- Volume 106, Issue 3, 2025

The present study was conducted to monitor the genotype diversity of circulating species A rotavirus (RVA) in Iran. A total of 300 faecal specimens were collected from children under 5 years of age hospitalized for acute gastroenteritis between October 2023 and October 2024. G3P[8] represented 72.91% (70/96) of all RVA-positive samples, further subdivided into equine-like G3P[8]-I2-E2 DS-1-like and human G3P[8]-I1-E1 Wa-like. A retrospective genetic analysis of G3P[8] strains isolated from 2015 to 2017 was also performed and showed that G3P[8] strains belong to the G3P[8]-E1-I1 Wa-like genetic pattern, which is typically similar to human G3P[8] Wa-like strains in this study. The emergence of equine-like G3P[8] DS-1-like strains in Iran may not be related to selection pressure from rotavirus vaccination, but rather to cross-border migration of rotavirus strains due to population movements.

Nuclear transport of proteins larger than 60 kDa occurs via energy-dependent active transport, whereas smaller proteins diffuse into the nucleus through nuclear pore complexes via passive nuclear transport. Although the dengue virus (DENV) replication cycle primarily takes place in the cytoplasm, the capsid protein and non-structural protein 5 (NS5) are imported into the nucleus through a nuclear localization sequence-dependent mechanism. However, given its small molecular weight (14 kDa), the DENV capsid protein may also enter the nucleus via passive diffusion. While some drugs primarily inhibit active nuclear transport, few are known to block passive diffusion. Notably, biguanides have been associated with inhibitory effects on passive nuclear transport. Since biguanides such as metformin (MET) exhibit anti-DENV properties, we investigated the effects of MET on the nuclear transport of DENV proteins. Our results suggest that MET induces changes in the nuclear membrane of Huh-7 cells and reduces capsid nuclear localization without affecting NS5 nuclear import. Furthermore, MET treatment did not alter capsid nuclear import in BHK-21 cells. Additionally, mimicking MET’s effects using a non-hydrolyzable ATP analogue increased capsid cytoplasmic retention and decreased DENV-2 replication. Finally, the inhibition of the classical active nuclear transport pathway did not block capsid nuclear transport, suggesting that DENV-2 capsid enters the nucleus in Huh-7 and Vero cells independently of this pathway.

Parainfluenza virus 5 (PIV5) is a paramyxovirus that has been isolated from numerous mammalian hosts and is notable for its ability to cause persistent infections. Although PIV5-infected cells are resistant to IFN due to the ability of the V protein to target STAT1 for degradation, PIV5 shows residual IFN sensitivity when infecting cells that have already been exposed to IFN. We have previously reported that the human IFN-stimulated gene with the greatest inhibitory effect on PIV5 is IFIT1. IFIT1 inhibits the translation of incompletely methylated mRNAs (Cap0) but not those 2′-O-methylated at the first transcribed nucleotide (Cap1). All Mononegavirales are thought to generate Cap1 mRNA, so the sensitivity of PIV5 to IFIT1 is surprising. Here, we show that PIV5 generates Cap0 mRNA but not Cap1 mRNA, thus explaining its sensitivity to IFIT1. Furthermore, the sensitivity of different PIV5 genes to IFIT1-mediated translation inhibition varies. In the absence of complete Cap methylation, we show that the presence or absence of 5′-terminal RNA hairpin structures in the 5′UTRs of PIV5 genes determines the extent to which they are sensitive to IFIT1. Notably, the genes involved in RNA synthesis are relatively resistant to IFIT1 inhibition. This presents a potential mechanism by which IFIT1 can regulate the outcome of PIV5 infection in response to IFN and may be important in allowing the virus to establish prolonged/persistent infections.

