Serum Virus Neutralization Test Research Articles

Serum Virus Neutralization Assay for Detection and Quantitation of Serum Neutralizing Antibodies to Influenza A Virus in Swine.

The serum virus neutralization (SVN) assay is a serological test used to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses (IAV) in swine to measure the titer of neutralizing antibodies postexposure, postvaccination, or after passive transfer of maternally derived antibody (MDA). Conventional SVN methods performed in vitro are based on inhibition of virus infectivity in cell culture in the presence of neutralizing antibodies in serum. Titer determination may be based on the presence or absence of cytopathic effect or evidence of viral infection using an immunoreactive technique. The SVN assay is relatively inexpensive using standard laboratory equipment, although it requires cell culture, more time and labor, and technical skill to conduct the assay compared to other serological methods. The SVN test may be used to evaluate the level of serological cross-reactivity between IAV exposure or vaccine antisera and heterologous influenza viruses that may correlate with cross-protection in the host.

Evidence of natural interspecific recombinant viruses between bovine alphaherpesviruses 1 and 5

Closely related bovine alphaherpesviruses 1 (BoHV-1) and 5 (BoHV-5) co-circulate in certain countries, rendering cattle co-infection possible. This is a prerequisite for BoHV recombination. Here, we report the first identification of homologous recombination between field isolates of BoHV-1 and BoHV-5, two alphaherpesviruses belonging to two distinct species with an average genomic similarity of 82.3%. Three isolates of BoHV-5, previously classified as subtype “BoHV-5b”, were phylogenetically studied and analyzed via eight PCR sequencing assays dispersed at regular intervals throughout the genome to discriminate between BoHV-1 and BoHV-5. In the phylogenetic analysis, differences of clustering were found in the UL27 gene which encodes the glycoprotein B (gB). We detected two recombination breakpoints in the open reading frame of the UL27 gene. We compared the amino acid sequences of the gB of BoHV-1.1 and 1.2, BoHV-5a and recombinant formerly named BoHV-5b (chimeric gB) and subsequently performed molecular modeling. All structures were alike and, simultaneously, similar to the chimeric gB. Neutralizing antibodies against BoHV-1, BoHV-5 and recombinant viruses were analyzed via serum virus neutralization test using polyclonal sera and a monoclonal antibody against gB to demonstrate an absence of viral escape for both assays.Our results show that homologous recombination between two related species of ruminant alphaherpesviruses can occur in natural field conditions. We found three recombinant field isolates, previously classified as BoHV-5b subtypes, between BoHV-1 and BoHV-5.

Immunization of horses with a polyvalent live-attenuated African horse sickness vaccine: Serological response and disease occurrence under field conditions

African horse sickness (AHS) is a non-contagious, insect-borne disease of equids caused by a RNA virus (AHSV), which belongs to the genus Orbivirus, family Reoviridae. The disease is endemic in sub-Saharan and western Africa, where prevention strictly depends upon vaccination. The present paper aims at evaluating the serological response and the occurrence of AHS in horses bred under field condition and regularly immunized using the commercially available live attenuated vaccine (LAV) produced by Onderstepoort Biological Products.The study was carried out in a farm located in the district of Windhoek (Namibia), where the disease is endemic. A total of 72 cross-breed horses, out of the 150 housed on the farm, were subdivided in six age groups, from 2 to 7years-old. Each group consisted of 12 heads which were born during the same breeding season and had undergone from four to nine vaccination courses. AHSV specific immune response was evaluated by serum-virus neutralization test. Data about the clinical occurrence of the AHS from 2006 to 2011 were made available. The immune response, in terms of number of seropositive horses and serum neutralizing titers, was quite variable among horses and against different serotypes. Neutralizing antibodies against all serotypes were recorded in all the horses only after eight vaccination courses at 6years of age onwards. Immune response to AHSV-5 and 9, which are not included in the LAV formulation, were also established. A severe AHS epidemic occurred in Namibia in 2011. On the farm under study, a total of 32 animals were clinically affected, 12 died, 11 of them were 2year-old or younger.Our data confirm that vaccination with LAV is a useful tool to reduce the severity of the disease in endemic areas. However, clinical and sometimes fatal AHS can still affect young vaccinated horses, thus highlighting the necessity to better understand the immune response to AHSV and to dispose of more effective vaccines.

