Search Results (70)

Enteroviruses are a group of highly prevalent human pathogens responsible for a wide range of illnesses, ranging from common cold symptoms to life-threatening diseases. A deep understanding of enterovirus biology, evolution, and host interaction is required for the development of effective vaccines and antivirals. Recombinant reporter viruses encoding luminescent or fluorescent proteins have been developed to facilitate such investigation. In this work, using coxsackievirus B3 as our model, we analyze how the insertion of fluorescent reporter genes at three distinct sites within the viral polyprotein affects viral fitness, identifying the most tolerant site for reporter insertion. We then describe a set of experimental workflows for measuring viral fitness, sera neutralization, antiviral efficacy, and recombination using fluorescent reporter viruses. The high homology between different enteroviruses suggests these assays can be readily adapted to study additional members of this medically and economically relevant group of viruses. Full article

Over the last 40 years, several studies have provided evidence demonstrating that viral vectors can result in effective gene targeting/insertions in a host’s genome. The traditional approaches of gene knock-down, -out, or -in involve an intensive transgenesis process that is plagued by extensive timescales. Plant viruses have the potential to target specific genes and integrate exogenous DNA molecules at the target locus. Their ability to manipulate a host’s genetic material and become a part of it makes them remarkable agents and helpful for molecular and synthetic biology. In this review, we describe how geminivirus-based vectors can be utilized to overcome traditional transgenesis. We highlight the progress that has been made so far and also discuss the hurdles that hinder the employment of geminivirus-based vectors. Furthermore, we conclude with a comparison of geminivirus-based vectors with other plant-derived vectors. Geminivirus-based vectors stand poised to revolutionize plant genome editing by making nucleic acid manipulation cheaper and easier to deploy, thus lessening the major technical constraints, including homology-directed repair (HDR)-mediated genome editing and time-inefficient tissue culture procedures. The insights given in this review illustrate a broader picture of geminiviral vectors, with an emphasis on engineering plant viruses to ease genome editing practices for crop improvements as well as boost experimental timescales from years to months. Full article

During the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SC2) infection was confirmed in various animal species demonstrating a wide host range of the virus. Prior studies have shown that the ACE2 protein is the primary receptor used by the virus to gain cellular entry and begin the replication cycle. In previous studies, we demonstrated that human and various bat ACE2 proteins can be utilized by SC2 viruses for entry. Bats are a suspected natural host of SC2 because of genetic homology with other bat coronaviruses. In this work, we demonstrate that expression of ACE2 genes from the common vampire bat (CVB) (Desmodus rotundus) and the pallid bat (PB) (Antrozous pallidus), supports infection and replication of some SC2 viruses in cell culture. Two cell lines were produced, CVB-ACE2 and PB-ACE2, expressing ACE2 from these bat species along with human TMPRSS2, in a model previously established using a non-permissive chicken DF-1 cell line. Results demonstrate that the original Wuhan lineage (WA1) virus and the Delta variant were able to infect and replicate in either of the bat ACE2 cell lines. In contrast, the Lambda and Omicron variant viruses infected both cell lines, but viral titers did not increase following infection. Viral detection using immunofluorescence demonstrated abundant spike (S) protein staining for the WA1 and Delta variants but little signal for the Lambda and Omicron variants. These studies demonstrate that while ACE2 from CVB and PB can be utilized by SC2 viruses to gain entry for infection, later variants (Lambda and Omicron) replicate poorly in these cell lines. These observations suggest more efficient human adaption in later SC2 variants that become less fit for replication in other animal species. Full article

Human cytomegalovirus (HCMV) is a high-risk pathogen in immunocompromised individuals, especially in transplant recipients. HCMV viremia must be monitored, and frequently, patients are treated with antiviral agents. HCMV has a variety of strategies to modulate host antiviral responses, and one important player is a viral homolog of the cellular interleukin-10 (cIL-10). The viral UL111A gene produces several HCMV IL-10 transcripts and protein isoforms through alternative splicing. The cmvIL-10 (isoform A) has similar properties to cIL-10, while LAcmvIL-10 (isoform B) has more restricted biological properties. Other isoforms are produced (C to H) but have unknown functions. Here, we investigated the expression of the most abundant transcripts, cmvIL-10 and LAcmvIL-10, in productively and latently infected cells and in peripheral blood mononuclear cells from renal transplant recipients up to 60 days post-transplantation. This study investigated HCMV cmvIL-10 and LAcmvIL-10 transcription profiles in vitro, in productive and latent infection, and in vivo, in peripheral blood mononuclear cells (PBMCs) of renal transplant patients. In vitro, both cmvIL-10 and LAcmvIL-10 transcripts were detected in both types at high levels and low levels in MRC-5 and latent infected (CD14+). When PBMCs from transplant patients were analyzed, LAcmvIL-10 was detected mostly sporadically and in only a few patients, while cmvIL-10 was found in all patients at all time points. Furthermore, it was observed in PBMCs that expression of cmvIL-10 was positively associated with an increase in viral DNA detection in the subsequently collected sample, indicating that expression of cmvIL-10 might precede viral DNA replication. These results contribute to the understanding of HCMV biology in different phases of infection. In addition, our initial analysis suggests that monitoring cmvIL-10, along with viral DNA, could improve early detection of HCMV reactivation in transplant recipients. Full article

