Journal of Virology最新文献

Pre-existing T-cell responses have been linked to reduced disease severity and better clinical outcomes during the 2009 influenza pandemic and the recent COVID-19 pandemic. We hypothesized that diversifying T-cell responses, particularly targeting conserved viral proteins such as the influenza A virus (IAV) nucleoprotein (NP), could protect against both epidemic and pandemic IAV strains. To test this, we created a mosaic nucleoprotein (MNP) by synthesizing a sequence that maximized the representation of 9-mer epitopes from 7422 NP sequences across human, swine, and avian IAVs. Notably, the MNP sequence showed high homology with the NP of the H5N1 strain affecting dairy cows in the ongoing outbreak. Mucosal immunization with the adjuvanted MNP vaccine induced robust CD8 and CD4 T-cell responses against both known immunodominant and in silico predicted subdominant epitopes. MNP-vaccinated mice challenged with epidemic H1N1 and H3N2 strains, which shared immunodominant CD8 and/or CD4 T-cell epitopes, showed a significant (~4 log) reduction in lung viral load. Importantly, MNP-vaccinated mice challenged with a pandemic H1N1 strain lacking shared immunodominant CD8 or CD4 epitopes exhibited a superior reduction in lung viral load, linked to T-cell responses targeting subdominant epitopes present in both the MNP and pandemic strain NP. These results suggest that a diversified T-cell response induced by the MNP vaccine could provide broad protection against severe disease from both current and emerging IAV strains.

Importance: The World Health Organization (WHO) estimates that seasonal influenza causes 3-5 million cases of severe illness annually. The influenza virus frequently undergoes genetic changes through antigenic drift and antigenic shift, resulting in annual epidemics and occasional pandemics. Consequently, a major public health objective is to develop a universal influenza vaccine that offers broad protection against both current and pandemic influenza A strains. In this study, we designed a nucleoprotein (NP) antigen (termed mosaic NP) comprising antigenic regions found in thousands of influenza viruses, aiming to use it as a vaccine to induce broad anti-influenza T-cell responses. Our findings indicate that the mosaic NP vaccine provided significant protection against seasonal H1N1 and H3N2, as well as the pandemic H1N1 strain, demonstrating its effectiveness across various influenza subtypes. These findings suggest that the mosaic NP is a potential universal influenza vaccine antigen, capable of protecting against diverse strains of influenza viruses.

Virion formation and egress are sophisticated processes that rely on the spatial and temporal organization of host cell membranes and the manipulation of host machineries involved in protein sorting, membrane bending, fusion, and fission. These processes result in the formation of infectious virions, defective particles, and various vesicle-like structures. In herpes simplex virus 1 (HSV-1) infections, virions and capsid-less particles, known as light (L)-particles, are formed. HSV-1 infection also stimulates the release of particles that resemble extracellular vesicles (EVs). In productively infected cells, most EVs are generated through the CD63 tetraspanin biogenesis pathway and lack viral components. A smaller subset of EVs, generated through the endosomal sorting complexes required for transport (ESCRT) pathway, contains both viral and host factors. Viral mechanisms tightly regulate EV biogenesis, including the inhibition of autophagy-a process critical for increased production of CD63+ EVs during HSV-1 infection. Mutant viruses that fail to suppress autophagy instead promote microvesicle production from the plasma membrane. Additionally, the viral protein ICP0 (Infected Cell Protein 0) enhances EV biogenesis during HSV-1 infection. The different types of particles can be separated by density gradients due to their distinct biophysical properties. L-particles and ESCRT+ EVs display a pro-viral role, supporting viral replication, whereas CD63+ EVs exhibit antiviral effects. Overall, these studies highlight that HSV-1 infection yields numerous and diverse particles, with their type and composition shaped by the ability of the virus to evade host responses. These particles likely shape the infectious microenvironment and determine disease outcomes.

