Characterization of a Cytopathogenic Reporter CSFV.

Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5'-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

Accuracy and efficacy of pre-dengue vaccination screening for previous dengue infection with five commercially available immunoassays: a retrospective analysis of phase 3 efficacy trials.

BACKGROUND: The tetravalent dengue vaccine (CYD-TDV) has been shown to provide protection against dengue disease over 5-year follow-up in participants with previous dengue infection, but increased the risk of dengue hospitalisation and severe dengue during long-term follow-up in those without previous dengue infection. WHO recommended pre-vaccination screening to identify those with previous dengue infection (ie, dengue seropositive) who would benefit from vaccination. We re-evaluated CYD-TDV efficacy in those identified as dengue seropositive using five commercially available immunoassays, and assessed immunoassay performance. METHODS: We included participants in the immunogenicity subsets of the phase 3 CYD14 (NCT01373281) and CYD15 (NCT01374516) CYD-TDV efficacy trials, which enrolled children aged 2-16 years in 2011-12 in five countries in the Asia-Pacific region (CYD14) and five Latin American countries (CYD15). Participants assessed had received at least one injection of study drug (CYD-TDV or placebo) and had baseline samples available. We tested baseline samples by IgG-based immunoassays to classify baseline dengue serostatus, using two ELISAs (EUROIMMUN and Panbio) and three rapid diagnostic tests (RDTs; TELL ME FAST, SD BIOLINE, and OnSite). Vaccine efficacy in preventing symptomatic, hospitalised, and severe virologically confirmed dengue was determined for participants who tested positive by each immunoassay. The specificity and sensitivity of each immunoassay was determined as percentage negative and positive agreement compared with the reference algorithm, which used dengue plaque reduction neutralisation test with 50% and 90% cutoffs and non-structural protein 1 IgG ELISA results to assign baseline serostatus. FINDINGS: Samples were available for 3967 participants, 2735 (69·0%) of whom were classified as seropositive by the reference algorithm. Vaccine efficacy against symptomatic virologically confirmed dengue in immunoassay-positive participants was high across all five immunoassays (EUROIMMUN ELISA 88·2% [95% CI 77·3 to 93·9], Panbio ELISA 87·6% [76·7 to 93·4], TELL ME FAST RDT 88·8% [67·0 to 96·2], SD BIOLINE RDT 82·8% [66·9 to 91·1], and OnSite RDT 89·7% [64·6 to 97·0]), as was vaccine efficacy against hospitalised virologically confirmed dengue (EUROIMMUN-ELISA 72·8% [38·9 to 87·9], Panbio ELISA 77·5% [52·8 to 89·3], TELL ME FAST RDT 92·4% [37·8 to 99·1], SD BIOLINE RDT 87·2% [54·5 to 96·4], and OnSite RDT 73·7% [-5·1 to 93·4]) and severe virologically confirmed dengue (EUROIMMUN ELISA 86·9% [-16·8 to 98·5], Panbio ELISA 91·3% [27·6 to 99·0], TELL ME FAST RDT 100·0% [not estimable to 100·0%], SD BIOLINE RDT 89·4% [9·6 to 98·8], and OnSite RDT 73·4% [-193·7 to 97·6]). The immunoassays exhibited high specificity (≥98·8% for all immunoassays apart from SD BIOLINE RDT) but variable sensitivities, with higher sensitivities observed for the ELISAs (EUROIMMUN 89·2% [87·9 to 90·3] and Panbio 92·5 [91·4 to 93·5]) than the RDTs (TELL ME FAST 52·5% [50·6 to 54·4], SD BIOLINE 71·1% [69·3 to 72·8], and OnSite 47·6% [45·7 to 49·5]). INTERPRETATION: Our findings suggest that these immunoassays could be used for pre-vaccination screening for CYD-TDV as tools to assist risk stratification until more sensitive and convenient tests become available. FUNDING: Sanofi Pasteur.

Predicting the presence and titre of rabies virus-neutralizing antibodies from low-volume serum samples in low-containment facilities.

