An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here, we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped vesicular stomatitis virus (VSV) neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase- and secreted embryonic alkaline phosphatase (SEAP)-expressing pseudotyped virus neutralizations, followed by green fluorescent protein (GFP)-expressing pseudotyped virus neutralization, and then the ELISAs. IMPORTANCE The ongoing COVID-19 pandemic is caused by infection with severe acute respiratory syndrome virus 2 (SARS-CoV-2). Prior infection or vaccination can be detected by the presence of antibodies in the blood. Antibodies in the blood are also considered to be protective against future infections from the same virus. The "gold standard" assay for detecting protective antibodies against SARS-CoV-2 is neutralization of authentic SARS-CoV-2 virus. However, this assay can only be performed under highly restrictive biocontainment conditions. We therefore characterized six antibody-detecting assays for their correlation with authentic virus neutralization. The significance of our research is in outlining the advantages and disadvantages of the different assays and identifying the optimal surrogate assay for authentic virus neutralization. This will allow for more accurate assessments of protective immunity against SARS-CoV-2 following infection and vaccination.

Induction of protective immune responses against a lethal Zika virus challenge post-vaccination with a dual serotype of recombinant vesicular stomatitis virus carrying the genetically modified Zika virus E protein gene.

The development of a vaccine to prevent Zika virus (ZIKV) infection has been one of the priorities in infectious disease research in recent years. There have been numerous attempts to develop an effective vaccine against ZIKV. It is imperative to choose the safest and the most effective ZIKV vaccine from all candidate vaccines to control this infection globally. We have employed a dual serotype of prime-boost recombinant vesicular stomatitis virus (VSV) vaccine strategy, to develop a ZIKV vaccine candidate, using a type 1 IFN-receptor knock-out (Ifnar-/-) mouse model for challenge studies. Prime vaccination with an attenuated recombinant VSV Indiana serotype (rVSVInd) carrying a genetically modified ZIKV envelope (E) protein gene followed by boost vaccination with attenuated recombinant VSV New Jersey serotype (rVSVNJ) carrying the same E gene induced robust adaptive immune responses. In particular, rVSV carrying the ZIKV E gene with the honeybee melittin signal peptide (msp) at the N terminus and VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the C terminus of the E gene induced strong protective immune responses. This vaccine regimen induced highly potent neutralizing antibodies and T cell responses in the absence of an adjuvant and protected Ifnar-/- mice from a lethal dose of the ZIKV challenge.

Recombinant adenovirus expressing vesicular stomatitis virus G proteins induce both humoral and cell-mediated immune responses in mice and goats.

BACKGROUND: Vesicular stomatitis (VS) is an acute, highly contagious and economically important zoonotic disease caused by the vesicular stomatitis virus (VSV). There is a need for effective and safe stable recombinant vaccine for the control of the disease. The human type 5 replication-defective adenovirus expression vector is a good way to construct recombinant vaccines. RESULTS: Three recombinant adenoviruses (rAd) were successfully constructed that expressed the VSV Indiana serotype glycoprotein (VSV-IN-G), VSV New Jersey serotype glycoprotein (VSV-NJ-G), and the G fusion protein (both serotypes of G [VSV-IN-G-NJ-G]) with potentiality to induce protective immunity. G proteins were successfully expressed with good immunogenicity. The rAds could induce the production of VSV antibodies in mice, and VSV neutralizing antibodies in goats, respectively. The neutralizing antibody titers could reach 1:32 in mice and 1:64 in goats. The rAds induced strong lymphocyte proliferation in mice and goats, which was significantly higher compared to the negative control groups. CONCLUSIONS: The three rAds constructed in the study expressed VSV-G proteins and induced both humoral and cellular immune responses in mice and goats. These results lay the foundation for further studies on the use of rAds in vaccines expressing VSV-G.

Recombinant vesicular stomatitis virus glycoprotein carrying a foot-and-mouth disease virus epitope as a vaccine candidate.

