Journal of Virology最新文献

The 2022 outbreak of monkeypox virus (MPXV), a double-stranded DNA virus, is remarkable for an unusually high number of single-nucleotide substitutions compared to earlier strains, with a strong bias toward C→T and G→A transitions consistent with the APOBEC3 cytidine deaminase activity. While APOBEC3-induced mutagenesis is well documented at the DNA level, its potential impact on MPXV RNA transcripts remains unclear. To assess whether APOBEC3 enzymes act on MPXV RNA, we analyzed RNA-seq data from infected samples. The enrichment of APOBEC signature substitutions among high-frequency mismatched positions led us to consider two possibilities: RNA editing at hotspots or fixed DNA mutations. Multiple lines of evidence support the conclusion that these substitutions arise from DNA-level mutagenesis rather than RNA editing. These include a substantial number of G→A substitutions remaining after normalization by gene strand direction, a largely neutral impact of substitutions on protein-coding sequences, the lack of positional correlation with transcriptional features or RNA secondary structure typically associated with APOBEC action hotspots, and an overlap with known genomic mutations in MPXV strains. Analysis of the nucleotide context of observed substitutions indicated that APOBEC3A or APOBEC3B was likely a driver of DNA-level mutagenesis.IMPORTANCEThe 2022 monkeypox virus (MPXV) outbreak showed an unusually high number of mutations thought to result from human antiviral enzymes of the APOBEC3 family. While such mutations have been clearly documented in the viral DNA, whether APOBEC3 also edits viral messenger RNA molecules remained unclear. In this study, we analyzed multiple publicly available MPXV RNA sequencing datasets to address this question. We found that the apparent APOBEC-like changes in RNA are best explained by fixed DNA mutations rather than active RNA editing. This finding helps clarify how MPXV evolves and adapts, suggesting that APOBEC3's role in shaping the virus likely operates at the DNA level. Understanding where and how these mutations occur provides insight into the virus's interaction with the human immune system and informs future studies on viral evolution and antiviral defenses.

Novel approaches to sensitize latently infected cells to apoptosis may provide additional methods to eliminate latent reservoirs. Prior research identified several retinoids as potential drugs that increase the sensitivity of HIV-infected cells to cell death. Retinoids are derivatives of vitamin A that target retinoid receptors causing antiproliferative and proapoptotic activity. Several are FDA-approved or in clinical trials. The aim of this study was to evaluate the ability of vitamin A, three of its natural metabolites, and nine synthetic derivatives to sensitize HIV-infected CD4 T cells to NK natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC). From the retinoids tested, alitretinoin, tazarotene acid, and AM80 significantly enhanced NK natural cytotoxicity in the presence of IL-15. Mechanistically, these retinoids increased NK degranulation upon target recognition in an HLA-F/KIR3DS1-dependent manner. Furthermore, these retinoids enhanced ADCC by transcriptionally increasing CD16 expression on NK cells. In conclusion, our study has identified at least three retinoids capable of enhancing NK natural cytotoxicity and ADCC against HIV-infected cells. These or other retinoids could be used to reduce HIV persistent reservoirs.IMPORTANCEThis study highlights how retinoids, compounds derived from vitamin A, can help the immune system target HIV-infected cells more effectively. HIV often hides in immune cells, making it difficult to fully eliminate the virus. We found that certain retinoids, including alitretinoin, tazarotene acid, and AM80, improve the function of natural killer (NK) cells-key immune cells that target infected cells. These retinoids boost NK cell activity by increasing their ability to release toxic molecules that kill infected cells and by enhancing their response to antibodies targeting HIV. This makes the infected cells more vulnerable to being eliminated. Since some of these retinoids are already approved for medical use, they could offer a promising way to reduce persistent HIV reservoirs in the body and improve efforts to cure the infection.