Platelet factor 4 (PF4) has been shown to regulate several viral infections. Our previous study demonstrated that PF4 inhibits the entry of enterovirus A 71 (EV71) and coxsackievirus A16 (CA16), which cause hand, foot, and mouth disease (HFMD). In this study, we report that PF4 also inhibits the circulating HFMD pathogen coxsackievirus A6 (CA6) and the re-emerging enterovirus D68 (EVD68). A 15-amino acid peptide, C15, at the C-terminus of PF4 confers anti-viral activity against multiple enteroviruses (EVs) besides CA6 and EVD68, including EV71 and CA16. Mechanistic studies revealed that wild-type C15 with a net-positive charge (+3), but not its mutants C15M and C15A (both -1), specifically binds to the VP3 capsid protein of CA6 and EVD68, thereby disrupting their attachment to the host cell surface. In addition, VP3 of EVs contains a conserved domain (residues 155-170) crucial for binding to C15. An aspartic acid residue at position 156 imparts a net-negative charge to this domain, which, when substituted with a neutrally charged amino acid, reduces the binding affinity of VP3 for C15. Additionally, C15 protects neonatal mice from lethal challenge upon a CA6 infection. These results suggest that C15 is a promising broad-spectrum anti-viral candidate against multiple EVs.

Importance: EVs, which pose a significant public health threat, can be classified into 15 species, with EV-A, -B, -C, and -D infecting humans and causing a wide range of diseases, from mild illnesses, such as HFMD, to more severe conditions, such as acute flaccid paralysis. The emergence of new and alternative strains highlights the urgent need for broad-spectrum anti-viral agents. In this study, we identified that the C15 of PF4 exhibits potent anti-viral activity against multiple EVs by binding to their surface and blocking their entry into host cells. Furthermore, C15 provides significant protection in vivo. These findings highlight the potential of C15 as a broad-spectrum anti-viral candidate. Our study opens a new avenue for developing treatments to combat the diverse and evolving threats posed by EVs.

Viral aseptic meningitis is a neuroinflammatory condition that occurs when viruses gain access to the central nervous system (CNS) and induce inflammation. The blood-brain barrier (BBB) is comprised of brain endothelial cells (BECs) that stringently regulate the passage of molecules, toxins, and pathogens from the circulation into the CNS. Through their unique properties, such as complex tight junctions, reduced rates of endocytosis, expression of efflux transporters, and restricted expression of leukocyte adhesion molecules, the BBB is often able to limit pathogen entry into the brain; however, certain neurotropic pathogens, such as coxsackievirus B3 (CVB3) are able to infect the CNS. We have previously demonstrated that CVB3 can infect and disrupt induced pluripotent stem cell-derived brain-like endothelial cells (iBECs), but the host response to this infection remains unknown. Here, we investigate global host transcriptional changes during CVB3 infection of iBECs using RNA sequencing. We validated our data set by exploring pathways altered by CVB3 using quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay of upregulated cytokines and interferon signaling molecules.

Importance: Coxsackievirus B3 (CVB3) is a leading cause of viral aseptic meningitis that can produce severe disease in susceptible individuals. To gain access to the central nervous system, CVB3 must cross central nervous system barriers, such as the blood-brain barrier. Previously, we have shown that CVB3 infects a human stem cell-derived brain-like endothelial cell model. Here, we report the global transcriptome of stem cell-derived brain-like endothelial cells to CVB3 infection and provide proof-of-concept validation of the dataset using molecular biology techniques. These data could inform novel mechanisms of CVB3-mediated blood-brain barrier dysfunction.

Neutralizing antibodies (nAbs) are important for the treatment of emerging viral diseases and for effective vaccine development. In this study, we generated and evaluated three nAbs (1H9, 2D7, and C4H4) against H7N9 influenza viruses and found that they differ in their ability to inhibit viral attachment, membrane fusion, and egress. We resolved the cryo-electron microscopy (cryo-EM) structures of H7N9 hemagglutinin (HA) alone and in complex with the nAb antigen-binding fragments (Fabs) and identified the HA head-located epitope for each nAb, thereby revealing the molecular basis and key residues that determine the differences in these nAbs in neutralizing H7N9 viruses. Moreover, we found that the humanized nAb CC4H4 provided complete protection in mice against death caused by a lethal H7N9 virus infection, even when nAb was given 3 days after the mice were infected. These findings provide new insights into the neutralizing mechanism and structural basis for the rational design of H7N9 virus vaccines and therapeutics.IMPORTANCEH7N9 viruses have caused severe infections in both birds and humans since their emergence in early 2013 in China. Their persistent presence and variation in avian populations pose a significant threat to both poultry and humans. There are no treatments for human infections. In this study, we thoroughly investigated the neutralization mechanisms, structural basis, and therapeutic effects of three nAbs (1H9, 2D7, and C4H4) against H7N9 viruses. We revealed the molecular determinants underlying the varied performances of the three nAbs in neutralizing H7N9 viruses and protecting H7N9-infected mice. These insights provide a solid foundation for the rational design of vaccines and therapeutics against H7N9 viruses.

