Recreating the biological steps of viral infection on a cell-free bioelectronic platform to profile viral variants of concern.

Viral mutations frequently outpace technologies used to detect harmful variants. Given the continual emergence of SARS-CoV-2 variants, platforms that can identify the presence of a virus and its propensity for infection are needed. Our electronic biomembrane sensing platform recreates distinct SARS-CoV-2 host cell entry pathways and reports the progression of entry as electrical signals. We focus on two necessary entry processes mediated by the viral Spike protein: virus binding and membrane fusion, which can be distinguished electrically. We find that closely related variants of concern exhibit distinct fusion signatures that correlate with trends in cell-based infectivity assays, allowing us to report quantitative differences in their fusion characteristics and hence their infectivity potentials. We use SARS-CoV-2 as our prototype, but we anticipate that this platform can extend to other enveloped viruses and cell lines to quantifiably assess virus entry.

Analyzing the factors affecting virus invasion by quantitative single-particle analysis.

Viral diseases are among the main threats to public health. Understanding the factors affecting viral invasion is important for antiviral research. Until now, it was known that most viruses have very low plaque-forming unit (PFU)-to-particle ratios. However, further investigation is required to determine the underlying factors. Here, using quantitative single-particle analysis methods, the invasion of Semliki Forest virus (SFV), Japanese encephalitis virus (JEV), and influenza A virus (IAV) containing attachment to the cell surface, entry into the cell, transport towards the cell interior, and fusion with endosomes to release nucleocapsids were quantitatively analysed in parallel. It was found that for SFV with an PFU-to-particle ratio of approximately 1:2, an entry efficiency of approximately 31% limited infection. For JEV, whose PFU-to-particle ratio was approximately 1:310, an attachment efficiency of approximately 27% and an entry efficiency of 10% were the main factors limiting its infection. Meanwhile, for IAV with PFU-to-particle ratios of 1:8100, 5% attachment efficiency, 9% entry efficiency, and 53% fusion efficiency significantly limited its infection. These results suggest that viruses with different infectivities have different limited steps in the invasion process. Moreover, there are significant differences in attachment efficiencies among viruses, emphasizing the pivotal role of attachment in viral invasion. The influence of the virus purification method on virus invasion was also investigated. This study, for the first time, reports the efficiencies of different stages of virus invasion, leading to a better understanding of virus invasion and providing a protocol to quantitatively analyse the virus invasion efficiency.

Seed coating with phages for sustainable plant biocontrol of plant pathogens and influence of the seed coat mucilage.

Pathogens resistant to classical control strategies pose a significant threat to crop yield, with seeds being a major transmission route. Bacteriophages, viruses targeting bacteria, offer an environmentally sustainable biocontrol solution. In this study, we isolated and characterized two novel phages, Athelas and Alfirin, which infect Pseudomonas syringae and Agrobacterium fabrum, respectively, and included the recently published Pfeifenkraut phage infecting Xanthomonas translucens. Using a simple immersion method, phages coated onto seeds successfully lysed bacteria post air-drying. The seed coat mucilage (SCM), a polysaccharide-polymer matrix exuded by seeds, plays a critical role in phage binding. Seeds with removed mucilage formed five to 10 times less lysis zones compared to those with mucilage. The podovirus Athelas showed the highest mucilage dependency. Phages from the Autographiviridae family also depended on mucilage for seed adhesion. Comparative analysis of Arabidopsis SCM mutants suggested the diffusible cellulose as a key component for phage binding. Long-term activity tests demonstrated high phage stability on seed surfaces and significantly increasing seedling survival rates in the presence of pathogens. Using non-virulent host strains enhanced phage presence on seeds but also has potential limitations. These findings highlight phage-based interventions as promising, sustainable strategies for combating pathogen resistance and improving crop yield.

Influence of bacterial swimming and hydrodynamics on attachment of phages.

Bacteriophages ("phages") are viruses that infect bacteria. Since they do not actively self-propel, phages rely on thermal diffusion to find target cells-but can also be advected by fluid flows, such as those generated by motile bacteria themselves in bulk fluids. How does the flow field generated by a swimming bacterium influence how it encounters phages? Here, we address this question using coupled molecular dynamics and lattice Boltzmann simulations of flagellated bacteria swimming through a bulk fluid containing uniformly-dispersed phages. We find that while swimming increases the rate at which phages attach to both the cell body and flagellar propeller, hydrodynamic interactions strongly suppress this increase at the cell body, but conversely enhance this increase at the flagellar bundle. Our results highlight the pivotal influence of hydrodynamics on the interactions between bacteria and phages, as well as other diffusible species, in microbial environments.

