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.

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.

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.

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 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.

A novel macluravirus, tentatively named "tsaoko stripe mosaic virus" (TkSMV), was identified in Amomum tsaoko through high-throughput sequencing. The complete genome sequence of TkSMV was determined using RT-PCR and RACE. The genome sequence consists of 8218 nucleotides, excluding the poly(A) tail, and contains a large open reading frame encoding a polyprotein of 2625 amino acids with a molecular weight of approximately 297.13 kDa. TkSMV is most closely related to Alpinia oxyphylla mosaic virus, sharing 71.5% nucleotide and 75.9% amino acid sequence identity. These values are below the species demarcation threshold for the family Potyviridae. These results suggest that TkSMV should be considered a distinct member of the genus Macluravirus.

In this study, we analyzed the dynamic molecular epidemiology of herpangina based on pharyngeal swabs and demographic data collected from children with herpangina monitored in Tongzhou district in China from January 2021 to December 2022. A total of 1022 herpangina cases were diagnosed. Out of 225 samples collected, 56.4% (127/225) were positive for non-polio enterovirus, with seven genotypes identified: coxsackievirus A4 (CV-A4), CV-A6, CV-A10, CV-A2, CV-A16, CV-B3, and CV-A8. The predominant genotypes associated with herpangina changed during and after the COVID-19 pandemic, with the predominant genotypes being CV-A4 and CV-A6 in 2021 and CV-A10 and CV-A6 in 2022.

Multidrug-resistant infections are becoming increasingly prevalent worldwide. One of the fastest-emerging alternative and adjuvant therapies being proposed is phage therapy. Naturally isolated phages are used in the vast majority of phage therapy treatments today. Engineered phages are being developed to enhance the effectiveness of phage therapy, but concerns over their potential escape remain a salient issue. To address this problem, we designed a biocontained phage system based on conditional replication using amber stop codon suppression. This system can be easily installed on any natural phage with a known genome sequence. To test the system, we individually mutated the start codons of three essential capsid genes in phage φX174 to the amber stop codon (UAG). These phages were able to efficiently infect host cells expressing the amber initiator tRNA, which suppresses the amber stop codon and initiates translation at TAG stop codons. The amber phage mutants were also able to successfully infect host cells and reduce their population on solid agar and liquid culture but could not produce infectious particles in the absence of the amber initiator tRNA or complementing capsid gene. We did not detect any growth-inhibiting effects on E. coli strains known to lack a receptor for φX174 and we showed that engineered phages have a limited propensity for reversion. The approach outlined here may be useful to control engineered phage replication in both the lab and clinic.

A new polerovirus, named "arachis mottle-associated virus" (ArMoV), was identified by high-throughput sequencing in a Pinto peanut (Arachis pintoi) plant. The genome sequence was confirmed by Sanger sequencing and contains 5775 nucleotides and seven predicted open reading frames (ORFs), showing a typical polerovirus genome structure. All of the proteins encoded by ArMoV showed less than 90% amino acid sequence identity to those of other poleroviruses, the threshold to establish a new species in the genus. Phylogenetic analysis based on P1-P2 fusion protein and coat protein amino acid sequences showed that tobacco polerovirus 1 and chickpea chlorotic stunt virus, respectively, were the most closely related to ArMoV. These data suggest that ArMoV is a member of a new species of the genus Polerovirus, for which the binomial name "Polerovirus ARMOV " is proposed.

Interleukin 1 receptor antagonist (IL1RN) is a competitive inhibitor of interleukin 1 (IL-1). Natural killer cells (NK cells) contribute to the elimination of viruses by their antiviral effector function, which depends on a balance between inhibitory and activating receptor genes such as NKG2D and NKG2A. Using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, the association of intronic single-nucleotide polymorphisms (SNPs) in these genes with viral infection were assessed in 111 patients with hepatitis E virus (HEV) infection and 222 HEV-naive healthy controls. An SNP in the IL1RN (VNTR) gene revealed allele 2 to be associated with protection against HEV infection (IL1RN *1/*1 vs. IL1RN *2/*2, OR = 0.26, 95% CI = 0.14-0.47, p < 0.001). Similarly, a polymorphism in the intronic region of NKG2A revealed an association with protection in a co-dominant model (A/A vs. A/G: OR = 0.40; 95% CI = 0.24-0.67; A/A vs. G/G: OR = 0.25; 95% CI = 0.10-0.57; p < 0.05) and an association with susceptibility in a dominant model (A/A + A/G vs. G/G: OR = 2.28; 95% CI = 1.06-4.93; p < 0.05) and a recessive model (AA vs. AG + GG: OR = 2.71; 95% CI = 1.66-4.48; p < 0.001). Our data suggest that genetic polymorphisms in host NKG2A and IL1RN have both protective and detrimental roles in HEV infection, although their impact on disease outcome remains unknown.