Serology is a core component of the surveillance and management of viral zoonoses. Virus neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of serum and high biosafety containment can limit widespread use. Here, focusing on Rabies lyssavirus, a globally important zoonosis, we developed a pseudotype micro-neutralization rapid fluorescent focus inhibition test (pmRFFIT) that overcomes these limitations. Specifically, we adapted an existing micro-neutralization test to use a green fluorescent protein-tagged murine leukaemia virus pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen detection and enabling use in low-containment laboratories. We further used statistical models to generate rapid, quantitative predictions of the probability and titre of rabies virus-neutralizing antibodies from microscopic imaging of neutralization outcomes. Using 47 serum samples from domestic dogs with neutralizing antibody titres estimated using the fluorescent antibody virus neutralization test (FAVN), pmRFFIT showed moderate sensitivity (78.79%) and high specificity (84.62%). Despite small conflicts, titre predictions were correlated across tests repeated on different dates both for dog samples (r = 0.93) and in a second data set of sera from wild common vampire bats (r = 0.72, N = 41), indicating repeatability. Our test uses a starting volume of 3.5 µl of serum, estimates titres from a single dilution of serum rather than requiring multiple dilutions and end point titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. The pmRFFIT enables high-throughput detection of rabies virus-neutralizing antibodies in low-biocontainment settings and is suited to studies in wild or captive animals where large serum volumes cannot be obtained.

Concept of sample-specific correction of immunoassay results for precise and accurate IgG quantification in horse plasma.

The hyperimmune horse plasma (HHP), prepared through active immunisation of horses with an antigen of interest, is the most common starting material for antitoxin (animal antibody-based therapeutics) production. Precise IgG quantification in plasma is a prerequisite for accurate estimation of the purification process efficiency. Although immunoglobulins from HHP have been purified for over a century, there is still no in vitro method for precise and accurate determination of IgG content in HHP. For this reason, the purification process efficiency has been assessed by antibody activity measurements, mostly performed in vivo. Here we describe the development of a precise and accurate in vitro immunoassay for IgG quantification in HHP. We showed and highlighted that any difference in composition of IgG population between the standard and the sample, with respect to both IgG subclass distribution and antigen-specific IgG content, leads to inaccurate IgG quantification. We demonstrated that caprylic acid precipitation as the method for IgG isolation from horse plasma renders the composition of IgG population unchanged. This very efficient, fast, simple and inexpensive method was used to prepare internal, sample-specific reference IgG for each plasma sample, which was tested simultaneously to a respective plasma sample. Deviation of IgG quantity determined by ELISA for each sample-specific reference from its nominal value was used for correction of the results of respective plasma sample, which led to accurate and precise IgG quantification as shown by method validation. The here presented novel concept of sample-specific correction of immunoassay results could be widely applicable and easily introduced in different immunoassays for more accurate and precise plasma IgG quantification.

Detection of Antibodies to HCV E1E2 by Lectin-Capture ELISA.

Enzyme-linked immunosorbent assays (ELISAs) enable rapid detection and quantitation of antibodies in samples. Such assays can be highly sensitive and can be performed in most laboratories with basic equipment. Although detecting binding antibodies to the surface proteins of most pathogens by ELISA is not always indicative of antibody function, i.e., neutralizing activity of antibodies, the results can be used as a first step toward more in-depth analysis of antibody responses. Here we describe a method that can be used to standardize ELISAs for the detection of HCV envelope antibodies across laboratories and provide adaptations of the method to further characterize antibody responses in serum samples.

In Vitro Neutralization Assay Using Cultured Hepatitis C Virus.

The method outlined here enables evaluation of the neutralization potency of monoclonal and polyclonal antibodies against in vitro cultured hepatitis C virus (HCV). The high variation in envelope protein sequence among HCV isolates necessitates the inclusion of several isolates, spanning the major genotypes of HCV, in order to make strong conclusions concerning the cross-reactive neutralization potential of a given antibody. This would be particularly relevant for any neutralization experiments aimed at uncovering novel therapeutic- or vaccine-relevant antibodies. In addition, these assays can also be used to compare neutralization sensitivity of novel cultured HCV to that of previously characterized isolates.

Standardized Method for the Study of Antibody Neutralization of HCV Pseudoparticles (HCVpp).

Hepatitis C virus (HCV) pseudoparticles (HCVpp) are generated by cotransfection of HCV envelope (E1 and E2) genes along with a retroviral packaging/reporter construct into HEK293T cells. Enveloped particles bearing HCV E1E2 proteins on their surface are released through a retroviral budding process into the supernatant. Viral E1E2 glycoproteins facilitate a single round of receptor-mediated entry of HCVpp into hepatoma cells, which can be quantified by reporter gene expression. These HCVpp have been employed to study mechanisms of HCV entry into hepatoma cells, as well as HCV neutralization by immune sera or HCV-specific monoclonal antibodies.