Role of core lipopolysaccharide biosynthetic genes in the infection and adsorption of broad-host-range bacteriophages of Rhizobium etli.

In this study, we examined the role of the lipopolysaccharide (LPS) core of Rhizobium etli in facilitating the adsorption and infection of phages with broad host range. When the plasmid-encoded LPS biosynthesis genes, wreU and wreV, were disrupted, distinct and contrasting effects on phage infection were observed. The wreU mutant strains exhibited wild-type adsorption and infection properties, whereas the wreV mutant demonstrated resistance to phage infection, but retained the capacity to adsorb phages. Complementation of the wreV mutant strains with a recombinant plasmid containing the wreU and wreV, restored the susceptibility to the phages. However, the presence of this recombinant plasmid in a strain devoid of the native lps-encoding plasmid was insufficient to restore phage susceptibility. These results suggest that the absence of wreV impedes the proper assembly of the complete LPS core, potentially affecting the formation of UDP-KdgNAg or KDO precursors for the O-antigen. In addition, a protein not yet identified, but residing in the native lps-encoding plasmid, may be necessary for complete phage infection.

Sequence polymorphisms in the reovirus σ1 attachment protein modulate encapsidation efficiency and replication in mice.

Many functions of viral attachment proteins are established, but less is known about the biological importance of viral attachment protein encapsidation efficiency. The mammalian orthoreovirus (reovirus) σ1 attachment protein forms filamentous trimers that incorporate into pentamers of the λ2 capsid protein. Reovirus strains vary in the efficiency of σ1 encapsidation onto progeny virions, which influences viral stability during entry into cells and the efficacy of tumor cell lysis. While the role of σ1 encapsidation has been evaluated in studies using cultured cells, the contribution of attachment protein encapsidation efficiency to viral infection in animals is less clear. Polymorphisms in reovirus σ1 at residues 22 and 249 have been implicated in viral dissemination in mice and susceptibility to proteolysis in the murine intestine, respectively. To determine whether these residues contribute to σ1 encapsidation efficiency, we engineered σ1 mutant viruses with single- and double-residue substitutions at sites 22 and 249. We found that substitutions at these sites alter the encapsidation of σ1 and that reoviruses encapsidating higher amounts of σ1 bind cells more avidly and have a modest replication advantage in a cell-type-specific manner relative to low σ1-encapsidating reoviruses. Furthermore, we found that a high σ1-encapsidating reovirus replicates and disseminates more efficiently in mice relative to a low σ1-encapsidating reovirus. These findings provide evidence of a relationship between viral attachment protein encapsidation efficiency and viral replication in cell culture and animal hosts. IMPORTANCE: Viral attachment proteins can serve multiple functions during viral replication, including attachment to host cells, cell entry and disassembly, and modulation of host immune responses. The relationship between viral attachment protein encapsidation efficiency and viral replication in cells and animals is poorly understood. We engineered and characterized a panel of reoviruses that differ in the capacity to encapsidate the σ1 attachment protein. We found that strains encapsidating σ1 with higher efficiency bind cells more avidly and replicate and spread more efficiently in mice relative to those encapsidating σ1 with lower efficiency. These results highlight a function for σ1 attachment protein capsid abundance in viral replication in cells and animals, which may inform future use of reovirus as an oncolytic therapeutic.

Novel insights into the host cell glycan binding profile of human metapneumovirus.

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galß1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.

Back to the Basics of SARS-CoV-2 Biochemistry: Microvascular Occlusive Glycan Bindings Govern Its Morbidities and Inform Therapeutic Responses.

