Virology

Mayaro virus (MAYV) is an emergent arthropod-borne virus that causes an acute febrile illness accompanied by arthralgia, similar to chikungunya virus. Increasing urbanization of MAYV outbreaks in the Americas has led to concerns that this virus could further expand its geographic range. Given the potential importance of this pathogen, we sought to fill some critical gaps in knowledge regarding MAYV infectivity and geographic variation. This study describes the cytopathogenicity of MAYV in human dermal fibroblasts, human skeletal muscle satellite cells, human embryonic kidney cells (HEK), peripherally derived human macrophages, and Vero cells. MAYV strain isolated from Bolivia (MAYV-U) infected cells more rapidly compared to MAYV strains isolated in Peru and Brazil (MAYV-P; MAYV-B), with high titers (1x108 pfu/ml) peaking at 37 hours post infection. MAYV-U also caused the most cytopathic effect in a time dependent manner. Furthermore, differently from the other two prototypic strains, MAYV-U harbors unique mutations in the E2 protein, D60G and S205F, likely to interact with the host cell receptor, which may explain the observed differences in infectivity. We further demonstrate that pre-treatment of cells with interferon-{beta} inhibited viral replication in a dose-dependent manner. Together, these findings advance our understanding of MAYV infection of human target cells and provide initial data regarding MAYV phenotypic variation according to geography. Author SummaryArthropod-borne viruses are of great public health concern, causing epidemics worldwide due to climate change, changes in land use, rapid urbanization, and the expanding geographic ranges of suitable vectors. Among these viruses, Mayaro is an emerging virus for which little is currently known. This study aims to answer fundamental questions of Mayaro virus biology using three geographically distinct viral strains to examine variability in infection kinetics and infectivity in susceptible cell types. We found one geographic isolate to have accelerated infection kinetics and increased cell damage because of infection. To better understand what was unique about this isolate, we compared their envelope protein, which is critical for entry into a cell. We found that the isolate with increased replication kinetics possessed mutations at sites that may promote viral entry, which could explain these findings. Together, these findings further our understanding of Mayaro virus biology and provide insight into factors that contribute to Mayaro transmission and infectivity.

Okra (bhendi) is a widely cultivated food crop in warm regions of the world, with India contributing approximately 60% of global production. However, okra cultivation in India is severely constrained by viral diseases, among which infections caused by the geminiviruses bhendi yellow vein mosaic virus (BYVMV) and okra enation leaf curl virus (OELCuV), in association with their satellites, represent major limitations to crop productivity. In recent years, the geminivirus-encoded C4 protein has emerged as a key pathogenicity determinant in this viral family, with functions that include suppression of multiple layers of plant antiviral defence and induction of disease symptoms. Here, we comparatively characterize the C4 proteins of BYVMV and OELCuV by determining their targeting signals and subcellular localization, and by assessing their ability to induce developmental abnormalities and suppress the cell-to-cell spread of RNA silencing. Our results reveal that the two C4 proteins display distinct subcellular localization patterns, yet both are capable of inducing developmental alterations, likely through different mechanisms, and of suppressing the intercellular spread of RNA silencing, possibly via interaction with a common host factor. Together, these findings suggest that C4 might be a critical virulence factor in okra-infecting geminiviruses and act as a symptom determinant. The C4 proteins encoded by BYVMV and OELCuV therefore emerge as promising targets for the development of antiviral management strategies in okra.

Munger black raspberry (Rubus occidentalis) has been a preferred indicator for Rubus viruses. The working hypothesis behind the Munger elevated susceptibility to viruses is its ability to sustain elevated virus titers; however, no research has yet explored this concept. To address this, we utilized an infectious clone of blackberry chlorotic ringspot virus to study the differences in virus expression between two Rubus species: R. occidentalis ( Munger) and R. L. subgenus Rubus Watson (blackberry, Natchez). Our data demonstrate that virus accumulation in Munger is over 47,300 times higher compared to Natchez, enhancing our understanding of virus-host dynamics and providing valuable insights into why Munger is a preferred indicator species for virus detection in Rubus spp.

