Baculovirus expression and purification of virion core and envelope proteins of goatpox virus to evaluate their diagnostic potential.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

Heterologous Prime-Boost Immunization Strategies Using Varicella-Zoster Virus gE mRNA Vaccine and Adjuvanted Protein Subunit Vaccine Triggered Superior Cell Immune Response in Middle-Aged Mice.

Purpose: Heterologous immunization using different vaccine platforms has been demonstrated as an efficient strategy to enhance antigen-specific immune responses. In this study, we performed a head-to-head comparison of both humoral and cellular immune response induced by different prime-boost immunization regimens of mRNA vaccine and adjuvanted protein subunit vaccine against varicella-zoster virus (VZV) in middle-aged mice, aiming to get a better understanding of the influence of vaccination schedule on immune response. Methods: VZV glycoprotein (gE) mRNA was synthesized and encapsulated into SM-102-based lipid nanoparticles (LNPs). VZV-primed middle-aged C57BL/6 mice were then subjected to homologous and heterologous prime-boost immunization strategies using VZV gE mRNA vaccine (RNA-gE) and protein subunit vaccine (PS-gE). The antigen-specific antibodies were evaluated using enzyme-linked immunosorbent assay (ELISA) analysis. Additionally, cell-mediated immunity (CMI) was detected using ELISPOT assay and flow cytometry. Besides, in vivo safety profiles were also evaluated and compared. Results: The mRNA-loaded lipid nanoparticles had a hydrodynamic diameter of approximately 130 nm and a polydispersity index of 0.156. Total IgG antibody levels exhibited no significant differences among different immunization strategies. However, mice received 2×RNA-gE or RNA-gE>PS-gE showed a lower IgG1/IgG2c ratio than those received 2×PS-gE and PS-gE> RNA-gE. The CMI response induced by 2×RNA-gE or RNA-gE>PS-gE was significantly stronger than that induced by 2×PS-gE and PS-gE> RNA-gE. The safety evaluation indicated that both mRNA vaccine and protein vaccine induced a transient body weight loss in mice. Furthermore, the protein vaccine produced a notable inflammatory response at the injection sites, while the mRNA vaccine showed no observable inflammation. Conclusion: The heterologous prime-boost strategy has demonstrated that an mRNA-primed immunization regimen can induce a better cell-mediated immune response than a protein subunit-primed regimen in middle-aged mice. These findings provide valuable insights into the design and optimization of VZV vaccines with the potentials to broaden varicella vaccination strategies in the future.

Structural basis for VLDLR recognition by eastern equine encephalitis virus.

Eastern equine encephalitis virus (EEEV) is the most virulent alphavirus that infects humans, and many survivors develop neurological sequelae, including paralysis and intellectual disability. Alphavirus spike proteins comprise trimers of heterodimers of glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) as cellular receptors for EEEV and a distantly related alphavirus, Semliki Forest virus (SFV). Here, we use single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain and found that EEEV and SFV interact with the same cellular receptor through divergent binding modes. Our studies suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.

A broadly protective antibody targeting glycoprotein Gn inhibits severe fever with thrombocytopenia syndrome virus infection.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes severe viral hemorrhagic fever and thrombocytopenia syndrome with a fatality rate of up to 30%. No licensed vaccines or therapeutics are currently available for humans. Here, we develop seven monoclonal antibodies (mAbs) against SFTSV surface glycoprotein Gn. Mechanistic studies show that three neutralizing mAbs (S2A5, S1G3, and S1H7) block multiple steps during SFTSV infection, including viral attachment and membrane fusion, whereas another neutralizing mAb (B1G11) primarily inhibits the viral attachment step. Epitope binning and X-ray crystallographic analyses reveal four distinct antigenic sites on Gn, three of which have not previously been reported, corresponding to domain I, domain II, and spanning domain I and domain II. One of the most potent neutralizing mAbs, S2A5, binds to a conserved epitope on Gn domain I and broadly neutralizes infection of six SFTSV strains corresponding to genotypes A to F. A single dose treatment of S2A5 affords both pre- and post-exposure protection of mice against lethal SFTSV challenge without apparent weight loss. Our results support the importance of glycoprotein Gn for eliciting a robust humoral response and pave a path for developing prophylactic and therapeutic antibodies against SFTSV infection.

Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane.

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

Fully human single-domain antibody targeting a highly conserved cryptic epitope on the Nipah virus G protein.

Nipah virus infection, one of the top priority diseases recognized by the World Health Organization, underscores the urgent need to develop effective countermeasures against potential epidemics and pandemics. Here, we identify a fully human single-domain antibody that targets a highly conserved cryptic epitope situated at the dimeric interface of the Nipah virus G protein (receptor binding protein, RBP), as elucidated through structures by high-resolution cryo-electron microscopy (cryo-EM). This unique binding mode disrupts the tetramerization of the G protein, consequently obstructing the activation of the F protein and inhibiting viral membrane fusion. Furthermore, our investigations reveal that this compact antibody displays enhanced permeability across the blood-brain barrier (BBB) and demonstrates superior efficacy in eliminating pseudovirus within the brain in a murine model of Nipah virus infection, particularly compared to the well-characterized antibody m102.4 in an IgG1 format. Consequently, this single-domain antibody holds promise as a therapeutic candidate to prevent Nipah virus infections and has potential implications for vaccine development.

A nucleoside-modified mRNA vaccine forming rabies virus-like particle elicits strong cellular and humoral immune responses against rabies virus infection in mice.

Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.

Expression and Purification of RVFV Glycoprotein C for Structural Studies Using Baculovirus Expression System.

The Rift Valley fever virus (RVFV), transmitted through mosquito bites, leads to severe illness in humans and livestock throughout Africa and the Arabian Peninsula, causing significant morbidity and mortality. As of now, there are no verified and efficacious drugs or licensed vaccines accessible for the prevention or treatment of RVFV infections in both humans and livestock. The mature RVFV virion has two envelope proteins on its surface: glycoprotein N (GN) and glycoprotein C (GC). These proteins play a significant role in facilitating the virus's entry into the host cell, making them prominent targets for entry mechanism research as well as targets for drugs and vaccine development. The initial stage in obtaining atomic-resolution structural and mechanistic information on viral entry as well as developing biochemical and biophysical research tools involves recombinant protein production. In this chapter, we describe a simplified and scalable protocol facilitating the generation of high-quality, high-titer baculovirus virus for expression and purification of RVFV GC, utilizing the baculovirus-mediated expression system in insect cells.

Characterization of human tibrovirus envelope glycoproteins.

Tibroviruses are novel rhabdoviruses detected in humans, cattle, and arthropods. Four tibroviruses are known to infect humans: Bas-Congo virus (BASV), Ekpoma virus 1 (EKV-1), Ekpoma virus 2, and Mundri virus. However, since none of them has been isolated, their biological properties are largely unknown. We aimed to characterize the human tibrovirus glycoprotein (G), which likely plays a pivotal role in viral tropism and pathogenicity. Human tibrovirus Gs were found to share some primary structures and display 14 conserved cysteine residues, although their overall amino acid homology was low (29%-48%). Multiple potential glycosylation sites were found on the G molecules, and endoglycosidase H- and peptide-N-glycosidase F-sensitive glycosylation was confirmed. AlphaFold-predicted three-dimensional (3D) structures of human tibrovirus Gs were overall similar. Membrane fusion mediated by these tibrovirus Gs was induced by acidic pH. The low pH-induced conformational change that triggers fusion was reversible. Virus-like particles (VLPs) were produced by transient expression of Gs in cultured cells and used to produce mouse antisera. Using vesicular stomatitis Indiana virus pseudotyped with Gs, we found that the antisera to the respective tibrovirus VLPs showed limited cross-neutralizing activity. It was also found that human C-type lectins and T-cell immunoglobulin mucin 1 acted as attachment factors for G-mediated entry into cells. Interestingly, BASV-G showed the highest ability to utilize these molecules. The viruses infected a wide range of cell lines with preferential tropism for human-derived cells whereas the preference of EKV-1 was unique compared with the other human tibroviruses. These findings provide fundamental information to understand the biological properties of the human tibroviruses. IMPORTANCE: Human tibroviruses are poorly characterized emerging rhabdoviruses associated with either asymptomatic infection or severe disease with a case fatality rate as high as 60% in humans. However, the extent and burden of human infection as well as factors behind differences in infection outcomes are largely unknown. In this study, we characterized human tibrovirus glycoproteins, which play a key role in virus-host interactions, mainly focusing on their structural and antigenic differences and cellular tropism. Our results provide critical information for understanding the biological properties of these novel viruses and for developing appropriate preparedness interventions such as diagnostic tools, vaccines, and effective therapies.