The USDA-ARS collection of insect viruses at Beltsville, MD, USA, contains samples of an alphabaculovirus from larvae of the tufted apple bud moth, Platynota idaeusalis Walker, as well as a presumptive betabaculovirus from the same host species. The viruses in these samples—Platynota idaeusalis nucleopolyhedrovirus isolate 2680 (PlidNPV-2680) and Platynota idaeusalis granulovirus isolate 2683 (PlidGV-2683)—were characterized by electron microscopy of their occlusion bodies (OBs) and determination and analysis of their genome sequences. Scanning and transmission electron microscopy of the OBs revealed morphologies typical for alphabaculoviruses and betabaculoviruses. Sequencing viral DNA resulted in circular genomes of 121,881 bp and 106,633 bp for PlidNPV-2680 and PlidGV-2683, respectively. Similar numbers of ORFs (128 for PlidNPV-2680, 125 for PlidGV-2683) were annotated, along with ten homologous regions (hrs) in the PlidNPV-2680 genome and five intergenic regions of tandem direct repeats (drs) in the PlidGV genome. Phylogenetic inference from core gene alignments suggested that PlidMNPV-2680 represents a unique lineage within the genus Alphabaculovirus, while PlidGV-2683 was grouped with clade b betabaculoviruses. A comparison of the PlidNPV-2680 and PlidGV-2683 genomes revealed a 1516 bp region in PlidNPV-2680 that exhibited 97.5% sequence identity to a region of the PlidGV-2683 genome, suggesting that recombination had occurred recently between viruses from these lineages. Full article

The feline panleukopenia virus (FPV) is a highly contagious virus that affects cats worldwide, characterized by leukopenia, high temperature and diarrhea. Recently, the continuous prevalence and variation of FPV have attracted widespread concern. The aim of this study was to investigate the isolation, genetic evolution, molecular characterization and epidemiological analysis of FPV strains among cats and dogs in China from 2019 to 2024. The 41 FPV strains, including 38 feline strains and 3 canine strains, were isolated from rectal swab samples by inoculating monolayer FK81 cells and performing a plaque purification assay. The viral and hemagglutination titers of these 41 FPV strains were 10^4.33~10^6.33 TCID₅₀/0.1 mL and 7.0 log₂~9.7 log₂, respectively. Based on the complete VP2 gene, the nucleotide homology of these FPV strains was 98.91~100%, and the homology with 24 reference FPV strains from different countries and hosts was 98.85~100%. The phylogenetic analysis revealed that 41 FPV strains were more closely related to the FPV strains of Asian origin (Asian FPV strain group) than those of European and American origin (European and American FPV strain group). Furthermore, 12 mutation sites of the VP2 protein were found in these FPV strains, of which 91 and 232 amino acid sites were previously reported. Moreover, the 91 amino acid site was found to be a positive selection site with the highest dN/dS value in the selection pressure analysis. Importantly, 35 FPV strains with 91S substitution in the VP2 protein (FPV-VP2-91S strains) had formed obvious evolutionary branches in the Asian FPV strain group. The analysis of all available VP2 protein sequences of Chinese FPV strains in the GenBank database showed that the occurrence rate of FPV-VP2-91S strains had been increasing from 15.63% to 100% during 2017~2024, indicating that the FPV-VP2-91S substitution in the VP2 protein was a noteworthy molecular characteristic of the dominant FPV strains in China. These results contribute to a better understanding of their genetic evolution and renew the knowledge of FPV molecular epidemiology. Full article