The PCR amplification of majority of the ssRNA of both genomic and non-genomic mRNA is accomplished by RT-PCR. The mRNA is subjected to cDNA synthesis using reverse transcriptase and either Oligo(dT)18, or random or gene specific reverse primers based priming strategies. The choice of primer largely depends on the nature of 3 prime terminus of mRNA and length of cDNA synthesized. In general, oligo(dT)18 is the preferred choice for mRNAs having poly(A) tail at 3 prime terminus. In general, tobamoviruses lack any poly(A) tail at their 3 prime untranslated region (UTR) which forms a tRNA like structure and upstream pseudoknot domain except tow viruses viz., Hibiscus latent Fort Pierce virus (HLFPV) and Hibiscus latent Singapore virus (HLSV) which accommodate internal poly(A) sequences of 46 and 87 nucleotides long, respectively in their 3 prime UTR. However, determination of full nucleotide sequence of Pepper mild mottle virus (PMMoV) using an oligo(dT)18 primed cDNA as template indicated the libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template which are known to lack poly(A) tail. at the end or internally at its 3 prime end. Moreover, coat protein (CP) gene of PMMoV and bean common mosaic virus (BCMV) (Potyvirus with a poly(A) tract at its 3 prime end) was amplified using cDNA primed with random primer as well as oligo(dT)18 was successfully amplified but with significant variation in the intensity of the amplification band in case of PMMoV but not in BCMV. This clearly indicated the presence of PMMoV mRNA with polyadenylated 3 prime tail in total RNA isolated from PMMoV infected capsicum leaves with abundance of non-polyadenylated PMMoV genomic RNA (gRNA). Hence, we hypothesize that the generation of polyadenylated RNA population during the infection cycle of PMMoV in pepper may be possible reason for libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template isolated from PMMoV infected leaves followed by amplification of entire PMMoV genome through RT-PCR. This is first study indicating the presence of polyadenylated or polyadenylated rich regions in PMMoV gRNA acquired during the infection cycle in pepper.

The Citrus yellow vein clearing virus (CYVCV) causes a viral disease that has been reported in specific citrus-growing regions in Euro-Asia including countries of Pakistan, India, Turkiye, Iran, China and south Korea. Recently, CYVCV was detected in a localized urban area in a town in heart of Californias citrus-growing region and marks the first occurrence of the virus in North America. CYVCV is spread by aphid and whitefly vectors and is graft and mechanically transmitted. Hence, it is an invasive disease that presents a significant threat to the California citrus industry, especially lemons which are highly susceptible to CYVCV. To elucidate the origin of the CYVCV California strain, we used long-read sequencing technology and obtained the complete genomes of three California CYVCV isolates, CA1, CA2, and CA3. The sequences of these isolates exhibited intergenomic similarities ranging from 95.4% to 97.4% to 54 publicly available CYVCV genome sequences which indicated a relatively low level of heterogeneity. However, CYVCV CA isolates formed a distinct clade from the other isolates when aligned against other CYVCV genomes and coat protein gene sequences. Based on a rooted Maximum Likelihood phylogenetic tree, CYVCV CA isolates shared the most recent common ancestor with isolates from India. Further examination of 79 coat protein gene sequences collected over a 31-year period that spanned regions from East and South Asia to the Middle East and California, Bayesian evolutionary inferences resulted in a spatiotemporal reconstruction that placed the origin of all CYVCV to the 1930s, with South Asia as the most plausible geographic source. This analysis also suggested that CYVCV CA isolates diverged from Indian lineages, possibly around the 2010s. Moreover, the spatiotemporal phylogenetic analysis indicated two additional virus diffusion pathways: one from South Asia to East Asia and another from South Asia to the Middle East. Collectively, our phylogenetic inferences offer insights into the probable dynamics of global CYVCV dissemination, emphasizing the need for citrus industries and regulatory agencies to closely monitor citrus commodities crossing state and international borders. Author SummaryA localized outbreak of CYVCV was detected in a central California town, marking its first appearance in North America. The study sequenced the complete genomes of three CYVCV isolates from California and employed statistical algorithms to investigate the population dynamics and origin of CYVCV. Upon comparing coat protein gene sequences, the CYVCV isolates from California formed a distinct group separate from those found in other geological regions. The studys spatiotemporal phylogenetic analysis highlighted that CYVCV likely originated in the 1930s, with South Asia as the most plausible source. Notably, the CYVCV isolates from California diverged from Indian lineages, possibly around the 2010s. This study contributes to a better understanding of CYVCVs genetic and molecular diversity, shedding light on virus ecology, evolution, and biology.