Serum virus neutralization assay for detection and quantitation of serum-neutralizing antibodies to influenza A virus in swine.

The serum virus neutralization (SVN) assay is a serological test to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses in swine to measure the titer of neutralizing antibodies post-infection or after vaccination. Conventional SVN methods performed in vitro are based on inhibition of virus infectivity in cell culture in the presence of neutralizing antibodies. Titer determination may be based on the presence or the absence of cytopathic effect or the evidence of viral infection using an immunoreactive technique. The SVN assay is relatively inexpensive using standard laboratory equipment although it requires cell culture, more time and labor, and technical skill to conduct the assay compared to other serological methods. The SVN test is useful to evaluate the level of serological cross-reactivity between vaccine antisera and variant influenza viruses that may correlate with cross-protection in the host.

Humoral and T cell-mediated immune responses to bivalent killed bovine viral diarrhea virus vaccine in beef cattle

The objective of this research project was to evaluate the antibody and cell-mediated immune responses to a multivalent vaccine containing killed bovine viral diarrhea virus (BVDV) types 1 and 2. Twenty castrated male crossbred beef cattle (350–420 kg body weight) seronegative to BVDV were randomly divided into two groups of 10 each. Group 1 served as negative mock-vaccinated control. Group 2 was vaccinated subcutaneously twice, 3 weeks apart, with modified live bovine herpesvirus 1, parainfluenza 3 virus and bovine respiratory syncytial virus diluted in diluent containing killed BVDV type 1 (strain 5960) and type 2 (strain 53637) in an adjuvant containing Quil A, Amphigen, and cholesterol. Serum samples were collected from all cattle at days −21, 0, and days 21, 28, 35, 56 and 70 post-vaccination. Standard serum virus neutralization tests were performed with BVDV type 1 (strain 5960) and type 2 (strain 125C). Anticoagulated blood samples were collected at day 0, and days 28, 35, 56 and 70 post-vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated, stimulated with live BVDV type 1 (strain TGAN) and type 2 (strain 890) and cultured in vitro for 4 days. Supernatants of cultured cells were collected and saved for interferon gamma (IFNγ) indirect enzyme-linked immunosorbent assay (ELISA). Four-color flow cytometry was performed to stain and identify cultured PBMC for three T cell surface markers (CD4, CD8, and γδ TCR) and to detect the activation marker CD25 (α chain of IL-2 receptor) expression. The net increase in %CD25+ cells (Δ%CD25+) of each T cell subset of individual cattle was calculated. The results of all post-vaccination weeks of each animal were plotted and the areas under the curve of each T cell subset were statistically analyzed and compared between groups. The mean area under the curve of the Δ%CD25+ data for days 0–70 of all subsets, except CD4−CD8+γδ TCR− (cytotoxic) T cell subset of both BVDV types 1 and 2 stimulated cells, of the vaccinated group were significantly higher than the control group ( P < 0.05). IFNγ production by PBMC from the vaccinated group showed significantly higher results ( P < 0.05) than the control group in the BVDV types 1 and 2 stimulated cells for at least some time points after vaccination. The vaccinated group also had significantly ( P < 0.0001) higher neutralizing antibody titers than the control group from day 28 onward.

Lesions and transmission of experimental adenovirus hemorrhagic disease in black-tailed deer fawns.

