Journal of General Virology最新文献

Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive and relatively cheap to detect infections with these pathogens early. Here, we use an amplification-free clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13)-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula virus (TULV) and Andes virus (ANDV), a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no consistent benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples, ANDV from hamster lung samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.

Hepatitis B virus (HBV) is a hepatotropic DNA virus that infects over 250 million people worldwide and causes serious liver diseases. HBV infection can modulate host cellular processes, potentially inducing proteomic changes in hepatocytes. In this study, we investigated how acute HBV infection alters the proteome and secretome of primary human hepatocytes, a physiologically relevant in vitro model that retains essential liver-specific functions. Protein-level changes in cell lysates and culture supernatants were quantified 8 days post-infection using data-independent acquisition MS. We used HBV infection in the presence of the entry inhibitor bulevirtide as a control to separate the effects of productive infection from those caused by inoculum-associated components. Despite robust infection, active HBV replication induced only subtle changes in host protein levels. Orthogonal validation of MS-identified candidates confirmed reticulocalbin-2 as a novel host factor downregulated during productive HBV infection. The functional role of candidate proteins identified by MS was assessed in vitro by siRNA-mediated knockdown and measurement of viral replication markers. Knockdown had no impact on viral RNA or antigen levels, suggesting that the observed proteomic changes may reflect stress responses or broader modulation of the hepatic microenvironment. Our findings support the concept of HBV as a stealth virus and underscore the importance of carefully controlled experimental systems for studying host responses to infection in vitro.

Defective viral genomes (DVGs) affect viral dynamics, pathogenicity and evolution, have been found in many in vivo viral infections, and in theory can be detected from sequencing data. We explored the utility of the currently available bioinformatic programs ViReMa, DI-tector, DVGfinder, DG-Seq and VODKA2 for identifying junction points in plant virus high-throughput sequencing data, looking at whether the outputs from these bioinformatic tools generally agree and exploring the possibility of using these tools to help us understand whether DVGs are consistently generated and maintained in a specific virus-host combination. We conducted a meta-analysis of eight previously published RNA sequencing datasets utilizing all five programs and compared the degree of output overlap, the most common junctions present in each output and whether these junctions match previously reported junctions for that virus. Our results demonstrate a low degree of agreement regarding identified junctions between programs, including the most frequently identified one, although the most frequently identified junctions typically corresponded to large, disruptive deletions. We found preliminary support for our prevalence hypothesis, although we ultimately conclude that a more robust dataset generated expressly for testing this hypothesis will be required for a convincing answer. Finally, we suggest that when using bioinformatic programs to search for DVGs, it is best to run the same dataset through multiple programs and look at the overlap to inform decisions on downstream characterization.

Hepatitis C virus (HCV) infection induces extensive rearrangements of host cytoplasmic membranes, leading to the formation of multiple membranous structures that facilitate RNA replication. Current knowledge of these membranous structures has largely relied on correlative light and electron microscopy techniques using chemical fixation and resin embedding. To overcome these limitations, cryo-preserved cells were prepared using cryo-focused ion beam (cryo-FIB) milling and cryo-ultramicrotomy. For the first time, the contents within the membranous structures have been observed in situ using cryo-electron tomography (cryo-ET) performed on lamellae (prepared via cryo-FIB) and on ultrathin sections (prepared via cryo-ultramicrotomy) from HCV subgenomic replicon-harbouring cells. Observations from 112 cryo-electron tomograms of cryo-FIB-derived samples revealed the presence of densities within the inner vesicles of a subset of single- and double-membrane vesicles, as well as within multi-vesicular bodies, which are consistent with the presence of the viral genome replication machinery. Notably, this study also presents the first direct visualization of densities within a multi-membrane vesicle observed by cryo-electron microscopy of vitreous sections. The cryo-ET methodologies established here lay the groundwork for future investigations into the architecture of the HCV replication complex, leveraging advanced computational tools for deeper structural and functional analyses.