In this study, a novel virus isolated from Nigrospora oryzae, tentatively named "Nigrospora oryzae mycovirus 1" (NoMyV1), was identified. NoMyV1 has a non-segmented dsRNA genome that is 2891 bp in length and contains two non-overlapping open reading frames (ORF1 and 2). ORF1 encodes a protein with sequence similarity to the putative capsid proteins or hypothetical proteins of other unclassified viruses, while ORF2 encodes an RNA-dependent RNA polymerase (RdRp). Sequence comparisons showed that NoMyV1 was most similar to Penicillium janczewskii Beauveria bassiana-like virus 1 (PjBblV1), with 76.12% amino acid sequence identity in the RdRp. In a phylogenetic analysis based on RdRp sequences, NoMyV1 was found to cluster with several other unclassified viruses for which a new genus, "Unirnavirus", which is distinct from the family Partitiviridae, has been proposed. Thus, we conclude that NoMyV1 is a novel member of the proposed genus "Unirnavirus".

Noroviruses (family Caliciviridae) are common causes of acute gastroenteritis worldwide. Multiple polymerase/capsid combinations have been identified among members of norovirus genogroup GII, at least 10 of which contain GII.P16 polymerase. During hospital-based surveillance (2003-2013) in Russia, we identified eight noroviruses with GII.P16 polymerase - five GII.3[P16], two GII.16[P16], and one GII.5[P16]. This is the first report of the nearly complete genome sequence of a rare recombinant GII.5[P.16] norovirus, which was found in the feces of a child in 2010. Phylogenetic analysis revealed that ORF1 and ORF2/3 of the strain GII.5[P.16]/RUS/Novosibirsk/Nsk-N490/2010 formed separate branches in clusters GII.P16 and GII.5, respectively.

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.

We present the genome sequence and organization and evidence of persistence of a new picorna-like virus infecting the flatworm Stenostomum leucops. The complete genome sequence belongs to a virus with a positive single-stranded RNA genome with two open reading frames (ORFs) flanked by untranslated regions and a polyadenylated C-terminus. The ORFs encode proteins with conserved motifs typical of members of the order Picornavirales. Phylogenetic analysis confirmed membership in this viral order, and it was found to be closely related to viruses found in Biomphalaria (Mollusca) in France and a virus detected in a metagenomic analysis of water sources from the USA, suggesting widespread distribution. RT-PCR analysis revealed that this virus can be detected in a laboratory-grown worm isolate for at least five years, suggesting persistent infection. However, no apparent deleterious effects were observed in the worms in culture, suggesting a possible commensal relationship between the virus and the worms.

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.

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.

Campylobacter jejuni is a leading cause of foodborne illness worldwide. The application of bacteriophages offers a promising approach to specifically target and reduce C. jejuni contamination in food products. In this study, two C. jejuni phages were characterized, and their ability to inhibit bacterial growth in combination with ethylenediaminetetraacetic acid (EDTA) was investigated. Both phages exhibited tolerance to a wide range of temperature (4-60 °C) and pH (3-9). Phage vB_CjeM-PC10 and vB_CjeM-PC22 were found to have a latent period of 30 min and 20 min and a burst size of 7 and 35 PFU/cell, respectively. Phage vB_CjeM-PC10 has a linear double-stranded DNA (dsDNA) genome of 51,148 bp with 77 ORFs and 29% GC content. Phage vB_CjeM-PC22 has a circular dsDNA genome of 32,543 bp with 56 ORFs and 28% GC content. At 42 °C, the combination of these phages (MOI = 10) and EDTA decreased the count of viable C. jejuni by 5.2 log and inhibited the regrowth of resistant cells for 48 h. At 4 °C, phage vB_CjeM-PC10 alone (MOI = 1000) reduced the count of viable C. jejuni by 3 log in brain heart infusion (BHI) broth and 2 log on chicken skin after incubation for 48 h. Although these phages were effective against C. jejuni, they cannot be utilized directly for food safety applications because they are lysogenic. Nevertheless, these findings expand the genome library of C. jejuni phages and enrich data resources by highlighting potential strategies for controlling C. jejuni 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.

In 2022-2024, three outbreaks of sheeppox (SPP) were reported in the European Union. These occurred in Spain, Bulgaria, and Greece and had serious economic consequences due to animal losses and trade restrictions. Five sheeppox virus (SPPV) whole-genome sequences (WGSs) were determined from samples collected during these outbreaks and analyzed in the context of all other published WGSs. Sheeppox virus strains can be divided in two, or possibly three, main groups. The isolates from the recent outbreaks belong to clade A2, which includes strains historically circulating in the Middle East and Northern Africa. Sequence divergence was low among the isolates that caused the recent European outbreaks. These results highlight the need for more regular and dense surveillance in under-sampled areas and the use of WGS to increase the chance of pinpointing the origin of an introduction, identifying potential introduction routes, and providing insights into SPPV evolution.