Adenovirus infection was the cause of an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California during the latter half of 1993. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. To study transmission of adenovirus infection in deer and susceptibility of black-tailed deer (Odocoileus hemionus columbianus) fawns to adenovirus infection, six 3-6-month-old black-tailed fawns were divided into two treatment groups. One group was inoculated intravenously and the other group was inoculated through the mucous membranes of the eyes, nose and mouth with purified adenovirus. Each treatment group also included two additional fawns (four total) that were not inoculated but were exposed to inoculated animals (contact animals). One fawn served as a negative control. Between 4 and 16 days postinoculation, 8/10 fawns developed systemic or localized infection with lesions identical to lesions seen in animals with natural disease that died during the epizootic. Transmission was by direct contact, and the route of inoculation did not affect the incubation period or the distribution of the virus (systemic or the localized infection). Immunohistochemical analysis using polyclonal antiserum against bovine adenovirus type 5 demonstrated staining in endothelial cells of vessels in numerous tissues in animals with systemic infection and endothelial staining only in vessels subtending necrotic foci in the upper alimentary tract in animals with the localized form of the disease. All inoculated or exposed animals had staining in the tonsillar epithelium. Transmission electron microscopic examination of lung and ileum from two fawns with pulmonary edema and hemorrhagic enteropathy demonstrated endothelial necrosis and adenovirus virions in endothelial cell nuclei. Adenovirus was reisolated in black-tailed deer pulmonary artery endothelial cells using lung homogenate of the first fawn that developed systemic adenovirus infection. Serum virus neutralization test results suggest that this deer adenovirus is a new serotype.

Antibody titers to pseudorabies virus in piglets immunized with gIII deleted pseudorabies vaccine in a pseudorabies infected herd

The decrease in titer of PRV antibodies in serum was evaluated at 10, 37, 67, 109 and 173 days of age in 16 non-vaccinated pigs and 43 pigs vaccinated at 3, 67 and 80 days of age with a modified live TK/gIII gene deleted pseudorabies virus (PRV) vaccine. Serum samples were analyzed for antibodies to PRV by the serum-virus neutralization test (SN), a commercial competitive ELISA (CELISA), and the CELISA OMNIMARK PRV differential (OMD) diagnostic kit. At 10 days of age, all pigs had SN titers ≥ 1:4 and were CELISA+/OMD+, indicating circulating antibodies to field strains of PRV. At 109 days, all non-vaccinated pigs had SN titers < 1:4. Forty-five percent of vaccinated pigs had SN titers ≥ 1:4, 56% were CELISA positive and most were CELISA+/OMD−, indicating antibodies due to vaccination. At 24 weeks of age, all pigs had SN titers ≥ 1:4 and were CELISA+/OMD+ due to exposure to field strains. Although circulating maternal antibodies interfere with the development of active immunity, vaccination at 3 days of age resulted in detectable antibodies by 67 days of age, and a limited immune response could be measured at 109 days of age.

Excretion of porcine reproductive and respiratory syndrome virus in semen after experimentally induced infection in boars

Summary Four boars intranasally inoculated with porcine reproductive and respiratory syndrome (PRRS) virus were monitored for 56 days after exposure for changes in semen characteristics and for the presence of virus in the semen. Clinically, 2 of 4 boars had mild respiratory signs of 1 day's duration after infection. Changes in appetite, behavior, or libido were not detected. All boars seroconverted on the indirect fluorescent antibody and serum virus neutralization tests by day 14 after inoculation. Virus was isolated from serum between days 7 and 14 after inoculation. During the monitoring period, semen volume decreased and pH correspondingly increased; however, this change began 7 to 10 days prior to infection. Differences in sperm morphologic features, concentration, or motility between the preinfection and postinfection samples were not observed. The PRRS virus was detected in semen at the first collection in each of the 4 boars (ie, 3 or 5 days after challenge exposure). Virus was detected in nearly all semen samples collected from the 4 infected boars through days 13, 25, 27, and 43, respectively. Neither gross nor microscopic lesions attributable to PRRS virus were observed in tissues collected at the termination of the experiment (day 56), and virus isolation results from reproductive tissues were negative.

Evaluation of the safety and efficacy of a thymidine kinase, inverted repeat, gI, and gpX gene-deleted pseudorabies vaccine.

