Virus Neutralization Test Research Articles

PSLBI-30 SARS-CoV-2 Screening and Detection: Assessment of surrogate virus neutralization test (svNT) diagnostic appropriateness in multispecies SARS-CoV-2 virus screening and detection

Abstract The National Veterinary Services Laboratories (NVSL) have been testing animal samples since the 2020 SARS-CoV-2 outbreak. The current “gold standard” for serodetection of SARS-CoV-2 is the virus neutralization (VN) test. This method requires specialized training, more time to complete, and specialized facilities (BSL 3). Therefore, the SARS-CoV-2 VN test is not widely run outside of NVSL. To facilitate increased testing capacity during the outbreak, the surrogate virus neutralization test (sVNT) was selected mid-2020. This commercially available kit uses an ELISA format, giving a percent inhibition (%I) cut-off value to indicate “negative” or “positive,” allowing for faster testing turnaround. Despite its widespread use in many species, an in-depth, multispecies analysis of the data comparing sVNT to VN results has not been reported. Such an analysis is useful to assess the diagnostic appropriateness of the sVNT test for SARS-CoV-2 virus screening and detection across multiple species. Results were considered from all serological samples submitted to NVSL from March 2020 to May 2023 for routine SARS-CoV-2 diagnostic testing. Of these, samples with results for both VN and sVNT testing were considered for further analysis (n = 942). Samples were sorted by the species and family levels, and were assessed for rates of “true positive,” “false positive,” “true negative,” and “false negative” by sVNT, using paired VN results as the indicator of the true status of the sample. Additionally, the increased sVNT cut-off threshold for %I (20%I to 30%I for “positive”) was assessed for appropriateness by species using mean %I values for VN-positive samples. Increasing sVNT %I cut-off threshold for “positive” results from 20%I to 30%I resulted in 58 samples being correctly re-classified as “negative” by sVNT, rather than “positive” at the 20%I cut-off threshold. The mean %I values for most species fell between 40 to 80%I, except for Felidae, which had a mean %I of 93%I, and a minimum %I of 40.6%I for VN-positive samples. The true negative (sVNT/VN-NEG) rates for most species fell between 97to 100%, with 0 to 3% being false negatives (svNT-NEG, VN-POS), with very few exceptions. However, the least false positive (sVNT-POS;VN-NEG) rate was 8.8%, ranging up to 100% false positives for Equidae and one species of Cervidae. While the mean %I for most species indicates the cut-off value is appropriate (for those species where sVNT is an appropriate diagnostic), overall, the results highlight a lack of precision and specificity of the sVNT for many animal species. This indicates a lack of diagnostic appropriateness of the sVNT as a SARS-CoV-2 screening tool for certain species and a need for refinement in cut-off values for other species. The sVNT as a screening tool varies considerably by species for serodetection of SARS-CoV-2, results should be interpreted with caution, and use of other confirmatory tests is recommended.

Evaluation of commercial quadrivalent foot-and-mouth disease vaccines against east African virus strains reveals limited immunogenicity and duration of protection

Foot-and-mouth disease virus (FMDV) causes a contagious disease (FMD) in cloven-hoofed animals. For FMD-endemic countries, vaccination is critical for controlling disease but is rarely monitored, despite substantial funds spent on vaccine purchases. We evaluated antibody responses in cattle to two commercial vaccines each containing antigens of four FMDV serotypes. Sampling was done over 360 days, with serology for each serotype performed using commercially available solid phase competition ELISAs (SPCE) and with virus neutralization tests (VNT) employing regionally relevant test viruses. A primary course of each vaccine was administered to 37 calves, some of which received a second dose after 28 days. Using new production batches of vaccines, all calves received a booster vaccination 180 days post vaccination, while 10 additional naïve calves were also vaccinated using the new batches and followed up for ∼180 days. Simple and general linear models were used to compare antibody responses which varied substantially according to vaccine, dose regime, serotype, and test, but were mostly insufficient to ensure a high likelihood of adequate or sustained probable protection. One of the vaccines administered as a two-dose primary course of vaccination was superior to other options, but even then, data trajectories from VNT responses suggested probable protection of 75 % of calves for 6 months for only one virus serotype. Calves administered with the other vaccine and those given a single primary dose developed low levels of antibodies, offering predicted likely protection lasting less than two months. Individual SPCE results were weakly correlated (r2 = 0.48) to neutralization and associated likelihoods of protection but SPCE and VNT agreed on which vaccine and dose regime performed best. Our findings highlight gaps in immunogenicity of FMD vaccines used in East Africa and reinforce the importance of independent quality control studies to evaluate and improve commercial FMD vaccines and vaccination regimes.

