Serum Neutralization Assay Research Articles

Validation of an improved competitive enzyme-linked immunosorbent assay to detect Equine arteritis virus antibody

The objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (cELISA) to detect antibody to Equine arteritis virus (EAV) based on GP5-specific nonneutralizing monoclonal antibody (mAb) 17B7(9) using the World Organization for Animal Health (OIE)-recommended protocol, which includes the following 5 in-house analyses. 1) The assay was calibrated with the OIE-designated reference serum panel for EAV; 2) repeatability was evaluated within and between assay runs; 3) analytical specificity was evaluated using sera specific to related viruses; 4) analytical sensitivity was evaluated with sera from horses vaccinated with an EAV modified live virus (MLV) vaccine; and 5) the duration of cELISA antibody detection following EAV vaccination was determined. The positive cELISA cutoff of ≥35% inhibition (%I) was confirmed by receiver operating characteristic plot analysis. Analytical sensitivity of the cELISA was comparable to the serum neutralization (SN) assay in that it detected EAV-specific antibody as early as 8 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over 5 years after the last vaccination. This cELISA could detect EAV-specific antibody in serum samples collected from horses infected with various EAV strains. In the field trial performed by American Association of Veterinary Laboratory Diagnosticians-accredited state laboratories and OIE laboratory, the diagnostic specificity of the cELISA was 99.5% and the diagnostic sensitivity was 98.2%. The data using various serum panels also had consistently significant positive correlation between SN titers and cELISA %I results. The results further confirm that the EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAV-specific antibodies in equine sera.

Induction of robust immunity response in mice by dual-expression-system-based recombinant baculovirus expressing the capsid protein of porcine circovirus type 2

BackgroundPorcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease that causes progressive weight loss, dyspnea, tachypnea, anemia, jaundice, and diarrhea in piglets. Although baculovirus is an enveloped virus that infects insects in nature, it has emerged as a vaccine vector, and we used it to develop a novel candidate vaccine for a preventive or therapeutic strategy to control PCV2 infections.MethodsImmunoblotting analysis of recombinant baculovirus and immunofluorescent staining of baculovirus-infected cells were followed using anti-ORF2 monoclonal antibodies. The BALB/c mice were immunized intramuscularly with this baculovirus. The titers of antibodies were mensurated with a Cap-protein-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The IFN-γ response in splenocytes harvested from immunized mice was measured by ELISA. Student's t-test was used to compare immune responses of different groups.ResultsIn this study, we successfully constructed a dual-expression-system-based recombinant baculovirus BV-GD-ORF2, which can display the PCV2 capsid (Cap) protein and VSV-G protein on the viral envelope and also expressing Cap protein on transduced mammalian cells, thereby functioning as both a subunit and a DNA vaccine. After infection, the Cap protein was expressed and displayed on the viral surface, as demonstrated with an indirect fluorescence assay and immunoblotting. The vaccination of mice with recombinant baculovirus BV-GD-ORF2 successfully induced robust Cap-protein-specific humoral and cellular immune responses.ConclusionsOur findings collectively demonstrate that the recombinant baculovirus BV-GD-ORF2 is a potential vaccine against PCV2 infections.

Structural, Antigenic, and Evolutionary Characterizations of the Envelope Protein of Newly Emerging Duck Tembusu Virus

Since the first reported cases of ducks infected with a previously unknown flavivirus in eastern China in April 2010, the virus, provisionally designated Duck Tembusu Virus (DTMUV), has spread widely in domestic ducks in China and caused significant economic losses to poultry industry. In this study, we examined in detail structural, antigenic, and evolutionary properties of envelope (E) proteins of six DTMUV isolates spanning 2010–2012, each being isolated from individual farms with different geographical locations where disease outbreaks were documented. Structural analysis showed that E proteins of DTMUV and its closely related flavivirus (Japanese Encephalitis Virus) shared a conserved array of predicted functional domains and motifs. Among the six DTMUV strains, mutations were observed only at thirteen amino acid positions across three separate domains of the E protein. Interestingly, these genetic polymorphisms resulted in no detectable change in viral neutralization properties as demonstrated in a serum neutralization assay. Furthermore, phylogenetic analysis of the nucleotide sequences of the E proteins showed that viruses evolved into two distinct genotypes, termed as DTMUV.I and DTMUV.II, with II emerging as the dominant genotype. New findings described here shall give insights into the antigenicity and evolution of this new pathogen and provide guidance for further functional studies of the E protein for which no effective vaccine has yet been developed.

