Characterization of infectious laryngotracheitis virus isolated from commercial layer chickens in Bangladesh during the year 2021-2022.

Objective: Infectious laryngotracheitis virus (ILTV) is responsible for causing infectious laryngotracheitis (ILT), which is a rapidly spreading and extremely transmissible disease in chickens. The current research aims to isolate and characterize ILTV from layer chickens in Bangladesh. Materials and Methods: A total of 345 samples (trachea, larynx, and lungs) were collected from ILT-suspected dead and sick layer chickens of 32 ILT-suspected farms in three different outbreak districts (Gazipur, Tangail, and Mymensingh) of Bangladesh during the outbreak year 2021-2022. Rapid detection kits examined the samples for avian influenza virus (AIV) and Newcastle disease virus (NDV). ILTV-specific primers were used to screen 72 NDV- and AIV-negative samples by polymerase chain reaction (PCR). Using chorioallantoic membrane (CAM), the study isolated the ILT virus from 9 to 10-day-old seronegative embryonated chicken eggs (ECEs) using selected PCR-positive samples. The virus was confirmed using nucleotide sequencing, agar gel immunodiffusion test (AGIDT), viral neutralization test (VNT), and pathogenicity evaluations using mortality index for chicken embryos (MICEs) and intra-tracheal pathogenicity index (ITPI). Results: The results indicated that among the PCR-positive 10 samples, only two (Alim_ILT_1001 and Alim_ILT_1,000) were found positive using ECEs. There were two field isolates of ILTVs, as shown by the amplicon size of the ICP4 gene-based PCR. A phylogenetic study of the ICP4 gene revealed that the recent isolates have a close similarity with the ILTV isolates of Turkey, Bangladesh, and Australia. AGIDT revealed strong precipitation lines due to ILTV-specific antibodies reacting with field viruses, while VNT neutralized both isolates with conventional ILTV antibodies. The pathogenicity testing indicated that Alim_ILT_1001 had MICE and ITPI values of 0.77 and 0.63, whereas Alim_ILT_1,000 had 0.71 and 0.57. Conclusion: Both the ILTV isolates have similarities with the isolates of Turkey, Bangladesh, and Australia, and they are highly virulent for chickens.

Different Neutralizing Antibody Responses of Heterologous Sera on Sheeppox and Lumpy Skin Disease Viruses.

Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.

Quantitative assay to analyze neutralization and inhibition of authentic Middle East respiratory syndrome coronavirus.

To date, there is no licensed vaccine for Middle East respiratory syndrome coronavirus (MERS-CoV). Therefore, MERS-CoV is one of the diseases targeted by the Coalition for Epidemic Preparedness Innovations (CEPI) vaccine development programs and has been classified as a priority disease by the World Health Organization (WHO). An important measure of vaccine immunogenicity and antibody functionality is the detection of virus-neutralizing antibodies. We have developed and optimized a microneutralization assay (MNA) using authentic MERS-CoV and standardized automatic counting of virus foci. Compared to our standard virus neutralization assay, the MNA showed improved sensitivity when analyzing 30 human sera with good correlation of results (Spearman's correlation coefficient r = 0.8917, p value < 0.0001). It is important to use standardized materials, such as the WHO international standard (IS) for anti-MERS-CoV immunoglobulin G, to compare the results from clinical trials worldwide. Therefore, in addition to the neutralizing titers (NT50 = 1384, NT80 = 384), we determined the IC50 and IC80 of WHO IS in our MNA to be 0.67 IU/ml and 2.6 IU/ml, respectively. Overall, the established MNA is well suited to reliably quantify vaccine-induced neutralizing antibodies with high sensitivity.

Seroprevalence of West Nile Virus among Equids in Bulgaria in 2022 and Assessment of Some Risk Factors.