The structure of immature tick-borne encephalitis virus supports the collapse model of flavivirus maturation.

We present structures of three immature tick-borne encephalitis virus (TBEV) isolates. Our atomic models of the major viral components, the E and prM proteins, indicate that the pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in a metastable conformation. Destabilization of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM topology and domain assignment in TBEV is similar to the mosquito-borne Binjari virus, but is in contrast to other immature flavivirus models. These results support that prM cleavage, the collapse of E protein ectodomains onto the virion surface, the large movement of the membrane domains of both E and M, and the release of the pr fragment from the particle render the virus mature and infectious. Our work favors the collapse model of flavivirus maturation warranting further studies of immature flaviviruses to determine the sequence of events and mechanistic details driving flavivirus maturation.

Antibody reactions of horses against various domains of the EHV-1 receptor-binding protein gD1.

Equid alphaherpesviruses 1 (EHV-1) and 4 (EHV-4) are closely related and both endemic in horses worldwide. Both viruses replicate in the upper respiratory tract, but EHV-1 may additionally lead to abortion and equine herpesvirus myeloencephalopathy (EHM). We focused on antibody responses in horses against the receptor-binding glycoprotein D of EHV-1 (gD1), which shares a 77% amino acid identity with its counterpart in EHV-4 (gD4). Both antigens give rise to cross-reacting antibodies, including neutralizing antibodies. However, immunity against EHV-4 is not considered protective against EHM. While a diagnostic ELISA to discriminate between EHV-1 and EHV-4 infections is available based on type-specific fragments of glycoprotein G (gG1 and gG4, respectively), the type-specific antibody reaction against gD1 has not yet been sufficiently addressed. Starting from the N-terminus of gD1, we developed luciferase immunoprecipitation system (LIPS) assays, using gD1-fragments of increasing size as antigens, i.e. gD1_83 (comprising the first 83 amino acids), gD1_160, gD1_180, and gD1_402 (the full-length molecule). These assays were then used to analyse panels of horse sera from Switzerland (n = 60) and Iceland (n = 50), the latter of which is considered EHV-1 free. We detected only one true negative horse serum from Iceland, whereas all other sera in both panels were seropositive for both gG4 (ELISA) and gD1 (LIPS against gD1_402). In contrast, seropositivity against gG1 was rather rare (35% Swiss sera; 14% Icelandic sera). Therefore, a high percentage of antibodies against gD1 could be attributed to cross-reaction and due to EHV-4 infections. In contrast, the gD1_83 fragment was able to identify sera with type-specific antibodies against gD1. Interestingly, those sera stemmed almost exclusively from vaccinated horses. Although it is uncertain that the N-terminal epitopes of gD1 addressed in this communication are linked to better protection, we suggest that in future vaccine developments, type-common antigens should be avoided, while a broad range of type-specific antigens should be favored.

Discovery of hepatitis B virus subviral particle biogenesis inhibitors from a bioactive compound library.