Bluetongue Virus in Lebanon

Since 2000, several incursions of bluetongue virus (BTV) occurred in the Mediterranean Basin involving European and surrounding Countries. The Middle East represents one of the most important gateways for the access of BTV in Europe. Limited data on the BTV situation in this area are available. In this perspective, an epidemiological survey on the presence of BTV in Lebanon was conducted. Of the 181 serum samples tested, 97 (mean=53.6%; 95% CI: 46.3-60.7) resulted positive when tested for the presence of BTV antibodies by c-ELISA, of these 42 (mean=42%; 95% CI: 32.8-51.8) serum samples were from sheep and 55 (mean=67.9%; 95% CI: 57.1-77.1) serum samples were from goats. Fourteen blood samples (14/110; mean=12.7%; 95% CI: 7.8-20.3), 6 (6/66; mean=9.1%; 95% CI: 4.4-18.5) from sheep and 8 (8/44; mean=18.2%; 95% CI: 9.6-32.0) from goats, were positive by qRT-PCR. The results with serum-neutralization assay and typing performed by RT-PCR confirmed that six BTV serotypes are currently circulating in Lebanon, and these serotypes are as follows: 1, 4, 6, 8, 16 and 24. This study is the first report that confirms the presence and circulation of BTV in Lebanon.

Prevalence of antibodies against Parainfluenza virus type 3, Respiratory syncitial virus and bovine Herpesvirus type 1 in sheep from Northern Prefectures of Japan.

Ovine sera collected in the Prefectures of Hokkaido, Aomori and Iwate in the Northern Japan were examined for the presence of antibodies against Respiratory syncytial virus (RSV), bovine Herpesvirus type 1 (infectious bovine rhinotracheitis: IBR) and Parainfluenza virus type 3 (PIV3) using serum neutralisation (SN) and enzyme-linked immunosorbent assay (ELISA) tests. Twenty-three animals (11.73%) out of the 196 tested were sero-positive to PIV3. Sixteen animals (8.69%) out of the 184 tested reacted to RSV. No animals were positive to IBR antigen. Sero-conversions to PIV3 were detected in Hokkaido and Iwate (14.92% and 8.82%, respectively). Antibodies against RSV were detected in Hokkaido (9.23%) and Aomori (14.28%). Although no diagnostic measures were in place, the infections did not appear to be related to any reduction in sheep productivity.

Serological and molecular evidence of bluetongue in sheep and goats in Uttar Pradesh, India

Cross-sectional experimental study was conducted with the objective to estimate the seroprevalence on the basis of antibodies to VP7 protein of bluetongue virus by competitive enzyme linked immunosorbent assay (c-ELISA), to test the neutralizing ability of the antibody to reference strains of 4 BTV serotypes (BTV-1, 2, 10 and 23) by micro serum neutralization assay (m-SNT), to check the presence of BTV dsRNA and to isolate and characterize bluetongue virus (BTV). A total of 91 serum and 26 whole blood samples were obtained from sheep and goat. The study was conducted between September and November, 2012 when the culicoides midges’ activity is maximal. The animals were observed for clinical signs of BTV infection and serum samples were obtained from all animals for c-ELISA and m-SNT. Further, blood samples were collected from the c-ELISA positive animals and subjected to virus isolation and nested RT-PCR. Out of 91 animals tested, 26 (28.6%) were found to be seropositive by c-ELISA and one sheep showed neutralizing antibody against BTV-1 serotype at a titer of 1.2 log10. Multivalent logistic regression analysis of risk factors like age, sex, body condition and species of animals were considered during the serological study and found that species of animal had significant influence (χ2 = 17.111, P 0.05) on seropositivity. It was worth enough to conclude that higher seroprevalence among goats indicated that goats would be the most important animals in the epidemiology of BTV with less clinical manifestation due to development of acquired immunity as the result of continuous exposure. Keywords : Bluetongue virus (BTV), competitive enzyme linked immunosorbent assay (c-ELISA), goat, seroprevalence, sheep, micro serum neutralization assay (m-SNT), nested reverse transcription polymerase chain reaction (RT-PCR) African Journal of Biotechnology Vol. 12(19), pp. 2699-2705

