A double antibody sandwich ELISA for rapid diagnosis of virus infection and to measure the humoral response against infectious bursal disease on clinical material

A double antibody sandwich ELISA (DAS-ELISA) was developed and employed for simultaneous direct detection of infectious bursal disease virus (IBDV) from bursal samples and to measure the humoral response, using the same basic immunoreagents, the purified and non-purified antigen, capture antibody and chicken hyperimmune sera were prepared, and standardized for this purpose, the DAS-ELISA was applied to both 80 bursal suspensions and 224 corresponding serum samples from vaccinated and non-vaccinated commercial hocks, Bursae samples were collected at 2 weeks of age, and submitted to histological examination, virus isolation in specific pathogen-free chickens embryos, and the DAS-ELISA technique, Serum titres obtained in indirect ELISA and serum neutralization test were compared with those in DAS-ELISA, the agreement was 80% between DAS-ELISA, and the conventional techniques, with high sensitivity (87%) and specificity (90%).

A liquid phase blocking ELISA for the detection of antibodies against infectious bronchitis virus

A liquid phase blocking ELISA (LPB-ELISA) was developed for the detection and measurement of antibodies against infectious bronchitis virus (IBV). The purified and nonpurified virus used as antigen, the capture and detector antibodies, and the chicken hyperimmune sera were prepared and standardized for this purpose. A total of 156 sera from vaccinated and 100 from specific pathogen-free chickens with no recorded contact with the virus were tested. The respective serum titers obtained in the serum neutralization test (SNT) were compared with those obtained in the LPB-ELISA. There was a high correlation (r2 = 0.8926) between the two tests. The LPB-ELISA represents a single test suitable for the rapid detection of antibodies against bronchitis virus in chicken sera, with good sensitivity (88%), specificity (100%) and agreement (95.31%).

Herpes Virus Bovino tipo 1 (BoHV-1) como posible causa de encefalitis en bovinos de la región del Magdalena Medio Colombiano. Estudio serológico y análisis epidemiológico

The bovine Herpesvirus type 1 and type 5 (BoHV-1 and BoHV-5), causing diseases and significant economic losses in farms of worldwide. Both affect the nervous system of cattle, although BoHV-5 has been the most associated with this type of pathogenesis. Given the death of animals with nervous symptoms and negative diagnoses for rabies virus in the area of study, this research focused on the detection of positive reactors to bovine herpes virus serum neutralization. We collected 518 blood samples from animals without Herpesvirus vaccine, in the municipalities of Caparrapi, Cimitarra, Honda and Victoria, in the Middle Magdalena River Region. In addition, epidemiological information useful to discuss neurological disease was collected through primary and secondary sources. For the analysis of data was used chi-square test by identification of relationship between evidence of viral infection and the variables recorded. The results revealed that 286 cases were positive for Herpesvirus infection, corresponding to a prevalence of 55.5%, however, there was no statistical relationship (p < 0.05) between the presence of antibodies and the variables analyzed. In conclusion, some cases of neurological disease in cattle in this region could be due to infection with herpes viruses. We discussed about the presence of BoHV-1 and BoHV-5 in the ambient, diagnosis and monitoring plans, as well as economic losses, which may cause in herds in this area.

Evaluation of an indirect ELISA method for the detection of chicken antibodies against infectious bronchitis virus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

First evidence of the emergence of novel putative infectious bronchitis virus genotypes in Cuba

The emergence of new infectious bronchitis virus (IBV) genotypes or serotypes along with the poor cross-protection observed among IBV serotypes have complicated the avian infectious bronchitis (IB) control programs in different geographic regions. In Cuba, the lack of genetic information regarding IBV and the increasing epidemiological importance of this virus in Cuban chicken flocks demand further characterization of IBV isolates. In the present work, studies of genetic diversity and phylogenetic relationships among recent IBV isolates from Cuban chicken flocks showing respiratory disorders were performed. Two putative genotypes genetically different to the Massachusetts genotype H120 strain used in the Cuban vaccination program were found in the flocks assessed. In addition, a potential nephropathogenic IBV isolate was found by first time in Cuba. (C) 2012 Elsevier Ltd. All rights reserved.