The Anti-SARS-CoV-2 S-Protein IgG, Which Is Detected Using the Chemiluminescence Microparticle Immunoassay (CMIA) in Individuals Having Either a History of COVID-19 Vaccination and/or SARS-CoV-2 Infection, Showed a High-Titer Neutralizing Effect.

Neutralizing antibodies plays a primary role in protective immunity by preventing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from entering the cells. Therefore, characterization of antiviral immunity is important for protection against SARS-CoV-2. In this study, the neutralizing effect of the anti-SARS-CoV-2 S1 protein IgG, which was detected using the chemiluminescence microparticle immunoassay (CMIA)-based SARS-CoV-2 IgG II Quant (Abbott, Waukegan, IL, USA) test in SARS-CoV-2 infected and/or vaccinated individuals, was investigated with a surrogate virus neutralization test (sVNT). In total, 120 Seropositive individuals were included in this study. They were divided into two groups: Vaccinated (n = 60) and Vaccinated + Previously Infected (n = 60). A commercial sVNT, the ACE2-RBD Neutralization Test (Dia.Pro, Milan, Italy), was used to assess the neutralizing effect. The assay is performed in two steps: screening and titration. The screening showed positive results in all seropositive samples. Low titration in 1.7%, medium titration in 5%, and high titration in 93.3% of the Vaccinated group, and medium titration in 1.7% and high titration in 98.3% of the other group, as obtained from the ACE2-RBD titration test. A strong positive and significant correlation was found between the SARS-CoV-2 IgG II Quant test and the ACE2-RBD titration test at the 1/32 titration level for both groups (p < 0.001 for both). This study shows that the SARS-CoV-2 IgG detected using the CMIA method after SARS-CoV-2 infection and/or vaccination has a high neutralizing titration by using the sVNT. In line with these data, knowledge that seropositivity determined by CMIA also indicates a strong neutralizing effect contributes to countrywide planning for protecting the population.

SARS-COV-2 SEROSURVEY OF HEALTHY, PRIVATELY OWNED CATS PRESENTING TO A NEW YORK CITY ANIMAL HOSPITAL IN THE EARLY PHASE OF THE COVID-19 PANDEMIC (2020-2021).

SARS-CoV-2, the cause of the ongoing COVID-19 pandemic, not only infects humans but is also known to infect various species, including domestic and wild animals. While many species have been identified as susceptible to SARS-CoV-2, there are limited studies on the prevalence of SARS-CoV-2 in animals. Both domestic and non-domestic cats are now established to be susceptible to infection by SARS-CoV-2. While serious disease in cats may occur in some instances, the majority of infections appear to be subclinical. Differing prevalence data for SARS-CoV-2 infection of cats have been reported, and are highly context-dependent. Here, we report a retrospective serological survey of cats presented to an animal practice in New York City, located in close proximity to a large medical center that treated the first wave of COVID-19 patients in the U.S. in the Spring of 2020. We sampled 79, mostly indoor, cats between June 2020 to May 2021, the early part of which time the community was under a strict public health "lock-down". Using a highly sensitive and specific fluorescent bead-based multiplex assay, we found an overall prevalence of 13/79 (16%) serologically-positive animals for the study period; however, cats sampled in the Fall of 2020 had a confirmed positive prevalence of 44%. For SARS-CoV-2 seropositive cats, we performed viral neutralization test with live SARS-CoV-2 to additionally confirm presence of SARS-CoV-2 specific antibodies. Of the thirteen seropositive cats, 7/13 (54%) were also positive by virus neutralization, and two of seropositive cats had previously documented respiratory signs, with high neutralization titers of 1/1024 and 1/4096; overall however, there was no statistically significant association of SARS-CoV-2 seropositivity with respiratory signs, or with breed, sex or age of the animals. Follow up sampling of cats showed that positive serological titers were maintained over time. In comparison, we found an overall confirmed positive prevalence of 51% for feline coronavirus (FCoV), an endemic virus of cats, with 30% confirmed negative for FCoV. We demonstrate the impact of SARS-CoV-2 in a defined feline population during the first wave of SARS-CoV-2 infection of humans, and suggest that human-cat transmission was substantial in our study group. Our study provide a new context for SARS-CoV-2 transmission events across species.