DETECTION OF CANINE DISTEMPER VIRUS SERUM NEUTRALIZING ANTIBODIES IN CAPTIVE U.S. PHOCIDS

Antibodies to morbilliviruses have been documented in free-ranging pinnipeds throughout populations in the Atlantic and Arctic Oceans, but not from the Pacific Ocean. As a symbolic geographic barrier between the exposed Atlantic and naive Pacific populations, the captive phocid population in North America had undocumented serologic status. In this study, canine distemper virus (CDV) serum neutralization assays were used to assess the prevalence of antibodies in this population with participation of 25 U.S. institutions from grey seals (Halichoerus grypus, n = 6) and harbor seals (Phoca vitulina, n = 108). Historic and environmental risk factors associated with the epidemiology of distemper virus were collected by survey. Based on antibodies to canine distemper virus, the prevalence of exposure in this population was 25.5%, with 28 seals (grey, n = 2; harbor, n = 26) demonstrating antibody titers > or = 1:16, and positive titers ranged from 1:4 to 1:1,536. By survey analysis, strong associations with seropositive status were identified for captive origin (P = 0.013) and movement among institutions (P = 0.024). Size of population has positive correlation with likelihood of seropositive seals at an institution (P = 0.020). However, no major husbandry or enclosure-based risk factors were identified in institutions with seropositive seals, and no interaction between individual or institutional risk factors was identified. Previously undocumented prior to this study, CDV antibodies were measured in harbor seals (n = 2) recently stranded from the Pacific coast.

Comparison of an improved competitive enzyme-linked immunosorbent assay with the World Organization for Animal Health–prescribed serum neutralization assay for detection of antibody to Equine arteritis virus

Equine arteritis virus (EAV) causes contagious equine viral arteritis, characterized by fever, anorexia, conjunctivitis, nasal discharge, dependent edema, abortion, infrequent death in foals, and establishment of the carrier state in stallions. The World Organization for Animal Health (OIE) defines a horse as seropositive if the serum neutralization (SN) antibody titer is ≥1:4 to EAV. However, determining the SN titer is time-consuming and requires specific laboratory facilities, equipment, and technical expertise to perform. Furthermore, interpretation of the SN titer of some sera can be difficult because of nonspecific cellular cytotoxicity of particular samples. Finally, the problem of interlaboratory variation also exists with SN assays. For these reasons, an alternative serologic test is desirable; however, none of the reported tests have equivalent sensitivity and specificity to the SN to be generally adopted. In an attempt to improve on a previously developed competitive enzyme-linked immunosorbent assay (cELISA) using EAV gp5-specific neutralizing monoclonal antibody (mAb) 4B2, the current study developed a modified protocol substituting the non-neutralizing mAb 17B7 for the neutralizing mAb 4B2; this along with several modifications of the test procedure improved the performance of the test. The relative specificity of the revamped cELISA was 99.8% when evaluated with 2,223 SN-negative sera. The relative sensitivity was 95.5% when evaluated with 246 SN-positive sera. This new cELISA was not affected by the presence of non-EAV-specific cytotoxicity in sera as observed in the SN assay. The results indicate that this new cELISA may be a viable alternative to the SN assay and merit additional validation.

Serosurvey for Antibody to Deerpox Virus in Five Cervid Species in Oregon, USA

Five cervid species in Oregon, USA were tested with a serum neutralization assay for antibody to deerpox virus (DPV). None of the 50 elk (Cervus elaphus ssp. roosevelti and nelsonii) had detectable antibody. Prevalence of antibody to DPV in the remaining species was: 52% (n=55) in black-tailed deer (Odocoileus hemionus columbianus), 32% (n= 59) in mule deer (O. hemionus hemionus), and 36% (n=50) in Columbian white-tailed deer (O. virginianus leucurus), with an overall antibody prevalence of 40.2% (n=164) for Odocoileus spp. Antibody-positive animals were identified throughout the state with no statistically significant differences among geographic regions. No statistically significant gender or age-related differences in antibody prevalence were demonstrated at either the genus or species level. This serosurvey indicates that exposure to DPV is common in Odocoileus populations in Oregon. Given the low rates of observed DPV-related disease, this high antibody prevalence suggests a pathogen of low virulence.

