A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike.

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here, we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We apply this platform to produce libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ∼7,000 distinct amino acid mutations in the context of up to ∼135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor-binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ∼105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants.

Virus-like particle (VLP) and live virus assays were used to investigate neutralizing immunity against Delta and Omicron SARS-CoV-2 variants in 259 samples from 128 vaccinated individuals. Following Delta breakthrough infection, titers against WT rose 57-fold and 3.1-fold compared with uninfected boosted and unboosted individuals, respectively, versus only a 5.8-fold increase and 3.1-fold decrease for Omicron breakthrough infection. Among immunocompetent, unboosted patients, Delta breakthrough infections induced 10.8-fold higher titers against WT compared with Omicron (p = 0.037). Decreased antibody responses in Omicron breakthrough infections relative to Delta were potentially related to a higher proportion of asymptomatic or mild breakthrough infections (55.0% versus 28.6%, respectively), which exhibited 12.3-fold lower titers against WT compared with moderate to severe infections (p = 0.020). Following either Delta or Omicron breakthrough infection, limited variant-specific cross-neutralizing immunity was observed. These results suggest that Omicron breakthrough infections are less immunogenic than Delta, thus providing reduced protection against reinfection or infection from future variants.

Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant.

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.

A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic. Dynamic tracking of variant frequencies revealed a recurrent pattern of G614 increase at multiple geographic levels: national, regional, and municipal. The shift occurred even in local epidemics where the original D614 form was well established prior to introduction of the G614 variant. The consistency of this pattern was highly statistically significant, suggesting that the G614 variant may have a fitness advantage. We found that the G614 variant grows to a higher titer as pseudotyped virions. In infected individuals, G614 is associated with lower RT-PCR cycle thresholds, suggestive of higher upper respiratory tract viral loads, but not with increased disease severity. These findings illuminate changes important for a mechanistic understanding of the virus and support continuing surveillance of Spike mutations to aid with development of immunological interventions.

Human Adaptation of Ebola Virus during the West African Outbreak.

The 2013-2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages.

Lamin B1 curtails early human papillomavirus infection by safeguarding nuclear compartmentalization and autophagic capacity.

Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.

Structure-guided mutations in CDRs for enhancing the affinity of neutralizing SARS-CoV-2 nanobody.

The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for the development of next generation therapeutics. This study delves into the refinement and engineering of complementarity-determining regions (CDRs) through in silico affinity maturation followed by binding validation using isothermal titration calorimetry (ITC) and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Protein-protein docking analysis identified 32 single mutants of which nine mutants were selected for molecular dynamics (MD) simulations. Subsequently, biophysical ITC studies provided insights into binding affinity, and consistent with in silico findings, six mutations that demonstrated better binding affinity than native nanobody were further tested in vitro for neutralization activity against SARS-CoV-2 pseudovirus. Leu106Thr mutant was found to be most effective in virus-neutralization with IC50 values of ∼0.03 µM, as compared to the native nanobody (IC50 ∼0.77 µM). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.

Cytoplasmic Tail Truncation Stabilizes S1-S2 Association and Enhances S Protein Incorporation into SARS-CoV-2 Pseudovirions.

Truncations of the cytoplasmic tail (CT) of entry proteins of enveloped viruses dramatically increase the infectivity of pseudoviruses (PVs) bearing these proteins. Several mechanisms have been proposed to explain this enhanced entry, including an increase in cell surface expression. However, alternative explanations have also been forwarded, and the underlying mechanisms for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein remain undetermined. Here, we show that the partial or complete deletion of the CT (residues 19 to 35) does not modify SARS-CoV-2 S protein expression on the cell surface when the S2 subunit is measured, whereas it is significantly increased when the S1 subunit is measured. We also show that the higher level of S1 in these CT-truncated S proteins reflects the decreased dissociation of the S1 subunit from the S2 subunit. In addition, we demonstrate that CT truncation further promotes S protein incorporation into PV particles, as indicated by biochemical analyses and cryo-electron microscopy. Thus, our data show that two distinct mechanisms contribute to the markedly increased infectivity of PVs carrying CT-truncated SARS-CoV-2 S proteins and help clarify the interpretation of the results of studies employing such PVs. IMPORTANCE Various forms of PVs have been used as tools to evaluate vaccine efficacy and study virus entry steps. When PV infectivity is inherently low, such as that of SARS-CoV-2, a CT-truncated version of the viral entry glycoprotein is widely used to enhance PV infectivity, but the mechanism underlying this enhanced PV infectivity has been unclear. Here, our study identified two mechanisms by which the CT truncation of the SARS-CoV-2 S protein dramatically increases PV infectivity: a reduction of S1 shedding and an increase in S protein incorporation into PV particles. An understanding of these mechanisms can clarify the mechanistic bases for the differences observed among various assays employing such PVs.