The aim of this study was to analyze the seroprevalence of West Nile virus (WNV) among equids in Bulgaria, confirm the results of a competitive ELISA versus the virus neutralization test (VNT) and investigate some predisposing factors for WNV seropositivity. A total of 378 serum samples from 15 provinces in northern and southern Bulgaria were tested. The samples originated from 314 horses and 64 donkeys, 135 males and 243 females, aged from 1 to 30 years. IgG and IgM antibodies against WNV protein E were detected by ELISA. ELISA-positive samples were additionally tested via VNT for WNV and Usutu virus. Thirty-five samples were WNV-positive by ELISA (9.26% [CI = 6.45-12.88]), of which 15 were confirmed by VNT; hence, the seroprevalence was 3.97% (CI = 2.22-6.55). No virus-neutralizing antibodies to Usutu virus were detected among the 35 WNV-ELISA-positive equids in Bulgaria. When compared with VNT, ELISA showed 100.0% sensitivity and 94.5% specificity. A statistical analysis showed that the risk factors associated with WNV seropositivity were the region (p < 0.0001), altitude of the locality (p < 0.0001), type of housing (p < 0.0001) and breed (p = 0.0365). The results of the study demonstrate, albeit indirectly, that WNV circulates among equids in northern and southern Bulgaria, indicating that they could be suitable sentinel animals for predicting human cases and determining the risk in these areas or regions of the country.

Evaluation of longitudinal passive immunity transfer against lumpy skin disease virus in calves by different serological methods.

To implement effective lumpy skin disease (LSD) control measures, such as timely vaccination, particularly in calves and serological monitoring, it is necessary to evaluate immune response after vaccination, both in adult cattle and in their calves. The aim of this study was to evaluate passive immunity transfer and duration of maternal antibodies against lumpy skin disease virus (LSDV) in calves born to vaccinated cows by two different serological methods. The longitudinal study was carried out on two farms in Serbia where no cases were reported during LSD outbreak in 2016. Fifteen cows on each farm were vaccinated and revaccinated with attenuated vaccine - Neethling strain. A total of 30 cows and 30 calves on both farms were included in the study. Serum samples from cows were collected on calving day and serum samples from their respective calves on days 10, 20, 30, 45, 60, 75, 90, 105 and 120 after birth. Colostrum samples were collected only from 15 cows on one farm. In order to determine the presence of antibodies against LSDV a total of 30 cow sera samples, 15 colostrum samples and 270 calf sera samples were examined by commercial enzyme-linked immunosorbent assay (ELISA) and modified virus neutralization test (VNT). Overall, the performance of both serological tests was very satisfactory. The results of this longitudinal study showed that persistence of passive immunity in calves is less than 4 months, and that most calves are not protected against LSDV at that age. Since the vaccination is the most important control measure against LSDV, the recommended age of six months for vaccination of calves born to vaccinated cows should be reassessed to achieve the most optimal protection against LSD.

Long-Term Immunological Alertness and Response to COVID-19 Vaccination-Conditions for Prevention in Early Palliative Oncological Care Patients.

Aside from the general population, the COVID-19 pandemic has also affected a group of patients in palliative oncology care. In this study, long-term immune responses against SARS-CoV-2 after vaccination were monitored in a cohort of patients in palliative oncology care. This non-randomized, prospective, and open-label pilot study recruited patients from the Palliative Oncology Program and included 147 patients, of which 80 were females (54.4%) and 67 males (45.6%). The overall evaluation included current health status, SARS-CoV-2 anti-S IgG titer, and neutralizing antibodies using the SARS-CoV-2 virus neutralization test (VNT). Anti-S IgG antibody analysis revealed high (H) antibody levels in 35.7% (n = 10) and very high (VH) levels in 39.3% (n = 11) of patients after the second vaccination dose. Similarly, after the third dose, H was found in 29.6% (n = 32) and VH in 55.5% (n = 60) of patients. High and very high anti-S IgG antibody levels were consistent with high VNT titers (>2560) and H antibody levels in 17.1% (n = 12) or VH in 82.9% (n = 58) of patients. Patients with two or more doses showed H and VH antibody levels at a median of 451 and 342 days after vaccination, respectively. In this clinical trial, patients showed high and very high levels of anti-S IgG antibodies over a longer period of time. These patients did not show reduced immunological responses to the COVID-19 vaccine challenge. We can assume that prevention through vaccination can reduce the risk of complications or death from COVID-19 in patients in early palliative oncology care.

Seroprevalence of anti-SARS-CoV-2 antibodies in household domestic ferrets (Mustela putorius furo) in Spain, 2019-2023.