Measuring bovine viral diarrhea virus vaccine response: Using a commercially available ELISA as a surrogate for serum neutralization assays

Genetic selection in livestock offers the opportunity to improve bovine viral diarrhea virus (BVDV) vaccine response, but first we must define how vaccine response should be measured. For measuring humoral vaccine response, serum neutralization (SN) measures antibodies that can neutralize BVDV, but relative to enzyme-linked immunosorbent assay (ELISA) is time consuming, technically demanding, and expensive. The ELISA, however, measures total BVDV-specific antibodies, regardless of whether the antibodies can neutralize BVDV. Our objective was to test whether a commercially available BVDV antibody ELISA could be used as a surrogate (or indicator trait) for neutralizing antibodies as measured by SN. Angus and Angus-cross calves (n=193) from two South Dakota research herds were vaccinated for BVDV-1 and BVDV-2. Sera and plasma samples (n=406) were collected from these calves at the time of vaccination and post-vaccination (20–72 days post-vaccination). The BVDV-specific antibody concentration was measured on each serum and plasma sample by (1) a commercially available total antibody ELISA, (2) BVDV-1 SN, and (3) BVDV-2 SN. Correlation between the ELISA and SN tests was estimated with a Spearman correlation coefficient. Higher BVDV ELISA sample-to-positive (S/P) ratios were positively correlated with higher BVDV-1 (ρ=0.809) and BVDV-2 (ρ=0.638) SN titers (P<0.0001), although the relationship was weaker when SN titers were <1:64. Higher BVDV-1 SN titers were also positively correlated with higher BVDV-2 SN titers (ρ=0.708; P<0.0001). The correlation between ELISA S/P ratios and SN titers was lower when calves were ≤2 months of age (ρ=0.344–0.566). Our results suggest that increased ELISA S/P ratios were associated with higher SN titers. We conclude that this BVDV antibody ELISA can be used as a surrogate for BVDV-1 and -2 SN titers when investigating genetic determinants of vaccine response, as long as samples are collected at 2 months of age or older.

Adenovirus-36 is associated with obesity in children and adults in Sweden as determined by rapid ELISA.

BackgroundExperimental and natural human adenovirus-36 (Adv36) infection of multiple animal species results in obesity through increasing adipogenesis and lipid accumulation in adipocytes. Presence of Adv36 antibodies detected by serum neutralization assay has previously been associated with obesity in children and adults living in the USA, South Korea and Italy, whereas no association with adult obesity was detected in Belgium/the Netherlands nor among USA military personnel. Adv36 infection has also been shown to reduce blood lipid levels, increase glucose uptake by adipose tissue and skeletal muscle biopsies, and to associate with improved glycemic control in non-diabetic individuals.Principal FindingsUsing a novel ELISA, 1946 clinically well-characterized individuals including 424 children and 1522 non-diabetic adults, and 89 anonymous blood donors, residing in central Sweden representing the population in Stockholm area, were studied for the presence of antibodies against Adv36 in serum. The prevalence of Adv36 positivity in lean individuals increased from ∼7% in 1992–1998 to 15–20% in 2002–2009, which paralleled the increase in obesity prevalence. We found that Adv36-positive serology was associated with pediatric obesity and with severe obesity in females compared to lean and overweight/mildly obese individuals, with a 1.5 to 2-fold Adv36 positivity increase in cases. Moreover, Adv36 positivity was less common among females and males on antilipid pharmacological treatment or with high blood triglyceride level. Insulin sensitivity, measured as lower HOMA-IR, showed a higher point estimate in Adv36-positive obese females and males, although it was not statistically significant (p = 0.08).ConclusionUsing a novel ELISA we show that Adv36 infection is associated with pediatric obesity, severe obesity in adult females and lower risk of high blood lipid levels in non-diabetic Swedish individuals.