Mouse Models for Studying HCV Vaccines and Therapeutic Antibodies.

In spite of the immense progress in hepatitis C virus (HCV) research, efforts to prevent infection, such as generating a vaccine, have not yet been successful. The high price tag associated with current treatment options for chronic infection and the spike in new infections concurrent with growing opioid abuse are strong motivators for developing effective immunization and understanding neutralizing antibodies' role in preventing infection. Humanized mice-both human liver chimeras as well as genetically humanized models-are important platforms for testing both possible vaccine candidates as well as antibody-based therapies. This chapter details the variety of ways humanized mouse technology can be employed in pursuit of learning how HCV infection can be prevented.

Test selection for antibody detection according to the seroprevalence level of Schmallenberg virus in sheep.

Schmallenberg virus (SBV), initially identified in Germany in 2011, spread rapidly throughout Europe causing significant economic losses in ruminant livestock. The ability to correctly detect emerging and re-emerging diseases such as SBV with reliable tests is of high importance. Firstly, we tested diagnostic performance, specificity, and sensitivity of three different assays used in SBV antibody detection using control sheep samples of determined status. After obtaining the results from the control samples, we assessed the potential of the assays to detect previously infected animals in field situations. The samples were investigated using IDEXX Schmallenberg virus Antibody Test Kit, ID Screen Schmallenberg virus Competition Multi-species ELISA and Serum Neutralisation Test (SNT). Analysis of control samples revealed that SNT was the most suitable test, which was therefore used to calculate concordance and test performance for the two other ELISA tests. To evaluate whether different assay performances had an impact under field conditions, sheep samples from two different contexts were tested: the emergence of SBV in Ireland and the re-emergence of SBV in Belgium. Comparing the results obtained from different assays to the non-reference standard assay SNT, we showed considerable differences in estimates of their sensitivity to detect SBV antibodies and to measure seroprevalence of the sheep flocks. Finally, a calculation of the number of randomly selected animals that needs to be screened from a finite flock, showed that SNT and ID.Vet are the most suitable to detect an introduction of the disease in low seroprevalence situations. The IDEXX ELISA test was only able to detect SBV antibodies in a higher seroprevalence context, which is not optimal for monitoring freedom of disease and surveillance studies.

A multi-laboratory study of diverse RSV neutralization assays indicates feasibility for harmonization with an international standard.

A current barrier to the standardized evaluation of respiratory syncytial virus (RSV) vaccine candidates is the wide variety of virus neutralization assay formats currently in use for assessing immunogenicity. Assay formats vary widely in labor intensiveness, duration, and sample throughput. Furthermore, the cell lines and virus strains used are not consistent among formats. The purpose of this multi-laboratory study was to assess the variability across a diverse array of assay formats that quantitate RSV neutralizing antibodies. Using a common specimen panel, the degree of overall agreement among existing assays was evaluated to inform on the need for harmonization of assay results. A total of 12 laboratories participated in the blinded survey study by testing a panel comprised of 57 samples chosen to span the reportable titer range of the assays. An independent statistical analysis was conducted to measure overall agreement of assay results. This analysis showed that precision was consistently high, whereas agreement varied widely among assays. To examine whether agreement could be improved, we conducted a harmonization exercise using a variety of sample types as pseudo standards. The results showed that the level of agreement could be improved, and provided information on the suitability of samples for developing an international standard.

Generation of a recombinant West Nile virus stably expressing the Gaussia luciferase for neutralization assay.

West Nile virus (WNV) is a neurotropic human pathogen that has caused increasing infected cases over recent years. There is currently no licensed vaccine or effective drug for prevention and treatment of WNV infection in humans. To facilitate antiviral drug discovery and neutralizing antibody detection, a WNV cDNA clone containing a luciferase reporter gene was constructed through incorporating Gaussia luciferase (Gluc) gene within the capsid-coding region of WNV genome. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated luciferase reporter WNV (WNV-Gluc) and the stable WNV-Gluc with high titers (>10(7)PFU/ml) was obtained through plaque purification. Luciferase activity was used to effectively quantify the viral production of WNV-Gluc. Using the reporter virus WNV-Gluc, we developed a luciferase based assay in a 12-well format for evaluating neutralizing antibodies. The reporter virus could be a powerful tool for epidemiological investigation of WNV, vaccine evaluation, antiviral drug screening, and the study of WNV replication and pathogenesis.