High levels of hepatitis B virus (HBV) surface antigen (HBsAg) in the blood of chronic HBV carriers are considered to drive the exhaustion of antigen-specific T and B lymphocytes and thus responsible for the persistence of infection. Accordingly, therapeutic elimination of HBsAg may facilitate the activation of adaptive antiviral immune responses against HBV and achieve a functional cure of chronic hepatitis B. We discovered recently that an amphipathic alpha helix spanning W156 to R169 of HBV small envelope (S) protein plays an essential role in the morphogenesis of subviral particles (SVPs) and metabolism of S protein. We thus hypothesized that pharmacological disruption of SVP morphogenesis may induce intracellular degradation of S protein and reduce HBsAg secretion. To identify inhibitors of SVP biogenesis, we screened 4417 bioactive compounds with a HepG2-derived cell line expressing HBV S protein and efficiently secreting small spherical SVPs. The screen identified 24 compounds that reduced intracellular SVPs and secreted HBsAg in a concentration-dependent manner. However, 18 of those compounds inhibited the secretion of HBsAg and HBeAg in HBV replicon transfected HepG2 cells at similar efficiency, suggesting each of those compounds may disrupt a common cellular function required for the synthesis and/or secretion of these viral proteins. Interestingly, lycorine more efficiently inhibited the secretion of HBsAg in HepG2 cells transfected with HBV replicons, HepG2.2.15 cells and HBV infected - HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). The structure activity relationship and antiviral mechanism of lycorine against HBV have been determined.

Immunogenicity study of a Novel DNA-Based HCV vaccine candidate.

In this study, we aimed to evaluate the immunogenic profile of a chimeric DNA-based hepatitis C virus (HCV) vaccine candidate encoding the full-length viral core-E1-E2 (HCV-CE) fragment. The vaccine candidate was designed to uniformly express the HCV genotype 4 core-E1-E2 protein. The recombinant HCV-CE protein was bacterially expressed in C41 (DE3) cells, and then BALB/c mice were immunized with different combinations of DNA/DNA or DNA/protein prime/boost immunizations. The proper construction of our vaccine candidate was confirmed by specific amplification of the encoded fragments and basic local alignment search tool (BLAST) results of the nucleotide sequence, which revealed a high degree of similarity with several HCV serotypes/genotypes. The platform for bacterial expression was optimized to maximize the yield of the purified recombinant HCV-CE protein. The recombinant protein showed high specific antigenicity against the sera of HCV-infected patients according to the ELISA and western blot results. The predicted B- and T-cell epitopes showed high antigenic and interferon-γ (IFN-γ) induction potential, in addition to cross-genotype conservation and population coverage. The mice antisera further demonstrated a remarkable ability to capture 100% of the native viral antigens circulating in the sera of HCV patients, with no cross-reactivity detected in control sera. In conclusion, the proposed HCV vaccination strategy demonstrated promising potential regarding its safety, immunogenicity, and population coverage.

Expression of dengue capsid-like particles in silkworm and display of envelope domain III of dengue virus serotype 2.

Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.

Viral Envelope Evolution in Simian-HIV-Infected Neonate and Adult-Dam Pairs of Rhesus Macaques.

We recently demonstrated that Simian-HIV (SHIV)-infected neonate rhesus macaques (RMs) generated heterologous HIV-1 neutralizing antibodies (NAbs) with broadly-NAb (bNAb) characteristics at a higher frequency compared with their corresponding dam. Here, we characterized genetic diversity in Env sequences from four neonate or adult/dam RM pairs: in two pairs, neonate and dam RMs made heterologous HIV-1 NAbs; in one pair, neither the neonate nor the dam made heterologous HIV-1 NAbs; and in another pair, only the neonate made heterologous HIV-1 NAbs. Phylogenetic and sequence diversity analyses of longitudinal Envs revealed that a higher genetic diversity, within the host and away from the infecting SHIV strain, was correlated with heterologous HIV-1 NAb development. We identified 22 Env variable sites, of which 9 were associated with heterologous HIV-1 NAb development; 3/9 sites had mutations previously linked to HIV-1 Env bNAb development. These data suggested that viral diversity drives heterologous HIV-1 NAb development, and the faster accumulation of viral diversity in neonate RMs may be a potential mechanism underlying bNAb induction in pediatric populations. Moreover, these data may inform candidate Env immunogens to guide precursor B cells to bNAb status via vaccination by the Env-based selection of bNAb lineage members with the appropriate mutations associated with neutralization breadth.

