Journal of Virology最新文献

Human seasonal H3 clade 3C3a influenza A viruses (IAV) were detected four times in U.S. pigs from commercial swine farms in Michigan, Illinois, and Virginia in 2019. To evaluate the relative risk of this spillover to the pig population, whole genome sequencing and phylogenetic characterization were conducted, and the results revealed that all eight viral gene segments were closely related to 2018-2019 H3N2 human seasonal IAV. Next, a series of in vitro viral kinetics, receptor binding, and antigenic characterization studies were performed using a representative A/swine/Virginia/A02478738/2018(H3N2) (SW/VA/19) isolate. Viral replication kinetic studies of SW/VA/19 demonstrated less efficient replication curves than all 10 swine H3N2 viruses tested but higher than three human H3N2 strains. Serial passaging experiments of SW/VA/19 in swine cells did not increase virus replication, but changes at HA amino acid positions 9 and 159 occurred. In swine transmission studies, wild-type SW/VA/19 was shed in nasal secretions and transmitted to all indirect contact pigs, whereas the human seasonal strain A/Switzerland/9715293/2013(H3N2) from the same 3C3a clade failed to transmit. SW/VA/19 induced minimal macroscopic and microscopic lung lesions. Collectively, these findings demonstrate that these human seasonal H3N2 3C3a-like viruses did not require reassortment with endemic swine IAV gene segments for virus shedding and transmission in pigs. Limited detections in the U.S. pig population in the subsequent period of time suggest a yet-unknown restriction factor likely limiting the spread of these viruses in the U.S. pig population.IMPORTANCEInterspecies human-to-swine IAV transmission occurs globally and contributes to increased IAV diversity in pig populations. We present data that a swine isolate from a 2018-2019 human-to-swine transmission event was shed for multiple days in challenged and contact pigs. By characterizing this introduction through bioinformatic, molecular, and animal experimental approaches, these findings better inform animal health practices and vaccine decision-making. Since wholly human seasonal H3N2 viruses in the United States were not previously identified as being transmissible in pigs (i.e., reverse zoonosis), these findings reveal that the interspecies barriers for transmission to pigs may not require significant changes to all human seasonal H3N2, although additional changes may be required for sustained transmission in swine populations.

Human coronavirus (CoV) HKU1 infection typically causes common cold but can lead to pneumonia in children, older people, and immunosuppressed individuals. Recently, human transmembrane serine protease 2 (hTMPRSS2) was identified as the functional receptor for HKU1, but its region and residues critical for HKU1 S binding remain elusive. In this study, we find that HKU1 could utilize human and hamster, but not rat, mouse, or bat TMPRSS2 for virus entry, displaying a narrow host range. Using human-bat TMPRSS2 chimeras, we show that the serine peptidase (SP) domain of TMPRSS2 is essential for entry of HKU1. Further extensive mutagenesis analyses of the C-terminal regions of SP domains of human and bat TMPRSS2s identify residues 417 and 469 critical for entry of HKU1. Replacement of either D417 or Y469 with asparagine in hTMPRSS2 abolishes its abilities to mediate entry of HKU1 S pseudovirions and cell-cell fusion, whereas substitution of N417 with D or N469 with Y in bat TMPRSS2 (bTMPRSS2) renders it supporting HKU1 entry. Our findings contribute to a deeper understanding of coronavirus-receptor interactions and cross-species transmission.IMPORTANCEThe interactions of coronavirus (CoV) S proteins with their cognate receptors determine the host range and cross-species transmission potential. Recently, human transmembrane serine protease 2 (hTMPRSS2) was found to be the receptor for HKU1. Here, we show that the TMPRSS2 of hamster, but not rat, mouse, or bat, can serve as a functional entry receptor for HKU1. Moreover, swapping the residues at the positions of 417 and 469 of bTMPRSS2 with the corresponding residues of hTMPRSS2 confers it supporting entry of HKU1 S pseudovirions, indicating the critical role of these residues in HKU1 entry. Our study identified the critical residues in hTMPRSS2 responsible for receptor interaction and host range of HKU1.

Japanese encephalitis virus (JEV) stands as a prominent vector-borne zoonotic pathogen, displaying neurotropism and eliciting Parkinson's disease (PD)-like symptoms among most symptomatic survivors. A characteristic feature of PD is the aggregation of mutated α-synuclein (α-syn) that damages the dopaminergic neurons. Considering this link between JEV-induced PD-like symptoms and α-syn pathogenesis, we explored the role of α-syn in JEV infectivity in neuronal cells. Our investigation revealed a significant increase in endogenous α-syn expression in JEV-infected cells. In addition, exogenous α-syn (Exoα-syn) treatment substantially reduced JEV replication, suggesting its anti-JEV effect. Furthermore, Exoα-syn treatment led to the upregulation of superoxide dismutase 1 (SOD1) and reduction in reactive oxygen species (ROS). The results were validated by endogenous α-syn-silencing, which decreased SOD1 and raised ROS levels in neuronal cells. Similarly, the SOD1 inhibition via LCS-1 also intensified ROS and JEV infection. Silencing of SOD1 in α-syn overexpressing neuro2a cells exhibited increased JEV replication. Overall, our results suggest that α-syn exerts an anti-JEV effect by regulating protein involved in oxidative stress inside neuronal cells. This study contributes valuable insights into the interplay between α-syn expression and JEV infectivity, shedding light on avenues further to investigate the potential role of α-syn in JEV pathogenesis.