Migration of the Swine Influenza Virus δ-Cluster Hemagglutinin N-Linked Glycosylation Site from N142 to N144 Results in Loss of Antibody Cross-Reactivity

Routine antigenic characterization of swine influenza virus isolates in a high-throughput serum neutralization (HTSN) assay found that approximately 20% of isolates were not neutralized by a panel of reference antisera. Genetic analysis revealed that nearly all of the neutralization-resistant isolates possessed a seasonal human-lineage hemagglutinin (HA; δ cluster). Subsequent sequencing analysis of full-length HA identified a conserved N144 residue present only in neutralization-resistant strains. N144 lies in a predicted N-linked glycosylation consensus sequence, i.e., N-X-S/T (where X is any amino acid except for proline). Interestingly, neutralization-sensitive viruses all had predicted N-linked glycosylation sites at N137 or N142, with threonine (T) occupying position 144 of HA. Consistent with the HTSN assay, hemagglutination inhibition (HI) and serum neutralization (SN) assays demonstrated that migration of the potential N-linked glycosylation site from N137 or N142 to N144 resulted in a >8-fold decrease in titers. These results were further confirmed in a reverse genetics system where syngeneic viruses varying only by predicted N-glycosylation sites at either N142 or N144 exhibited distinct antigenic characteristics like those observed in field isolates. Molecular modeling of the hemagglutinin protein containing N142 or N144 in complex with a neutralizing antibody suggested that N144-induced potential glycosylation may sterically hinder access of antibodies to the hemagglutinin head domain, allowing viruses to escape neutralization. Since N-linked glycosylation at these sites has been implicated in genetic and antigenic evolution of human influenza A viruses, we conclude that the relocation of the hemagglutinin N-linked glycosylation site from N142 to N144 renders swine influenza virus δ-cluster viruses resistant to antibody-mediated neutralization.

Serological responses and clinical outcome after vaccination of mares and foals with equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) vaccines

Equine herpesvirus type 1 and type 4 (EHV-1 and EHV-4) cause infections of horses worldwide. While both EHV-1 and EHV-4 cause respiratory disease, abortion and myeloencephalopathy are observed after infection with EHV-1 in the vast majority of cases. Disease control is achieved by hygiene measures that include immunization with either inactivated or modified live virus (MLV) vaccine preparations. We here compared the efficacy of commercially available vaccines, an EHV-1/EHV-4 inactivated combination and an MLV vaccine, with respect to induction of humoral responses and protection of clinical disease (abortion) in pregnant mares and foals on a large stud with a total of approximately 3500 horses. The MLV vaccine was administered twice during pregnancy (months 5 and 8 of gestation) to 383 mares (49.4%), while the inactivated vaccine was administered three times (months 5, 7, and 9) to 392 mares (50.6%). From the vaccinated mares, 192 (MLV) and 150 (inactivated) were randomly selected for serological analyses. There was no significant difference between the groups with respect to magnitude or duration of the humoral responses as assessed by serum neutralization assays (median range from 1:42 to 1:130) and probing for EHV-1-specific IgG isotypes, although neutralizing responses were higher in animals vaccinated with the MLV preparation at all time points sampled. The total number of abortions in the study population was 55/775 (7.1%), 9 of which were attributed to EHV-1. Seven of the abortions were in the inactivated and two in the MLV vaccine group (p=0.16). When foals of vaccinated mares were followed up, a dramatic drop of serum neutralizing titers (median below 1:8) was observed in all groups, indicating that the half-life of maternally derived antibody is less than 4 weeks.

Vaccination against Feline Panleukopenia: implications from a field study in kittens

BackgroundFeline Panleukopenia (FPL) is a serious disease of cats that can be prevented by vaccination. Kittens are routinely vaccinated repeatedly during their first months of life. By this time maternally derived antibodies (MDA) can interfere with vaccination and inhibit the development of active immunity. The efficacy of primary vaccination under field conditions was questioned by frequent reports to the Paul-Ehrlich-Institut on outbreaks of FPL in vaccinated breeding catteries. We therefore initiated a field study to investigate the development of immunity in kittens during primary vaccination against FPL.64 kittens from 16 litters were vaccinated against FPL at the age of 8, 12 and 16 weeks using three commercial polyvalent vaccines. Blood samples were taken before each vaccination and at the age of 20 weeks. Sera were tested for antibodies against Feline Panleukopenia Virus (FPV) by hemagglutination inhibition test and serum neutralisation assay in two independent diagnostic laboratories.ResultsThere was a good correlation between the results obtained in different laboratories and with different methods. Despite triple vaccination 36.7% of the kittens did not seroconvert. Even very low titres of MDA apparently inhibited the development of active immunity. The majority of kittens displayed significant titres of MDA at 8 and 12 weeks of age; in some animals MDA were still detected at 20 weeks of age. Interestingly, the vaccines tested differed significantly in their ability to overcome low levels of maternal immunity.ConclusionsIn the given situation it is recommended to quantify antibodies against FPV in the serum of the queen or kittens before primary vaccination of kittens. The beginning of primary vaccination should be delayed until MDA titres have declined. Unprotected kittens that have been identified serologically should be revaccinated.