Foot-and-mouth disease (FMD) is one of the most highly contagious animal diseases. In an effort to overcome the drawbacks of the currently used inactivated foot-and-mouth disease virus vaccine, a novel recombinant protein carrying foot-and-mouth disease virus VP1 GH loop epitope linked to vesicular stomatitis virus glycoprotein was expressed in a baculovirus system. Its antigenicity was confirmed with ELISA using monoclonal antibody against foot-and-mouth disease virus. Twice immunizations one month apart in field pigs resulted in a significant antibody increase compared to the glutathione S-transferase carrier containing the same epitope and the commercial vaccine. To my knowledge, this is the first report that the recombinant protein vaccine was superior to the current vaccine. Although further studies are required to examine their immunogenicity in a large number of animals, this study sheds light on the development of a novel recombinant protein vaccine that could be easily produced in a general laboratory as an alternative to the current FMD vaccine, which requires a biosafety level 3 containment facility for vaccine production.

Spatiotemporal regulation of type I interferon expression determines the antiviral polarization of CD4+ T cells.

Differentiation of CD4+ T cells into either follicular helper T (TFH) or type 1 helper T (TH1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into TH1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4+ T cell differentiation and might instruct vaccine design strategies.

Epidemiological situation of vesicular stomatitis virus infection in cattle in the state of Paraíba, semiarid region of Brazil.

The aim of this survey was to estimate the apparent herd-level and animal-level prevalences, as well as to identify risk factors and spatial clustering of vesicular stomatitis virus (VSV) positive herds in the state of Paraíba, semiarid of Brazil. The state was divided into three sampling strata: Sertão, Borborema and Zona da Mata/Agreste. For each sampling stratum, herd-level and animal-level prevalences were estimated by a two-stage sampling survey. First, a pre-established number of herds (primary sampling units) were randomly selected; second, within each herd, a pre-established number of cows aged ≥ 24 months were systematically selected (secondary sampling units). In total, 2279 animals were sampled from 468 herds. Serum samples were submitted to virus neutralization (VN) test for detection of antibodies to VSV using three viral strains: VSIV-3 2013SaoBento/Paraiba E, strain Indiana (VSIV-1) and VSNJV. A herd was considered positive for VSV if it included at least one positive animal in herds of up to 10 females, two positive animals in herds of 11-99 females, and three positive in herds with more than 99 females. The spatial clustering was assessed using the Cuzick-Edwards' k-nearest neighbor method and spatial scan statistics. The apparent herd-level prevalence in the state of Paraíba was 38.5% (95% CI = 35.5-41.6%), 80.6% (95% CI = 73.6-86.2%) in the region of Sertão, 7.0% (95% CI = 3.9-12.2%) in Borborema, and 2.6% (95% CI = 1.0-6.7%) in Agreste/Zona da Mata. The apparent animal-level prevalence was 26.2% (95% CI = 20.6-32.8%) in the state of Paraíba, 48.2% (95% CI = 41.5-54.9%) in Sertão, 6.3% (95% CI = 2.7-14%) in Borborema, and 3.2% 1.9% (95% CI = 0.4-8.4%) in Agreste/Zona da Mata. The risk factors identified were as follows: mixed production (milk/beef) (OR = 4.54), herd size > 23 animals (OR = 3.57), presence of cervids (OR = 15.24), rental of pastures (OR = 3.02), sharing of water sources (OR = 2.57) and presence of horses (OR = 1.69). Two significant clusters of positive herds were detected: the primary cluster covered the Sertão region and the secondary cluster covered part of the Sertão and Borborema regions. Our results suggest high VSV circulation in the bovine population of the state of Paraíba, semiarid region of Brazil, mainly in the Sertão mesoregion, and based on risk factor analysis it was possible to identify important associations that deserve more investigation on causal factors.

Virus recognition by Toll-7 activates antiviral autophagy in Drosophila.

Innate immunity is highly conserved and relies on pattern recognition receptors (PRRs) such as Toll-like receptors (identified through their homology to Drosophila Toll) for pathogen recognition. Although Drosophila Toll is vital for immune recognition and defense, roles for the other eight Drosophila Tolls in immunity have remained elusive. Here we have shown that Toll-7 is a PRR both in vitro and in adult flies; loss of Toll-7 led to increased vesicular stomatitis virus (VSV) replication and mortality. Toll-7, along with additional uncharacterized Drosophila Tolls, was transcriptionally induced by VSV infection. Furthermore, Toll-7 interacted with VSV at the plasma membrane and induced antiviral autophagy independently of the canonical Toll signaling pathway. These data uncover an evolutionarily conserved role for a second Drosophila Toll receptor that links viral recognition to autophagy and defense and suggest that other Drosophila Tolls may restrict specific as yet untested pathogens, perhaps via noncanonical signaling pathways.