Importance: Japanese encephalitis virus (JEV) poses a significant threat, particularly to children. Despite extensive research efforts, the development of effective treatments against JEV has been impeded. One of the major setbacks is a lack of comprehensive understanding of neurotropism. The study focuses on alpha-synuclein (α-syn), a neuronal protein, and aims to determine its role in JEV pathogenesis. The present study reveals that the host cell upregulates α-syn in response to JEV infection. α-syn restrains JEV propagation by modulating superoxide dismutase 1 (SOD1) expression which further blocks JEV-induced ROS generation. Endogenous α-syn silencing led to a decrease in SOD1 expression and increased viral titer. α-syn plays a crucial role in counteracting oxidative stress through SOD1, which is essential for limiting JEV replication. This study provides broader implications for antiviral strategies and their possible role in neurodegenerative diseases; however, there is still much to explore, particularly regarding α-syn aggregation kinetics in JEV infection.

During the process by which human immunodeficiency virus (HIV-1) enters cells, the envelope glycoprotein (Env) trimer on the virion surface engages host cell receptors. Binding to the receptor CD4 induces Env to undergo transitions from a pretriggered, "closed" (State-1) conformation to more "open" (State 2/3) conformations. Most broadly neutralizing antibodies (bNAbs), which are difficult to elicit, recognize the pretriggered (State-1) conformation. More open Env conformations are recognized by poorly neutralizing antibodies (pNAbs), which are readily elicited during natural infection and vaccination with current Env immunogens. Env heterogeneity likely contributes to HIV-1 persistence by skewing antibody responses away from the pretriggered conformation. The conformationally flexible gp160 Env precursor on the infected cell or virion surface potentially presents multiple pNAb epitopes to the host immune system. Although proteolytic cleavage to produce the functional, mature Env trimer [(gp120/gp41)₃] stabilizes State-1, many primary HIV-1 Envs spontaneously sample more open conformations. Here, we establish inducible cell lines that produce replication-defective HIV-1 particles with Env trimers stabilized in a pretriggered conformation. The mature Env is enriched on virus-like particles (VLPs). Using complementary approaches, we estimate an average of 25-50 Env trimers on each VLP. The stabilizing changes in Env limit the natural conformational heterogeneity of the VLP Env trimers, allowing recognition by bNAbs but not pNAbs. These defective VLPs provide a more homogeneous source of pretriggered Env trimers in a native membrane environment. Thus, these VLPs may facilitate the characterization of this functionally important Env conformation and its interaction with the immune system.IMPORTANCEA major impediment to the development of an effective HIV/AIDS vaccine is the inefficiency with which human immunodeficiency virus (HIV-1) envelope glycoproteins elicit antibodies that neutralize multiple virus strains. Neutralizing antibodies recognize a particular shape of the envelope glycoproteins that resides on the viral membrane before the virus engages the host cell. Here, we report the creation of stable cell lines that inducibly produce non-infectious HIV-like particles. The normally flexible envelope glycoprotein spikes on these virus-like particles have been stabilized in a conformation that is recognized by broadly neutralizing antibodies. These virus-like particles allow the study of the envelope glycoprotein conformation, its modification by sugars, and its ability to elicit desired neutralizing antibodies.