SARS-CoV-2 is the causal agent of Coronavirus Disease 2019 (COVID-19) in humans that emerged in late 2019. This virus is able to infect humans and different animal species. Among pets, cats and ferrets are more susceptible to be infected by the SARS-CoV-2. Epidemiological studies are an important tool to provide information under natural conditions of exposure to SARS-CoV-2 virus. In comparison to cats, limited epidemiological studies have been performed in domestic ferrets (Mustela putorius furo) reporting the presence of antibodies in this species. This study analysed the presence of anti-SARS-CoV-2 antibodies in 432 cliend-owned ferrets from different geographical areas of Spain during the different waves of COVID-19 outbreaks from December 2019 to May 2023 (42 months). For this purpose, anti-SARS-CoV-2 antibodies were detected by an enzyme-linked immunosorbent method (ELISA) using the receptor binding domain (RBD) of Spike antigen and confirmed by serum virus neutralization assay. Eighteen of the 432 ferrets included were seroreactive by the in-house ELISA (4.17%, 95% Confidence Interval (CI): 2.65-6.49). In this sense, the wave of COVID-19 with the higher number of seropositive ferrets occurred during the seventh wave when the different Omicron subvariants were the dominant virus variants. Our results suggest that the risk of SARS-CoV-2 transmission in domestic ferrets in natural conditions is low. Further research is need to evaluate the potential risk of transmission of SARS-CoV-2 from human to pets.

Domestic and wild animal samples and diagnostic testing for SARS-CoV-2.

From the first cases in 2019, COVID-19 infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have resulted in over 6 million human deaths in a worldwide pandemic. SARS-CoV-2 is commonly spread from human to human through close contact and is capable of infecting both humans and animals. Worldwide, there have been over 675 animal outbreaks reported that resulted in over 2000 animal infections including domestic and wild animals. As the role of animal infections in the transmission, pathogenesis, and evolution of SARS-CoV-2 is still unfolding, accurate and reliable animal diagnostic tests are critical to aid in managing both human and animal health. This review highlights key animal samples and the three main diagnostic approaches used for animal testing: PCR, serology, and Next Generation Sequencing. Diagnostic results help inform (often difficult) clinical decision-making, but also possible ways to mitigate spread among pets, food supplies, or wildlife. A One Health approach has been key to monitoring the SARS-CoV-2 pandemic, as consistent human-animal interactions can lead to novel variants. Having multiple animal diagnostic tests for SARS-CoV-2 available is critical to ensure human, animal, and environmental health.

Broad immunogenic spectrum of monovalent and trivalent foot-and-mouth disease virus vaccines containing O1 campos, A24 cruzeiro and A Argentina 2001 strains against circulating viral lineages in cattle and pigs.

FMD remains endemic in many Asian and African countries where multiple variants of serotypes O and A, among others, currently circulate. Due to lack of cross-protection between serotypes and incomplete protection between some strains even within a serotype, an important challenge for the application of effective vaccination programs is to select highly immunogenic and widely cross-reactive vaccine strains. Adaptation of a candidate field virus for use as a vaccine can be quite complex, so that whenever possible, the use of well-established vaccine viruses could have enormous advantages. FMD vaccine strains harmonized for use in South America have shown excellent results in FMD control, not only in the region, where it is still used systematically as a preventive measure, but also more recently in some Asian countries. To gain further insight into the immunogenic spectrum of these strains, VN tests (VNT) were performed with sera from cattle and/or pigs vaccinated with monovalent (type O) or trivalent (types O and A) formulations against 122 type O and 32 type A field viruses isolated from 35 countries in Asia and Africa, belonging to different lineages. Almost all VNT titers obtained were within the expected protective level, indicating the wide immunogenic spectrum of high potency FMD vaccines formulated with O1 Campos, A24 Cruzeiro and A Argentina 2001 South American vaccine strains belonging to EURO-SA topotypes against currently active viruses from other topotypes. These in vitro results are in line with previously reported in vivo challenge tests in pigs against three A/ASIA/Sea-97 isolates and two isolates belonging to type O lineages O/SEA/Mya-98 and O/ME-SA/Ind-2001e.

Investigation of Potency and Safety of Live-Attenuated Peste des Petits Ruminant Virus Vaccine in Goats by Detection of Cellular and Humoral Immune Response.