Phylogenetic analyses and antigenic characterization of foot-and-mouth disease virus PanAsia lineage circulating in China between 1999 and 2023

Foot-and-mouth disease (FMD) is one of the most important transboundary animal diseases caused by foot-and-mouth disease virus (FMDV), leading to significant economic losses worldwide. The first report of PanAsia lineage of FMDV in China was in 1999. Since 2011, 18 outbreaks attributed to PanAsia lineage viruses have been reported across 7 provinces or municipality in China. Phylogenetic analysis indicated that these PanAsia strains were clustered into three distinct clades (clade 1, clade 2, and clade 3), with nucleotide homology ranging from 91.4% to 100%. The outbreaks of FMD caused by clade 1 strains occurred around 1999 when this lineage was prevalent globally. Clade 2 strains dominated from 2011 to 2013, while clade 3 strains were prevalent during 2018–2019, sharing only 93% homology with clade 2 strains and 91% with clade 1 strains. Tracing analysis showed that these outbreaks represented 3 distinct introductions of PanAsia viruses into China. Virus neutralization tests (VNT) have demonstrated that current commercial vaccines are effective to protect susceptible animals against these strains (r1 ​> ​0.3). However, the growing demand for livestock has promoted animal movement and encouraged the exchange of products, services, and materials between countries, thereby heightening the risk of exotic strain incursions. Therefore, it is imperative to reinforce border controls and limit animal movements among various Asian countries continually to reduce the risk of new transboundary diseases, such as FMD incursion. Additionally, PanAsia-2 strains need to be taken seriously to prevent its incursions, and the relevant vaccines against PanAsia-2 strains need to be stockpiled in preparation for any possible incursion.

Detection and Genetic Characterization of Border Disease Virus (BDV) Isolated from a Persistently Infected Sheep in a Migratory Flock from Rajasthan State, Northwestern India.

Border disease virus (BDV) causes significant economic losses in sheep farming worldwide. In India, BDV has not yet been studied in sheep migrating for summer pasturing. This study aimed to determine the extent of BDV infection in migratory sheep and provide genetic characteristics of BDV. Blood and serum samples from 90 lambs of a migratory sheep flock (600) in Central India were collected and subjected to molecular detection, phylogenetic analysis and virus neutralization test (VNT). We detected BDV in two lambs through real-time RT-PCR, while 64.4% (58/90) of in-contact lambs had BDV neutralizing antibodies. One apparently healthy lamb was found to be persistently infected with BDV. Phylogenetic analysis of 5'-UTR and Npro genes and the concatenated datasets typed the BDV isolate from PI sheep as BDV-3 genotype. However, it showed a closer relationship with BDV-3 strains from China than the previously reported Indian BDV-3 strains. This is the first report on the detection of BDV persistently infected migratory sheep in India. Additionally, we provided evidence of genetic variability among BDV-3 strains in India. The findings improve our understanding of epidemiology and genetic characteristics of BDV in India and highlight the potential risks associated with the traditional practice of sheep migration for summer pasturing.

Post-Hoc Analysis of Potential Correlates of Protection of a Recombinant SARS-CoV-2 Spike Protein Extracellular Domain Vaccine Formulated with Advax-CpG55.2-Adjuvant.