The respiratory syncytial virus (RSV) fusion protein (F) facilitates virus-cell membrane fusion, which is critical for viral entry, and cell-cell fusion. In contrast to many type I fusion proteins, RSV F must be proteolytically cleaved at two distinct sites to be fusogenic. Cleavage at both sites results in the release of a 27 amino-acid fragment, termed Pep27. We examined proteolytic processing and the role of Pep27 for RSV F from both RSV A2 and RSV B9320 laboratory-adapted strains, allowing important comparisons between A and B clade F proteins. F from both clades was cleaved at both sites, and pulse-chase analysis indicated that cleavage at both sites occurs early after synthesis, most likely within the secretory pathway. Mutation of either site to alter the furin recognition motif blocked cell-cell fusion activity. To assess the role of Pep27 in F processing and expression, we deleted the Pep27 fragment, but preserved the cleavage sites. Deletion of Pep27 reduced F surface expression and cell-cell fusion. Two conserved N-linked glycosylation sites within Pep 27 are present in both the RSV A2 and RSV B9320 F. Randomization of the Pep27 sequence, while conserving the two N-liked glycosylation sites, did not significantly change surface expression, and only modestly reduced cell-cell fusion. However, the disruption of either Pep27 glycosylation site reduced cell-cell fusion. This work clarifies the timing of RSV F proteolytic cleavage and offers insight into the crucial role the N-linked glycosylation sites within Pep27 play in the biological function of F.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections, with no currently available small-molecule drugs that are both safe and effective. A major obstacle in antiviral drug development is the rapid emergence of drug-resistant viral strains. Targeting multiple viral compounds may help mitigate the development of resistance. Herein, we conducted a drug screening using the Antiviral Traditional Chinese Medicine Active Compound Library, aiming to identify compounds that simultaneously target the RSV fusion (F) protein, glycoprotein (G), and the host heparan sulfate proteoglycans (HSPGs). From this screening, 10 candidate compounds were identified for their ability to interact with all three targets. Among these 10 candidates, chebulagic acid (CHLA) and punicalagin (PUG) demonstrated the most potent inhibition of RSV replication. In vitro dose-response assays confirmed the antiviral efficacy of CHLA (IC50: 0.07864 µM) and PUG (IC50: 0.08065 µM). Further experiments revealed both CHLA and PUG disrupt RSV attachment and membrane fusion by targeting the RSV-F and G proteins, rather than HSPG. Notably, CHLA and PUG were found to bind to the CX3C motif of the RSV-G protein, with docking assays predicting their binding sites at cysteines 176 and 182. Additionally, CHLA enhanced the conformational stability of the RSV-F protein before fusion. In an in vivo study, both CHLA and PUG were shown to alleviate RSV-induced pulmonary pathology by reducing viral titers, mitigating lung injury, and suppressing the inflammatory responses in the lungs. Our findings suggest that CHLA and PUG hold potential as therapeutic agents for RSV infection.IMPORTANCEA significant challenge in developing anti-respiratory syncytial virus (RSV) agents is the rapid emergence of resistant viral strains. Designing drugs that target multiple viral components can effectively reduce the likelihood of developing resistant strains. In this study, we screened compounds from the Antiviral Traditional Chinese Medicine Active Compound Library, aiming to simultaneously targe the RSV fusion (F) protein, glycoprotein (G), and host heparan sulfate proteoglycans (HSPGs). Our findings revealed that chebulagic acid (CHLA) and punicalagin (PUG) significantly inhibited RSV replication both in vitro and in vivo and interacted with all three targets. Both CHLA and PUG were able to disrupt RSV attachment and membrane fusion. Mechanistically, CHLA and PUG were found to bind to the CX3C motif of the RSV-G protein, with CHLA also enhancing the conformational stability of the RSV-F protein before fusion. In conclusion, our study suggests that CHLA and PUG hold promise as therapeutic agents against RSV infection.

The immunoproteasome (IP) is a predominantly inducible component of the ubiquitin proteasome system that plays key roles in multiple aspects of immune function, inflammation, and protein homeostasis. We used murine hepatitis virus strain 1 (MHV-1), a mouse coronavirus, to define the role of IP activity during acute coronavirus respiratory infection. Expression of the β5i subunit of the IP and cytokines that induce IP activity, including IFN-γ, TNF-α, and IFN-β, increased in lungs and livers of CH3/HeJ mice following intranasal infection with MHV-1. IP inhibition using ONX-0914 did not affect MHV-1 replication in bone marrow-derived dendritic cells in vitro. IP inhibition in vivo exacerbated virus-induced weight loss and mortality but had no effect on virus replication in lungs or livers. IP inhibition had minimal effect on virus-induced pulmonary inflammation but led to substantially increased liver pathology, including greater upregulation of pro-inflammatory cytokines and histological evidence of inflammation and necrosis. Those findings were associated with evidence of increased endoplasmic reticulum stress although not with accumulation of ubiquitinated protein. Our results indicate that the IP is a protective host factor during acute MHV-1 infection.

Importance: Inflammatory responses triggered by acute infection by respiratory viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drive morbidity and mortality. Infection of mice with murine hepatitis virus strain 1 (MHV-1), a mouse coronavirus, is a useful model to study the pathogenesis of coronavirus respiratory infections. The immunoproteasome is an inducible component of the ubiquitin proteasome system that is poised to contribute to multiple aspects of immune function, inflammation, and protein homeostasis during an infection. We used the MHV-1 model to define the role of the immunoproteasome in coronavirus pathogenesis. We found that immunoproteasome subunit expression increases in the lungs and the liver during acute MHV-1 respiratory infection. Inhibition of immunoproteasome activity did not affect MHV-1 replication but increased MHV-1-induced weight loss, mortality, and inflammation in lungs and livers. Thus, our findings indicate that the immunoproteasome is a critical protective host factor during coronavirus respiratory infection.