The peste des petits ruminant (PPR) virus is a transboundary virus found in small domestic ruminants that causes high morbidity and mortality in naive herds. PPR can be effectively controlled and eradicated by vaccinating small domestic ruminants with a live-attenuated peste des petits ruminant virus (PPRV) vaccine, which provides long-lasting immunity. We studied the potency and safety of a live-attenuated vaccine in goats by detecting their cellular and humoral immune responses. Six goats were subcutaneously vaccinated with a live-attenuated PPRV vaccine according to the manufacturer's instructions, and two goats were kept in contact. Following vaccination, the goats were monitored daily, and we recorded their body temperature and clinical score. Heparinized blood and serum were collected for a serological analysis, and swab samples and EDTA blood were collected to detect the PPRV genome. The safety of the used PPRV vaccine was confirmed by the absence of PPR-related clinical signs, a negative pen-side test, a low virus genome load as detected with RT-qPCR on the vaccinated goats, and the lack horizontal transmission between the in-contact goats. The strong humoral and cellular immune responses detected in the vaccinated goats showed that the live-attenuated PPRV vaccine has a strong potency in goats. Therefore, live-attenuated vaccines against PPR can be used to control and eradicate PRR.

The dynamics of neutralizing antibodies against SARS-CoV-2 in cats naturally exposed to virus reveals an increase in antibody activity after re-infection.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causative agent of Coronavirus Disease 2019 in humans. To date, little is known about the persistence of antibodies against SARS-CoV-2 in animals under natural conditions, in particular susceptible pets such as cat. This study reports the detection and monitoring of the humoral response against SARS-CoV-2 including the detection of immunoglobulins G specific for receptor binding domain of SARS-CoV-2 spike protein by an enzyme-linked immunosorbent assay and neutralizing antibodies by virus neutralization assay. Results showed that these antibodies last longer than 16 months in two naturally apparently healthy infected cats with the absence of clinicopathological findings during the follow-up. Moreover, re-infection is also possible with an important increase in virus neutralization test titers in both animals with no evident systemic signs found during each physical examination and with values of hematologic and biochemical parameters inside the normal reference intervals. Our results confirm a slow but progressive decrease of the kinetics and immunity of neutralizing antibodies in cats after the infection. Furthermore, similar to humans SARS-CoV-2 reinfection can stimulate an increase of the neutralizing antibodies determined by these two serological techniques in domestic cats.

Serological evidence of circulation of West Nile virus in equids in Algerian eastern drylands and its epidemiological risk factors.

In order to determine the prevalence of equine infectious anemia virus (EIAV), Usutu virus (USUV), and West Nile virus (WNV) in eastern Algerian drylands, 340 sera from distinct equids have been collected from 2015 to 2017. Serological analysis for the presence of antibodies against EIAV and flaviviruses was performed using commercially available ELISAs. Sera detected positive, doubtful, or negative close to the doubtful threshold in flavivirus ELISA were tested by the virus neutralization test (VNT), using WNV and USUV strains. The prevalence of WNV antibodies with ELISA was 11.47% (39/340) against 13.53% (46/340) by WNV VNT. EIAV antibodies were not detected in any samples. WNV seroprevalence varies with species, breed and location of horses. Only, one equid was positive for both WNV and USUV neutralizing antibodies. This is the first screening on equids sera of EIAV and USUV in Algeria. This study indicate that WNV and possibly USUV have circulated/are circulating in the Algerian equine population, unlike EIAV does not seem to be present.