Epstein-Barr virus (EBV) infects more than 90% of adults worldwide and causes a range of diseases, including multiple malignancies and autoimmune disorders. However, due to a host range restriction, EBV cannot infect commonly used experimental animals, posing a significant obstacle to developing EBV-specific prophylactic and therapeutic agents. Rhesus lymphocryptovirus (rhLCV), an ortholog of EBV, naturally infects rhesus macaques, which is a surrogate model for EBV research. In this study, we demonstrate that cynomolgus macaque (Macaca fascicularis), a primate closely related to rhesus macaque, is susceptible to rhLCV infection. rhLCV can immortalize B cells of cynomolgus macaques to develop cy-LCLs. We developed a high rhLCV-producing cy-LCL cell line, LCL111, and optimized the induction conditions to increase viral production, surpassing the original rhLCV producer LCL8664. Importantly, EBV gHgL-specific monoclonal antibody (mAb) AMMO1 and gB-specific mAb 3A5 can cross-react with rhLCV proteins and block the formation of cy-LCLs. Overall, we established an efficient rhLCV-producing cell line, and rhLCV infection of cynomolgus macaques represents a promising alternative surrogate model for efficiency evaluation of EBV vaccines and mAbs.

Importance: Epstein-Barr virus (EBV) naturally infects only humans, creating a major barrier to evaluating the efficiency of vaccines and therapies in vivo. As an EBV ortholog, rhesus lymphocryptovirus (rhLCV) offers a biologically relevant surrogate system. However, its application has been primarily limited to rhesus macaques. Here, we demonstrate that cynomolgus macaque lymphocytes are also susceptible to rhLCV in vitro, and the newly transformed cy-LCL111 shows superior and sustained rhLCV production ability. rhLCV infection of cynomolgus macaque lymphocytes can be efficiently neutralized by anti-EBV gH/gL nAbs AMMO1 and anti-EBV gB mAbs 3A5, highlighting the potential of cynomolgus macaques as an in vivo model to assess anti-EBV mAb and vaccine efficacy. Our findings support the use of cynomolgus macaques as an additional model for EBV research and offer a useful platform for evaluating EBV-specific prophylactic or therapeutic strategies.

Neuronal injury and death contribute to long-term impairments and lethality during viral encephalitis. Rift Valley fever virus (RVFV), an arbovirus with epidemic potential, can manifest as late-onset encephalitis in humans, yet this disease outcome remains understudied. A lethal rodent model of RVF encephalitis is characterized by a dysregulated immune response, neuronal necrosis, and blood-brain barrier breakdown that precedes lethality. In this study, we built upon this prior work using both in vivo and in vitro models to interrogate the mechanism of cell death in neurons during RVFV infection. We found an increase in proteins associated with apoptosis, pyroptosis, and necroptosis in the brains of animals that succumb to lethal RVFV encephalitis. We then focused on identifying the primary cell death pathways in primary cortical neurons, which were highly susceptible to infection by pathogenic and attenuated viral strains. Using immunoblotting, immunocytochemistry, and in-cell western assays, we found that neurons infected with RVFV resulted in the activation of multiple cell death pathways, leading to neuron cell death. These findings further our understanding of the impact of RVFV infection, which is critical to identifying therapeutics that support neuron integrity and minimize injury during viral encephalitis.IMPORTANCERift Valley fever may be accompanied by late-onset encephalitis in humans. Our lab has studied the in vivo mechanisms of neurological disease, yet the precise mechanisms of cell death in the central nervous system have been elusive. An understanding of the how and why of cell death from Rift Valley fever virus (RVFV) infection may guide the design of therapeutic interventions. Here, we use primary neurons to probe the mechanism of cell death following RVFV infection. We found that RVFV triggers multiple cell death pathways both in the brains of animals that succumb to lethal RVFV encephalitis as well as in ex vivo neuronal cultures. Induction of cell death occurs even with infection by an attenuated vaccine strain. These findings provide a platform for understanding cell death mechanisms caused by RVFV infection and identifying therapeutics that support neuron integrity during viral encephalitis.