COMPARING ONRAB® AND RABORAL V-RG® ORAL RABIES VACCINE FIELD PERFORMANCE IN RACCOONS AND STRIPED SKUNKS, NEW BRUNSWICK, CANADA, AND MAINE, USA

Control of rabies in mesocarnivore reservoirs through oral rabies vaccination (ORV) requires an effective vaccine bait. Oral rabies vaccine performance in the field may be affected by a variety of factors, including vaccine bait density and distribution pattern, habitat, target species population density, and the availability of competing foods. A field study in which these covariates were restricted as much as possible was conducted along the international border of the state of Maine (ME), USA, and the province of New Brunswick (NB), Canada, to compare the performance of two oral rabies vaccines in raccoons (Procyon lotor) and striped skunks (Mephitis mephitis). RABORAL V-RG(®) (vaccinia-rabies glycoprotein recombinant oral vaccine in fishmeal-coated sachet) or ONRAB(®) (adenovirus-rabies glycoprotein recombinant oral vaccine in Ultralite bait matrix) were distributed in ME and NB, respectively, by fixed-wing aircraft at a density of 75 baits/km(2) along parallel flight lines spaced 1.0 km apart. Sera were collected from live-trapped raccoons and skunks 5-7 wk post-ORV and assayed to determine antibody prevalence in each area. Duplicate serum samples were provided blind to two different laboratories for analyses by rabies virus serum neutralization assays (at both laboratories) and a competitive enzyme-linked immunosorbent assay (at one laboratory). There was no significant difference in the proportion of antibody-positive animals determined by the three serologic methods, nor was there a significant difference between ONRAB and RABORAL V-RG in the proportion of antibody-positive striped skunks observed post-ORV. In contrast, the proportion of antibody-positive raccoons was significantly higher in the ONRAB- versus the RABORAL V-RG-baited areas (74% vs. 30%; χ(2)=89.977, df=5, P<0.0001). These data support that ONRAB may serve as an effective tool for raccoon rabies control.

Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: Sensitive and high-throughput analysis utilizing secreted alkaline phosphatase

Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.

Genetic and antigenic characterization of recent human-like H1 (δ-cluster) swine influenza virus isolates

Objective: To assess genetic and antigenic properties of contemporary human-like H1 (δ-cluster) swine influenza virus (SIV) isolates circulating in US swine herds. Materials and methods: The hemagglutinin genes from 37 δ-cluster SIV isolates were fully sequenced. The isolates were antigenically categorized using a high throughput serum neutralization (HTSN) assay incorporating antisera generated against the four currently circulating clusters of H1 SIV and a commercial vaccine containing a δ-cluster SIV isolate. A subset of the isolates were further characterized with the hemagglutination inhibition (HI) assay using H1 SIV antisera. Results: Genetic analysis of the hemagglutinin gene of 2009-2010 δ-cluster isolates identified five distinct subclusters with 97% to 100% sequence similarity within subclusters and 95% to 97% similarity between subclusters. Antisera generated against SIV representing α, β, and γ clusters failed to neutralize any of 37 δ-cluster viruses in an HTSN assay. Only 46% of the δ-cluster isolates were neutralized by at least one δ-cluster antiserum. Hemagglutination inhibition assay results on a subset of thirteen 2010 isolates were in good agreement with the HTSN assay, with 38% of the isolates positive (HI titer &gt; 40) to at least one δ-cluster antiserum. There was no measurable antibody titer to antiserum generated from the commercial vaccine or the 13 isolates. Implications: Significant genetic and antigenic heterogeneity exists among δ-cluster H1 SIV isolates, suggesting that either multiple representatives of this cluster may be required in commercial vaccines or that herd-specific vaccines may be required to protect swine from influenza virus infection.