Chronic infection with hepatitis B virus (HBV) (chronic HBV infection) places patients at increased risk for liver cirrhosis and hepatocellular carcinoma. Although nucleos(t)ide analogues are mainly used for the treatment of HBV, they require long-term administration and may lead to the emergence of drug-resistant mutants. Therefore, to identify targets for the development of novel anti-HBV drugs, we screened for HBV-suppressive host factors using a plasmid expression library of RNA-binding proteins (RBPs). We tested the effect of 132 RBPs on HBV replication by ectopically expressing these proteins along with HBV in hepatocellular carcinoma and evaluated the intracellular capsid-associated HBV DNA level. Our screen identified NEDD4-binding protein 1 (N4BP1) as having an anti-HBV effect. In hepatocellular carcinoma cell lines transfected or infected with HBV, the overexpression of N4BP1 decreased core-associated HBV DNA levels, while knockdown or knockout of the gene encoding N4BP1 rescued core-associated HBV DNA levels. N4BP1 possesses the KH-like and RNase domains, and both were required for the anti-HBV effect of N4BP1. Additionally, we measured levels of HBV pregenomic RNA (pgRNA) and covalently closed circular DNA in the RBP-transfected cells and confirmed that N4BP1 binds pgRNA directly and regulates both the 3.5 and 2.4/2.1 kb HBV RNA. In summary, N4BP1 is a newly identified host factor able to counteract HBV production by regulating 3.5 and 2.1/2.4 kb HBV RNA.

Previously, we had developed synthetic genomics methods to assemble an infectious clone of herpes simplex virus type-1 (HSV-1) strain KOS. To do this, the genome was assembled from 11 separate cloned fragments in yeast using transformation-associated recombination. Using this method, we generated null mutations in five tegument protein-coding genes as well as different combinations of these mutants. The single-locus mutants were all able to plaque on Vero cells. However, one multi-locus combination, ∆UL16/UL21, proved lethal for virus replication in non-permissive cells. The proteins encoded by the genes UL16 and UL21 are of interest because they are known to physically interact and are constituents of the tegument structure. Furthermore, their roles in HSV-1-infected cells are unclear. Both are dispensable for HSV-1 replication; however, in HSV-2, their mutation results in nuclear retention of assembled capsids and has activities that impact nuclear membrane integrity as well as activities of proteins that function in nuclear egress. We thus characterized these HSV-1 viruses that carry the single and double mutants. What we found was that the single mutants could replicate within cells and spread from infected to uninfected cells, albeit at significantly reduced levels. However, the double mutant (∆16/21) could not produce infectious progeny in a 24 h growth cycle and could not spread from cell to cell. Confocal microscopy of VP16-Venus expressed by these viruses as well as immunofluorescence assays for glycoprotein B showed perturbation of the nuclear membrane, which was pronounced in ∆21 and ∆16/21 infected cells. All the mutants assembled DNA-filled capsids as judged by ultrastructural analyses and sedimentation studies. Electron microscopy revealed the presence of numerous mature viruses in WT-infected cells but fewer such particles in the ∆16- and ∆21-infected cells. What we discovered is that in cells where both pUL16 and pUL21 are absent, cytoplasmic capsids were evident, but mature enveloped particles were not detected. The capsid particles isolated from all the single- and multi-locus mutant-infected cells showed significantly lower levels of incorporation of both VP16 and pUL37 when compared to the WT capsids. This reduced incorporation may be related to the loss of the integrity of the architecture of the nuclear membrane. Interestingly, the incorporation of pUL16 was not affected by the absence of pUL21 and vice versa, as judged by immunoblots. These data now show that of the tegument proteins, like the essential pUL36, pUL37 and VP16, the complex of pUL16 and pUL21 should be considered as important mediators of maturation and cell-to-cell spread of the particle.

Bacilladnaviruses are single-stranded DNA viruses that infect diatoms that, so far, have been primarily identified in marine organisms and environments. Using a viral metagenomics approach, we discovered 13 novel bacilladnaviruses originating from samples of mud-flat snail (Amphibola crenata; n=3 genomes) and benthic sediments (n=10 genomes) collected from the Avon-Heathcote Estuary in New Zealand. Comparative genomics and phylogenetic analysis of the new bacilladnavirus sequences in the context of the previously classified members of the family helped refine and further expand the Bacilladnaviridae taxonomy. Here, based on the replication-associated protein phylogeny and pairwise identities, we established 4 new genera – Aberdnavirus, Keisodnavirus, Puahadnavirus and Seawadnavirus – and 13 new species within the family. Comparison of the bacilladnavirus capsid protein sequences suggests that the positively charged N-terminal region (R-arm) is required for encapsidation of the larger genomes, whereas the smaller bacilladnavirus genomes can be packaged in the absence of the R-arm subdomain. Furthermore, analysis of the bacilladnavirus genomes revealed that members of three genera encode a highly derived variant of a phospholipase A1, which is predicted to be involved in the lysis of the infected diatoms and/or facilitates the entry of the virions into the host cells. Collectively, our results allow refining of the taxonomy of bacilladnaviruses and provide new insights into the biology and evolution of this understudied group of diatom viruses.