Serum neutralization with different types and subtypes of bovine herpesvirus 1 and 5 / Soroneutralização com diferentes tipos e subtipos de herpesvírus bovinos 1 e 5

The serum neutralization (SN) test is the gold standard method to measure neutralizing antibodies to bovine herpesviruses. However, in view of the further subdivisions of bovine herpesviruses in types/subtypes, defining which virus to use at challenge in SN tests may be difficult. In view of that, this study was carried out to re-evaluate (SN) sensitivity with different types/subtypes of bovine herpesviruses types 1 (BoHV-1) and 5 (BoHV-5) as challenge viruses. Bovine sera (n=810) were collected from two distinct geographic regions and tested by SN with three type 1 viruses (BoHV-1.1 strains "Los Angeles" and "EVI123/98"; BoHV-1.2a strain "SV265/96") and three type 5 viruses (BoHV-5a strain "EVI88/95"; BoHV-5b strain "A663" and BoHV-5c "ISO97/95"). SN tests were performed with a 1 hour incubation of the serum-virus mixtures at 37ºC against 100 TCID50 of each of the viruses. SN sensitivity varied greatly depending on the challenge virus used in the test. The highest sensitivity (327 positive/810 total sera tested; 40.37 percent) was attained when the positive results to the six viruses were added together. No association could be found between any particular type or subtype of virus and the sensitivity of the test. When positive results to each single strain were considered, SN sensitivity varied from 41.7 percent to 81.7 percent, depending on the virus and the geographic region of origin of the sera. Variation was detected even when challenge viruses belonged to the same subtype, where disagreement between positive results reached 41 percent...

O teste de soroneutralização (SN) é o método padrão para a mensuração de anticorpos neutralizantes para herpesvírus bovinos. Entretanto, com as subdivisões propostas destes agentes em tipos e subtipos, a definição de qual amostra utilizar como virus de desafio à SN pode ser difícil. Em vista disso, este estudo foi realizado para re-avaliar a sensibilidade de testes de SN utilizando diferentes tipos e subtipos de herpesvírus bovinos tipos 1 (BoHV-1) e 5 (BoHV-5) como amostras de desafio. Soros bovinos (n=810) foram coletados de duas regiões geográficas distintas e testados frente a amostras do tipo 1 (BoHV-1.1: amostras "Los Angeles" e "EVI123/98", BoHV-1.2a: amostra "SV265/96") e três amostras do tipo 5 (BoHV-5a: "EVI88/95"; BoHV-5b: "A663" e BoHV-5c "ISO97/95"). Os testes de SN foram realizados com incubação de 1 hora a 37ºC da mistura soro-vírus, frente a 100 doses infectantes para 50 por cento dos cultivos celulares (DICC50) de cada um dos vírus. A sensibilidade da SN variou grandemente em função do vírus utilizado no teste. A maior sensibilidade (327 soros positivos/810 soros testados; 40.37 por cento) foi alcançada quando os resultados positivos frente aos seis diferentes vírus foram somados. Nenhuma associação foi detectada entre determinado tipo/subtipo de vírus e a sensibilidade do teste. Quando resultados positivos frente a cada vírus foram considerados isoladamente, a sensibilidade da SN variou entre 41,7 por cento a 81,7 por cento, dependendo do vírus de desafio e da região geográfica de origem das amostras de soro. Variação foi detectada mesmo quando as amostras de desafio pertenciam a um mesmo subtipo; a discrepância entre os resultados positivos atingiu até 41 por cento. Estes resultados indicam que testes de SN contra amostras isoladas de vírus podem apresentar uma sensibilidade notadamente baixa; o emprego de diferentes amostras de vírus de desafio pode aumentar consideravelmente a sensibilidade da prova...

A rapid serum neutralization test in microplates for the detection of antibodies to hog cholera virus.

The fluorescent antibody serum neutralization (FASN) test for the detection of antibodies to hog cholera virus was developed utilizing 96-well and Terasaki microplates. This microtechnique, especially when performed in Terasaki plates, offers some advantage if compared with conventional FASN in coverslip cell cultures, being easier and more rapid, saving of reagents and allowing simple microscopic observation.

Automated microtransfer technique for the assay of poliovirus- and mumps virus-neutralizing antibodies.

Use of an automated apparatus to quantitate mumps virus- and poliovirus-neutralizing antibody is described. The automated titration equipment affords savings in effort, time, and reagents in conducting large-scale surveys for the determination of mumps- and poliovirus-neutralizing antibodies. This technique has been found to be reproducible and gives results comparable to other antibody assay methods.