A thymidine kinase (TK), inverted repeat, glycoprotein I (gI) and glycoprotein X (gpX) gene-deleted modified live virus pseudorabies vaccine was evaluated for safety in swine and for efficacy in protecting swine against challenge with pseudorabies virus (PRV). Safety was evaluated by inoculating pregnant gilts intravenously and 3-day-old pigs intracerebrally with the vaccine. Efficacy was evaluated by 1) vaccinating 3-day-old pigs with a minimal protective dose intranasally and then challenging with PRV 3 weeks postvaccination or 2) vaccinating weaned pigs with a standard field dose intramuscularly and then challenging with PRV 4 weeks postvaccination. The pigs vaccinated intranasally remained clinically normal following vaccination and challenge with PRV. The pigs vaccinated intramuscularly remained clinically normal following vaccination, but mild respiratory signs were seen in some of the vaccinated pigs following challenge with PRV. Humoral immune response was evaluated with enzyme-linked immunosorbent assays (ELISAs) and a serum virus neutralization test. All of the intramuscularly vaccinated pigs became gI and gpX positive on differential ELISAs following challenge. All of the intranasally vaccinated pigs were seropositive on the indirect gI ELISA following challenge, but not all of the pigs were seropositive on the blocking gI ELISA or the gpX ELISA 3 weeks postchallenge.

A Study Comparing the Immunologic Responses of Swine to Pseudorabies Viral Antigens Based on the ELISA, Serum Virus Neutralization, and Latex Agglutination Tests

A study of pseudorabies virus (PRV)-vaccinated pigs comparing the immune responses detected by the latex agglutination test (LAT) with responses detected by other routine tests for pseudorabies antibodies indicated that LAT was more sensitive than either the enzyme-linked immunosorbent assay (ELISA) or the serum virus neutralization test (SVNT). The LAT detected antibodies sooner than ELISA and SVNT in unvaccinated pigs after challenge with virulent PRV. The specificities of the 3 tests were found to be near 100%. The LAT is a good alternative to SVNT or ELISA for detection of PRV-specific antibodies.

ANTIGENIC COMPARISONS BETWEEN HERPESVIRUSES ISOLATED FROM FALLOW DEER IN ALBERTA AND THE VIRUSES OF INFECTIOUS BOVINE RHINOTRACHEITIS, EQUINE RHINOPNEUMONITIS AND DN-599, A NON-IBR BOVINE HERPESVIRUS

Antigenic comparison studies of three herpesviruses isolated from fallow deer (Dama dama) in Alberta and herpesviruses from some domestic species were carried out by the alpha serum-virus neutralization test. Complete cross neutralization was demonstrated among the deer herpesviruses and equine herpesvirus type 1.

Agar-gel immunodiffusion assay for pseudorabies virus antibody

Soluble antigen prepared from pseudorabies virus-infected embryonic pig kidney cells was used to demonstrate the presence of pseudorabies virus antibodies in sera taken from naturally and experimentally infected swine. Antibody could be detected by agar-gel immunodiffusion as early as 14 days postexposure and was demonstrable at least 3 months after experimental infection (the longest period tested). The reliability of the agar-gel immunodiffusion test was comparable to that of the microtitration serum-virus neutralization test commonly used in diagnostic laboratories. One advantage of the agar-gel immunodiffusion test was that severely hemolyzed and cytotoxic serum samples could be tested with confidence. The test is simple, rapid, and inexpensive and could be easily adopted by diagnostic laboratories that receive requests for pseudorabies virus antibody determinations in swine sera.

Vaccination of Turkeys against Newcastle Disease

Turkeys given primary vaccination with an inactivated Newcastle (ND) vaccine failed to respond satisfactorily in the serumvirus neutralization test and challenge of immunity via the respiratory tract. Revaccination did not effect a satisfactory anamnestic response. LaSota strain Newcastle disease virus (NDV), however, applied by eyedrop or in drinking water, stimulated excellent immunity by the same criteria. Response was unsatisfactory when LaSota NDV was given intramuscularly. Bl-strain NDV given by eyedrop or drinking water induced good immunity but of lower magnitude than from the LaSota strain. Cell-culture ND vaccine resulted in relatively poor protection from infection of the respiratory tract, and the virus-neutralizing antibody titers were relatively low or marginal. The potential use of lentogenic NDV strains in turkey vaccination programs was discussed.