Unveiling the seroprevalence of human papillomavirus in Guangdong, China: Implications for vaccination strategies.

Seroepidemiological characteristics of human papillomavirus (HPV) in community residents reflect natural infection and can guide the reform of vaccination programs. A population-based serological survey was conducted in Guangdong Province. Serum anti-HPV IgG antibody levels were determined by an ELISA. Neutralizing antibodies against HPV6, 11, 16, and 18 were detected via a pseudovirus-based neutralization assay (PBNA). A total of 5122 serum samples were collected from community residents, including 1989 males and 3133 females, in three cities of Guangdong Province. The rate of HPV IgG antibody positivity in females was 5.39% (95% CI: 4.6-6.2), which was greater than that in males (2.36%; 95% CI: 1.7-3.1). HPV IgG antibodies were more frequently detected in females aged 51-60 years (11.30%; 95% CI: 7.6-16.0), whereas in males, the detection increased with age and reached 4.94% (95% CI: 2.8-6.9) in the group aged ≥71 years. The seropositivity of neutralizing antibodies against HPV6 and 11 was greater than that against HPV16 and 18. The serum neutralizing antibody titers in individuals who received three doses of a vaccine were 7- to 12-fold greater than those in individuals who did not receive the vaccine. The neutralizing antibody titers slightly decreased within 40 months and ranged from 0.038 to 0.057 log ED50 per month. A moderate consistency between the HPV ELISA and PBNA results was observed (Kappa score = 0.49, r = 0.249, 0.635, 0.382, and 0.466 for HPV6, 11, 16, and 18, respectively). The HPV seropositivity rate among healthy residents of Guangdong Province was found to be low among children and adolescents and to increase with age. The serum neutralizing antibody titers were significantly greater in the vaccine group than that in the control group, and this difference persisted over time, which indicated promising protection against HPV infection.

Vaginal pH value can affect the susceptibility to human papillomavirus infection.

BACKGROUND: Cervical cancer is the fourth most common cancer among women, with persistent high-risk human papillomavirus (HPV) infection being responsible for its progression. In healthy, pre-menopausal women, the vaginal pH value is maintained at 3.8-4.5, but various factors can affect it. Previous studies have suggested the relationship between vaginal pH value and HPV infection. In this study, we aimed to explore the relationship between vaginal pH and susceptibility of HPV infection. METHODS: In our study, we retrospectively collected medical information from women who underwent leukorrhea examination at our hospital. We excluded women with infectious diseases or cancer, those who were pregnant or within 6 months post-delivery, and those without HPV test results within 6 months. The association between percentage of HPV infection and vaginal pH value was analyzed. Furthermore, we prepared HPV pseudovirus (PsVs) by co-transfecting structure plasmids and report plasmids in 293FT cells. In vitro, we changed the pH value of cell culture medium to investigate its influence on HPV PsVs infection. In vivo, we changed mouse's vaginal pH value to investigate its influence on HPV PsVs infection. RESULTS: Our retrospective study included 3115 women aged 20-78, including 2531 women with HPV negative and 584 women with HPV positive. The percentages of both HPV infection and high-risk HPV infection were higher in women with a vaginal pH value ≥5.0 compared to those with a pH value < 5.0. In vitro, HPV PsVs infection rate was higher in cell culture medium of higher pH value, dominantly due to the influence of pH value on the stage of HPV PsVs adhering to cell surface. Neither of the cell surface HPV receptors Syndecan-1 nor integrin α6 was found to be changed obviously in different pH values. In vivo, more HPV PsVs were adhered to the mouse's vaginal epithelial cells with the increase of the vaginal pH value. CONCLUSIONS: Our study suggests a possible association between vaginal pH value and HPV infection. The pH value can influence the susceptibility of HPV PsVs infection by affecting the adhering of HPV PsVs to cells in vivo and in vitro. Additionally, the cell surface HPV receptors Syndecan-1 and Integrin α6 do not seem to be affected by pH value, and the specific mechanism needs to be further explored.

