Characterization of plasma membrane-associated proteins from Aedes albopictus mosquito (C6/36) cells that mediate West Nile virus binding and infection

This study isolated and characterized the West Nile virus (WNV) putative receptor molecule(s) from Aedes albopictus mosquito (C6/36) cells. The binding of WNV to C6/36 cells was saturated with 5000 particles per cell. The entry of WNV into C6/36 cells was strongly inhibited when pretreated with proteinase K and to a lesser extent with sodium periodate. However, pretreatment of C6/36 cells with phospholipases, glycosidases, heparinases and neurimidase had no effect on virus entry. By using virus overlay protein blot assay, WNV was observed to bind to the 140-kDa, 95-kDa, 70-kDa and 55-kDa plasma membrane-associated molecules isolated from C6/36 cells. Murine antibodies generated against the 95-kDa and 70-kDa membrane proteins effectively blocked WNV, Japanese encephalitis virus (JEV) and Dengue virus (DV) serotype 2 infection in C6/36 cells. In addition, the binding of the recombinant-WNV envelope domain III protein to C6/36 cells can be inhibited by the anti-95-kDa and anti-70-kDa membrane protein antibodies. These data strongly supported the possibility that the 95-kDa and 70-kDa plasma membrane-associated proteins are part of a receptor complex for mosquito-borne flaviviruses (WNV, JEV and DV) on mosquito cells.     

Immunity to West Nile virus

Over the past five years, West Nile (WN) virus has emerged as an important public health concern in the United States. Recent studies from experimental models of WN virus infection have increased our understanding of its pathogenesis and immunity. These include the demonstration that the gene encoding 2'-5'oligoadenylate synthetase is responsible for murine susceptibility to WN virus, the elucidation of the contributions of B, CD8(+) and gamma T cells in the control of murine WN virus infection, and the use of active immunization with envelope protein and passive transfer of immunoglobulin for immunotherapy. These efforts will facilitate the development of effective vaccines and therapies to combat WN virus.     

Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4 virus

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G(2337) C (Met(457) Ile) in the E gene and A(6751) G (Lys(125) Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this article could be useful for monitoring the molecular consistency and quality control of vaccine strains.     

Receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion

Porcine epidemic diarrhea virus (PEDV) infection in Vero cells is facilitated by trypsin through an undefined mechanism. The present study describes the mode of action of trypsin in enhancing PEDV infection in Vero cells during different stage of the virus life cycle. During the viral entry stage, trypsin increased the penetration of Vero-cell-attached PEDV by approximately twofold. However, trypsin treatment of viruses before receptor binding did not enhance infectivity, indicating that receptor binding is essentially required for trypsin-mediated entry upon PEDV infection. Trypsin treatment during the budding stage of virus infection induces an obvious cytopathic effect in infected cells. Furthermore, we also show that the PEDV spike (S) glycoprotein is cleaved by trypsin in virions that are bound to the receptor, but not in free virions. These findings indicate that trypsin affects only cell-attached PEDV and increases infectivity and syncytium formation in PEDV-infected Vero cells by cleavage of the PEDV S protein. These findings strongly suggest that the PEDV S protein may undergo a conformational change after receptor binding and cleavage by exogenous trypsin, which induces membrane fusion.     

The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.     

The kinetics of proinflammatory cytokines in murine peritoneal macrophages infected with envelope protein-glycosylated or non-glycosylated West Nile virus

The envelope (E) protein glycosylation status of the New York strain of West Nile (WN) virus is an important determinant of virus neuroinvasiveness. To elucidate the determinant of the difference between E protein-glycosylated and non-glycosylated WN virus infections, the cytokine expression of murine peritoneal macrophages infected with each virus was examined. Tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta were up-regulated with replication of the E protein-glycosylated virus. Interferon (IFN) beta and IL-6 were up-regulated with the clearance of both viruses. These results suggest that TNFalpha and IL-1beta expression are related to the virulence of E protein-glycosylated WN virus.     

Low-neurovirulence Theiler's viruses use sialic acid moieties on N-linked oligosaccharide structures for attachment

Low-neurovirulence BeAn and DA Theiler's murine encephalomyelitis viruses (TMEV) cause persistent infection in the central nervous system (CNS) of susceptible mouse strains, leading to an inflammatory demyelinating process. A role for a specific virus-cell receptor interaction has been posited to explain why only low- and not high-neurovirulence TMEV cause persistent CNS infections. Low- but not high-neurovirulence TMEV use sialic acid for attachment to mammalian cells, which may contribute to neurovirulence attenuation and viral persistence. Analysis of BeAn virus binding and infection in cells with altered (mutated) cell-surface expression of sialic acid containing glyconjugates indicated that both binding and infection are mediated entirely by N-linked glycoproteins. By contrast, GDVII virus binding and infection appears to be dependent only in part on N-linked glycoproteins and not on O-linked glycoproteins or glycolipids. These results indicate that low-neurovirulence BeAn virus uses a sialic acid moiety expressed on an N-linked carbohydrate of a glycoprotein that serves as the protein entry receptor.     