SpikoGen® vaccine is a subunit COVID-19 vaccine composed of an insect cell expressed recombinant spike protein extracellular domain formulated with Advax-CpG55.2™ adjuvant. A randomized double-blind, placebo-controlled Phase II clinical trial was conducted in 400 adult subjects who were randomized 3:1 to receive two intramuscular doses three weeks apart of either SpikoGen® vaccine 25 μg or saline placebo, as previously reported. This study reports a post hoc analysis of the trial data to explore potential immune correlates of SpikoGen® vaccine protection. A range of humoral markers collected pre- and post-vaccination, including spike- and RBD-binding IgG and IgA, surrogate (sVNT), and conventional (cVNT) virus neutralization tests were compared between participants who remained infection-free or got infected over three months of follow-up. From 2 weeks after the second vaccine dose, 21 participants were diagnosed with SARS-CoV-2 infection, 13 (4.2%) in the SpikoGen® group and 8 (9%) in the placebo group. Those in the vaccinated group who experienced breakthrough infections had significantly lower sVNT titers (GMT 5.75 μg/mL, 95% CI; 3.72-8.91) two weeks after the second dose (day 35) than those who did not get infected (GMT 21.06 μg/mL, 95% CI; 16.57-26.76). Conversely, those who did not develop SARS-CoV-2 infection during follow-up had significantly higher baseline sVNT, cVNT, spike-binding IgG and IgA, and RBD-binding IgG, consistent with a past SARS-CoV-2 infection. SpikoGen® further reduced the risk of re-infection (OR 0.29) in baseline seropositive (previously infected) as well as baseline seronegative participants. This indicates that while SpikoGen vaccine is protective in seronegative individuals, those with hybrid immunity have the most robust protection.

Evaluation of a qualified MDCK cell line for virus isolation to develop cell-based influenza vaccine viruses with appropriate antigenicity

We established a qualified Madin-Darby canine kidney cell line (qMDCK-Cs) and investigated its suitability for source virus isolation to develop cell-based seasonal influenza vaccine viruses using vaccine manufacturer cells (Manuf-Cs). When inoculated with 81 influenza-positive clinical specimens, the initial virus isolation efficiency of qMDCK-Cs was exceeded 70%. Among the qMDCK-C isolates, 100% of the A/H1N1pdm09, B/Victoria and B/Yamagata strains and >70% of the A/H3N2 strains showed antigenicity equivalent to that of the contemporary vaccine or relevant viruses in haemagglutination inhibition (HI) or virus neutralization (VN) tests using ferret antisera. These qMDCK-C isolates were propagated in Manuf-Cs (MDCK and Vero cells) (Manuf-C viruses) to develop vaccine viruses. In reciprocal antigenicity tests, ferret antisera raised against corresponding reference viruses and Manuf-C viruses recognized 29 of 31 Manuf-C viruses and corresponding reference viruses, respectively at HI or VN titres more than half of the homologous virus titres, which is the antigenicity criterion for cell culture seasonal influenza vaccine viruses specified by the World Health Organization. Furthermore, ferret antisera against these Manuf-C viruses recognized ≥95% of the viruses circulating during the relevant influenza season with HI or VN titres greater than one-quarter of the homologous virus titres. No cell line-specific amino acid substitutions were observed in the resulting viruses. However, polymorphisms at positions 158/160 of H3HA, 148/151 of N2NA and 197/199 of B/Victoria HA were occasionally detected in the qMDCK-C and Manuf-C viruses but barely affected the viral antigenicity. These results indicated that qMDCK-Cs are suitable for isolating influenza viruses that can serve as a source of antigenically appropriate vaccine viruses. The use of the qMDCK-C isolates will eliminates the need for clinical sample collection, virus isolation, and antigenicity analysis every season, and is expected to contribute to the promotion of vaccine virus development using manufacturer cells.

Seroepidemiology of bovine herpesvirus-1 in goats in south-western Iran.

Widely regarded as one of the chief causes of diseases in cattle population, bovine herpesvirus-1 (BoHV-1) has the potential to infect sheep and goat, making them potential reservoirs or hosts for this virus. Thus, preventive measures against BoHV-1 in cattle should not overlook the ability of this virus to infect other animals. Therefore, the focal point of this study was to ascertain the seroprevalence of BoHV-1 in 300 healthy goats, the relationship between host and the environmental determinants of infection, and the contributing role of goats in the epidemiology of the BoHV-1. In order to pinpoint the existing antibodies to BoHV-1, the obtained sera were analyzed by Virus Neutralization test. According to this test, the seroprevalence of BoHV-1 appeared to be 64.33% in southwestern Iran. What logistic regression disclosed was that the odds ratio between age and infection with BoHV-1 was 0.83 (p=0.01), representing a decrease of 17% as goats grew one year older. In addition, females manifested a higher relative frequency of infection compared to males, with the odds of infection in female goats being registered at 1.88, compared to those in males (p=0.2). Moreover, contrasted with goats lacking any history of abortion, those with a history of abortion featured 1.1 as the odds ratio (p=0.87). The seroprevalence in Hendijan, Ahvaz, Shushtar and Dasht e Azadegan was detected to stand at 73.24, 71.30, 55.56 and 47.06 percent, respectively, with 6% of fluctuation in the infection rates being attributed to various geographical locations under the scrutiny of this study (p=0.003). Having attested the marked seroprevalence of BoHV-1, the definitive role of goats in the epidemiology of this virus as a secondary host or reservoir was confirmed by the present study, necessitating the strict monitoring of BoHV-1 in goats by animal health authorities in areas where BoHV-1 abounds in cattle.