Consistent with the biochemistry of coronaviruses as well established over decades, SARS-CoV-2 makes its initial attachment to host cells through the binding of its spike protein (SP) to sialylated glycans (containing the monosaccharide sialic acid) on the cell surface. The virus can then slide over and enter via ACE2. SARS-CoV-2 SP attaches particularly tightly to the trillions of red blood cells (RBCs), platelets and endothelial cells in the human body, each cell very densely coated with sialic acid surface molecules but having no ACE2 or minimal ACE2. These interlaced attachments trigger the blood cell aggregation, microvascular occlusion and vascular damage that underlie the hypoxia, blood clotting and related morbidities of severe COVID-19. Notably, the two human betacoronaviruses that express a sialic acid-cleaving enzyme are benign, while the other three-SARS, SARS-CoV-2 and MERS-are virulent. RBC aggregation experimentally induced in several animal species using an injected polysaccharide caused most of the same morbidities of severe COVID-19. This glycan biochemistry is key to disentangling controversies that have arisen over the efficacy of certain generic COVID-19 treatment agents and the safety of SP-based COVID-19 vaccines. More broadly, disregard for the active physiological role of RBCs yields unreliable or erroneous reporting of pharmacokinetic parameters as routinely obtained for most drugs and other bioactive agents using detection in plasma, with whole-blood levels being up to 30-fold higher. Appreciation of the active role of RBCs can elucidate the microvascular underpinnings of other health conditions, including cardiovascular disease, and therapeutic opportunities to address them.

Murine norovirus mutants adapted to replicate in human cells reveal a post-entry restriction.

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors. IMPORTANCE: Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.

Novel Anti-Viral Properties of the Herbal Extract of Davallia mariesii against Influenza A Virus.

Gu-Sui-Bu, the dried rhizome of Davallia mariesii, is a traditional Chinese herbal remedy with a significant history of treating osteoporosis and inflammatory conditions. However, its potential as an anti-influenza agent and its underlying mechanisms of action remain unexplored. To obtain a more potent extract from D. mariesii and gain insights into its mechanism of action against influenza A virus (IAV), we utilized a partitioning process involving organic solvents and water, resulting in the isolation of butanolic subfractions of the D. mariesii extract (DMBE). DMBE exhibited a broad anti-viral spectrum, effectively inhibiting IAV, with an EC50 of 24.32 ± 6.19 µg/mL and a selectivity index of 6.05. We subsequently conducted a series of in vitro assays to evaluate the antiviral effects of DMBE and to uncover its mechanisms of action. DMBE was found to inhibit IAV during the early stages of infection by hindering the attachment of the virus onto and its penetration into host cells. Importantly, DMBE was observed to hinder IAV-mediated cell-cell fusion. It also inhibited neuraminidase activity, plaque size, and the expression levels of phospho-AKT. In summary, this study provides evidence for the effectiveness of D. mariesii as a complementary and alternative herbal remedy against IAV. Specifically, our data highlight DMBE's capabilities in inhibiting viral entry and the release of virions.

Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection.

The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.

Phosphatidylserine-exposing extracellular vesicles in body fluids are an innate defence against apoptotic mimicry viral pathogens.

Some viruses are rarely transmitted orally or sexually despite their presence in saliva, breast milk, or semen. We previously identified that extracellular vesicles (EVs) in semen and saliva inhibit Zika virus infection. However, the antiviral spectrum and underlying mechanism remained unclear. Here we applied lipidomics and flow cytometry to show that these EVs expose phosphatidylserine (PS). By blocking PS receptors, targeted by Zika virus in the process of apoptotic mimicry, they interfere with viral attachment and entry. Consequently, physiological concentrations of EVs applied in vitro efficiently inhibited infection by apoptotic mimicry dengue, West Nile, Chikungunya, Ebola and vesicular stomatitis viruses, but not severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus 1, hepatitis C virus and herpesviruses that use other entry receptors. Our results identify the role of PS-rich EVs in body fluids in innate defence against infection via viral apoptotic mimicries, explaining why these viruses are primarily transmitted via PS-EV-deficient blood or blood-ingesting arthropods rather than direct human-to-human contact.

Bacterial-derived sialidases inhibit porcine rotavirus OSU replication by interfering with the early steps of infection.