The outbreak of highly pathogenic avian H5 influenza (HPAI) clade 2.3.4.4b in cattle has spread across the United States. Mice with pre-existing immunity to H1N1 virus or with a live-attenuated influenza vaccine showed protection against a lethal bovine-derived HPAI H5N1 viral challenge. Notably, ferrets with mixed immunity also demonstrated protection against a feline-derived H5N1 virus, independent of cross-reactive neutralization titers, but antibodies to whole virus were observed. To investigate protective factors, we conducted T cell epitope mapping using published H1N1 viral sequences and found high conservation of key T cell epitopes in the bovine HPAI H5N1 strain. Depletion of T cells in mice prior to and during primary H1N1 infection impacted cross-protective antibodies to H5N1 virus, with CD4 depletion increasing mortality and CD8 depletion mildly impacting morbidity upon H5N1 viral challenge. This underscores the need to investigate memory T cell responses alongside antibodies in assessing preexisting cross-protection to HPAI H5N1 viruses.IMPORTANCEThe rapid spread of highly pathogenic avian H5 influenza (HPAI) clade 2.3.4.4b in U.S. cattle represents an urgent and evolving public health threat. Our findings reveal that pre-existing immunity, whether from seasonal H1N1 infection or live-attenuated vaccination, can confer substantial protection against lethal bovine- and feline-derived HPAI H5N1 viruses, even in the absence of strong cross-neutralizing antibody titers. By integrating T cell epitope mapping with mechanistic depletion studies, we demonstrate that conserved CD4 and CD8 T cell epitopes across H1N1 and H5N1 strains underpin this cross-protection. Critically, loss of CD4 T cell help during primary H1N1 infection disrupts the development of cross-reactive antibody responses and markedly worsens outcomes after H5N1 challenge. These results identify memory T cell responses as important determinants of heterosubtypic immunity and highlight the need to incorporate T cell-focused metrics into risk assessment, vaccine evaluation, and preparedness strategies for emerging HPAI H5N1 viruses.

During the COVID-19 pandemic, multiple SARS-CoV-2 variants emerged, each with distinct pathogenicity and transmissibility. This study investigated the role of the viral spike (S) protein in disease progression, focusing on the highly virulent S variant. The Delta S protein exhibited enhanced cleavage efficiency at the S1/S2 junction, resulting in partial dissociation of the S1 subunit, with detectable levels of extracellular S1. Unexpectedly, transient expression of Ancestral and Delta S protein induced by a recombinant vesicular stomatitis viral vector caused mild pulmonary inflammation, neutrophil activation, microthrombosis, and ~40% mortality in transgenic K18-hACE2 mice between 8 and 16 days, similar to post-COVID sequelae. The diseased mice displayed splenic atrophy and systemic inflammation, with elevated serum IGFBP-1 and CXCL13 levels. Consistent with the animal findings, serum samples from long COVID patients showed significantly elevated IGFBP-1 levels. CXCL13 levels were particularly elevated in patients with more severe long COVID symptoms. Notably, treatment with the antiplatelet agent aspirin significantly reduced both mortality and weight loss in mice exposed to Delta S protein expression. These findings suggest that SARS-CoV-2 S protein-associated coagulation and systemic inflammation during infection may contribute to the development of post-acute sequelae of COVID-19.IMPORTANCEOur study investigates the distinctive pathogenic properties of the SARS-CoV-2 spike (S) protein from highly virulent variants, with a particular focus on its delayed pathological effects in mice. Using a vesicular stomatitis virus (VSV) vector to transiently express the Ancestral and Delta variant S proteins in K18-hACE2 mice, we observed minimal acute symptoms initially; however, approximately 40% of the mice developed mild pulmonary inflammation, neutrophil activation, and microthrombosis, leading to death between 8 and 16 days post-infection. This delayed pathology was accompanied by elevated circulating levels of CXCL13 and IGFBP-1. Consistent with these findings, serum samples from long COVID patients also showed significantly increased IGFBP-1 levels, while CXCL13 levels were particularly elevated in individuals with more severe long COVID symptoms. These findings provide important observational evidence that may guide future mechanistic studies on long COVID and inform the development of potential therapeutic approaches.