The Protection Level of S-RBD SARS-CoV-2 Immunoglobulin G Antibodies Using the Chemiluminescent Immunoassay Compared to the Surrogate Virus Neutralization Test Method.

COVID-19 infection in high-risk populations is fatal and has a poor prognosis, necessitating a test to determine the protectiveness of immune response. Antibody testing is necessary to determine the body's immune response to COVID-19 infection and also vaccination strategies. Among the various methods available, the chemiluminescent immunoassay (CLIA) test is more widely used and accessible to determine antibody levels. This study aimed to determine the protection level of S-RBD SARS-CoV-2 IgG using CLIA compared to the Surrogate Virus Neutralization Test (SVNT). The population of this study comprised all healthcare professionals who experienced S-RBD SARS-CoV-2 IgG antibody level examinations. S-RBD SARS-CoV-2 IgG antibody levels were examined using CLIA and SVNT. The cut-off was determined using a receiver operating characteristic (ROC) curve, and area under the curve (AUC) measurements were evaluated. The result showed a strong positive correlation between S-RBD SARS-CoV-2 IgG CLIA and SVNT, with a value of r = 0.933 and p < 0.001. The value ≥ 37.29 BAU/mL was determined as the cut-off based on SVNT 30% inhibition level with sensitivity, specificity, and positive and negative predictive values of 96.5%, 90.9%, 96.5%, and 90.9%, respectively. A titer of antibodies greater than or equal to 37.29 BAU/mL with CLIA showed the presence of protective antibodies compared to SVNT.

Rapid Detection of Getah Virus Antibodies in Horses Using a Recombinant E2 Protein-Based Immunochromatographic Strip.

The prevalence and impact of Getah virus (GETV) are significant concerns in China. GETV can infect a wide range of animals, including horses, pigs, sheep, cattle, birds, and humans, resulting in substantial losses in the livestock and agricultural industries. GETV infection can cause the development of ulcers and inflammation in the mouth and gums of horses, which result in pain and discomfort and lead to symptoms such as reduced appetite, drooling, and difficulty chewing. As a result, there is a pressing need for efficient and rapid disease diagnosis methods. However, the currently available diagnostic methods have limitations in terms of operational time, equipment, and the experience of the individuals using them. In this study, a rapid, specific, and sensitive detection method was developed using a colloidal gold-based immunochromatographic strip (ICS) for the detection of antibodies against GETV in horses. To prepare the ICS, the antigen domain of the E2 glycoprotein of GETV was expressed using the Escherichia coli expression system after analysis with DNAstar v7.1 software. The nitrocellulose membrane was coated with rE2 protein or SPA to form the test line and control line, respectively. After optimizing the reaction conditions, the sensitivity, specificity, and repeatability of the strip were verified. The results showed that the test strip had a detection limit of up to 1:320 dilutions for GETV-positive serum, with no cross-reactivity observed with other equine-susceptible pathogens such as equine arteritis virus (EAV), equine herpesvirus-1 (EHV-I), equine infectious anemia virus (EIAV), equine influenza virus (EIV), African horse sickness virus (AHSV), and Japanese encephalitis virus (JEV). Furthermore, the ICS exhibited a concordance rate of 94.0% when testing 182 clinical serum samples compared to the virus neutralization test. Overall, this ICS diagnosis method will be an effective tool for the rapid detection of GETV in the field.