Pandemic preparedness requires vaccine platforms that are fast to produce, thermostable and suitable for broad deployment. DNA vaccines are well suited to this task but have historically suffered from poor immunogenicity when delivered by conventional intramuscular (IM) injection. Here, we evaluated high-density microarray patch (HD-MAP) delivery of a DNA vaccine encoding the influenza A/California/01/2009 (H1N1pdm09) haemagglutinin (HA) antigen. In vivo imaging of a luciferase reporter construct demonstrated earlier and higher expression following HD-MAP application compared to IM injection. HD-MAP delivery of the HA vaccine induced strong HA-specific IgG responses, whereas IM delivery did not. Upon challenge with a homologous H1N1 virus, all HD-MAP-vaccinated mice were protected from weight loss, while 50% of intramuscularly vaccinated mice met humane endpoints. These findings support the use of HD-MAPs to overcome delivery limitations of DNA vaccines and enhance their utility for future outbreak and pandemic response.

Over the last decade, heterologous prime-boost vaccination regimens have been established as a promising strategy to enhance immune responses and make optimal use of the advantages of different vaccine platforms. Modified vaccinia virus Ankara (MVA), a replication-deficient poxviral vector with an established safety profile, is under clinical investigation as a versatile recombinant vaccine platform against various infectious diseases. In the context of coronavirus disease 2019 (COVID-19), a recombinant MVA-based vaccine candidate expressing the prefusion-stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (MVA-ST) has demonstrated safety, immunogenicity and protection in preclinical studies using different animal models. Furthermore, a phase Ib clinical trial in healthy adults showed that MVA-ST is safe, well-tolerated and immunogenic when used as a booster following mRNA priming. In this study, we evaluated heterologous prime-boost vaccination regimens using MVA-ST as a booster in Syrian hamsters. Hamsters were primed with an mRNA vaccine (BNT162b2, BioNTech/Pfizer) or the adenoviral vector vaccine Ad26.COV2.S (Janssen) and subsequently boosted with MVA-ST at a dose of 10⁸ p.f.u. These heterologous vaccination regimens induced robust protection against severe SARS-CoV-2 disease, with superior immunogenicity compared to homologous MVA-ST vaccination. Notably, even a lower booster dose (10⁷ p.f.u.) of MVA-ST following mRNA priming conferred strong protection against SARS-CoV-2 challenge infection, while still associated with limited viral shedding from the upper respiratory tract. These findings highlight the potential of MVA-ST as a heterologous booster to enhance the immunogenicity and protective efficacy of existing COVID-19 vaccines and also to improve vaccination strategies against other emerging pathogens.

Tomato spotted wilt virus (TSWV) is a highly destructive plant pathogen transmitted by thrips, including Frankliniella occidentalis, in a circulative and propagative manner. To counter viral infections, thrips activate antiviral defences through C20 oxygenated polyunsaturated fatty acids (PUFAs), known as eicosanoids. However, at later stages of infection, C18 PUFAs, including epoxyoctadecamonoenoic acids (EpOMEs), modulate immune responses by preventing excessive and unnecessary activation. Our previous study demonstrated that TSWV elevates EpOME levels in thrips to suppress antiviral responses and enhance viral replication, with its nonstructural protein S (NSs) playing a key role in this process. In this study, we investigated the impact of NSs protein variation on vector immunity and virus-vector interactions. We assessed relative TSWV titres in thrips larvae and examined the role of eicosanoids, specifically 12,13-EpOME and PGE₂, in regulating viral load and apoptosis. Our results revealed that 12,13-EpOME significantly increased viral titres, whereas PGE₂ reduced the viral accumulation by promoting apoptosis in the vector insect. Phylogenetic analysis identified distinct NSs variations among TSWV isolates, with resistance-breaking (RB) and WT strains, which modulated differential infection patterns in thrips gut tissues, as visualized through fluorescence in situ hybridization. RB strains exhibited significantly higher viral titres, along with increased expression of EpOME biosynthetic gene (Fo-CYP24) and decreasing expression of EpOME degradation gene (Fo-sEH2). Apoptosis assays using the terminal deoxynucleotidyl transferase dUTP nick-end labelling assay further indicated that RB strains suppressed the gut epithelial cell death in thrips by antagonizing a process regulated by PGE₂. Additionally, in vivo transient expression of the NSs gene in a nontarget insect, Spodoptera exigua, demonstrated the immunosuppressive effects by inducing EpOME level through upregulation of Se-CYP expression and downregulation of Se-sEH expression. Indeed, RB strains suppressed cellular immune responses more effectively than WT strains in S. exigua. These findings provide novel insight into the role of NSs genetic variation in TSWV transmission in the insect vector as well as in the host plants.