Baculoviruses, the largest studied insect viruses, are highly pathogenic to host insects. Bombyx mori nucleopolyhedrovirus (BmNPV) is the main cause of nuclear polyhedrosis of silkworm, a viral disease that causes significant economic losses to the sericulture industry. The anti-BmNPV mechanism of the silkworm has not yet been characterized. Carboxypeptidase is an enzyme that is involved in virtually all life activities of animals and plants. Studies have shown that the carboxypeptidase family is related to insect immunity. There are few reports on the role of carboxypeptidase in the defense of silkworms against pathogen invasion. In this study, we identified the homologous gene Bombyx mori metal carboxypeptidases12 (BmMCP12) related to mammalian carboxypeptidase A2 (CPA2) and found that BmMCP12 had a Zn-pept domain. The BmMCP12 gene was primarily located in the cytoplasm and was highly expressed in the midgut of silkworms, and the expression level in BmN-SWU1 cells was upregulated after infection with BmNPV. After overexpression of the BmMCP12 gene, quantitative real-time (qRT)-PCR and Western blots showed that BmMCP12 could inhibit BmNPV replication, whereas knockout of the gene had the opposite effect. In addition, we constructed transgenic silkworm strains with a knockout of BmMCP12, and the transgenic strains had reduced resistance to BmNPV. These findings deepen the functional study of silkworm carboxypeptidase and provide a new target for BmNPV disease prevention in silkworms. Full article

Epidemiological surveys revealed that 33 of the 93 samples were positive for FHV-1, with the gD gene of these 33 samples exhibiting low variation, high homology, and no critical amino acid mutation. Feline herpesvirus type 1 (FHV-1), also known as feline viral rhinotracheitis (FVR) virus, is one of the main causes of URT disease in cats. All cats can become hosts of FHV-1, and the spread of this disease affects the protection of rare feline animals. Nasal swabs from cats with URT disease were collected at five veterinary clinics in Yanji City from 2022 to 2024. The purpose of this study was to isolate and investigate the epidemiology of FHV-1. The gD gene of the FHV-1 strain was cloned and inserted into the pMD-18T vector and transformed into a competent Escherichia coli strain. Subsequently, the gD gene of the positive samples was sequenced and phylogenetic analysis was performed to determine the genetic evolution relationship between the strains. We successfully isolated the FHV-1 strain YBYJ-1 in Yanji City for the first time. The diameter of the virus is approximately 150–160 nm. After 48 h of virus inoculation, the cells were round, isolated, and formed grape-like clusters. The gD gene of the virus was sequenced, and the length was 1125 bp, which proved the isolate was FHV-1. This study found that the genetic evolution of the FHV-1 gD gene was stable, expanding the molecular epidemiological data on FHV-1 in cats in Yanji City. Full article

Endogenous viral elements (EVEs) have been reported to exist widely in the genomes of eukaryotic organisms, and they are closely associated with the growth, development, genetics, adaptation, and evolution of their hosts. In this study, two methods—homologous sequence search and genome alignment—were used to explore the endogenous viral sequences in the genomes of Fragaria species. Results revealed abundant endogenous pararetroviruses (EPRVs) in the genomes of Fragaria species, including 786 sequences belonging to five known taxa such as Caulimovirus and other unclassified taxa. Differences were observed in the detected EPRVs between the two methods, with the homologous sequence search having a greater number of EPRVs. On the contrary, genome alignment identified various types and sources of virus-like sequences. Furthermore, through genome alignment, a 267-bp sequence with 95% similarity to the gene encoding the aphid-transmitted protein of Strawberry vein banding virus (Caulimovirus venafragariae) was discovered in the F. chiloensis genome, which was likely a recent insertion. In addition, the statistical analysis of the genome alignment results indicated a remarkably higher abundance of virus-like sequences in the genomes of polyploid strawberries compared with diploid ones. Moreover, the differences in virus-like sequences were observed between the genomes of Fragaria species and those of their close relatives. This study enriched the diversity of viruses that infect strawberries, and laid a theoretical foundation for further research on the origin of endogenous viruses in the strawberry genome, host–virus interactions, adaptation, evolution, and their functions. Full article

Rhinovirus C (RV-C) infects airway epithelial cells and is an important cause of acute respiratory disease in humans. To interrogate the mechanisms of RV-C-mediated disease, animal models are essential. Towards this, RV-C infection was recently reported in wild-type (WT) mice, yet, titers were not sustained. Therefore, the requirements for RV-C infection in mice remain unclear. Notably, prior work has implicated human cadherin-related family member 3 (CDHR3) and stimulator of interferon genes (STING) as essential host factors for virus uptake and replication, respectively. Here, we report that even though human (h) and murine (m) CDHR3 orthologs have similar tissue distribution, amino acid sequence homology is limited. Further, while RV-C can replicate in mouse lung epithelial type 1 (LET1) cells and produce infectious virus, we observed a significant increase in the frequency and intensity of dsRNA-positive cells following hSTING expression. Based on these findings, we sought to assess the impact of hCDHR3 and hSTING on RV-C infection in mice in vivo. Thus, we developed hCDHR3 transgenic mice, and utilized adeno-associated virus (AAV) to deliver hSTING to the murine airways. Subsequent challenge of these mice with RV-C15 revealed significantly higher titers 24 h post-infection in mice expressing both hCDHR3 and hSTING—compared to either WT mice, or mice with hCDHR3 or hSTING alone, indicating more efficient infection. Ultimately, this mouse model can be further engineered to establish a robust in vivo model, recapitulating viral dynamics and disease. Full article