An outbreak of SARS-CoV-2 omicron variant and deaths of three lions in a zoo

There have been reports of the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to various mammalian species. Some infected animals show clinical signs and may even die in rare cases. Outbreaks of SARS-CoV-2 have been reported in zoos where susceptible animals are bred in high population densities. However, there have been few reports of omicron variant outbreaks in zoo animals. From late 2022 to 2023, an outbreak of the SARS-CoV-2 omicron variant occurred in one Japanese zoo. A total of 24 lions were housed in the zoo; 13 of them showed respiratory symptoms, and the three oldest lions died. Molecular and histopathological analyses revealed that the deceased lions were infected with SARS-CoV-2 omicron BF.7.15. Virus-neutralization tests showed that all 21 lions were positive for antibodies against the omicron variant, but not against the delta variant. In addition, three tigers and one bear in the same or neighboring building as the lions possessed antibodies against the omicron variant. This is a very rare report on the outbreak of a SARS-CoV-2 omicron variant infection that resulted in the death of animals. This finding demonstrates the importance of continuous countermeasures to protect non-vaccinated animals from SARS-CoV-2 infection.

Detection of IgM antibodies against bovine viral diarrhea virus using IgM capture ELISA on farms with persistently infected cattle

Bovine viral diarrhea (BVD) is a serious disease in cattle and causes economic losses in the livestock industry. Bovine viral diarrhea virus (BVDV) is the causative agent of BVD and spreads among herds via persistently infected (PI) animals that shed large amounts of the virus throughout their lives. Hence, identifying, and culling PI animals and assessing the immune status against BVDV on farms are important strategies for controlling BVD. Additionally, estimating the time when individuals around PI animals were infected with the virus could also be supportive information to interpret a farm status. We herein constructed a BVDV-specific IgM capture ELISA using recombinant E2 antigen and applied it to detecting BVDV-specific IgM antibodies on farms with identified PI cattle. The IgM ELISA detected anti-BVDV IgM antibodies during approximately 2–3 weeks post infection and identified IgM-positive cattle on two farms with recognized PI cattle. Virus neutralization tests showed that almost all adult cattle had high virus neutralization antibodies against BVDV, and sero-positive and -negative cattle coexisted in young herds. In this situation, most of the IgM-positive cattle were in relatively young animals, implying that BVDV had been recently spreading in these young herds. Thus, our findings demonstrated that detecting IgM antibodies could be useful to know recent BVDV infection on farm on which PI cattle were identified.

Persistence of maternal antibodies against goat pox virus in goat kids.

In goat kids, choosing the appropriate age to administer the first dose of goat pox disease (GTP) vaccine requires knowing when maternal antibody decline concentrations. Determine the persistence of maternal antibodies against goat pox virus (GTPV) in goat kids. Twenty Saanen goat kids from birth to 120 days old. In 2 groups, including: control (receiving colostrum from nonvaccinated does) and treatment (receiving colostrum from vaccinated does). On zero, 3, 7, 14, 21, 28, 42, 56, 70, 100 and 120 days after the birth, virus neutralization test was used to measure the serum concentration of antibodies against GTPV. At the age of 56 days, the first seronegative goat kids (n = 2) were recorded in the treatment group. At the age of 120 days, all the goat kids in the treatment group were seronegative. The average virus neutralization index (VNI) of the goat kids became negative at the age of 100 to 120 days. All goat kids in the control group were negative at all times. One hundred to 120 days of the age seems to be the time to administer the first GTP vaccine in the goat kids with passive immunity against goat pox.

Surveillance of SARS-CoV-2 Infection in Rodent Populations in Tokyo, Japan.

The maintenance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among wildlife populations poses a potential risk for the emergence of novel variants. Therefore, monitoring SARS-CoV-2 infection among animals is crucial. As urban rodents live in close proximity to human habitats, there is concern that they may be a potential source of zoonoses. To examine the prevalence of SARS-CoV-2 in rodent populations, we analyzed 128 serum samples and 129 oral swabs collected from 128 brown rats (Rattus norvegicus) and 2 black rats (Rattus rattus) captured for pest control purposes in Tokyo, Japan, between May and December 2023. A virus-neutralizing test using the Omicron variant revealed no evidence of SARS-CoV-2 infection in these populations. Real-time RT-PCR from oral swabs did not detect any SARS-CoV-2 RNA-positive rats. These results indicate the low probability of SARS-CoV-2 circulation among rat populations in Tokyo.