Borealpox virus (BRPV; formerly Alaskapox) is an orthopoxvirus that has caused seven reported human infections in Alaska since 2015, including a fatal case in 2023. The natural reservoir of BRPV is unknown, although previous investigations have raised the possibility of wild small mammals transmitting the virus to humans, either through direct contact or via domestic cats and dogs. To understand which species may be involved in the maintenance and/or spillover of BRPV in Alaska, we trapped and sampled wild small mammals (including voles, shrews, and squirrels) in 2021 and 2024 near reported human case locations in Fairbanks and the Kenai Peninsula, respectively. We found evidence of previous exposure to orthopoxviruses in five species (including the House Mouse, Mus musculus) and detected BRPV DNA as well as viable virus in Northern Red-backed Voles (Clethrionomys rutilus). Further, screening of tissues from historical museum specimens revealed BRPV DNA in C. rutilus specimens collected in Denali National Park and Preserve in 1998 and 1999, 17 years before the first reported human case of BRPV. Phylogenomic analysis of all human and animal BRPV isolates strongly supports the hypothesis of local human infections through multiple spillover events. These findings suggest C. rutilus as a possible reservoir species for BRPV and indicate that BRPV has been present in Alaskan wild small-mammal populations for at least 25 years. Our study highlights the potential of museum collections to elucidate the temporal, spatial, and host ranges of emerging pathogens. Further museum- and field-based sampling will clarify the true geographic range of BRPV, which is closely related to Old World orthopoxviruses and may be circulating beyond North America.

Mpox (formerly monkeypox) is caused by monkeypox virus (MPXV) and has prompted two recent global health emergencies. Clade IIb MPXV, a recently recognized subclade, has been associated with oral and genital lesions and transmission among men who have sex with men (MSM); however, mechanisms of genital pathogenesis and sexual transmission are not understood. We investigated several routes of MPXV Clade IIb virus infection (intranasal, oral, anal, and intraperitoneal) and found high and prolonged viral titres in the testes after IP inoculation still detectable at 21 days. The testes had significant changes to tissue architecture including loss of spermatogenesis, disorganization of spermatozoa, loss of Leydig cells, and breakdown of the seminiferous tubule membranes. Viral antigen positive cells were present in the interstitial spaces between the seminiferous tubules with macrophage infiltration also evident. This work provides insights into potential of sexual transmission for MPXV viruses as well as mechanisms of Mpox disease which may significantly impact long-term fertility or sex organ health of infected males. Author SummaryMpox (formerly monkeypox) is a painful disease similar to smallpox caused by the monkeypox virus (MPXV) which is an emerging/re-emerging virus. Recently the WHO has declared two global health emergencies due to human-to-human transmission of MPXV. Clade IIb MPXV is a newly recognized subclade that has been associated with genital lesions and sexual transmission/contact; however, how the virus causes disease in sex-organs or how it is passed from person-to-person is not understood. We investigated several routes of MPXV Clade IIb inoculation and found IP inoculation in male mice led to high and prolonged viral titres in testes. Additionally, the testes were significantly affected having loss of sperm and cellular organization. Evidence of inflammatory cell infiltration, specifically, macrophages, are suspected to be the cause of testes specific disease. This work provides insights into potential of sexual transmission for MPXV viruses as well as mechanisms of Mpox disease.