An epidemiological study spanning twelve years has revealed that sheeppox disease is both widespread and endemic, predominantly surging during the winter and summer seasons. This investigation focused on sheeppox across 11 field outbreaks, involving 889 animals from non-migratory flocks across six districts in Karnataka, in the southern peninsula of India. Among these, 105 animals exhibited clinical signs suggestive of sheeppox, such as lesions on the body, and 95 cases were confirmed through PCR testing. The overall positivity rate for sheeppox stood at 10.68% (95 out of 889 animals). The incidence of sheeppox was notably higher in animals aged between 1 and 2 years and was more prevalent in females. Affected animals displayed symptoms including respiratory distress, weakness, fever, loss of appetite, depression, and various skin lesions ranging from papular to pock lesions across their bodies. There was a significant increase in total leukocyte count, while hemoglobin levels, red blood cell counts, and hematocrit values significantly decreased. On gross examination, sheeppox lesions, varying from vesicular to nodular forms, were predominantly found on hairless areas of the body. Microscopic examination of skin lesions revealed extensive changes, such as hyperkeratosis, parakeratosis, acanthosis, hydropic degeneration, and necrosis of epithelial cells, along with characteristic intracytoplasmic viral inclusions. The lungs exhibited type-II pneumocyte hyperplasia and proliferative bronchiolitis, also with intracytoplasmic inclusions. Confirmation of the sheeppox virus was achieved through PCR and subsequent sequence analysis. Phylogenetic analysis of the full-length P32 and RPO30 gene demonstrated homology with sheeppox isolates from various parts of India and neighboring countries, indicating that Indian sheeppox viruses are highly lineage-specific and correlate with the host of origin. Based on these findings, it is recommended to implement a homologous vaccination strategy, utilizing selective host/viral strains to enhance protection in susceptible animals. Full article

Carnivore protoparvovirus-1, feline parvovirus (FPV), and canine parvovirus (CPV) continue to spread in companion animals all over the world. As a result, FPV and CPV underwent host-to-host transfer in carnivorous wild-animal hosts. Here, a total of 82 fecal samples of suspected cat FPV infections were collected from Henan Province from 2020 to 2022. The previously published full-length sequence primers of VP2 and NS1 genes were used to amplify the targeted genes of these samples, and the complete gene sequences of 11 VP2 and 21 NS1 samples were obtained and analyzed. Analysis showed that the amino acid homology of the VP2 and NS1 genes of these isolates was 96.1–100% and 97.6–100%, respectively. The phylogenetic results showed that the VP2 and NS1 genes of the local isolates were mainly concentrated in the G1 subgroup, while the vaccine strains were distributed in the G3 subgroup. Finally, F81 cells were inoculated with the local endemic isolate Luoyang-01 (FPV-LY strain for short) for virus amplification, purification, and titer determination, and the pathogenesis of FPV-LY was detected. After five generations of blind transmission in F81 cells, cells infected with FPV-LY displayed characteristic morphological changes, including a round, threadlike, and wrinkled appearance, indicative of viral infection. The virus titer associated with this cytopathic effect (CPE) was measured at 1.5 × 10⁶ TCID₅₀/mL. Subsequent animal regression tests confirmed that the virus titer of the PFV-LY isolate remained at 1.5 × 10⁶ TCID₅₀/mL, indicating its highly pathogenic nature. Cats exposed to the virus exhibited typical clinical symptoms and pathological changes, ultimately succumbing to the infection. These results suggest that the gene mutation rate of FPV is increasing, resulting in a complex pattern of gene evolution in terms of host preference, geographical selection, and novel genetic variants. The data also indicate that continuous molecular epidemiological surveillance is required to understand the genetic diversity of FPV isolates. Full article