Orthoflavivirus surveillance in the Netherlands: Insights from a serosurvey in horses & dogs and a questionnaire among horse owners.

Zoonotic arboviruses (arthropod-borne) of the Orthoflavivirus genus, such as West Nile virus (WNV), Usutu virus (USUV) and Tick-borne encephalitis virus (TBEV), are emerging in Northwestern Europe and pose a threat to both human and animal health. In the Netherlands, passive symptomatic surveillance (notification of clinical cases) in horses is one of the main pillars for the early detection of WNV. For such passive surveillance to work properly, horse owners and veterinarians need to recognize symptoms and report suspected cases to the authorities. Currently, little is known about the seroprevalence of orthoflaviviruses in domestic animals in the Netherlands. Therefore, this study aims at identifying the seroprevalence of WNV and USUV in horses and dogs in the Netherlands. Additionally, this study seeks to evaluate the knowledge and perceptions of Dutch horse owners towards mosquito-borne viruses. A cross-sectional serosurvey in horses and dogs was conducted between May 2021 and May 2022. Serum samples were screened using an ELISA and doubtful and positive samples were confirmed by Virus Neutralization Tests for WNV, USUV and TBEV. A validated questionnaire, the MosquitoWise survey, was used to assess the knowledge and perceptions of Dutch horse owners towards mosquito-borne viruses between July and October 2022. The serosurvey revealed a low seroprevalence for WNV in horses and no WNV-positive dogs were found. Similarly, a low USUV seroprevalence was found in dogs. The MosquitoWise survey revealed a high knowledge level for horse owners and high awareness of WNV vaccination but a more limited intent to vaccinate. The low seroprevalences of WNV and USUV indicate many dogs and horses remain susceptible, offering opportunities for trend analysis and surveillance. However, despite multiple recent detections of WNV, USUV, and TBEV in humans, the role of dogs and horses in early detection of human cases is debatable. High awareness among horse owners and the absence of detected equine WNV cases highlight this uncertainty. Continued surveillance is crucial for detecting increased virus circulation and protecting both animal and human health.

Characterization of an engineered ACE2 protein for its improved biological features and its transduction into MSCs: A novel approach to combat COVID-19 infection

Transduced MSCs that express engineered ACE2 could be highly beneficial to combat COVID-19. Engineered ACE2 can act as decoy targets for the virus, preventing its entry into healthy lung cells. To this end, genetic engineering techniques were used to integrate the ACE2 gene into the MSCs genome. The MSCs were evaluated for proper expression and functionality. The mutated form of ACE2 was characterized using various techniques such as protein expression analysis, binding affinity against spike protein, thermal stability assessment, and enzymatic activity assays. The functionality of the mACE2 was assessed on SARS-CoV-2 using the virus-neutralizing test. The obtained results indicated that by introducing specific mutations in the ACE2 gene, the resulting mutant ACE2 had enhanced interaction with viral spike protein, its thermal stability was increased, and its enzymatic function was inhibited as a decoy receptor. Moreover, the mACE2 protein showed higher efficacy in the neutralization of the SARS-CoV-2. In conclusion, this study proposes a novel approach with potential benefits such as targeted drug delivery and reduced side effects on healthy tissues. These transduced MSCs can also be used in combination with other anti-COVID-19 treatments. Design of similar engineered biomolecules with desired properties could also be used to target other diseases.

Foot and Mouth Disease Virus Strain-specific Antibody Titres in Naturally Infected or Vaccinated Bulls in Kenya