Apparent disappearance of Vesicular Stomatitis New Jersey Virus from Ossabaw Island, Georgia.

Ossabaw Island, Georgia, is the only reported endemic focus of Vesicular Stomatitis New Jersey Virus (VSNJV) in the United States. Based on recent negative serologic results of white-tailed deer and feral swine and the failure to isolate VSNJV from Lutzomyia shannoni, it appears that VSNJV is no longer present at this site. This apparent disappearance does not appear to be related to a change in L. shannoni habitat, specifically to the density of tree holes in the maritime and mixed hardwood forests. We believe that the disappearance of VSNJV from Ossabaw Island is directly related to a reduction in the feral swine population and a subsequent increase in the utilization of white-tailed deer by the known vector, L. shannoni.

Lesion development and replication kinetics during early infection in cattle inoculated with Vesicular stomatitis New Jersey virus via scarification and black fly (Simulium vittatum) bite.

Vesicular stomatitis viruses are the causative agents of vesicular stomatitis, an economically important contagious disease of livestock that occurs in North, Central, and South America. Little is known regarding the early stages of infection in natural hosts. Twelve adult Holstein steers were inoculated with Vesicular stomatitis New Jersey virus (VSNJV) on the coronary bands (CB) of the feet via scarification (SC) or by VSNJV-infected black fly (Simulium vittatum) bite (FB). Three additional animals were inoculated on the neck skin using FB. Clinical disease and lesion development were assessed daily, and animals were euthanatized from 12 hours post inoculation (HPI) through 120 HPI. The animals inoculated in the neck failed to develop any clinical signs or gross lesions, and VSNJV was detected neither by in situ hybridization (ISH) nor by immunohistochemistry (IHC). Lesions on the CB were more severe in the animals infected by FB than by SC. In both groups, peak VSNJV replication occurred between 24 and 48 HPI in keratinocytes of the CB, as evidenced by ISH and IHC. There was evidence of viral replication limited to the first 24 HPI in the local draining lymph nodes, as seen through ISH. Successful infection via FB required logarithmically less virus than with the SC technique, suggesting that components in black fly saliva may facilitate VSNJV transmission and infection in cattle. The lack of lesion development in the neck with the same method of inoculation used in the CB suggests that specific characteristics of the CB epithelium may facilitate VSNJV infection.

Estimation of the time of seroconversion to the New Jersey serotype of vesicular stomatitis virus in sentinel cattle of dairy herds located at high and low elevations in southern Mexico.

OBJECTIVE: To estimate the time of seroconversion to the New Jersey serotype of vesicular stomatitis virus (VSNJV) in sentinel cattle of dairy herds located at high and low elevations in southern Mexico and to determine the factors associated with an increase in VSNJV transmission. ANIMALS: 471 dairy cattle in 4 free-ranging dairy herds located at high and low elevations in southern Mexico. PROCEDURES: Serum samples from all cattle were screened by use of serum neutralization (SN) tests for antibodies against VSNJV. Cattle with SN titers<1:20 were designated as sentinel cattle and tested every 10 weeks for seroconversion to VSNJV (SN titer≥1:80). A Cox proportional hazards regression model was used to compare the hazard for seroconversion between sentinel cattle located at high and low elevations and kept under similar management and nutritional conditions. RESULTS: Hazard of VSNJV seroconversion was significantly higher for sentinel cattle located at high elevations, compared with the hazard for sentinel cattle located at low elevations. Dairy cattle located at high elevations seroconverted to VSNJV more frequently during the rainy season and the beginning of the dry season. CONCLUSIONS AND CLINICAL RELEVANCE: Seroconversion to VSNJV was more likely in dairy cattle in southern Mexico located at high elevations than in dairy cattle located at low elevations. These findings should contribute to understanding the dynamics of VSNJV infection in endemic areas and should be useful in the design of effective preventive and control strategies to decrease the impact of future VSV incursions.

Subcapsular sinus macrophages prevent CNS invasion on peripheral infection with a neurotropic virus.