Mutations in the West Nile prM protein affect VLP and virion secretion in vitro

Mutation of the West Nile virus-like particle (WN VLP) prM protein (T20D, K31A, K31V, or K31T) results in undetectable VLP secretion from transformed COS-1 cells. K31 mutants formed intracellular prM-E heterodimers; however these proteins remained in the ER and ER-Golgi intermediary compartments of transfected cells. The T20D mutation affected glycosylation, heterodimer formation, and WN VLP secretion. When infectious viruses bearing the same mutations were used to infect COS-1 cells, K31 mutant viruses exhibited delayed growth and reduced infectivity compared to WT virus. Epitope maps of WN VLP and WNV prM were also different. These results suggest that while mutations in the prM protein can reduce or eliminate secretion of WN VLPs, they have less effect on virus. This difference may be due to the quantity of prM in WN VLPs compared to WNV or to differences in maturation, structure, and symmetry of these particles.     

Isolation of homologous arbovirus cultures from heterologous mixtures using limit dilution and virus-specific enzyme immunoassays

Viral cultures were identified recently that contained both Kunjin virus and the closely related flavivirus West Nile. The observation that the KUN virus population grew more efficiently in a mosquito cell line (C6/36) while the WN population replicated more effectively in mammalian cells (Vero) allowed enrichment for either virus by culturing the mixture in the appropriate cell line. Limit dilution of the enriched virus preparations was then performed by infecting microtitre cultures with serial ten fold dilutions. Culture wells that contained a pure population of virus were then identified by immunostaining fixed cell monolayers with virus-specific monoclonal antibodies. Subsequent passage of the 'cloned' viruses in either C6/36 or Vero cells and analysis of the infected cultures by specific monoclonal antibody staining, PCR and nucleotide sequencing confirmed the identity of the virus and that in each case an homogeneous virus population had been obtained. This procedure is particularly useful for isolating virus populations from heterogeneous mixtures that fail to develop discrete plaques in infected cell monolayers.     

Vertical transmission of West Nile Virus by three California Culex (Diptera: Culicidae) species

Three California Culex species previously identified as efficient laboratory vectors of West Nile (WN) virus were tested for their capability to vertically transmit WN virus. Wild-caught Culex pipiens pipiens L., Culex pipiens quinquefasciatus Say, and two populations of Culex tarsalis Coquillett females were inoculated intrathoracically with 10(2.7 +/- 0.1) plaque-forming units of WN virus. F1 progeny were reared at 18 degrees C and subsequently tested as adults for infectious virus on Vero cell culture. Virus was not detected in 197 pools comprising 4,884 Cx. p. pipiens. The minimum filial infection rate (MFIR) for Cx. p. quinquefasciatus was approximately 3.0/1,000 for 665 progeny tested in 28 pools. There was no virus detected in 102 pools of 2,453 progeny from Cx. tarsalis collected in Riverside County. The MFIR for Cx. tarsalis collected in Yolo County was approximately 6.9/1,000 for 2,165 progeny tested in 86 pools. Mosquito progeny infected vertically during the fall could potentially serve as a mechanism for WN virus to overwinter and initiate horizontal transmission the following spring.     

Mosquito cells bind and replicate hepatitis C virus

Several studies have demonstrated some hepatitis C virus (HCV) replication in lymphocyte and hepatocyte cell lines such as in African green monkey Vero cells. The aim of the present study was to select other cell lines able to bind and replicate HCV. Human hepatoma PLC/PRF/5 cells, human lymphoma Namalwa cells, Vero and mosquito AP61 cells were inoculated with HCV-positive plasma, washed six times and examined for the presence of the viral genome at different times post infection, using an RT-PCR method. Binding of HCV to cells was estimated by HCV RNA detection in cells 2 hr after inoculation and in the last wash of these cells. Successive virus passages in cells were carried out. All the cells studied were able to bind HCV but only AP61 and Vero cells provided evidence of replication and production of infectious virus: virus RNA was detected during 28 days post-infection in four successive virus passages. CD81 molecules, a putative HCV receptor, were detected by cytofluorometric analysis. Vero cells express CD81 molecules whereas these molecules were not detected on AP61 cells. It is suggested that other receptors are involved in HCV binding to Vero and AP61 cells.     

