H.A. Westbury

Self-assembly, antigenicity, and immunogenicity of the rabbit haemorrhagic disease virus (Czechoslovakian strain V-351) capsid protein expressed in baculovirus

SummaryRabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein demonstrated that the capsid protein expressed in insect cells self-assembled to form empty virus-like particles (VLP) which are similar in size and morphology to that of native virus. These particles were immunoreactive with polyclonal anti-RHDV antibodies and with four monoclonal antibodies which recognise conformational epitopes of the virus. The results indicated that the VLPs were morphologically and antigenically indistinguishable from native virus. The recombinant VLPs induced high levels of RHDV-specific antibodies in rabbits and mice following immunisation. The immune response to the VLPs protected the rabbits following challenge with the virulent RHDV. In haemagglutination assays, the VLPs bound to human red blood cells similar to the native virus particles. The recombinant protein and or VLPs is suitable for the development of a rapid, sensitive and reliable test for detection of antibodies to RHDV and for use as a vaccine for domestic rabbits.

Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and E rns using phage-displayed random peptide library

Summary.Infection of cells with classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoproteins E2 and Erns with receptor molecules on the cell surface. These proteins are also the major antigens for eliciting neutralizing antibodies and conferring protective immunity. Here we report the identification of multiple neutralizing epitopes on these proteins by screening a phage-displayed random peptide library with CSFV-specific neutralizing monoclonal antibodies. Two different E2-specific neutralizing mAbs (a18 and 24/10) were found to bind to a common motif SPTxL, which is similar to the sequence SPTTL of the E2 protein (aa 289–293), indicating that this is likely to be an immunodominant epitope. Similarly, an immunodominant epitope corresponding to the sequence DKN of Erns (aa 117–119) was identified for two independent Erns-specific neutralizing antibodies, b4-22 and 24/16, respectively. Another binding motif, CxNNxTC, was identified for mAb 24/16, but not for b4-22. Sequencing analysis of the genes coding for the light chain of these mAbs was conducted to ensure that all mAbs were derived from different hybridomas, rather than from different subclones of a common parent line. Inhibition studies using immunofluorescent antibody assay and virus neutralization test demonstrated that the mimotope peptides truly mimicked the antibody binding determinants on the viral proteins. The detailed mapping data for these neutralizing epitopes will be useful for development of improved diagnostic tests and perhaps a peptide-based vaccine for this important swine disease.

The presence of cross-reactive antibodies to rabbit haemorrhagic disease virus in Australian wild rabbits prior to the escape of virus from quarantine

Summary. Sera collected from Australian wild rabbits prior to the escape of rabbit haemorrhagic disease virus (RHDV) from Wardang Island were examined for RHDV antibodies using purified recombinant capsid protein VP60 expressed from baculovirus. A VP60-based indirect ELISA showed that 196 of 392 wild rabbit sera reacted (OD450>0.15) with VP60. Twenty sera (OD450 ranging from 0.15–2.47), randomly chosen from the 196 positive sera, recognized the 64 kDa VP60 in Western blot analysis, indicating that the reactivity detected in ELISA is indeed specific to the VP60 antigen. In a separate study, sera of 23 rabbits from an RHD-free area after the escape of RHDV were tested by ELISA and 21 of the 23 rabbits were found to be positive. When these rabbits were challenged with a lethal dose of RHDV, 11 out of the 23 rabbits survived. The presence of RHDV-protective antibodies in some of these rabbits suggested that they had been exposed to a pre-existing non-virulent rabbit calicivirus closely related to RHDV. These results highlight the need to study the prevalence of, and to characterize, this viral agent in order to effectively control rabbit populations in Australia and New Zealand.

Detection of hog cholera virus antigens in experimentally-infected pigs using an antigen-capture ELISA

An antigen-capture ELISA was used to detect hog cholera virus (HCV) antigens in blood and tissues taken from pigs infected with 2 different strains of virus. Specific antigens were demonstrated in peripheral blood leucocytes (PBLs) and a wide range of tissue samples 4-6 days after infection of pigs with a moderate-high virulent HCV strain (Weybridge virus). Strong signal to noise (S/N) ratios were obtained in the ELISA for PBLs and lymphoid tissues such as spleen, tonsil and mesenteric lymph nodes at 5-7 days after infection with the Weybridge virus, S/N ratios varying between 8.1-19.7 for blood samples and 4.3-19.1 for spleen samples. High positive ELISA results were also obtained for duodenum and ileum samples (S/N ratios 10.3-18.6) taken from these pigs, reflecting severe pathological changes observed in the gut at post mortem. In contrast, the antigen-capture ELISA gave strong positive results for PBLs and spleen samples only at 7-9 days after infection of pigs with a low virulent strain of HCV (New South Wales virus). The ELISA S/N ratios averaged 9.5 for PBLs and 8.9 for spleen samples in these animals. Although virus isolation detected infection earlier in the infected pigs, the ELISA returned positive results on PBLs and spleen samples around the time all of the animals first showed typical signs of classical swine fever. The technique does not require tissue culture and takes less than 36 h to return a definitive result.

