cause of the inflammatory response. It also provides new ideas for the study of therapeutic targets of ARV.