A cross-sectional serosurvey of SARS-CoV-2 and co-infections in stray cats from the second wave to the sixth wave of COVID-19 outbreaks in Spain.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causative agent of Coronavirus Disease 2019 in humans. Among domestic animals, cats are more susceptible to SARS-CoV-2 than dogs. The detection of anti-SARS-CoV-2 antibodies in seemingly healthy cats and/or infected cats which are in close contact with infected humans has been described. The presence of animals that tested positive by serology or molecular techniques could represent a potential transmission pathway of SARS-CoV-2 that can spill over into urban wildlife. This study analyses the seroprevalence variation of SARS-CoV-2 in stray cats from different waves of outbreaks in a geographical area where previous seroepidemiological information of SARS-CoV-2 was available and investigate if SARS-CoV-2-seropositive cats were exposed to other co-infections causing an immunosuppressive status and/or a chronic disease that could lead to a SARS-CoV-2 susceptibility. For this purpose, a total of 254 stray cats from Zaragoza (Spain) were included. This analysis was carried out by the enzyme-linked immunosorbent assay using the receptor binding domain of Spike antigen and confirmed by serum virus neutralization assay. The presence of co-infections including Toxoplasma gondii, Leishmania infantum, Dirofilaria immitis, feline calicivirus, feline herpesvirus type 1, feline leukemia virus and feline immunodeficiency virus, was evaluated using different serological methods. A seropositivity of 1.57% was observed for SARS-CoV-2 including the presence of neutralizing antibodies in three cats. None of the seropositive to SARS-CoV-2 cats were positive to feline coronavirus, however, four SARS-CoV-2-seropositive cats were also seropositive to other pathogens such as L. infantum, D. immitis and FIV (n = 1), L. infantum and D. immitis (n = 1) and L. infantum alone (n = 1).Considering other pathogens, a seroprevalence of 16.54% was detected for L. infantum, 30.31% for D. immitis, 13.78%, for T. gondii, 83.86% for feline calicivirus, 42.52% for feline herpesvirus type 1, 3.15% for FeLV and 7.87% for FIV.Our findings suggest that the epidemiological role of stray cats in SARS-CoV-2 transmission is scarce, and there is no increase in seropositivity during the different waves of COVID-19 outbreaks in this group of animals. Further epidemiological surveillances are necessary to determine the risk that other animals might possess even though stray cats do not seem to play a role in transmission.

Evaluation of diagnostic performance of H-based blocking ELISA for specific detection of peste des petits ruminants in domestic sheep, goats, cattle and camels.

INTRODUCTION: Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats, peste des petits ruminants (PPR), which is targeted for global control and eradication by 2030. The serological diagnostic tool kits for accurate diagnosis of PPR have inherent strengths and weaknesses that require parallel validation and optimization across animal species. Thus, the objective of this study was to evaluate diagnostic performance of haemagglutinin based PPR blocking ELISA (HPPR- b-ELISA), that was developed by Africa Union Pan African Veterinary Vaccine Center for specific detection of anti- PPRV antibodies. METHODS: In preliminarily investigation, diagnostic performance of the HPPR-b-ELISA®, commercial PPR competition ELISA (c-ELISA) and virus neutralization test (VNT) were compared for the detection of anti-PPRV antibodies in goats, sheep, cattle and camels. RESULTS: The sensitivity and specificity of HPPR- b-ELISA® were 79.55 and 99.74%, respectively, compared to c-ELISA. The HPPR- b-ELISA® was in perfect agreement (κ = 0.86) with the c-ELISA in all sera collected from goats, sheep and cattle. There was almost perfect agreement between the species of goats (κ = 0.82) and sheep (κ = 0.98), while the agreement was substantial in cattle (κ = 0.78) and no agreement was observed in camels (κ = 0.00). Similarly, the sensitivity and specificity of the HPPR b-ELISA were 80 and 96.36%, respectively compared to VNT with almost perfect agreement in goats (κ = 0.83) and sheep (κ = 0.89), moderate in cattle (κ = 0.50) and none in camels (κ = 0.00). CONCLUSION: Our study revealed that HPPR- b-ELISA is a suitable and valid method that can alternatively be used for screening and monitoring of PPR in sheep, goats and cattle except for camels.

COVID-19 serum can be cross-reactive and neutralizing against the dengue virus, as observed by the dengue virus neutralization test.

OBJECTIVES: Observing the serological cross-reactivity between SARS-CoV-2 and dengue virus (DV), we aimed to elucidate its effect on dengue serodiagnosis and infectivity in a highly dengue-endemic city in India. METHODS: A total of 52 COVID-19 (reverse transcription-polymerase chain reaction [RT-PCR] positive) serum samples were tested in rapid lateral flow immunoassays and DV immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) to detect DV or SARS-CoV-2 IgG/immunoglobulin M. The COVID-19 antibody (Ab) positive samples were subjected to a virus neutralization test (Huh7 cells) using DV type 1 (DV1) clinical isolate. RESULTS: Most (93%) of the SARS-CoV-2 Ab-positive serum samples cross-reacted with DV in rapid or ELISA tests. All were DV RNA and nonstructural protein 1 (NS1) antigen-negative. COVID-19 serum samples that were DV cross-reactive neutralized DV1. Of these, 57% had no evidence of DV pre-exposure (DV NS1 Ab-negative). The computational study also supported potential interactions between SARS-CoV-2 Ab and DV1. CONCLUSION: DV serodiagnosis will be inconclusive in areas co-endemic for both viruses. The COVID-19 pandemic appears to impart a protective response against DV in DV-endemic populations.

