Archives of Virology最新文献

In this study, a continuous cell line (KM cells) derived from koi (Cyprinus carpio koi) muscle was established and characterized. The KM cells were subcultured for more than 70 passages and showed high viability after long-term cryopreservation. The KM cell line was optimally cultured in medium 199 containing 10% foetal bovine serum at 25°C. A chromosome analysis indicated that the cell line remained diploid, with a mean chromosome count of 100. DNA sequencing and comparative analysis of the 16S rRNA and cytochrome oxidase I gene sequences showed that the KM cell line originated from koi. In transfection experiments using the plasmid pEGFP, KM cells demonstrated a high level of transfection efficiency, suggesting their potential for use in foreign gene expression studies. Inoculation with spring viraemia of carp virus (SVCV) resulted in a substantial cytopathic effect, and the level of production of SVCV in KM cells was higher than that in the epithelioma papulosum cyprinid (EPC) cell line that is normally used to produce the virus. However, no cytopathic effect was observed when these cells were inoculated with koi herpesvirus, carp oedema virus, or grass carp reovirus. These observations suggest that the newly established KM cell line will be a valuable tool for investigating the pathogenesis of infection with spring viraemia of carp virus.

In this study, a multiplex PCR method was developed for the detection of four diarrhea-associated viruses of canines, including canine bocavirus (CBoV), canine circovirus (CCV), torque teno canis virus (TTCV), and canine kobuvirus (CKV). Four pairs of compatible primers, one specific for each virus, were designed based on conserved sequences. After optimization of parameters such as primer concentration and annealing temperature in single and multiple amplifications, four specific fragments were amplified simultaneously with high sensitivity and specificity in one PCR reaction. The fragments amplified were 165 bp (CBoV), 345 bp (CCV), 506 bp (TTCV), and 666 bp (CKV) in length. The sensitivity of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There was no cross-reaction with the canine pathogens canine parvovirus (CPV), canine distemper virus (CDV), or canine coronavirus (CCoV). Testing of canine fecal samples from China using the multiplex PCR assay revealed the presence of CBoV, CCV, TTCV, and CKV in 10.1%, 6.2%, 2.8%, and 1.7% of the samples, respectively. The results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100%. In addition, the complete genome sequences of the viruses in three CKV-positive samples were determined and found to be 95.7 − 96.6% identical to the reference strain US-PC0082 and genetically more distant from other animal kobuvirus. The multiplex PCR method established in this study is convenient, with high specificity and sensitivity, which will be helpful for the rapid differential diagnosis of CBoV, CCV, TTCV, and CKV infections.

Epstein–Barr virus nuclear antigen 1 (EBNA1) contains two arginine-glycine (RG) repeats that contain symmetric/asymmetric dimethylarginine (SDMA/ADMA) and monomethylarginine (MMA) residues. We generated mouse monoclonal antibodies directed against a monomethylated GRGRGG-containing repeat located between amino acids 328 and 377 of EBNA1. In addition to detecting MMA-modified EBNA1, we also had the goal of identifying cellular proteins that bind to MMA-modified EBNA1 in EBV-positive Raji cells. Furthermore, we hypothesized that antibodies against MMA-modified EBNA1 might also recognize cell factors that use an MMA-modified surface structure similar to that of EBNA1 to bind to their common targets. Using a combination of immunoprecipitation and mass spectrometry, we identified a number of such cellular proteins, including SNRPD1-3, ALY/REF, RPS15, DIDO1, LSM12, LSM14A, DAP3, and CPSF1. An NACA complex protein that was shown previously to bind to the glycine-alanine repeat of EBNA1 was also identified. The proteins identified in this study are involved in splicing, tumorigenesis, transcriptional activation, DNA stability, and RNA processing or export.