Infectious Bursal Disease Virus (IBDV) (Avibirnavirus genus, Birnaviridae family) is a non-enveloped virus with a double-stranded RNA (dsRNA) genome. IBDV causes a highly contagious and immunosuppressive disease in domestic chickens (Gallus gallus), representing a major threat to the global poultry industry. Apoptotic cell death and exacerbated innate immune responses have been implicated in IBDV pathogenesis. Previous studies from our laboratory demonstrated the crucial role of type I interferon (IFN) in triggering apoptosis in IBDV-infected cell cultures. Genomic IBDV dsRNA is recognized by the cytoplasmic pattern recognition receptor (PRR) melanoma differentiation-associated gene 5 (MDA5) in chicken cells, triggering type I IFN responses. However, the contribution of the endosomal PRR Toll-like receptor 3 (TLR3) dsRNA sensor on type I IFN production upon IBDV infection has not been studied, despite several studies have demonstrated that its expression is significantly upregulated upon IBDV infection. Here, we demonstrate that ablation of TLR3 gene expression in DF-1 chicken fibroblasts results in a complete blockade of IBDV-induced apoptosis, a marked reduction in IFN production, and a significant enhancement of virus progeny yields. Notably, this effect appears to be specific to IBDV, as similar outcomes were not observed with other RNA viruses. Our findings also suggest that TLR3 may also play a role in viral release into the extracellular space. Additionally, receptor interacting protein kinase 1 (RIPK1), a protein that interacts with TLR3 through the adaptor Toll/IL-1 receptor (TIR) domain-containing adaptor-inducing interferon-{beta} (TRIF), was shown to contribute to both IFN production and apoptosis in response to IBDV infection or dsRNA stimulation in DF-1 cells. Overall, this study provides new insights into the innate immune recognition of IBDV, highlighting the central role of TLR3 in mediating antiviral responses in chicken cells. Author summaryInfectious Bursal Disease Virus (IBDV) is the etiological agent of a devastating syndrome known as IBD or Gumboro disease, which affects domestic chickens (Gallus gallus), leading to significant economic losses in the poultry industry worldwide. The virus primarily targets immature B lymphocytes causing their killing by apoptosis, resulting in severe immunosuppression. Diverse studies have suggested that exacerbation of the innate immune response is related to IBDV pathogenesis. Here, we have investigated the role of the cellular sensor of double-stranded RNA (dsRNA) Toll-like receptor 3 (TLR3) on the fate of IBDV-infected cells. Deletion of the TLR3 gene completely blocks apoptosis induced in IBDV-infected DF-1 cells, drastically reduces interferon (IFN) production and improves viral replication efficiency. We also demonstrated the participation of receptor interacting protein kinase 1 (RIPK1), a known downstream mediator of TLR3, in both, IFN production and apoptosis induction in response to IBDV infection. These findings provide new molecular insights into the mechanisms underlying the robust type I IFN response observed during IBDV infection and contribute to a deeper understanding of IBDV pathogenesis.

Nipah virus (NiV) is a zoonotic highly pathogenic Paramyxovirus inducing lethal outbreaks of encephalitis and severe acute respiratory syndrome (SARS) with an average case-fatality rate of 75%. Two viral strains, NiV-Malaysia (NiV-Mal) and NiV-Bangladesh (NiV-Ban), associated to distinct geographical distribution, route of transmission, symptoms and lethality have been described. Due to the permanent threat of these emerging infections and the lack of approved therapeutics, it is crucial to improve our understanding regarding NiV-associated pathogenesis. Mice represent a small and accessible animal model, provided with numerous biological tools for the functional assessment of different genes related to antiviral response. Here, we analyze the susceptibility of mice deficient for type I interferon receptor (IFNAR KO) to infection with either NiV-Mal or NiV-Ban through intraperitoneal or intranasal routes. Our results show that IFNAR KO mice are susceptible to NiV-Ban infection via intraperitoneal route, although to a lesser extent than NiV-Mal, and develop encephalitis and pulmonary syndrome with viral propagation to different organs and lethal outcome in 60% of infected animals. In addition, intranasal administration of both viral strains led to a subclinical infection with viral replication in brain and lungs and production of virus-specific neutralizing antibodies. These results indicate IFNAR KO mice as a small animal model permitting comparative studies of the immunopathogenesis perpetrated by both NiV-Mal and NiV-Ban infections. Author summaryAvailability of small animal models represents a major issue to characterize virus-associated pathogenesis to further implement therapeutic strategies. Previous studies performed with NiV-Mal and NiV-Ban strains in wild-type (WT) mice did not indicate signs of NiV disease. However, when challenged with NiV-Mal, IFNAR KO mice suffered fatal outcomes, thus providing useful information on viral immunopathogenesis. Surprisingly, while being the more frequently re-emerging virus strain, no infection studies with NiV-Ban have been performed in IFNAR KO mice so far. Here, we sought comparing pathogenesis after NiV-Mal- and NiV-Ban-infection following IP and IN inoculations in IFNAR KO mice. Indeed, our results highlighted that contrary to fatal IP challenge, IN inoculation lead to a subclinical infection with both NiV strains. Moreover, we determined that NiV-Mal is more pathogenic than NiV-Ban following IP infection. Finally, distinct histopathological manifestations and tropism were associated with each viral strain and specific route of infection. Overall, our study implies that IFNAR KO mice represent a useful animal model to study strain-specific NiV pathophysiology.