Avian coronavirus Spike Glycoprotein Ectodomain Shows a Low Codon Adaptation to Gallus gallus with Virus-Exclusive Codons in Strategic Amino Acids Positions

This is a study on the Avian coronavirus IBV and chicken host-relationship from the codon usage point of view based on fifty-nine non-redundant IBV S1 sequences (nt 1-507) from strains detected worldwide and chicken tissue-specific protein genes sequences from IBV-replicating sites. The effective number of codons (ENC) values ranged from 36 to 47.8, indicating a high-to-moderate codon usage bias. The highest IBV codon adaptation index (CAI) value was 0.7, indicating a distant virus versus host synonymous codons usage. The ENC x GC3 % curve indicates that both mutational pressure and natural selection are the driving forces on codon usage pattern in S1. The low CAI values agree with a low S protein expression and considering that S protein is a determinant for attachment and neutralization, this could be a further mechanism besides mRNA transcription attenuation for a low expression of this protein leading to an immune camouflage.

Humoral response to 2 inactivated bluetongue virus serotype-8 vaccines in South American camelids

BACKGROUND: Bluetongue virus serotype 8 (BTV-8) has caused disease in domestic ruminants in several countries of northern Europe since 2006. In 2008 a mass-vaccination program was launched in most affected countries using whole virus inactivated vaccines. OBJECTIVE: To evaluate 2 inactivated vaccines (Bovilis BTV 8; BTVPUR AlSap8) for immunogenicity and safety against BTV-8 in South American camelids (SAC) in a field trial. ANIMALS: Forty-two SAC (25 Alpacas, 17 Llamas) aged between 1 and 16 years. METHODS: The animals were vaccinated twice at intervals of 21 days. They were observed clinically for adverse local, systemic, or both reactions throughout the trial. Blood samples collected on days 0, 14, 21, 43, and 156 after vaccination were tested for the presence of BTV-8 virus by real time-polymerase chain reaction and of specific antibodies by competitive ELISA and a serum neutralization test. RESULTS: All vaccinated animals developed antibodies to BTV-8 after the 2nd administration of the vaccine. No adverse effects were observed except for moderate local swellings at the injection site, which disappeared within 21 days. Slightly increased body temperatures were only observed in the first 2 days after vaccination. The BTV was not detected in any of the samples analyzed. CONCLUSIONS AND CLINICAL IMPORTANCE: The administration of the 2 inactivated commercial vaccines was safe and induced seroconversion against BTV-8 in all vaccinated animals. The results of this study suggest that 2 doses injected 3 weeks apart is a suitable vaccination regimen for SAC.

Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and/or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive anti-VHSV-AB titres, but was negative in virus isolation. With IHNV, RT-PCR detected two positive samples not identified by virus isolation while in these fish the SPNT result had been questionable. One of the IHNV-positive samples represents the first detection of IHNV-RNA in wild brown trout in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus isolation. However, in a titration experiment under laboratory conditions, the sensitivity of RT-PCR was not significantly higher when compared with virus isolation.

Immunohistochemical diagnosis of persistent infection with Bovine Viral Diarrhea Virus (BVDV) on skin biopsies

Detection of persistent infection with BovineViral Diarrhea Virus (BVDV) is essential for both epidemiological and clinical reasons. In addition to the classical virological methods such as virus isolation in tissue culture, ELISA and RT-PCR, immunohistochemistry of skin biopsies has become a useful and reliable tool. Assuming that the presence of BVDV antigen in skin structures is restricted to persistent infection, this method could differentiate from transient infection. In order to answer this question, 6 calves were experimentally infected orally with a non-cytopathic genotype 1 BVDV strain belonging to the subtype k.The calves developed fever, mucopurulent nasal discharge, coughing and leucopenia with relative lymphopenia. Immunohistochemistry of skin biopsies taken daily up to day 13-post infection did not reveal any evidence of BVDV infection. BVDV was, however, isolated from blood samples on cell cultures. Anti-NS3-antibody-ELISA and serum neutralization tests showed that all six calves seroconverted. We conclude that in acute BVDV infections, with genotype 1 and the subtypes found in Switzerland (b, e, h and k) viral antigen is not found in epidermal structures of the skin. In contrast, persistently infected animals test positive for BVD viral antigen by immunohistochemistry of the skin.