Vaccinia virus exhibits cell-type-dependent entry characteristics

Differing and sometimes conflicting data have been reported regarding several aspects of vaccinia virus (VV) entry. To address this, we used a β-galactosidase reporter virus to monitor virus entry into multiple cell types under varying conditions. Entry into HeLa, B78H1 and L cells was strongly inhibited by heparin whereas entry into Vero and BSC-1 cells was unaffected. Bafilomycin also exhibited variable and cell-type-specific effects on VV entry. Entry into B78H1 and BSC-1 cells was strongly inhibited by bafilomycin whereas entry into Vero and HeLa cells was only partially inhibited suggesting the co-existence of both pH-dependent and pH-independent VV entry pathways in these cell types. Finally, entry into HeLa, B78H1, L and BSC-1 cells exhibited a lag of 6-9 min whereas this delay was undetectable in Vero cells. Our results suggest that VV exploits multiple cell attachment and entry pathways allowing it to infect a broad range of cells.     

Transstadial transfer of West Nile virus by three species of ixodid ticks (Acari: Ixodidae)

Larvae and/or nymphs of four species of ixodid ticks, Ixodes scapularis Say, Amblyomma americanum (L.), Dermacentor andersoni Stiles, and Dermacentor variabilis Say, were fed to completion on laboratory hamsters or mice which had been inoculated with a West Nile (WN) virus isolate from Culex pipiens L. captured in Connecticut USA. Maximum titers in mice and hamsters were approximately 5 and two logs, respectively, lower than recorded (10 logs) in a naturally infected American crow, Corvus brachyrhynchos Brehm. WN virus was isolated in Vero cell culture from ticks and detected by TaqMan RT-polymerase chain reaction (PCR) in ticks that had completed their feeding as larvae or nymphs, and in I. scapularis, D. andersoni, and D. variabilis that had molted into the next stage of development. Naive hosts, fed upon by nymphs that as larvae had fed on viremic hosts, did not become infected. WN virus was isolated in Vero cell culture from one female I. scapularis and was detected by TaqMan RT-PCR in 24 adult I. scapularis, one D. andersoni, and two D. variabilis adults that had fed to completion as larvae on viremic hosts and as nymphs on naive mice or hamsters. Three species of ixodid ticks acquired WN virus from viremic hosts and transstadially passed the virus, but vector competency was not demonstrated.     

Actin filaments participate in West Nile (Sarafend) virus maturation process

West Nile (Sarafend) virus has previously been shown to egress by budding at the plasma membrane of infected cells, but relatively little is known about the mechanism involved in this mode of release. During the course of this study, it was discovered that actin filaments take part in the virus maturation process. Using dual-labeled immunofluorescence and immunoelectron microscopy at late infection (10 hr p.i.), co-localization of viral structural (envelope and capsid) proteins with actin filaments was confirmed. The virus structural proteins were also immunoprecipitated with anti-actin antibody, further demonstrating the strong association between the two components. Perturbation of actin filaments by cytochalasin B strongly inhibited the release of West Nile virus (approximately 10,000-fold inhibition) when compared with the untreated cells. Infectious virus particles were recovered after the removal of cytochalasin B. Further confirmation was obtained when nucleocapsid particles were found associated with disrupted actin filaments at the periphery of cytochalasin B-treated cells. Together, these results showed that actin filaments do indeed have a key role in the release of West Nile (Sarafend) virions.     

Cellular glycosaminoglycans and low density lipoprotein receptor are involved in hepatitis C virus adsorption

The initial binding of Hepatitis C virus (HCV) to the cell membrane is a critical determinant of pathogenesis. Two putative HCV receptors have been identified, CD81 and low-density lipoprotein receptor (LDLr). CD81 interacts in vitro with the HCV E2 envelope glycoprotein, and LDLr interacts with HCV present in human plasma. In order to characterize these potential receptors for HCV, virus from plasma, able to replicate in cell culture, was inoculated on Vero cells or human hepatocarcinoma cells. HCV adsorption was assessed by quantitating cell-associated viral RNA by a real-time RT-PCR method. Anti-LDLr antibody, low and very low density lipoproteins inhibited significantly HCV adsorption, confirming the role of LDLr as HCV receptor. Only one out of the two anti-CD81 antibodies used in this study led to a partial inhibition of HCV binding. This study also highlights a role for glycosaminoglycans (GAGs) in HCV adsorption: treatment of virus with heparin led to 70% inhibition of attachment, as did desulfation of cellular GAGs. Treatment of Vero cells with heparin-lyase significantly inhibited virus attachment but by only 30%. These results demonstrate the complexity of the HCV binding step in which LDLr interacts strongly with HCV, whereas the interaction of HCV with GAGs and particularly with CD81 seem to be more moderate.     

The anti-HIV drug nelfinavir mesylate (Viracept) is a potent inhibitor of cell fusion caused by the SARSCoV-2 spike (S) glycoprotein warranting further evaluation as an antiviral against COVID-19 infections

Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is the causative agent of the coronavirus disease-2019 (COVID-19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV-2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID-19. These results suggest that SARS CoV-2 is able to spread from cell-to-cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA-approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S-n- and S-o-mediated cell fusion with complete inhibition at a 10-μM concentration. In-silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S-n- and S-o-mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV-2 symptoms appear.