Cystoviridae is a family of double stranded RNA (dsRNA) phage that infects various strains of Pseudomonas syringae, a Gram-negative soil bacteria known to infect various crops. Surrounding the icosahedral capsids of these phages is a bacterial derived phospholipid membrane. Embedded within this membrane is a multi-component protein complex, referred to as the spike complex. The spike complex is responsible for host recognition and membrane fusion. We studied the ability of two members of the Cystivirdae family to infect cells in the presence of purified outer membrane vesicles (OMVs) and lipopolysaccharide (LPS) derived from distinct sources. In this study we determined that OMVs from the host Pseudomonas pseudoalcaligenes strain: East River isolate A (ERA) inhibit Phi 8 and Phi 12 host infection. These OMVs range in size from 30 to 60 nm and bind to Phi 8 and Phi 12. However, OMV purified from P. syringae pv. phaseolicola LM2691 and E. coli {Delta}yciB {Delta}dcrB did not inhibit Phi 8 or Phi 12 host infection. However, LPS derived from ERA and LM2691 inhibited Phi 8 and Phi 12 infection, demonstrating that LPS is the receptor for these two viruses, and that OMV biogenesis is selective of LPS. LPS derived from other non-Cystoviridae Gram-negative bacteria, did not inhibit infection. We confirmed that host proteins are not required for Phi 8 or Phi 12 host interaction. Our results also suggest that differences in lipid A and the core polysaccharide in LPS may influence Phi 8 and Phi 12 host binding. IMPORTANCEMost phage families studied to date use a tailed appendage, composed of a multitude of proteins, for cellular recognition, membrane penetration, and genome injection. This contrasts with members of the Cystoviridae family which possess a phospholipid membrane bilayer with embedded proteins responsible for cellular recognition and membrane fusion. Thus, the Cystoviridae are akin to enveloped viruses which also use protein complexes embedded into their membrane for cellular recognition and membrane fusion. Examples of such viruses include the Retroviridae, Coronoviridae, Herpesviridae, and Orthomyxoviridae families. The binding specifics of Cystoviridae to the host outer membrane are unknown. Using Cystoviridae-OMV interaction we began to uncover the host requirements for binding Cystoviridae. The results presented determine that only lipid A and the core polysaccharide of LPS are required for Cystoviridae outer membrane binding.

Grapevine Red Blotch Virus is a major grapevine pathogen and is associated with reduced carbon assimilation and delayed berry ripening in Vitis vinifera L. Recent work suggests that the virus alters leaf carbon metabolism prior to emergence of visible symptoms. Therefore, diurnal and seasonal measurements were conducted to quantify changes in leaf carbon balance and to elucidate the chronology of symptom progression in leaves and fruit. Healthy and infected vines were assayed in a commercial vineyard during which leaf water relations, photosynthesis, and nonstructural carbohydrates were measured. Additionally, sugar and anthocyanin accumulation in the fruit were monitored at the end of the season to characterize the impact of the virus on ripening. Virus infection significantly reduced carbon assimilation pre- and postveraison, but the impact was more pronounced postveraison and during the afternoon when vine water status was the lowest. Similarly, virus infection significantly increased leaf starch concentration pre- and postveraison, but increased leaf starch in infected vines was detected two weeks prior to veraison. Virus infection had the greatest impact on obstructing leaf carbon export postveraison, especially during the afternoon. The virus had no impact on chlorophyll fluorescence, indicating there was no sustained photosystem impairment and suggesting that changes in chlorophyll fluorescence were a transient response to reduced carbon assimilation and export. This study provides evidence that reduced carbon export constitutes a feedback inhibition response to accumulation of leaf starch prior to the appearance of visible symptoms or impacts to ripening, which may aid earlier detection of the virus.