Diagnostic gap in Bovine viral diarrhea virus serology during the periparturient period in cattle

Detection of antibodies against Bovine viral diarrhea virus (BVDV) in serum and milk by enzyme-linked immunosorbent assay (ELISA) is a crucial part of all ongoing national schemes to eradicate this important cattle pathogen. Serum and milk are regarded as equally suited for antibody measurement. However, when retesting a seropositive cow 1 day after calving, the serum was negative in 6 out of 9 different ELISAs. To further investigate this diagnostic gap around parturition, pre- and postcalving serum and milk samples of 5 cows were analyzed by BVDV antibody ELISA and serum neutralization test (SNT). By ELISA, 3 out of the 5 animals showed a diagnostic gap in the serum for up to 12 days around calving but all animals remained positive in SNT. In milk, the ELISA was strongly positive after birth but antibody levels decreased considerably within the next few days. Because of the immunoglobulin G (IgG)1-specific transport of serum antibodies into the mammary gland for colostrum production, the IgG subclass specificity of the total and the BVDV-specific antibodies were determined. Although all 5 animals showed a clear decrease in the total and BVDV-specific IgG1 antibody levels at parturition, the precalving IgG1-to-IgG2 ratios of the BVDV-specific antibodies were considerably lower in animals that showed the diagnostic gap. Results showed that BVDV seropositive cows may become "false" negative in several ELISAs in the periparturient period and suggest that the occurrence of this diagnostic gap is influenced by the BVDV-specific IgG subclass response of the individual animal.

Infection of cattle with Border disease virus by sheep on communal alpine pastures

The purpose of this study was to investigate whether sheep grazing communal alpine pastures with cattle can transmit Border disease virus (BDV) to cattle. A total of 1170 sheep and 923 cattle were tested for BDV using RT-PCR (sheep) and for pestivirus antibodies using an ELISA (cattle), respectively, before being moved to one of 4 pastures (A, B, C and D). Eight sheep from pasture C were viraemic. 396 of 923 cattle examined before the pasture season were seronegative. The latter were re-examined after the pasture season and 99 were seropositive or indeterminate. Antibody specificity was determined in 25 of these using a serum neutralization test (SNT). BDV infection was confirmed in 10 cattle and was considered likely in 8 others. BVDV infection was confirmed in 4 cattle and considered likely in 3 after pasturing. The study has shown that the transmission of BDV from sheep to cattle is possible on communal alpine pastures.

Investigation of border disease and bovine virus diarrhoea in sheep from 76 mixed cattle and sheep farms in eastern Switzerland

The purpose of this study was to examine the occurrence of sheep persistently infected with Border disease virus (BDV) on 76 mixed cattle and sheep farms and whether seroconversion to BDV infection occurred in cattle of these farms. Seroprevalence of BDV and bovine viral disease virus (BVDV) infection in sheep was also investigated. Quantitative RT-PCR for pestivirus detection and an ELISA to detect pestivirus antibodies were used in 2'384 and 2'291 ovine blood samples, respectively. Another 27 seropositive sheep from ten flocks underwent serum neutralization testing to differentiate between BDV and BVDV antibodies. A BDV titre that was at least four times higher than the BVDV titre was interpreted as the result of BDV infection. Titres against BVDV were interpreted in an analogous fashion. All examined sheep were pestivirus-negative, 310 sheep were seropositive, 119 had an indeterminate titre and 1'862 were seronegative. The flock seroprevalence ranged from 0.0 to 73.9 %. Three of the 27 flocks that underwent serum neutralization testing were interpreted as BDV-infected because of 6 sheep with higher BDV titres, and 6 flocks were interpreted as BVDV-infected because of 14 sheep with higher BVDV titres.

Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

Early events in poliovirus infection: virus-receptor interactions.

The interaction of poliovirus with its cell receptor initiates conformational changes that lead to uncoating of the viral RNA. Three types of genetic analyses have been used to study the poliovirus-receptor interaction: (i) mutagenesis of the poliovirus receptor (PVR), (ii) selection of viral mutants resistant to neutralization with soluble PVR, and (iii) selection of viral variants adapted to use mutant PVRs. The results of these studies show that a small portion of the first immunoglobulin-like domain of PVR contacts viral residues within a deep depression on the surface of the capsid that encircles the fivefold axis of symmetry. Viral capsid residues that influence the interaction with PVR are also found in locations such as the capsid interior that cannot directly contact PVR. These mutations might influence the ability of the capsid to undergo receptor-mediated conformational transitions that are necessary for high-affinity interactions with PVR.