Construction and biological characterisation of recombinant porcine circovirus type 2 expressing the V5 epitope tag

Porcine circovirus type 2 (PCV2) is a major causal agent of post-weaning multisystemic wasting syndrome in piglets. To investigate the feasibility of PCV2 expressing an exogenous epitope, a 14-amino-acid V5 epitope derived from simian parainfluenza virus type 5, was inserted into the C terminus of the capsid protein. Recombinant PCV2 expressing the V5 epitope, recPCV2/CL-V5, was rescued by transfecting an infectious clone into PK-15 cells and was characterised by an immunoperoxidase monolayer assay (IPMA), a serum neutralisation assay (SNA), a capture enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. The V5 epitope was detected in the recombinant marker virus by IPMA and capture ELISA. Furthermore, there was no detectable difference in the antigenicity of the recombinant marker virus compared with the parental virus by IPMA and SNA using PCV2-positive serum and the neutralising monoclonal antibody 1D2. However, recPCV2/CL-V5 marker virus could be differentiated from the parental virus by PCR, IPMA and capture ELISA. The recombinant marker virus was stable on multiplication through 10 passages in PK-15 cells, with a maximum titre of 10 6.25 50% tissue culture infective dose (TCID 50)/ml. BALB/c mice were inoculated with the recombinant or parental virus via the intranasal and intraperitoneal routes. The parental and recombinant viruses both could replicate in mice, cause microscopic pathological changes, and induce mice to generate anti-PCV2 antibodies. Furthermore, the recombinant marker virus could also induce anti-V5 epitope tag antibodies. These results indicated that V5 epitope could be displayed on the surface of the capsid protein by inserting its gene just before stop codon of open reading frame 2. More importantly, insertion of the V5 epitope did not seem to interfere with biological characterisation of the recPCV2/CL-V5 marker virus.

Association of human adenovirus-36 in overweight Korean adults

Although human adenovirus-36 (Ad-36) has been reported to be associated with obesity in US adults and children, Korean children and the Italian population, the association has not been found in Dutch or Belgian populations or in US military subjects. Therefore, we examined whether Ad-36 infection is associated with obesity in Korean adults. A total of 540 age- and sex-matched individuals, who were normal weight, overweight or obese, were selected from participants in routine health examinations at the Ewha Womans University Medical Center. Overweight participants were defined as those with a body mass index (BMI) of 23 ≤ BMI<25 kg m(-2) and obese subjects were those with BMI ≥ 25 kg m(-2), according to the International Obesity Task Force definition. Ad-36 antibody was measured using a serum neutralization assay. Although more overweight participants than normal or obese subjects tested positive for the Ad-36 antibody (40%, 32.8% and 30%, respectively), the differences were not significant. The participants who tested positive for Ad-36 antibody had lower levels of triglycerides (TG) in each of the three groups, higher total cholesterol (TC) in the obese group and higher high-density lipoprotein-cholesterol (HDL-C) in both the normal and obese groups. The odds ratio (OR) for Ad-36 antibody positivity was greater in overweight than in normal subjects (OR=2.03; 95% confidence interval (CI), 1.16-3.55) after adjusting for age, sex and waist circumference. However, this OR was non-significant in the obese group (OR=1.56; 95% CI, 0.67-3.67). Ad-36 seems to be strongly associated with overweight, but not obese, Korean adults.

Oncolysis of malignant human melanoma tumors by Coxsackieviruses A13, A15 and A18

Many RNA viruses are displaying great promise in the field of oncolytic virotherapy. Previously, we reported that the picornavirus Coxsackievirus A21 (CVA21) possessed potent oncolytic activity against cultured malignant melanoma cells and melanoma xenografts in mice. In the present study, we demonstrate that three additional Group A Coxsackieviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15) and Coxsackievirus A18 (CVA18), also have similar oncolytic activity against malignant melanoma. Each of the viruses grew quickly to high titers in cancer cells expressing ICAM-1 and intratumoral injection of preformed subcutaneous SK-Mel-28 xenografts in mice with CVA13, CVA15 and CVA18 resulted in significant tumor volume reduction.As preexisting immunity could potentially hinder oncolytic virotherapy, sera from stage IV melanoma patients and normal controls were tested for levels of protective antibody against the panel of oncolytic Coxsackieviruses. Serum neutralization assays revealed that 3 of 21 subjects possessed low levels of anti-CVA21 antibodies, while protective antibodies for CVA13, CVA15 and CVA18 were not detected in any sample. Serum from individuals who were seropositive for CVA21 failed to exhibit cross-neutralization of CVA13, CVA15 and CVA18. From these studies it can be concluded that the administration of CVA13, CVA15 or CVA18 could be employed as a potential multivalent oncolytic therapy against malignant melanoma.