The yellow mosaic disease (YMD) caused by begomoviruses is a major constraint for the production of pole bean (Phaseolus vulgaris L.) in India. Survey was carried out in the eastern dry zone of Karnataka during 2019-20 to record the incidence of yellow mosaic disease in pole bean which revealed the ubiquitous prevalence of YMD in pole bean ranging from 6.02 to 80.74 per cent. Leaf samples collected (symptomatic and asymptomatic) were subjected for begomovirus detection using specific primers. Twelve samples, representing all the 12 taluks in the surveyed region were considered for full genome amplification by RCA, cloned and sequenced. Genome length of 12 current isolates ranged from 2718 - 2744 and 2668 - 2671 nucleotides for DNA-A and DNA-B, respectively. Sequence analysis using Sequence Demarcation Tool (SDT) showed >91 per cent nucleotide identity of current isolates (DNA-A) with other horsegram yellow mosaic virus (HgYMV) isolates available in the GenBank. As per existing ICTV criteria, all the current isolates can be considered as strains of HgYMV. Further, DNA-B associated with all the 12 isolates also shared >91 per cent nucleotide identity with DNA-B of HgYMV isolates, indicating absence of component re-assortment in HgYMV. Variation in the pairwise nucleotide identity and phylogenetic analysis confirmed the existence of new strains within the current HgYMV isolates. GC plot analysis reveals potential recombination in the low GC rich regions. Further, recombination breakpoint analysis indicated intra-species recombination in both DNA-A and DNA-B, which might have driven the origin of new strains in HgYMV. This is the first comprehensive study on begomoviruses ioslates associated with the yellow mosaic disease of pole bean based on complete genome sequencing in the world.

Hepatitis E virus (HEV) is an expanding zoonotic viral disease threat. Although HEV causes acute viral hepatitis, it is increasingly being recognized as a systemic pathogen with detection and damage in extrahepatic tissues. The presence of HEV RNA in the semen of chronically infected human patients in the absence of viremia and fecal shedding and presence of HEV in the sperm head underscores the need to understand the interaction of HEV within the male reproduction system. Male accessory glands secrete biofluids necessary for sperm nourishment and to neutralize the acidity of the vagina. The role of male accessory glands in the dissemination and persistence of HEV infection have not been studied. Using an immunosuppressed pig model for chronic HEV infection, we demonstrate infectious HEV in mature sperm cells altering the sperm motility and morphology. HEV isolated from sperm cells remained infectious in human hepatoma cells. Spermatic fluid contained lower virus titers than the sperm cells from chronically infected pigs highlighting that the sperm cells themselves can associate with the virus. Evaluation of the male accessory glands demonstrated viral replication, infiltration of CD45 leukocytes, and apoptosis associated with HEV infection. A decrease in serum testosterone levels was evident in the HEV infected pigs. Even though a lower viral RNA titer was seen in serum and feces of chronically infected, immunosuppressed and ribavirin treated pigs, high viral RNA and infectious particles in sperm is a concern. Our findings necessitate further studies defining the mechanism of sperm cell invasion by HEV, length of HEV survival in sperm cells during chronic HEV infection, and risk of sexual transmission of HEV during both acute and chronic phases of infection. Author SummaryHepatitis E virus, a leading cause of acute viral hepatitis, causes both acute and chronic infection in humans. Recent advances within the HEV field have demonstrated extrahepatic diseases associated with HEV. More recent findings have revealed infectious HEV in the vagina, Sertoli cells, and ejaculate of humans, and sperm cells of pigs. We demonstrate that the male accessory sex glands may have a role in the persistence of HEV infection during chronic infections. We utilized an established immunosuppressed pig model and treated pigs with ribavirin to study the presence of virus in the sperm cells. We demonstrated high viral RNA loads and infectious particles associated with sperm cells. Our study further highlights the importance of the testis, as an immune privileged site, in the maintenance of chronic HEV infection. New studies to evaluate the mechanisms by which HEV associates with sperm cells, the length of HEV survival in sperm cell fractions, and consideration of the testes as a potential HEV reservoir are necessary.

Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routs by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. The interaction of the virus with GM1a was studied using fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), imaging FCS (ImFCS) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), and the effect on infectivity and movement of the virus during infection was explored using plaque assays and fluorescence-based imaging and single particle tracking (SPT). GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a ten-fold increase and seven-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems respectively. The interaction of virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection, and functions as a coreceptor/ attachment factor for DENV during infection in mammalian systems. Author SummaryDengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, no successful antivirals or treatments have been isolated to this date. This may be due to the incomplete understanding of the virus infection mechanism, including a lack of an identified true receptor and entry related attachment factors or co-receptors responsible for internalization of the virus. This work focuses on the early infection stage of DENV1 and DENV2 strains, to identify how the virus moves on cell surfaces in its search for its receptors, and identifies the critical role of the sialic acid ganglioside GM1a during internalization of the virus.