Application of Noninvasive Tools to Decide the Need for Beta-Blockers for Variceal Bleeding Prophylaxis in Compensated Advanced Liver Disease: A Decision Curve Analysis.

Background and aims: Noninvasive tools (NITs) reliably categorise patients with compensated advanced chronic liver disease (cACLD) into high-risk and low-risk group for harbouring varices needing treatment. Here, we assess the ability of these NITs to predict the need for nonselective beta-blockers at baseline based on risk of variceal bleeding (VB) on follow-up. Methods: This was a retrospective multicentre analysis of patients with cACLD categorised at baseline into different risk groups by NITs (Baveno-VI, expanded Baveno-VI, platelet-albumin, platelet-model for end-stage liver disease (MELD) and anticipate study platelet criteria) and by endoscopy (high risk vs low risk/no varices). VB event rates on follow-up were estimated in different risk strata. Decision curve analysis (DCA) was used to estimate the benefit of administering nonselective beta-blockers (NSBB) using NITs over endoscopic classification at different threshold probabilities of VB event rates and estimating the number needed to treat (NNT) to identify one additional bleeder over endoscopy. Results: A total of 1284 patients (mean age: 44.7 ± 13.5 years, 72.4% males) of hepatitis B (29.2%), nonalcoholic fatty liver disease (24.9%), hepatitis C (20.1%), and alcohol (17.5%)-related cACLD were included with 323 (25.2%) having high-risk varices. Ninety-eight (7.6%) patients developed VB over a median follow-up of 20 (9-35) months. The 1-year and 3-year rate of VB with all NITs was 5.7-7.4% and 13.2-16.4% among high-risk and 0-2.3% and 0-5% among low-risk subgroups, respectively (P < 0.001) in both viral and nonviral aetiologies. Among patients classified as low risk on Baveno-VI criteria, none developed VB on follow-up. At thresholds of <3% event rate of VB, Baveno-VI (NNT-176), platelet-albumin (NNT-576) and anticipate platelet (NNT-233) criteria were superior, whereas endoscopic stratification was superior above this event rate on DCA. Conclusions: The use of both elastography and blood-based NITs at baseline can accurately identify the need for NSBB for VB prophylaxis in patients of cACLD on follow-up.

Borderline and weakly positive antibody levels against the S-protein of SARS-CoV-2 exhibit limited agreement with virus neutralization titres.

The presence of neutralizing antibodies against SARS-CoV-2 in a large number of people is - besides cellular immunity - important to overcome the SARS-CoV-2 pandemic. While determination of neutralizing antibodies via virus neutralization tests are laborious, assays to determine the antibody levels serologically are fully automated and widely available. Correlations between these methodologies were recently given by the manufacturers, however performance in samples close to the cut off value have not yet been fully validated. Thus, we analysed 22 borderline and low positive (<100 BAU/ml) samples and 9 high positive (≥ 100 BAU/ml) from infected and/or vaccinated individuals and compared the SARS-CoV-2 IgG II Quant assay (Abbott), LIAISON SARS-CoV-2 TrimericS IgG (Diasorin), Elecsys Anti-SARS-CoV-2 S (Roche), and SARS-CoV-2 IgG (Siemens) with results obtained from a virus neutralization test. Based on the cut off values given by Abbott, Diasorin, Roche, and Siemens, the positive serologic results were concordant with the virus neutralization test in 100%, 76%, 88%, and 71%, respectively, while in turn, negative ones were in agreement in 29%, 79%, 93%, and 86%, respectively. In conclusion, weakly positive, serologic results are challenging to correctly predict the presence of neutralizing antibodies. Our study suggests, that different cut off values (for positivity vs. presence of neutralizing antibodies) could improve the test's performance, but determination thereof requires more samples to be analysed.

Serological anti-SARS-CoV-2 neutralizing antibodies association to live virus neutralizing test titers in COVID-19 paucisymptomatic/symptomatic patients and vaccinated subjects.