IFNAR1, one of the type I IFN receptors, is crucial to mammalian host defense against viral invasion. However, largely unknown is the immunological role of the fish teleost protein IFNAR1, also known as CRFB5. We have successfully cloned the whole cDNA of the Japanese eel’s (Anguilla japonica) CRFB5a homolog, AjCRFB5a. The two fibronectin-3 domains and the transmembrane region (238–260 aa) of AjCRFB5a are normally present, and it shares a three-dimensional structure with zebrafish, Asian arowana, and humans. According to expression analyses, AjCRFB5a is highly expressed in all tissues found, particularly the liver and intestine. In vivo, Aeromonas hydrophila, LPS, and the viral mimic poly I:C all dramatically increased AjCRFB5a expression in the liver. Japanese eel liver cells were reported to express AjCRFB5a more strongly in vitro after being exposed to Aeromonas hydrophila or being stimulated with poly I: C. The membranes of Japanese eel liver cells contained EGFP-AjCRFB5a proteins, some of which were condensed, according to the results of fluorescence microscopy. Luciferase reporter assays showed that AjCRFB5a overexpression strongly increased the expression of immune-related genes in Japanese eel liver cells, such as IFN1, IFN2, IFN3, IFN4, IRF3, IRF5, and IRF7 of the type I IFN signaling pathway, as well as one of the essential antimicrobial peptides LEAP2, in addition to significantly inducing human IFN-promoter activities in HEK293 cells. Additionally, RNA interference (RNAi) data demonstrated that knocking down AjCRFB5a caused all eight of those genes to drastically lower their expression in Japanese eel liver cells, as well as to variable degrees in the kidney, spleen, liver, and intestine. Our findings together showed that AjCRFB5a participates in the host immune response to bacterial infection by inducing antimicrobial peptides mediated by LEAP2 and favorably modulates host antiviral immune responses by activating IRF3 and IRF7-driven type I IFN signaling pathways. Full article

Soil Sinorhizobium phage AP-16-3, a strain phylogenetically close to Rhizobium phage 16-3, was isolated in a mountainous region of Dagestan, belonging to the origin of cultivated plants in the Caucasus, according to Vavilov N.I. The genome of phage AP-16-3 is 61 kbp in size and contains 62 ORFs, of which 42 ORFs have homologues in the genome of Rhizobium phage 16-3, which was studied in the 1960s–1980s. A search for Rhizobium phage 16-3-related sequences was performed in the genomes of modern strains of root nodule bacteria belonging to different species, genera, and families. A total of 43 prophages of interest were identified out of 437 prophages found in the genomes of 42 strains, of which 31 belonged to Sinorhizobium meliloti species. However, almost all of the mentioned prophages contained single ORFs, and only two prophages contained 51 and 39 ORFs homologous to phages related to 16-3. These prophages were detected in S. meliloti NV1.1.1 and Rh. leguminosarum OyaliB strains belonging to different genera; however, the similarity level of these two prophages did not exceed 14.7%. Analysis of the orphan genes in these prophages showed that they encoded predominantly virion structural elements, but also enzymes and an extensive group of hypothetical proteins belonging to the L, S, and E regions of viral genes of phage 16-3. The data obtained indicate that temperate phages related to 16-3 had high infectivity against nodule bacteria and participated in intragenomic recombination events involving other phages, and in horizontal gene transfer between rhizobia of different genera. According to the data obtained, it is assumed that the repetitive lysogenic cycle of temperate bacteriophages promotes the dissolution of the phage genetic material in the host bacterial genome, and radical updating of phage and host bacterial genomes takes place. Full article

Adenovirus vectors possess a good safety profile, an extensive genome, a range of host cells, high viral yield, and the ability to elicit broad humoral and cellular immune responses. Adenovirus vectors are widely used in infectious disease research for future vaccine development and gene therapy. In this study, we obtained a fowl adenovirus serotype 4 (FAdV-4) isolate from sick chickens with hepatitis–hydropericardium syndrome (HHS) and conducted animal regression text to clarify biological pathology. We amplified the transfer vector and extracted viral genomic DNA from infected LMH cells, then recombined the mixtures via the Gibson assembly method in vitro and electroporated them into EZ10 competent cells to construct the FAdV-4 infectious clone. The infectious clones were successfully rescued in LMH cells within 15 days of transfection. The typical cytopathic effect (CPE) and propagation titer of FAdV-4 infectious clones were also similar to those for wild-type FAdV-4. To further construct the single-cycle adenovirus (SC-Ad) vector, we constructed SC-Ad vectors by deleting the gene for IIIa capsid cement protein. The FAdV4 infectious clone vector was introduced into the ccdB cm expression cassette to replace the IIIa gene using a λ-red homologous recombination technique, and then the ccdB cm expression cassette was excised by PmeI digestion and self-ligation to obtain the resulting plasmids as SC-Ad vectors. Full article