Molecular computing was used to investigate the possible causal agents of chilli crop samples showing mixed symptoms of yellow leaf curl and little leaf type diseases in the Uttar Pradesh province, India. Total genomic DNA was extracted from twenty-five samples and amplified by PCR using a universal primer pair for begomovirus and phytoplasma. Mixed infection samples show positive amplified products for begomovirus (DNA-A and betasatellite) and phytoplasma (16S rRNA and Sec A). The identified begomovirus from chilli samples was identified as a strain isolate of the previously described Chilli Leaf Curl Virus (94.2% nucleotide sequence identity), which is known to infect Solanum lycopersicon, in Oman, whereas the 16S rRNA was identified from the source Candidatus Phytoplasma trifolii (99.04% nucleotide sequence identity), which is known to infect Helichrysum flowering plants in India. Subsequently, molecular computing research based on phylogenetic interweaves, putative recombination, amino acid selection, and genetic diversity were investigated, revealing divergent evolutionary patterns with significant variation and recombination events. The majority of the sequence variations observed in begomovirus and phytoplasma were caused via inter- and intra-specific recombination. These findings could be the first in silico combined infection analysis of ChiLCV and Ca.P.trifolii in a chilli crop in India, revealing the potential adaption and evolution of begomovirus and phytoplasma to a new geographic range and crop.

Although Australian marsupials are characterised by unique biology and geographic isolation, little is known about the viruses present in these iconic wildlife species. The Dasyuromorphia are an order of marsupial carnivores found only in Australia that include both the extinct Tasmanian tiger (Thylacine) and the highly threatened Tasmanian devil. Several other members of the order are similarly under threat of extinction due to habitat loss, hunting, disease, and competition and predation by introduced species such as feral cats. We utilised publicly available RNA-seq data from the NCBI Sequence Read Archive (SRA) database to document the viral diversity within four Dasyuromorphia species. Accordingly, we identified 15 novel virus species from five DNA virus families (Adenoviridae, Anelloviridae, Herpesviridae, Papillomaviridae and Polyomaviridae) and three RNA virus taxa: the order Jingchuvirales, the genus Hepacivirus, and the delta-like virus group. Of particular note was the identification of a marsupial specific clade of delta-like viruses that may indicate an association of deltaviruses and with marsupial species dating back to their origin some 160 million years ago. In addition, we identified a highly divergent hepacivirus in a numbat liver transcriptome that falls outside of the larger mammalian clade, as well as the first detection of the Jingchuvirales in a mammalian host - a chu-like virus in Tasmanian devils - thereby expanding the host range beyond invertebrates and ectothermic vertebrates. As many of these Dasyuromorphia species are currently being used in translocation efforts to reseed populations across Australia, understanding their virome is of key importance to prevent the spread of viruses to naive populations.

Thogotoviruses are a group of tick-borne, six-segmented, negative-sense single-stranded RNA viruses. These viruses encode an RNA-dependent RNA polymerase that recognizes promoter sequences located at the genomic termini to initiate RNA synthesis. The 5' and 3' ends of the genome bind to the polymerase and function as a promoter. Outside the catalytic center, they base-pair with each other to form a double-stranded RNA structure. This structure is referred to as the distal duplex and plays an important role in RNA synthesis. In this study, we investigated how the RNA sequence of the distal duplex influences polymerase activity using minigenome systems of two thogotoviruses, Oz virus (OZV) and Dhori virus (DHOV). Each virus exhibits distinct activities among its six segments. In OZV, one determinant of these differences is the base pair at positions 5'12 and 3'11 within the distal duplex, where promoter activity varies depending on whether the base pair is G:C or A:U. In contrast, the DHOV polymerase is not affected by this difference. These results indicate that, even within the genus Thogotovirus, viruses differ in whether they possess a mechanism that modulates promoter activity based on subtle sequence differences within the distal duplex. Furthermore, phylogenetic analysis and comparison of promoter sequences suggest that thogotoviruses can be divided into groups that do or do not regulate intersegment promoter activity via the base pair at positions 5'12 and 3'11. HighlightsO_LIMinigenome systems of Oz virus and Dhori virus reveal segment-specific differences in promoter activity C_LIO_LIThe distal duplex sequence modulates RNA synthesis in a virus-dependent manner C_LIO_LIThe base pair at positions 5'12/3'11 determines promoter activity in Oz virus but not in Dhori virus C_LIO_LIThogotoviruses can be divided into groups that do or do not regulate promoter activity via distal duplex sequence variation at positions 5'12/3'11 C_LI