A large number of immunoassays have been developed to detect specific anti-SARS-CoV-2 antibodies; however, not always they are functional to neutralize the virus. The reference test for the anti-spike neutralizing antibodies (nAbs) ability to counteract the viral infection is the virus neutralization test (VNT). Great interest is developing on reliable serological assays allowing antibodies concentration and antibody protective titer correlation. The aim of our study was to detect nAbs serum levels in paucisymptomatic, symptomatic and vaccinated subjects, to find a cut-off value able to protect from virus infection. nAbs serum levels were detected by a competitive automated immunoassay, in association to VNT with the SARS-CoV-2 original and British variant strains. The median nAbs concentrations were: 281.3 BAU/ml for paucisymptomatics; 769.4 BAU/ml for symptomatics; 351.65 BAU/ml for the vaccinated cohort; 983 BAU/ml considering only the second dose vaccinated individuals. The original strain VNT analysis showed 1:80 median neutralization titers in paucisymptomatic and vaccinated subjects; 1:160 in symptomatic patients; 1:160 in the second dose groups. The British variant VNT analysis showed lower neutralization titers in paucisymptomatic and vaccinated groups (1:40); the same titer in symptomatic patients (1:160); the second dose group confirmed the original strain titer (1:160). In conclusion, our data showed optimal correlations with a proportional increase between neutralizing activity and antibody concentration, making nAbs detection a good alternative to virus neutralization assays, difficult to carry out in routine laboratories. Finally, ROC curve analysis established a cut-off of 408.6 BAU/ml to identify subjects with a low risk of infection.

Seroprevalence of Equine Herpesvirus 1 (EHV-1) and Equine Herpesvirus 4 (EHV-4) in the Northern Moroccan Horse Populations.

This study reports the first equine herpesvirus-1 (EHV-1) and equine herpesvirus-4 (EHV-4) seroprevalence investigation in horse populations of Morocco in 24 years. It also aims to determine antibody titers in horses vaccinated under field conditions with a monovalent EHV-1 vaccine. Blood samples were collected from 405 horses, including 163 unvaccinated and 242 vaccinated animals. They were tested using a commercial type-specific enzyme-linked immunosorbent assay (ELISA) and a virus neutralization test (VNT). Overall, 12.8% unvaccinated, and 21.8% vaccinated horses were positive for EHV-1. All samples were positive for EHV-4 when tested with the type-specific ELISA. In the vaccinated group, the VNT revealed a mean antibody titer of 1:49 for EHV-1 and 1:45 for EHV-4. The present study demonstrates that EHV-1 and EHV-4 are endemic in the horse populations in the north of Morocco, with prevalence differences between regions. Furthermore, horses vaccinated with a monovalent EHV-1 vaccine had low antibodies titers. This study highlights the necessity to establish and/or support efficient biosecurity strategies based on sound management of horses and characterize further and potentially improve the efficiency of the EHV vaccines and vaccination protocol used in the field.

Development and validation of an IgM antibody capture ELISA for early detection of Hendra virus.

Zoonotic transmission of Hendra virus (HeV) from primary hosts (pteropid bats) to horses, and, occasionally, onward adventitious spread to humans, is associated with high mortality rates in both affected secondary species. The introduction of an effective recombinant G protein vaccine for use in horses has been a major advance for the suppression of disease risk. However, equine HeV vaccination induces neutralising antibody that is indistinguishable from a post infection immune response when using most first line serology assays (eg. VNT and some ELISAs). We have constructed and evaluated an IgM antibody capture (MAC) ELISA which employs yeast expressed HeV nucleoprotein (N). All other serology tests use the G protein which does not detect early infection and is present in the current Hendra virus vaccine and may cause ambiguity in interpretation of results. Thus, this is the first test developed using a N protein which can successfully detect a recent (primarily within the last four weeks) infection of horses with HeV and is not affected by vaccination induced antibody. Testing a limited panel (21 samples) of post infection sera, a normal serum panel (288 samples) and a post vaccination panel (163 samples), we have estimated DSe to be 100 % (95 % CI, 83.9-100.0 %) and DSp to be 98.4 % (95 % CI, 96.8-99.4 %) relative to assigned serology results (VNT, ELISA and Luminex) for the test panels. The HeV IgM MAC ELISA is intended to supplement other molecular and serology test results, with selective use, and is the only serology test which can provide an indication for recent infection which is otherwise not available.