This is the first description of the complete genome sequence of a newly characterized monopartite begomovirus isolated from an asymptomatic uncultivated plant, Melochia tomentosa, collected in Burkina Faso. The sequence was obtained through rolling-circle amplification, cloning, and Sanger sequencing. The provisional species name “Begomovirus melochiae” and common virus name “melochia associated virus” (MeAV) are proposed. The MeAV genome was found to share the most nucleotide sequence similarity with three African monopartite begomoviruses: tomato curly stunt virus (74%), pepper yellow vein Mali virus (73%), and tomato leaf curl Cameroon virus (73%). Phylogenetic analysis confirmed its relationship to Old World monopartite begomoviruses. The discovery of MeAV in an uncultivated and asymptomatic plant provides a further example of the high diversity of begomoviruses in sub-Saharan African ecosystems.

NLR family member X1 (NLRX1) is an important member of the NOD-like receptor (NLR) family and plays unique roles in immune system regulation. Patients with hepatitis B virus (HBV) infection are more likely to have the NLRX1 mutation p.Arg707Cys than healthy individuals. It has been reported that NLRX1 increases the infection rate of HBV in HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). However, the role of NLRX1 mutation (p.Arg707Cys) in hepatitis remains unclear. We constructed Huh7 cells that stably overexpressed NTCP, using LV003 lentivirus. First, wild-type (WT) and mutant (MT) NLRX1 overexpression plasmids were constructed. The MT plasmid contained a point mutation at position 707 of the WT overexpression plasmid. Then, Huh7-NTCP cells were transfected with the WT or MT NLRX1 overexpression plasmid, and subsequent NLRX1 expression was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. HBV RNA levels were determined using RT-qPCR. HBsAg and HBcAg levels were confirmed immunohistochemically. Interferon alpha (IFN-α), interleukin 6 (IL-6), and type I interferon beta (IFN-β) levels were determined using enzyme-linked immunosorbent assay kits. p-p65, p-interferon regulatory factor (IRF) 3, and p-IRF7 expression levels were examined using western blot. The interaction of NLRX1 and retinoic acid-inducible gene (RIG)-1/mitochondrial antiviral signaling (MAVS) protein was confirmed by coimmunoprecipitation. The interaction of NLRX1 with IFN-α, IL-6, or IFN-β was analyzed by dual luciferase reporter gene assay. The levels of HBV RNA, HBsAg, and HBcAg in infected cells transfected with the WT NLRX1 or MT NLRX1 expression plasmid were higher than those in the untransfected control group; and these levels were lower in the cells transfected with MT NLRX1 than in those transfected with WT NLRX1. The levels of IFN-α, IFN-β, IL-6, p-p65, p-IRF3, and p-IRF7 were lower in cells transfected with WT NLRX1 or MT NLRX1 than in control cells. The levels of IFN-β, p-p65, p-IRF3, and p-IRF7 were higher in cells transfected with MT NLRX1 than in those transfected with WT NLRX1. Moreover, NLRX1 competitively inhibited RIG1 binding to MAVS, but the mutation in MT NLRX1 reduced this inhibitory effect. In addition, NLRX1 decreased the promoter activity of IFN-α, IFN-β, and IL-6. Our findings revealed that NLRX1 is a regulatory factor that inhibits the anti-HBV ability of hepatocytes and that the mutation p.Arg707Cys in NLRX1 suppresses HBV infection and activates the IFN/nuclear factor κB pathway.

Hepatitis B virus (HBV) infection is a major public health issue and is responsible for considerable morbidity and mortality globally. In Pakistan, the prevalence of chronic HBV infection varies from 2% to 4%, with an estimated exposure rate of approximately 34%. The major objective of this study was to determine the prevalence of HBV genotypes and the pattern of escape mutations in the HBV S gene in two major provinces of Pakistan: Punjab and Khyber Pakhtunkhwa. A total of 146 serum/plasma samples collected from hepatitis B patients were sequenced by the Sanger method. Phylogenetic analysis indicated the intermixed circulation of closely related strains of HBV genotype D and subgenotypes HBV/D1, HBV/D2, and HBV/A2 in both provinces, and various escape mutations (Y100C, P120S/T, G119R, C121S/Y, R122K, T126I, A128V, Q129H/R, G130R, T131N/I, M133T/I, Y134N, C137W, S143L, and D144E) were identified. These findings provide important insights into the current prevalence of HBV genotypes in Pakistan and will help in the establishment of more-efficient disease interventions and patient management strategies.