A competition ELISA for the detection of antibodies to rabbit haemorrhagic disease virus

Monoclonal antibodies (mAbs) were raised to a preparation of rabbit haemorrhagic disease virus (RHDV) purified from the livers of experimentally infected rabbits. Rabbit antisera to RHDV significantly blocked the binding of two mAbs (2D3(3) and 2D4(5)) to RHDV-coated microplate wells in a competition ELISA. The virus-specific nature of these mAbs was confirmed by immunoperoxidase and immunofluorescence assays on formalin-fixed and fresh infected liver tissue. Utilization of one of these mAbs (2D3(3)) in a competition ELISA, resulted in an RHDV antibody assay which proved more specific than an indirect ELISA and more rapid and reliable than a haemagglutination inhibition assay for screening serum samples from wild and experimental rabbits.

Molecular epidemiology of foot-and-mouth disease viruses from South East Asia 1998–2006: The Lao perspective

Foot-and-mouth disease (FMD) causes sporadic disease outbreaks in the Lao Peoples Democratic Republic (Lao PDR) and appears to be endemic within a livestock population largely susceptible to infection. As Lao PDR is a major thoroughfare for transboundary animal movement, regular FMD outbreaks occur causing economic hardship for farmers and their families. The dominant serotype causing outbreaks between 1998 and 2006 was type O. Using phylogenetic analysis, type O isolated viruses were divided into two topotypes: South East Asia (SEA) and the Middle East-South Asia (ME-SA). Type A virus was reported only in 2003 and 2006 and type Asia 1 only in 1996 and 1998.

A comparison of enzyme-linked immunosorbent assay, complement fixation and virus isolation for foot and mouth disease diagnosis

A total of 205 epithelial tissue samples were examined for the presence of foot and mouth disease virus by either the complement-fixation (CF) test, enzyme-linked immunosorbent assay (ELISA) and/or by virus isolation in bovine thyroid or kidney cell cultures. The virus was isolated from 134 of the 201 (67%) specimens. Samples, from which virus was isolated, were termed virus-positive samples. The CF test detected viral antigen in 30 (24%) of 123 virus-positive samples, whereas the ELISA detected it in 100 (81%) of these specimens. The ELISA was thus at least 3 times more efficient than the CF test in detecting the virus in epithelial-tissue samples. There were 5 samples from which virus was not isolated but which were positive with the ELISA procedure. The ELISA was particularly useful for testing samples from pigs and for assessing specimens from animals with resolving lesions. The ELISA gave virus type-specific results in 89% of 63 virus-positive cases compared to 40% for the CF test. The ELISA was thus a very useful, accurate and sensitive method for the direct testing of epithelial tissues of affected animals.

Genetic typing of classical swine fever viruses from Lao PDR by analysis of the 5' non-coding region

The 5′ non-coding region (5′-NCR) of 27 classical swine fever virus (CSFV) isolates from Lao People’s Democratic Republic (Lao PDR) during 1997 and 1999 were amplified by RT-PCR. A 150-bp region of the 5′-NCR amplicons was analysed and compared with reference CSFV of European and Asian origin and a phylogenetic dendrogram constructed. Following analysis, all viruses were determined to belong to genogroup 2. Viruses from Lao PDR grouped on a geographical basis with the majority of northern/central isolates falling into subgroup 2.1 and southern/central isolates falling into subgroup 2.2. These results concur with previous studies of CSF viruses from Lao PDR, although this study recognized the first occurrence of subgroup 2.1 in southern Lao PDR.

Comparative susceptibility of indigenous and improved pig breeds to Classical swine fever virus infection: practical and epidemiological implications in a subsistence-based, developing country setting.

This study investigated the comparative susceptibility of indigenous Moo Laat and improved Large White/Landrace pig breeds to infection with classical swine fever virus (CSFV) under controlled conditions in the Lao People’s Democratic Republic (Lao PDR). The Moo Laat (ML) and Large White/Landrace crossbreed (LWC) pigs were inoculated with a standard challenge strain designated Lao/Kham225 (infectivity titre of 102.75 TCID50/ml). The results demonstrated that both the native breed and an improved pig breed are fully susceptible to CSFV infection and the mortality rate is high. LWC pigs demonstrated lower (or shorter) survival times (50% survival time: 11 days), earlier and higher pyrexia and earlier onset of viraemia compared to ML pigs (50% survival time: 18 days). In the context of village-based pig production, the longer time from infection to death in native ML pigs means that incubating or early sick pigs are likely to be sold once an outbreak of CSF is recognized in a village. This increased longevity probably contributes to the maintenance and spread of disease in a population where generally the contact rate is low.

Development of a polymerase chain reaction to detect Vietnamese isolates of duck virus enteritis.

A polymerase chain reaction (PCR) method for the detection of duck virus enteritis (DVE) virus in tissues of infected and affected ducks, and in cell culture was developed. This required us to obtain specific nucleotide sequence information as we could not find any specific data about the genome of the virus. We found the assay to be highly effective in detecting the virus under experimental conditions and to be easily transferred to laboratories in Vietnam where it is being used in studies on the epidemiology of the disease. We have applied this simple and rapid diagnostic method to the detection of DVE isolates grown in cell culture and tissues from infected birds. The assay was also able to differentiate DVE from other avian herpesviruses, such as Mareks disease, infectious laryngotracheitis virus and goose herpesvirus.