Pseudovirus-Based Systems for Screening Natural Antiviral Agents: A Comprehensive Review.

Since the outbreak of COVID-19, researchers have been working tirelessly to discover effective ways to combat coronavirus infection. The use of computational drug repurposing methods and molecular docking has been instrumental in identifying compounds that have the potential to disrupt the binding between the spike glycoprotein of SARS-CoV-2 and human ACE2 (hACE2). Moreover, the pseudovirus approach has emerged as a robust technique for investigating the mechanism of virus attachment to cellular receptors and for screening targeted small molecule drugs. Pseudoviruses are viral particles containing envelope proteins, which mediate the virus's entry with the same efficiency as that of live viruses but lacking pathogenic genes. Therefore, they represent a safe alternative to screen potential drugs inhibiting viral entry, especially for highly pathogenic enveloped viruses. In this review, we have compiled a list of antiviral plant extracts and natural products that have been extensively studied against enveloped emerging and re-emerging viruses by pseudovirus technology. The review is organized into three parts: (1) construction of pseudoviruses based on different packaging systems and applications; (2) knowledge of emerging and re-emerging viruses; (3) natural products active against pseudovirus-mediated entry. One of the most crucial stages in the life cycle of a virus is its penetration into host cells. Therefore, the discovery of viral entry inhibitors represents a promising therapeutic option in fighting against emerging viruses.

Functional Characterisation of Surfactant Protein A as a Novel Prophylactic Means against Oncogenic HPV Infections.

Human papillomavirus (HPV) infection poses a significant health challenge, particularly in low- and middle-income countries (LMIC), where limited healthcare access and awareness hinder vaccine accessibility. To identify alternative HPV targeting interventions, we previously reported on surfactant protein A (SP-A) as a novel molecule capable of recognising HPV16 pseudovirions (HPV16-PsVs) and reducing infection in a murine cervicovaginal HPV challenge model. Building on these findings, our current study aimed to assess SP-A's suitability as a broad-spectrum HPV-targeting molecule and its impact on innate immune responses. We demonstrate SP-A's ability to agglutinate and opsonise multiple oncogenic HPV-PsVs types, enhancing their uptake and clearance by RAW264.7 murine macrophages and THP-1 human-derived immune cells. The SP-A opsonisation of HPV not only led to increased lysosomal accumulation in macrophages and HaCaT keratinocytes but also resulted in a decreased infection of HaCaT cells, which was further decreased when co-cultured with innate immune cells. An analysis of human innate immune cell cytokine profiles revealed a significant inflammatory response upon SP-A exposure, potentially contributing to the overall inhibition of HPV infection. These results highlight the multi-layered impact of SP-A on HPV, innate immune cells and keratinocytes and lay the basis for the development of alternative prophylactic interventions against diverse HPV types.

Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis.

Coronavirus disease represents a real threat to the global population, and understanding the biological features of the causative virus, that is, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is imperative for mitigating this threat. Analyses of proteins such as primary receptors and coreceptors (cofactors), which are involved in the entry of SARS-CoV-2 into host cells, will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates present in proximity to the attachment sites of SARS-CoV-2 spike proteins, using proximity labeling and proteomic analysis. The identified proteins represent key candidate factors that may be required for viral entry. We found SARS-CoV-2 host protein DPP4, cell adhesion protein Cadherin 17, and glycoprotein CD133 colocalized with cell membrane-bound SARS-CoV-2 spike proteins in Caco-2 cells and thus showed potential as candidate factors. Additionally, our analysis of the experimental infection of HEK293T cells with a SARS-CoV-2 pseudovirus indicated a 2-fold enhanced infectivity in the CD133-ACE2-coexpressing HEK293T cells compared to that in HEK293T cells expressing ACE-2 alone. The information and resources regarding these coreceptor labeling and analysis techniques could be utilized for the development of antiviral agents against SARS-CoV-2 and other emerging viruses.