Multiple viruses that are highly pathogenic in humans are known to have evolved in bats. How bats tolerate infection with these viruses, however, is poorly understood. As viruses engage in a wide range of interactions with their hosts, it is essential to study bat viruses in a system that resembles their natural environment like bat-derived in vitro cellular models. However, stable and accessible bat cell lines are not widely available for the broader scientific community. Here, we generated in vitro reagents for the Sebas short-tailed bat (Carollia perspicillata), tested multiple methods of immortalization, and characterized their susceptibility to virus infection and response to immune stimulation. Using a pseudotyped virus library and authentic virus infections, we show that these C. perspicillata cell lines derived from a diverse array of tissues are susceptible to viruses bearing the glycoprotein of numerous orthohantaviruses, including Andes and Hantaan virus and are also susceptible to live hantavirus infection. Furthermore, stimulation with synthetic double-stranded RNA prior to infection with VSV and MERS-CoV induced a protective antiviral response, demonstrating the suitability of our cell lines to study the bat antiviral immune response. Taken together, the approaches outlined here will inform future efforts to develop in vitro tools for virology from non-model organisms and these C. perspicillata cell lines will enable studies on virus-host interactions in bats.

IntroductionSemliki forest virus (SFV) is a model virus used to investigate the Alphavirus genus, which includes human pathogens Chikungunya virus and Ross River virus. Viruses harness cellular machinery to facilitate various steps of their replicative cycles. Ion channels are one group of cellular proteins required for the efficient replication of some viruses, including Influenza A viruses, Ebola virus and members of the Betacoronavirus genus. This study focussed on understanding SFVs requirement for functional ion channels during replication. MethodsThe effect of ion channel inhibitors on in vitro SFV infections was measured to investigate the contribution of ion channels in its replication cycle. ResultsIn vitro SFV infections carried out in the presence or absence of different ion channel inhibitors showed broad-range K+ channel inhibitors reproducibly attenuated virus replication and reduced its cytotoxicity in two mammalian cell lines. These broad-range K+ channel inhibitors disrupted an early, post-entry step causing a delay or reduction in SFV protein and RNA synthesis. Screens using inhibitors of specific K+ channel families showed that two-pore domain K+ channel (2pK) inhibitors attenuated SFV replication. Confocal microscopy revealed decreased detection of dsRNA and SFV protein in the presence of inhibitor but no change in RNA and protein colocalisation, which would indicate disruption of replication complexes. Broad-range K+ and 2pK inhibitors decreased viral RNA replication and transcription from the subgenomic promoter. ConclusionsK+ channel inhibitors attenuate in vitro SFV replication by inhibiting an early, post-entry step of virus replication, potentially RNA synthesis. ImportanceNo antiviral therapies have been approved for clinical use against diseases caused by members of the Alphavirus genus. Work presented in this manuscript shows for the first time that SFV genome replication and virus induced cytotoxicity can be reduced in vitro by treating infected cells with K+ channel inhibitors. This work provides the basis for investigating the effectiveness of K+ channel inhibitors against other alphaviruses both in vitro and in vivo and, because many ion channel inhibiting drugs are already in clinical use, rapid repurposing against alphavirus infections would be possible.

Public health concerns about recent viral epidemics have motivated researchers to seek transdisciplinary understanding of infection in wildlife hosts. With its deep history devoted to explaining the abundance and distribution of organisms, ecology can augment current methods for studying viral dynamics. However, datasets allowing ecological explorations of viral communities are lacking, and common methods for delineating viral operational taxonomic units (OTUs), or "species", are subjective. Here, we comprehensively sampled 1,086 bats from two Puerto Rican caves and tested them for infection with herpesviruses. Using percent identity of nucleotides and a machine learning algorithm, we categorized herpesviruses into 41 OTUs, representing approximately 80% of all herpesviruses in the host community. Although 13 OTUs were detected in multiple host species, OTUs generally exhibited host specificity by infecting a core host species at a significantly higher prevalence than in all other species combined. Only two OTUs showed significantly different prevalence between host sexes. This work is the first exploration of viral community ecology in a community of wildlife hosts.