Lymph nodes (LNs) capture microorganisms that breach the body's external barriers and enter draining lymphatics, limiting the systemic spread of pathogens. Recent work has shown that CD11b(+)CD169(+) macrophages, which populate the subcapsular sinus (SCS) of LNs, are critical for the clearance of viruses from the lymph and for initiating antiviral humoral immune responses. Here we show, using vesicular stomatitis virus (VSV), a relative of rabies virus transmitted by insect bites, that SCS macrophages perform a third vital function: they prevent lymph-borne neurotropic viruses from infecting the central nervous system (CNS). On local depletion of LN macrophages, about 60% of mice developed ascending paralysis and died 7-10 days after subcutaneous infection with a small dose of VSV, whereas macrophage-sufficient animals remained asymptomatic and cleared the virus. VSV gained access to the nervous system through peripheral nerves in macrophage-depleted LNs. In contrast, within macrophage-sufficient LNs VSV replicated preferentially in SCS macrophages but not in adjacent nerves. Removal of SCS macrophages did not compromise adaptive immune responses against VSV, but decreased type I interferon (IFN-I) production within infected LNs. VSV-infected macrophages recruited IFN-I-producing plasmacytoid dendritic cells to the SCS and in addition were a major source of IFN-I themselves. Experiments in bone marrow chimaeric mice revealed that IFN-I must act on both haematopoietic and stromal compartments, including the intranodal nerves, to prevent lethal infection with VSV. These results identify SCS macrophages as crucial gatekeepers to the CNS that prevent fatal viral invasion of the nervous system on peripheral infection.

Experimental infection of Didelphis marsupialis with vesicular stomatitis New Jersey virus.

Although vesicular stomatitis has been present for many years in the Americas, many aspects of its natural history remain undefined. In this study, we challenged five adult Virginia opossums (Didelphis marsupialis) with vesicular stomatitis New Jersey serotype virus (VSNJV). Opossums had no detectable antibodies against VSNJV prior to being inoculated with 10(6.5) median tissue culture infective doses (TCID(50)) of VSNJV by two routes; intraepithelial/subepithelial (IE/SE) inoculation and scarification in the muzzle (SM). Clinical response was monitored daily and animals were tested for viral shedding. All infected animals developed vesicles and ulcers on the tongue and inflammation of the nasal alar folds. Virus was isolated from esophagus-pharynx, nasal, and from ocular swabs and lesions samples. The failure to detect viremia in these animals indicates that a source other than blood may be required for transmission to insect vectors. Our results suggest that D. marsupialis could play a role in the maintenance of VSNJV outside of domestic animal populations and could provide a model to study vesicular stomatitis virus pathogenesis.

Development of a blocking ELISA using a recombinant glycoprotein for the detection of antibodies to vesicular stomatitis New Jersey virus.

A recombinant glycoprotein (R-GP) of vesicular stomatitis New Jersey virus (VSV-NJ) was expressed in insect cells by a baculovirus system. Its utility as a diagnostic antigen in a blocking ELISA was investigated as an alternative to the current native GP extracted from VSV-NJ. With the cut-off value of 73% inhibition, the R-GP ELISA exhibited 99.1% specificity for naive sera from cattle and horses. It did not cross-react with VSV-Indiana (VSV-IN) positive sera and differentiated from foot-and-mouth disease and swine vesicular disease. Taken together, this is the first report that the R-GP has a potential to be used as a diagnostic antigen in place of the native GP for the detection of antibodies to VSV-NJ in cattle and horses.

Experimental transmission of vesicular stomatitis New Jersey virus from Simulium vittatum to cattle: clinical outcome is influenced by site of insect feeding.

Vesicular stomatitis New Jersey virus (VSNJV) is an insect-transmitted Rhabdovirus causing vesicular disease in domestic livestock including cattle, horses, and pigs. Natural transmission during epidemics remains poorly understood, particularly in cattle, one of the most affected species during outbreaks. This study reports the first successful transmission of VSNJV to cattle by insect bite resulting in clinical disease. When infected black flies (Simulium vittatum Zetterstedt) fed at sites where VS lesions are usually observed (mouth, nostrils, and foot coronary band), infection occurred, characterized by local viral replication, vesicular lesions, and high neutralizing antibody titers (> 1: 256). Viral RNA was detected up to 9 d postinfection in tissues collected during necropsy from lesion sites and lymph nodes draining those sites. Interestingly, when flies were allowed to feed on flank or neck skin, viral replication was poor, lesions were not observed, and low levels of neutralizing antibodies (range, 1:8-1:32) developed. Viremia was never observed in any of the animals and infectious virus was not recovered from tissues on necropsies performed between 8 and 27 d postinfection. Demonstration that VSNJV transmission to cattle by infected black flies can result in clinical disease contributes to a better understanding of the epidemiology and potential prevention and control methods for this important disease.