Rotavirus infections in suckling and weaning piglets cause severe dehydration and death, resulting in significant economic losses in the pig breeding industry. With the continuous emergence of porcine rotavirus (PoRV) variants and poor vaccine cross-protection among various genotypes, there is an urgent need to develop alternative strategies such as seeking effective antiviral products from nature, microbial metabolites and virus-host protein interaction. Sialidases play a crucial role in various physiopathological processes and offer a promising target for developing antivirus drugs. However, the effect of bacterial-derived sialidases on the infection of PoRVs remains largely unknown. Herein, we investigated the impact of bacterial-derived sialidases (sialidase Cp and Vc) on PoRV strain OSU(Group A) infection, using differentiated epithelial monkey kidney cells (MA104) as a model. Our results indicated that the pretreatment of MA104 with exogenous sialidases effectively suppressed PoRV OSU in a concentration-dependent manner. Notably, even at a concentration of 0.01 µU/mL, sialidases significantly inhibited the virus (MOI = 0.01). Meanwhile, we found that sialidase Vc pretreatment sharply reduced the binding rate of PoRV OSU. Last, we demonstrated that PoRV OSU might recognize α-2,3-linked sialic acid as the primary attachment factor in MA104. Our findings provide new insights into the underlying mechanism of PoRV OSU infections, shedding lights on the development of alternative antivirus approaches based on bacteria-virus interaction.

C1q enables influenza hemagglutinin stem binding antibodies to block viral attachment and broadens the antibody escape repertoire.

Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.

Glucosylceramide in bunyavirus particles is essential for virus binding to host cells.

Hexosylceramides (HexCer) are implicated in the infection process of various pathogens. However, the molecular and cellular functions of HexCer in infectious cycles are poorly understood. Investigating the enveloped virus Uukuniemi (UUKV), a bunyavirus of the Phenuiviridae family, we performed a lipidomic analysis with mass spectrometry and determined the lipidome of both infected cells and derived virions. We found that UUKV alters the processing of HexCer to glycosphingolipids (GSL) in infected cells. The infection resulted in the overexpression of glucosylceramide (GlcCer) synthase (UGCG) and the specific accumulation of GlcCer and its subsequent incorporation into viral progeny. UUKV and several pathogenic bunyaviruses relied on GlcCer in the viral envelope for binding to various host cell types. Overall, our results indicate that GlcCer is a structural determinant of virions crucial for bunyavirus infectivity. This study also highlights the importance of glycolipids on virions in facilitating interactions with host cell receptors and infectious entry of enveloped viruses.

C1QTNF5 is a novel attachment factor that facilitates the entry of influenza A virus.

Influenza A virus (IAV) binds sialic acid receptors on the cell surface to enter the host cells, which is the key step in initiating infection, transmission and pathogenesis. Understanding the factors that contribute to the highly efficient entry of IAV into human cells will help elucidate the mechanism of viral entry and pathogenicity, and provide new targets for intervention. In the present study, we reported a novel membrane protein, C1QTNF5, which binds to the hemagglutinin protein of IAV and promotes IAV infection in vitro and in vivo. We found that the HA1 region of IAV hemagglutinin is critical for the interaction with C1QTNF5 protein, and C1QTNF5 interacts with hemagglutinin mainly through its N-terminus (1-103 aa). In addition, we further demonstrated that overexpression of C1QTNF5 promotes IAV entry, while blocking the interaction between C1QTNF5 and IAV hemagglutinin greatly inhibits viral entry. However, C1QTNF5 does not function as a receptor to mediate IAV infection in sialic acid-deficient CHO-Lec2 cells, but promotes IAV to attach to these cells, suggesting that C1QTNF5 is an important attachment factor for IAV. This work reveals C1QTNF5 as a novel IAV attachment factor and provides a new perspective for antiviral strategies.

Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.

Direct interaction of the molecular chaperone GRP78/BiP with the Newcastle disease virus hemagglutinin-neuraminidase protein plays a vital role in viral attachment to and infection of culture cells.