Vertical transmission of human cytomegalovirus (HCMV) during pregnancy is a major cause of congenital disease. In the absence of robust vaccination strategies, antiviral drug regimens are being developed to inhibit the vertical transmission of the virus. Recent clinical data have indicated that valaciclovir, an orally bioavailable form of aciclovir, was effective at limiting vertical transmission of HCMV when administered during pregnancy. However, there is no widely cited in vitro analysis of valaciclovir's antiviral effect against HCMV, and it is possible, like aciclovir, that valaciclovir has poor anti-HCMV activity. The antiviral effects of aciclovir and valaciclovir against HCMV were compared to the widely used anti-HCMV drug ganciclovir. Compared to ganciclovir, the anti-HCMV effects of either aciclovir or valaciclovir were poor, and robust anti-HCMV activity in all cell lines tested (adult fibroblast, foetal fibroblast or trophoblast cells) was only observed at high drug concentrations. All drugs had no obvious effects on the viability of uninfected cells. Overall, valaciclovir had poor anti-HCMV activity, and its anti-HCMV efficacy upon administration during pregnancy may rely on a combination of factors. These data argue for the continued development of valaciclovir and anti-HCMV compounds to inhibit vertical virus transmission.

Rabies is a fatal zoonosis that impairs host immune function, yet effects on peripheral lymphoid architecture are poorly defined. During a 2021-2022 rabies virus (RABV) outbreak in South Africa, we collected cervical lymph nodes from 36 rabies-suspect dogs; RABV RNA was detected in 27. Canine distemper virus RNA was detected in a subset across both RABV-positive (RABV+) and RABV-negative (RABV-) groups and was not associated with clinical-sign count. We set up a computer-assisted histological analysis tool to quantify germinal-centre (GC) nucleus density and immunohistochemistry for CD20, PNA and IBA1 to profile B cells, GC activity and macrophages. Within the outbreak cohort, GC density and marker-based metrics did not differ between RABV+ and RABV- dogs. Two healthy dogs were included as reference tissues; values in outbreak dogs were generally lower, but these contrasts are contextual given the limited, non-matched controls. This study provides a reproducible framework for quantifying immune cell organization in field-collected tissues during natural RABV exposure and highlights the need for larger, geographically matched control groups and complementary functional immune measurements.

Plant virus infections pose a substantial threat to crop quality and productivity, contributing to considerable economic losses in global agriculture annually. Traditionally, laboratories have widely adopted serological techniques, such as ELISA, and molecular methods, including quantitative PCR, for virus diagnostics. More recently, sophisticated next-generation sequencing approaches have been introduced to improve the efficiency and reliability of virus detection and identification. However, the development of sensitive, rapid and low-cost methods for the on-site detection, quantification and identification of plant viruses remains an ongoing challenge and is still in its early days. Point-of-care technologies have not fully realized their potential in agriculture due to numerous challenges, such as the elevated cost of development, lack of standardized validation and insufficient field testing. Therefore, future success depends on addressing these technical, economic and regulatory hurdles, as well as considering the specific user needs within the agricultural context. In this mini-review, recent advancements in biosensing for on-site plant virus monitoring, involving nanotechnology-based sensors, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, electrochemical and modern field-effect transistor-based sensors offering high sensitivity, speed and portability, are discussed. These technologies, when integrated with smartphone applications and/or machine learning modules, could enable real-time, field-deployable diagnostics for early disease management and sustainable agriculture. The aim is to raise awareness among plant virologists about this panel of emerging diagnostic concepts that could help improve current methods, ultimately facilitating the management of plant viral diseases.