The Typing of Bluetongue Virus

SUMMARY Typing of bluetongue virus has been carried out by applying the technique of plaque inhibition. This method has advantages over serum virus neutralization tests in eggs, tubes or petri dishes.

Studies on the fluorescent and neutralizing antibodies to bluetongue virus in sheep.

The sensitivity of the indirect fluorescent antibody technique (FAT) was compared with the serum-virus neutralization test (S-VNT) in the detection of bluetongue virus antibodies.

Experimental Infection of Cattle with Pantropic Rift Valley Fever Virus

When the titre of pantropic RVF virus was estimated in cattle, the results compared favourably with those obtained in adult mice and tissue cultures. The intensity of response to experimental infection did not depend on the amount of virus inoculated. The response of individual cattle to experimental injection varied considerably when judged by pyrexia, viraemia and circulating neutralizing antibody. The susceptibility of cattle could not be judged solely by the absence of neutralizing antibody in their pre-challenge sera, as assessed by the serum-virus neutralization test carried out in adult mice.

Antigenic differences between strains of foot-and-mouth disease virus of type SAT 1.

Antigenic differences between the strains RV 11 and SA 13/61 of foot-and-mouth disease virus (type SAT 1) were studiedin vivoby cross-protection tests. Cattle inoculated with formolized antigen of either strain developed good immunity to experimental infection with the identical strain but little resistance to the other strain.In vitrothe results of complement fixation tests and of serum-virus neutralization tests in tissue culture were consistent with the observations madein vivo. The results of studies on the serological relationships between four strains of type SAT 1 are presented.The importance of strain differences in the epizootiology and control of the disease is discussed briefly.The authors wish to thank Dr I. A. Galloway and Dr J. B. Brooksby for their advice and criticism, and to acknowledge the valuable technical assistance of Mr K. Herniman, Mr R. L. G. King and Mr E. Scoates.

Loss of immunogenic properties of vaccinia virus inactivated by formaldehyde.

In the experiments here reported, vaccinia virus in the form of partly purified elementary body suspensions was exposed to 0.006 M formaldehyde in the presence of 0.02 M glycine at 20–24 °C. Samples were taken at intervals to determine the rate of inactivation. Assays were carried out by intracutaneous inoculation of rabbits and by the plaque count method in monkey kidney cell monolayers. In addition, the virus suspensions so treated were injected into rabbits and the resulting immune response was studied by serum–virus neutralization tests performed on the rabbit skin and in tissue cultures.It was found that the rate of inactivation by HCHO decreased with time in accordance with the usual dynamics of disinfection. Similarly, the immunogenic properties decreased in proportion to the fall in infectivity. Minimal quantities of virus could still be detected after 150–250 hours; and samples removed at these times were capable of producing a feeble immune response. Later samples were neither infective nor immunogenic. These experiments lend no support to the belief that HCHO-inactivated vaccinia virus could be employed as a substitute for living virus in immunization against smallpox.

Cytopathogenicity in tissue culture by a tumor virus from mice.

SummaryCytopathogenic changes were observed in mouse embryo cell cultures that had been inoculated with tissue culture-grown virus isolated from mouse tumor tissues. Quantitative assays of the virus, referred to as the SE polyoma virus, and serum-virus neutralization tests were correlated with the induction of tumors in hamsters.

Laboratory Techniques in Rabies

A World Health Organisation meeting for the South-East Asia region dealing with problems of rabies and its control was held at the Pasteur Institute of Southern India, Coonoor, India, in July 1952. Medical and Veterinary officials from 23 different countries participated. WHO consultants on rabies arranged demonstrations and laboratory courses and prepared laboratory instructions for use in the training course. The WHO Expert Committee on Rabies at its second session in Rome, September 1953, recommended that the laboratory instructions and lecture material prepared for the Coonoor meeting be expanded and revised for publication.This technical manual covers five phases of laboratory work with rabies virus. Part I describes the rapid diagnosis of rabies by demonstration of Negri bodies, the mouse inoculation test for the isolation of rabies virus and the identification of rabies virus by the serumvirus neutralization test.