Echovirus type 18 (E18) is a member of the genus Enterovirus of the family Picornaviridae. In this study, we investigated the characteristics of E18 infections in hospitalized adults with meningoencephalitis that occurred during an unusual epidemic in south-western Hungary in mid-2023. Five (6.1%) out of 82 cerebrospinal fluid specimens that were tested were positive for an enterovirus, four of which were E18 (OR372160 and PP861087-PP861090). Headache (100%), fever (75%), retrobulbar pain (50%), nausea (50%), joint/limb pain (50%), exanthema, photophobia, and vomiting were the most common symptoms. Sequence analysis showed that these viruses were related to unpublished emerging E18 strains from France (2022/2023) and China (2019/2020). Further study is necessary to monitor the circulation of epidemic/pandemic E18 variants over time.

This article reports changes to virus taxonomy and taxon nomenclature that were approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in April 2024. The entire ICTV membership was invited to vote on 203 taxonomic proposals that had been approved by the ICTV Executive Committee (EC) in July 2023 at the 55th EC meeting in Jena, Germany, or in the second EC vote in November 2023. All proposals were ratified by online vote. Taxonomic additions include one new phylum (Ambiviricota), one new class, nine new orders, three new suborders, 51 new families, 18 new subfamilies, 820 new genera, and 3547 new species (excluding taxa that have been abolished). Proposals to complete the process of species name replacement to the binomial (genus + species epithet) format were ratified. Currently, a total of 14,690 virus species have been established.

Two Ralstonia phages, FLC1-1B and FLC4-3B, were isolated from leaf litter compost, using Ralstonia pseudosolanacearum, which is a causal agent of bacterial wilt disease, as a host. The genomic DNA sequences of FLC1-1B and FLC4-3B were determined and found to be 290,008 bp and 291,257 bp in length, respectively, and they were therefore classified as jumbo phages. However, they did not show high similarity to any jumbo phage genomic sequence in the NCBI nt database. The closest hit in a BLAST search was the jumbo phage ripduovirus RP12, with only 35% coverage and 77% sequence identity, whereas the FLC1-1B and FLC4-3B sequences were 99.0% identical. Based on these findings, FLC1-1B and FLC4-3B should be classified as members of a new genus in the order Caudoviricetes. FLC4-3B was found to suppress wilt disease in tomato plants, suggesting that it has potential as a biocontrol agent for managing R. pseudosolanacearum infections.

Viruses have undergone evolutionary adaptations to tune their utilization of carbon sources, enabling them to extract specific cellular substrates necessary for their replication. The lack of a reliable cell culture system and a small-animal model has hampered our understanding of the molecular mechanism of replication of hepatitis E virus (HEV) genotype 1. Our recent identification of a replicative ensemble of mutant HEV RNA libraries has allowed us to study the metabolic prerequisites for HEV replication. Initial assessments revealed increased glucose and glutamine utilization during HEV replication. Inhibition of glycolysis and glycolysis + glutaminolysis reduced the levels of HEV replication to similar levels. An integrated analysis of protein-metabolite pathways suggests that HEV replication markedly alters glycolysis, the TCA cycle, and glutamine-associated metabolic pathways. Cells supporting HEV replication showed a requirement for fructose-6-phosphate and glutamine utilization through the hexosamine biosynthetic pathway (HBP), stimulating HSP70 expression to facilitate virus replication. Observations of mannose utilization and glutamine dependence suggest a crucial role of the HBP in supporting HEV replication. Inhibition of glycolysis and HSP70 activity or knockdown of glutamine fructose-6-phosphate amidotransferase expression led to a substantial reduction in HEV RNA and ORF2 expression accompanied by a significant decrease in HSP70 levels. This study demonstrates that glucose and glutamine play critical roles in facilitating HEV replication.