SARS-CoV-2 Vaccine Immunogenicity in Patients with Gastrointestinal Cancer Receiving Systemic Anti-Cancer Therapy.

INTRODUCTION: Patients with gastrointestinal (GI) cancers have an increased risk of serious complications and death from SARS-CoV-2 infection. The immunogenicity of vaccines in patients with GI cancers receiving anti-cancer therapies is unclear. We conducted a prospective study to evaluate the prevalence of neutralizing antibodies in a cohort of GI cancer patients receiving chemotherapy following SARS-CoV-2 vaccination. MATERIALS AND METHODS: Between September 2020 and April 2021, patients with cancer undergoing chemotherapy were enrolled. At baseline (day 0), days 28, 56, and 84, we assessed serum antibodies to SARS-CoV-2 spike (anti-S) and anti-nucleocapsid (anti-NP) and concomitantly assessed virus neutralization using a pseudovirus neutralization assay. Patients received either the Pfizer/BioNTech BNT162b2, or the Oxford/AstraZeneca ChAdOx1 vaccine. RESULTS: All 152 patients enrolled had a prior diagnosis of cancer; colorectal (n = 80, 52.6%), oesophagogastric (n = 38, 25.0%), and hepato pancreatic biliary (n = 22, 12.5%). Nearly all were receiving systemic anti-cancer therapy (99.3%). Of the 51 patients who did not receive a vaccination prior to, or during the study, 5 patients had detectable anti-NP antibodies. Ninety-nine patients received at least one dose of vaccine prior to, or during the study. Within 19 days following the first dose of vaccine, 30.0% had anti-S detected in serum which increased to 70.2% at days 20-39. In the 19 days following a second dose, anti-S positivity was 84.2% (32/38). However, pseudovirus neutralization titers (pVNT80) decreased from days 20 to 39. CONCLUSION: Despite the immunosuppressive effects of chemotherapy, 2 doses of SARS-CoV-2 vaccines are able to elicit a protective immune response in patients' ongoing treatment for gastrointestinal cancers. Decreases in pseudoviral neutralization were observed after 20-39 days, re-affirming the current recommendation for vaccine booster doses. CLINICAL TRIAL REGISTRATION NUMBER: NCT04427280.

Whole genome CRISPR screening strategy to identify genes contributing to SARS-CoV-2 spike and VSV-G mediated entry.

Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole-genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using live viruses and cellular lethality for selection, these screens can identify an overwhelming number of genes without specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole-genome screen and counter-screen strategy based on a pseudoviral platform that allowed identification of genes specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and vesicular stomatitis virus glycoprotein (VSV-G) mediated entry. Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed the identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein packaged into the pseudovirus was used to bypass retro-transcription and integration by directly activating a floxed fluorescent protein reporter upon entry  reduced the number of gene hits and increase specificity for viral entry. Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and low-density lipoprotein receptors, respectively, and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry-associated genes. Validation of a few hits demonstrates that this approach distinguishes envelope-specific host factors from genes affecting reporter expression. Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication.

The neutralization effect of montelukast on SARS-CoV-2 is shown by multiscale in silico simulations and combined in vitro studies.

Small molecule inhibitors have previously been investigated in different studies as possible therapeutics in the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. We initially demonstrated the dual inhibition profile of montelukast through multiscale molecular modeling studies. Next, we characterized its effect on both targets by different in vitro experiments including the enzyme (main protease) inhibition-based assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T/hACE2+TMPRSS2, and virus neutralization assay using xCELLigence MP real-time cell analyzer. Our integrated in silico and in vitro results confirmed the dual potential effect of montelukast both on the main protease enzyme inhibition and virus entry into the host cell (spike/ACE2). The virus neutralization assay results showed that SARS-CoV-2 virus activity was delayed with montelukast for 20 h on the infected cells. The rapid use of new small molecules in the pandemic is very important today. Montelukast, whose pharmacokinetic and pharmacodynamic properties are very well characterized and has been widely used in the treatment of asthma since 1998, should urgently be completed in clinical phase studies and, if its effect is proved in clinical phase studies, it should be used against coronavirus disease 2019 (COVID-19).