Viruses represent a diverse pool of obligate parasites that infect virtually every known organism, as such, their study is incredibly valuable for a range of fields including public health, medicine, agriculture, and ecology, and the development of biomedical technologies. Having evolved over millions of years, each virus has a unique and often complicated biology, that must be characterized on a case-by-case basis, even between strains of the same taxon. Owing to its nanoscale spatial resolution, atomic force microscopy (AFM) represents a powerful tool for exploring virus biology, including structural features, kinetics of binding to host cell ligands, virion self-assembly, and budding behaviors. Through the availability of numerous chemistries and advances in imaging modes, AFM is able to explore the complex web of host-virus interactions and life-cycle at a single virus level, exploring features at the level of individual bonds and molecules. Due to the wide array of techniques developed and data analysis approaches available, AFM can provide information that cannot be furnished by other modalities, especially at a single virus level. Here, we highlight the unique methods and information that can be obtained through the use of AFM, demonstrating both its utility and versatility in the study of viruses. As the technology continues to rapidly evolve, AFM is likely to remain an integral part of research, providing unique and important insight into many aspects of virology.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Previous studies from other groups showed that CD163 is required for PRRSV uncoating and genome release. However, CD163 does not interact with nucleocapsid (N) protein. In this study, the neonatal Fc receptor (FcRn) was demonstrated to be irreplaceable for PRRSV infection by knockdown, overexpression, antibodies or IgG blocking, knockout, and replenishment assays. FcRn was further revealed to be involved in PRRSV uncoating for the first time rather than viral attachment and internalization. In detail, FcRn was determined to colocalize with CD163 and PRRSV virions in early endosomes and specially interact with N protein in early endosomes. Taken together, these results provide evidence that FcRn is an essential cellular factor for PRRSV uncoating, which will be a promising target to interfere with the viral infection.IMPORTANCEPRRSV infection results in a severe swine disease affecting pig farming in the world. Although CD163 has been implicated as the uncoating receptor for PRRSV but the uncoating mechanism of PRRSV remains unclear. Here, we identified that FcRn facilitated virion uncoating via interaction with viral N protein in early endosomes. Our work actually expands the knowledge of PRRSV infection and provides an attractive therapeutic target for the prevention and control of PRRS.

The papillomavirus (PV) E2 protein is highly conserved, consisting of an N-terminal transactivation domain linked to a C-terminal DNA binding and dimerization domain (DBD) by a flexible hinge region. The E2 DBD exhibits a helix-turn-helix structure that dimerizes into a beta barrel prior to binding DNA; the first helix, α1, is responsible for recognition of the palindromic E2 binding site. The DNA recognition helix consists of a tract of basic amino acids with a highly conserved central cysteine residue. Previous mutational analysis studies on this conserved cysteine have found that it is not required for viral replication or DNA binding. To investigate the function of this conserved cysteine in vitro and in vivo, we generated point mutations in MmuPV1 E2 at cysteine 307. We report here that this cysteine in the DNA recognition helix is required for transient viral replication and transactivation of proximal promoters, but C307 point mutants are still capable of enhancing the activation of distant upstream promoters in vitro. MmuPV1 genomes with the C307 mutation failed to produce warts when injected into mice, suggesting that the DNA recognition cysteine is required for viral replication in vivo.

Importance: Papillomaviruses are the etiological agents of cancers of the oropharynx and anogenital tract. Understanding the mechanisms underlying PV pathogenesis is complicated by the strict species tropism displayed by the virus. The research presented here is significant because it links in vitro and in vivo models investigating the role of a conserved cysteine in the MmuPV1 E2 protein. This work elucidates the molecular mechanisms that regulate PV transcription and DNA replication and how these contribute to disease progression.