Antibodies targeting the glycan cap of Ebola virus glycoprotein are potent inducers of the complement.

Antibodies to Ebola virus glycoprotein (EBOV GP) represent an important correlate of the vaccine efficiency and infection survival. Both neutralization and some of the Fc-mediated effects are known to contribute the protection conferred by antibodies of various epitope specificities. At the same time, the role of the complement system remains unclear. Here, we compare complement activation by two groups of representative monoclonal antibodies (mAbs) interacting with the glycan cap (GC) or the membrane-proximal external region (MPER) of GP. Binding of GC-specific mAbs to GP induces complement-dependent cytotoxicity (CDC) in the GP-expressing cell line via C3 deposition on GP in contrast to MPER-specific mAbs. In the mouse model of EBOV infection, depletion of the complement system leads to an impairment of protection exerted by one of the GC-specific, but not MPER-specific mAbs. Our data suggest that activation of the complement system represents an important mechanism of antiviral protection by GC antibodies.

Pseudorabies virus tegument protein US2 antagonizes antiviral innate immunity by targeting cGAS-STING signaling pathway.

Background: The cGAS-STING axis-mediated type I interferon pathway is a crucial strategy for host defense against DNA virus infection. Numerous evasion strategies developed by the pseudorabies virus (PRV) counteract host antiviral immunity. To what extent PRV-encoded proteins evade the cGAS-STING signaling pathway is unknown. Methods: Using US2 stably expressing cell lines and US2-deficient PRV model, we revealed that the PRV tegument protein US2 reduces STING protein stability and downregulates STING-mediated antiviral signaling. Results: To promote K48-linked ubiquitination and STING degradation, US2 interacts with the LBD structural domain of STING and recruits the E3 ligase TRIM21. TRIM21 deficiency consistently strengthens the host antiviral immune response brought on by PRV infection. Additionally, US2-deficient PRV is less harmful in mice. Conclusions: Our study implies that PRV US2 inhibits IFN signaling by a new mechanism that selectively targets STING while successfully evading the host antiviral response. As a result, the present study reveals a novel strategy by which PRV evades host defense and offers explanations for why the Bartha-K61 classical vaccine strain failed to offer effective defense against PRV variant strains in China, indicating that US2 may be a key target for developing gene-deficient PRV vaccines.

Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage.

A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed.

Recombination of variable and host range regions of glycoprotein gp85 in different avian leukosis virus subgroup K isolates.

Given the high prevalence of avian leukosis virus subgroup K (ALV-K) in chickens in China, the positive rate of ALV-K in local chickens in Henan province was investigated, and the genetic region encoding the glycoprotein gp85 of isolates from positive chickens was analyzed. The positive rate of ALV-K in local chickens in Henan was found to be 87.2% (41/47). Phylogenetic analysis of gp85 sequences revealed six clusters that differed in their host range regions (hr1 and hr2) and variable regions (vr1, vr2, and vr3). Evidence of recombination of hr1, hr2, vr1, vr2, and vr3 was observed between the different clusters. The isolate HN23LS02 appears to have obtained its hr1 and hr2 regions from separate lineages via recombination but without having a significant affect on the replication capacity of the virus.

Immunological characteristics of a recombinant alphaherpesvirus with an envelope-embedded Cap protein of circovirus.

Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.