2,4-Dihydroxycinnamic acid as spike ACE2 inhibitor and apigenin as RdRp inhibitor in Nimbamritadi Panchatiktam Kashayam against COVID-19: an in silico and in vitro approach.

Nimbamritadi Panchatiktam Kashayam (NPK) is an ayurvedic formulation composed of ingredients with potent anti-viral activities. We studied the interaction energy of 144 phytoconstituents present in NPK against spike receptor-binding domain (RBD) complexed with ACE2 protein (PDB ID: 6LZG) and RNA-dependent RNA polymerase protein (PDB ID: 7BTF) using Biovia Drug Discovery studio. The result indicated that 2,4-hydroxycinnamic acid exerts more significant binding affinities (28.43 kcal/mol) than Umifenovir (21.24 kcal/mol) against spike ACE2. Apigenin exhibited the highest binding affinities (54.63 kcal/mol) compared with Remdesivir (24.52 kcal/mol) against RdRp. An in vitro analysis showed a reduction in the number of lentiviral particles on transfected HEK293T-hACE2 cells as assessed by pseudovirus inhibition assay. At the same time, the tested compounds showed non-toxic up to 100 µg/ml in normal cells by MTT assay. The study highlights the plausible clinical utility of this traditional medicine against SARS CoV2.

Establishment of a neutralization assay for Nipah virus using a high-titer pseudovirus system.

OBJECTIVE: To construct a high-titer Nipah pseudovirus packaging system using the HIV lentivirus backbone vector and establish a safe neutralization assay for Nipah pseudovirus in biosafety level 2 facilities. METHODS: Nipah virus (NiV) fusion protein (F) and glycoprotein (G) recombinant expression plasmids, psPAX2, and pLenti CMV Puro LUC (w168-1) were transiently transfected into 293T cells for 72 h for the generation of a NiV pseudovirus. The neutralization ability of Nipah virus F and G protein antibodies was assessed using the pseudovirus. RESULTS: A NiV pseudovirus was constructed using 293T cells. The ideal mass ratio of plasmid psPAX2: w168-1: F: G for transfection was determined to be 4:4:1:1. The specificity of recombinant F and G protein expression was indicated by indirect immunofluorescence and western blotting. The pseudovirus particles showed obvious spikes under a transmission electron microscope. The NiV pseudovirus titer was 4.73 × 105 median tissue culture infective dose per mL, and the pseudovirus could be effectively neutralized by polyclonal antibodies specifically targeting the F and G proteins respectively. CONCLUSIONS: A NiV pseudovirus was successfully generated using HIV vector systems, and was used as a platform for a safe and reliable pseudovirus-based neutralizing assay that can be performed in biosafety level 2 facilities.

Pseudotyped Viruses for Lyssavirus.

Lyssaviruses, which belong to the family Rhabdoviridae, are enveloped and bullet-shaped ssRNA viruses with genetic diversity. All members of Lyssavirus genus are known to infect warm-blooded animals and cause the fatal disease rabies. The rabies virus (RABV) in lyssavirus is the major pathogen to cause fatal rabies. The pseudotyped RABV is constructed to study the biological functions of G protein and evaluation of anti-RABV products including vaccine-induced antisera, rabies immunoglobulins (RIG), neutralizing mAbs, and other antiviral inhibitors. In this chapter, we focus on RABV as a representative and describe the construction of RABV G protein bearing pseudotyped virus and its applications. Other non-RABV lyssaviruses are also included.

Pseudotyped Virus for Flaviviridae.

Members of Flaviviridae are enveloped single positive-stranded RNA viruses including hepacivirus, pestivirus, pegivirus, and mosquito-transmitted flavivirus, which are important pathogens of infectious diseases and pose serious threats to human health. Pseudotyped virus is an artificially constructed virus-like particle, which could infect host cells similar to a live virus but cannot produce infectious progeny virus. Therefore, pseudotyped virus has the advantages of a wide host range, high transfection efficiency, low biosafety risk, and accurate and objective quantification. It has been widely used in biological characteristics, drug screening, detection methods, and vaccine evaluation of Flaviviridae viruses like hepatitis C virus, Japanese encephalitis virus, dengue virus, and Zika virus.