Foot-and-mouth disease (FMD) is a global viral infection that causes vesicular lesions in and around the mouth and feet, causing reluctance of animals to eat or move. In Kenya, bulls raised for AI receive vaccinations against FMD, but it is unclear if these animals experience vaccine-induced immunity. No research has been conducted to determine if animals in endemic areas develop natural immunity or whether animals in disease-free regions might be seropositive. This study aimed to determine the prevalence and levels of foot and mouth disease virus infection-triggered and vaccine-induced antibodies. A cross-sectional study was conducted on bulls farmed for AI production and vaccinated against FMD. Antibodies were quantified using a virus-neutralization test. One-way analysis of variance (ANOVA) and the Kruskal-Wallis test with Tukey and Dunn post-tests, respectively, were used to examine the data using the GraphPad InStat program. Additionally, the Spearman test was employed for correlation analysis and the t-test for intergroup differences analysis. A statistically significant P value was defined as less than 0.05. Findings showed protective antibody levels were present in 23%, 10.3%, 2.6%, and 7.7% of the animals in the FMD non-endemic region against the FMD virus strains O, A, SAT 1, and SAT 2, respectively. The protection provided by the O strain virus was significantly greater than that of SAT 1 (P = 0.01). In the FMD endemic area, all sampled animals showed protection levels at 100%, 100%, 100% and 29% for virus strains O, A, SAT 1, and SAT 2 respectively with the antibody titres showing significant differences (P &lt; 0.05) for all the intergroup analysis except between strains O vs SAT 1 and A vs SAT 1 (P &gt; 0.05). To conclude, the current research suggests that FMD may be making a comeback in the areas where the illness is not established. Furthermore, it seems that sperm recovery upon freezing is somewhat mitigated by FMDV-specific antibodies. The study advises monitoring FMD in areas where the illness is not endemic and confirms the current findings with larger sample sizes to enable more informed decision-making.

Survey of severe acute respiratory syndrome coronavirus 2 in captive and free-ranging wildlife from Spain

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), considered a zoonotic agent of wildlife origin, can infect various animal species, including wildlife in free-range and captive environments. Detecting susceptible species and potential reservoirs is crucial for preventing the transmission, spread, genetic evolution, and further emergence of viral variants that are major threats to global health. This study aimed to detect exposure or acute infection by SARS-CoV-2 in 420 animals from 40 different wildlife species, including terrestrial and aquatic mammals, from different regions of Spain during the 2020–2023 coronavirus disease 19 (COVID-19) pandemic. In total, 8/137 animals were positive for SARS-CoV-2 antibodies against the receptor binding domain and/or viral nucleoprotein according to independent ELISAs. However, only one ELISA-positive sample of a captive bottlenose dolphin (Tursiops truncatus) tested positive for SARS-CoV-2 neutralizing antibodies with a low titre (SNT50 38.15) according to a virus neutralization test. Cetaceans are expected to have a high risk of infection with SARS-CoV-2 according to early predictive studies due to the similarity of their angiotensin converting enzyme 2 cell receptor to that of humans. Moreover, of 283 animals analysed for SARS-CoV-2 RNA using RT-qPCR, none tested positive. Our results reinforce the importance of considering cetaceans at risk for SARS-CoV-2 infection and support taking preventive biosecurity measures when interacting with them, especially in the presence of individuals with suspected or confirmed COVID-19. Although most animals in this study tested negative for acute infection or viral exposure, ongoing surveillance of wildlife species and potentially susceptible animals is important to prevent future spillover events and detect potential novel reservoirs.

A recombinant Getah Virus expressing a GFP gene for rapid neutralization testing and antiviral drug screening assay

Getah virus (GETV) is a re-emerging mosquito-borne RNA virus that induces fever, hind limb edema, swollen submandibular lymph nodes, and urticaria in horses. In pigs, the virus often results in stillbirths among pregnant sows, and neurological symptoms leading to death in piglets. Currently, there are no specific treatments or drugs available for GETV infection. The use of reporter viruses to monitor viral replication and spread in real-time within infected cells and animals provides a powerful tool for targeting antiviral drugs throughout the viral life cycle. Their fluorescence-tracked characteristics greatly facilitate virus neutralization tests (VNTs). In this study, we engineered two recombinant viruses by inserting different reporter protein genes at the 3′ end of the structural protein gene, an unreported location that can accommodate exogenous genes. The rGEEiLOV and rGEEGFP viruses demonstrated genetic stability for at least five passages and replicated at a rate similar to that of the parental virus in BHK-21 cells. The rGEEGFP virus facilitated viral neutralization testing. Additionally, we used the reporter virus rGEEGFP to confirm ivermectin, a broad-spectrum antiparasitic agent, as a potential inhibitor of GETV in vitro. Ivermectin appears to inhibit the early replication stages of the virus and can block cell-to-cell viral transmission. In conclusion, rGEEGFP holds significant potential for antiviral screening to identify specific inhibitors against GETV and for use in viral neutralization tests.