Introduction: Glucose Regulated Proteins/Binding protein (GRP78/Bip), a representative molecular chaperone, effectively influences and actively participates in the replication processes of many viruses. Little is known, however, about the functional involvement of GRP78 in the replication of Newcastle disease virus (NDV) and the underlying mechanisms. Methods: The method of this study are to establish protein interactomes between host cell proteins and the NDV Hemagglutinin-neuraminidase (HN) protein, and to systematically investigate the regulatory role of the GRP78-HN protein interaction during the NDV replication cycle. Results: Our study revealed that GRP78 is upregulated during NDV infection, and its direct interaction with HN is mediated by the N-terminal 326 amino acid region. Knockdown of GRP78 by small interfering RNAs (siRNAs) significantly suppressed NDV infection and replication. Conversely, overexpression of GRP78 resulted in a significant increase in NDV replication, demonstrating its role as a positive regulator in the NDV replication cycle. We further showed that the direct interaction between GRP78 and HN protein enhanced the attachment of NDV to cells, and masking of GRP78 expressed on the cell surface with specific polyclonal antibodies (pAbs) inhibited NDV attachment and replication. Discussion: These findings highlight the essential role of GRP78 in the adsorption stage during the NDV infection cycle, and, importantly, identify the critical domain required for GRP78-HN interaction, providing novel insights into the molecular mechanisms involved in NDV replication and infection.

In silico prediction of interaction between Nipah virus attachment glycoprotein and host cell receptors Ephrin-B2 and Ephrin-B3 in domestic and peridomestic mammals.

Nipah virus (NiV) is a lethal bat-borne zoonotic virus that causes mild to acute respiratory distress and neurological manifestations in humans with a high mortality rate. NiV transmission to humans occurs via consumption of bat-contaminated fruit and date palm sap (DPS), or through direct contact with infected individuals and livestock. Since NiV outbreaks were first reported in pigs from Malaysia and Singapore, non-neutralizing antibodies against NiV attachment Glycoprotein (G) have also been detected in a few domestic mammals. NiV infection is initiated after NiV G binds to the host cell receptors Ephrin-B2 and Ephrin-B3. In this study, we assessed the degree of NiV host tropism in domestic and peridomestic mammals commonly found in Bangladesh that may be crucial in the transmission of NiV by serving as intermediate hosts. We carried out a protein-protein docking analysis of NiV G complexes (n = 52) with Ephrin-B2 and B3 of 13 domestic and peridomestic species using bioinformatics tools. Protein models were generated by homology modelling and the structures were validated for model quality. The different protein-protein complexes in this study were stable, and their binding affinity (ΔG) scores ranged between -8.0 to -19.1 kcal/mol. NiV Bangladesh (NiV-B) strain displayed stronger binding to Ephrin receptors, especially with Ephrin-B3 than the NiV Malaysia (NiV-M) strain, correlating with the observed higher pathogenicity of NiV-B strains. From the docking result, we found that Ephrin receptors of domestic rat (R. norvegicus) had a higher binding affinity for NiV G, suggesting greater susceptibility to NiV infections compared to other study species. Investigations for NiV exposure to domestic/peridomestic animals will help us knowing more the possible role of rats and other animals as intermediate hosts of NiV and would improve future NiV outbreak control and prevention in humans and domestic animals.

CD4 is an important host factor for Japanese encephalitis virus entry and replication in PK-15 cells.

Japanese encephalitis virus (JEV) is a flavivirus that is spread through mosquito bites and is the leading cause of viral encephalitis in Asia. JEV can infect a variety of cell types; however, crucial receptor molecules remain unclear. The purpose of this study was to determine whether porcine CD4 protein is a receptor protein that impacts JEV entry into PK15 cells and subsequent viral replication. We confirmed the interaction between the JEV E protein and the CD4 protein through Co-IP, virus binding and internalization, antibody blocking, and overexpression and created a PK-15 cell line with CD4 gene knockdown by CRISPR/Cas9. The results show that CD4 interacts with JEV E and that CD4 knockdown cells altered virus adsorption and internalization, drastically reducing virus attachment. The level of viral transcription in CD4 antibody-blocked cells, vs. control cells, was decreased by 49.1%. Based on these results, we believe that CD4 is a receptor protein for JEVs. Furthermore, most viral receptors appear to be associated with lipid rafts, and colocalization studies demonstrate the presence of CD4 protein on lipid rafts. RT‒qPCR and WB results show that virus replication was suppressed in PK-15-CD4KD cells. The difference in viral titer between KD and WT PK-15 cells peaked at 24 h, and the viral titer in WT PK-15 cells was 5.6 × 106, whereas in PK-15-CD4KD cells, it was only 1.8 × 106, a 64% drop, demonstrating that CD4 deficiency has an effect on the process of viral replication. These findings suggest that JEV enters porcine kidney cells via lipid raft-colocalized CD4, and the proliferation process is positively correlated with CD4.