Powassan virus (POWV) is a tick-borne flavivirus endemic to the United States, Canada, and parts of Russia. POWV remains an under-studied pathogen, despite the potential for serious and life-threatening neurologic complications following infection. While prior studies have characterized viral diversity due to single nucleotide polymorphisms, little is known about POWV recombination, defective RNAs (D-RNAs), and functional structural variants (SVs). Understanding POWV recombination in its natural vector can provide important insights into its replication and evolution. We analyzed POWV sequence data from 53 ticks collected from the Northeastern United States to characterize and quantify recombination patterns in naturally infected ticks. We then compared these results to single-passage isolates. Deletions were common in POWV RNA from ticks, and several areas of the genome were enriched for recombination junctions. Deletions were often associated with areas of microhomology. While most deletions were sample-specific, two major deletion archetypes were observed across multiple tick samples. The first consisted of small 19-50 base deletions in the methyltransferase domain of the ns5 RNA-dependent RNA-polymerase coding sequence, resulting in a mixture of putative SVs and D-RNAs. The second consisted of approximately 1,600 base deletions spanning the ns2a-ns3 coding sequences, resulting in putative D-RNAs with abrogated viral protease function. Deletions in ns2a-ns3 were significantly enriched after one passage in baby hamster kidney cells, despite a decrease in overall deletions. These results demonstrate that POWV RNA recombines frequently, with certain variants more common than others. These findings may carry implications for virus immune evasion and persistence in ticks.IMPORTANCEPowassan virus is a tick-borne flavivirus that can cause serious, life-threatening neurological disease. Understanding how Powassan virus replicates and evolves within its tick vector may elucidate factors important for persistence, transmission, and human disease. Defective RNAs (D-RNAs) are replication-incompetent viral genomes generated through internal deletions. D-RNAs are associated with disease severity and persistent infection in other viruses but have not been described for Powassan virus. Here, we show that Powassan virus produces abundant putative D-RNAs in field-collected ticks and that patterns of D-RNA expression change after one passage in mammalian cells. Although the function of these D-RNAs remains unknown, this work demonstrates that they occur under natural conditions and establishes a critical framework for investigating the role of D-RNAs in Powassan virus replication and transmission.

Adaptor protein complex 2 (AP2), a central regulator of clathrin-mediated endocytosis and intracellular cargo trafficking, is hijacked by numerous viruses to complete their infectious cycles. This review systematically synthesizes the multifaceted roles of AP2 across the entire viral life cycle, from entry and replication to assembly and release, as well as in immune evasion. By delineating how diverse viruses exploit this key host machinery, we further consolidate the rationale and current progress in developing broad-spectrum antiviral strategies that target AP2 and its regulatory pathways. This work aims to provide a unified perspective on AP2 as a critical host-pathogen interface, offering new insights into viral pathogenesis and antiviral drug discovery.

Bovine viral diarrhea virus (BVDV) is a major animal pathogen with a broad host range, causing gastrointestinal, respiratory, and reproductive diseases in cattle worldwide. BVDV exists as two biotypes: cytopathic (cp) and non-cytopathic (ncp). Although both cpBVDV and ncpBVDV have developed sophisticated strategies to evade or subvert host antiviral innate immune response, the underlying mechanisms remain incompletely understood. Autophagy, a process essential for maintaining cellular homeostasis, plays an important role in regulating viral replication and antiviral immunity. In this study, we demonstrated that the induction of autophagy with rapamycin enhanced the production of infectious progeny for both cpBVDV and ncpBVDV, whereas pharmacological inhibition of autophagy with 3-MA reduced viral yields. We further showed that modulating autophagy significantly influenced the early stages of the viral life cycle and the production of type I IFN (IFN-I). Notably, overexpression of BECN1 suppressed the synthesis of IFN-α and IFN-β, thereby promoting the replication of both cpBVDV and ncpBVDV. Conversely, RNA interference-mediated knockdown of BECN1 potentiated the antiviral innate immune response and restricted viral replication. Mechanistically, BECN1 was found to inhibit RIG-I-MAVS pathway activation by promoting ubiquitination and subsequent degradation of mitochondrial antiviral signaling (MAVS) protein, leading to suppression of IFN-I production. Additionally, both cpBVDV and ncpBVDV were shown to induce autophagy via the ROS-endoplasmic reticulum stress axis. These findings deepen our understanding of how BVDV evades host immunity and may inform the development of preventive strategies against BVDV infection.

Importance: Bovine viral diarrhea virus (BVDV), the causative agent of bovine viral diarrhea-mucosal disease, is a major global threat to cattle health. BVDV employs sophisticated strategies to evade host defense and facilitate its replication. Understanding these mechanisms is crucial for developing effective vaccines and antiviral agents. Our study elucidates how cytopathic BVDV and non-cytopathic BVDV subvert the host's antiviral innate immune response by exploiting autophagy to inhibit the RIG-I-MAVS pathway. A key finding is that BECN1-mediated autophagy directly targets MAVS protein for degradation via a specific BECN1 and MAVS interaction. Furthermore, we demonstrate that BVDV activates autophagy through ROS-ER stress axis to promote its replication. These insights reveal a novel immune evasion mechanism of BVDV and highlight the therapeutic potential of autophagy inhibition in treating BVDV-related diseases.