Expression, cellular localization and antibody responses of the African swine fever virus genes B602L and K205R.

Previously, we identified serological immunodeterminants of African swine fever virus (ASFV), including pK205R and pB602L, without homologues in the database. pK205R is expressed as a 33-kD protein from 4 h post-infection onward, initially diffusely distributed throughout cells, and subsequently in viral factories. pK205R was not found in purified virus. Both pK205R and pB602L are recognised by hyperimmune antisera from domestic pigs and bushpigs at late time points after infection, suggesting they may be useful diagnostically to distinguish animals persistently infected with virus.

Geographical and age-stratified distributions of foot-and-mouth disease virus-seropositive and probang-positive cattle herds in the Adamawa province of Cameroon.

Six of the seven known serotypes of foot-and-mouth disease (FMD) virus occur in Africa. This paper describes the results of a population-based cross-sectional study of the seroprevalence of FMD and the persistence of the virus in cattle herds and associated sheep flocks in the Adamawa province of Cameroon. Antibody titres measured by the virus neutralising test indicated that serotypes O, A and SAT2 viruses had been circulating in the province. The estimates of apparent seroprevalence in cattle herds, based on five juvenile animals (eight to 24 months old) per herd, were 74.8 per cent for serotype SAT2, 30.8 per cent for serotype A and 11.2 per cent for serotype O, indicating recent exposure; the estimates based on animals more than 24 months of age were 91.1 per cent for SAT2, 83.6 per cent for A and 34.2 per cent for serotype O. Epithelial and oropharyngeal samples were collected from cattle and small ruminants, cultured and typed by ELISA; serotypes A and SAT2 were isolated from both types of sample. The herd-level estimate of apparent prevalence of probang-positive herds was 19.5 per cent and the animal-level estimate of apparent prevalence was 3.4 per cent. The geographical distribution of the seropositive herds based on juveniles suggested that recent SAT2 exposure was widespread and particularly high in the more northern and western parts of the province, whereas recent exposure to serotype A was patchy and more concentrated in the south and east. This distribution corresponded very closely with the distribution of herds from which virus was recovered by probang, indicating recent exposure or infection. No serotype O viruses were recovered from cattle, and the distribution of seropositive herds suggested very localised recent exposure. The apparent prevalence of probang-positive animals declined with the age of the animal and the period since the last recorded outbreak in the herd.

Comparison of two 3ABC enzyme-linked immunosorbent assays for diagnosis of multiple-serotype foot-and-mouth disease in a cattle population in an area of endemicity.

The development of a serological test for foot-and-mouth disease virus (FMDV) which is quick and easy to use, which can identify all seven serotypes, and which can differentiate vaccinated from convalescing or potential virus carriers would be a major advance in the epidemiological toolkit for FMDV. The nonstructural polyprotein 3ABC has recently been proposed as such an antigen, and a number of diagnostic tests are being developed. This paper evaluates the performance of two FMDV tests for antibodies to nonstructural proteins in an unvaccinated cattle population from a region of Cameroon with endemic multiple-serotype FMD. The CHEKIT-FMD-3ABC bo-ov (CHEKIT) enzyme-linked immunosorbent assay (ELISA) (Bommeli Diagnostics/Intervet) is a commercially available test that was compared with a competitive 3ABC ELISA (C-ELISA) developed in Denmark. The tests were compared with the virus neutralization test as the "gold standard." Diagnostic sensitivity and specificity were examined over a range of test cutoffs by using receiver operating characteristic curves, which allowed comparison of the overall performance of each test. The results indicated that the CHEKIT ELISA kit was 23% sensitive and 98% specific and the Danish C-ELISA was 71% sensitive and 90% specific at the recommended cutoff. These results have important implications if the tests are to be used to screen herds or individual cattle in surveillance programs, at border crossings for import-export clearance, or following emergency vaccination in an outbreak situation.

A serological survey of ruminant livestock in Kazakhstan during post-Soviet transitions in farming and disease control.

The results of a serological survey of livestock in Kazakhstan, carried out in 1997--1998, are reported. Serum samples from 958 animals (cattle, sheep and goats) were tested for antibodies to foot and mouth disease (FMD), bluetongue (BT), epizootic haemorrhagic disease (EHD), rinderpest (RP) and peste des petits ruminants (PPR) viruses, and to Brucella spp. We also investigated the vaccination status of livestock and related this to changes in veterinary provision since independence in 1991. For the 2 diseases under official surveillance (FMD and brucellosis) our results were similar to official data, although we found significantly higher brucellosis levels in 2 districts and widespread ignorance about FMD vaccination status. The seroprevalence for BT virus was 23%, and seropositive animals were widespread suggesting endemicity, despite the disease not having being previously reported. We found a few seropositives for EHDV and PPRV, which may suggest that these diseases are also present in Kazakhstan. An hierarchical model showed that seroprevalence to FMD and BT viruses were clustered at the farm/village level, rather than at a larger spatial scale. This was unexpected for FMD, which is subject to vaccination policies which vary at the raion (county) level.

Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody.

Protective immunity to African swine fever virus (ASFV) may involve a combination of both serological and cellular mechanisms. This work is focused on the identification of the possible relevant serological immunodeterminants of immunity. Thus, 14 serological immunodeterminants of ASFV have been characterized by exhaustive screening of a representative lambda phage cDNA expression library of the tissue culture-adapted Ba71V strain of ASFV. The library was constructed using RNA extracted from Vero cells infected for 3, 6, 9 and 12 h. A total of 150 clones was selected arbitrarily by antibody screening of the library with a polyclonal antiserum from a domestic pig surviving infection with the virulent Malta isolate of ASFV. Sequencing of these clones permitted identification of 14 independent viral proteins that stimulated an antibody response. These included six proteins encoded by previously unassigned open reading frames (ORFs) (B602L, C44L, CP312R, E184L, K145R and K205R) as well as some of the more well-studied structural (A104R, p10, p32, p54 and p73) and non-structural proteins (RNA reductase, DNA ligase and thymidine kinase). Immunogenicity of these proteins was confirmed by demonstrating the corresponding antibodies in sera from pigs infected either with the Malta isolate or with the OURT88/3-OURT88/1 isolate combination. Furthermore, the majority of these ORFs were also recognized by immune antiserum from the natural host, the bush pig, following secondary challenge with the virulent Malawi (SINT90/1) isolate of ASFV. Thus, it is possible that some of these determinants may be important in protection against virus infection.

Protection of cattle against rinderpest by intranasal immunisation with a dry powder tissue culture vaccine.

Dry powder tissue culture rinderpest vaccine containing 10(2.5) TCID(50) of virus per dose administered intranasally to cattle induced high titre circulating antibody responses and protection against challenge with a virulent strain of rinderpest virus. A reduction in the dose of virus to 10(1.1) TCID(50) resulted in a failure to elicit detectable antibody responses and a lack of protection. Intranasal powder vaccine offers several advantages over conventional needle-administered aqueous rinderpest vaccine, including greater stability in the absence of a cold chain, reduced risk of 'needle transfer' of other microbial agents present in the vaccinated herd and lower cost.

Heterotypic recognition of recombinant FMDV proteins by bovine T-cells: the polymerase (P3Dpol) as an immunodominant T-cell immunogen.

In this study we have examined the recognition of VP0, VP1, VP2, VP3 and P3Dpol by PBMC and CD4+ T-cells from infected, vaccinated-challenged, and multiply-vaccinated (O1, A24, C1 or ASIA1) cattle using recombinant proteins of an O1 serotype virus. The structural protein VP1 was recognised in an homotypic context whereas VP2, VP3, VP4 and P3Dpol were also recognised by T-cells from animals exposed to heterotypic viruses. Only the non-structural protein P3Dpol was consistently recognised by T-cells from the majority of animals examined and heterotypic recognition correlated with the presence of serologically detectable P3Dpol in purified virus. Thus, P3Dpol is a major cross-reactive immunodeterminant of FMDV, eliciting heterotypic T-cell responses and, therefore, with possible potential for inclusion in a subunit vaccine.

Expression of biologically active recombinant porcine GM-CSF by baculovirus gene expression system.

The full length porcine granulocyte/macrophage colony stimulating factor (GM-CSF) cDNA, including secretion signal peptide coding region was recloned into baculovirus transfer vector pAcYM1. The vector was then transfected with Autographica californica nuclear polyhedrosis virus (AcNPV) DNA into SF21AE cells and the recombinant virus AcPGM was recovered. Recombinant porcine GM-CSF (rpGM-CSF) was obtained from the serum-free culture medium of Tn5 cells infected with the AcPGM virus, and was shown to be a glycosylated 21 kDa protein as confirmed by tunicamycin treatment and [3H]-glucosamine uptake. The biological activities of rpGM-CSF in AcPGM-infected cell culture supernatants were demonstrated by porcine bone marrow cell proliferation and haematopoietic cell colony formation assays. The use of rpGM-CSF enabled us to culture porcine monocytes/macrophage and dendritic-like cells, derived from either porcine bone marrow or peripheral blood, for up to 4 months.

Monoclonal antibodies putatively identifying porcine B cells.

Comparison was made of the binding of 38 test and three standard monoclonal antibodies (mAbs) to B cells from various pig lymphoid tissues by flow cytometry (FCM) and immunohistochemistry. Some mAbs were also tested on B cells from foetal pig tissues. Twenty of the new mAbs bound, though to variable degrees, to porcine B cells but only three were given cluster assignations: C35 (#147) and BB6-11C9 (#167) were assigned to wCD21 and 2F6/8 (#057) was assigned to SWC7.

Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.

Clinical signs and humoral immune response in horses following equine herpesvirus type-1 infection and their susceptibility to equine herpesvirus type-4 challenge.

A group of three horses was experimentally infected with equine herpesvirus type 1 (EHV-1) and showed clinical signs characterised by a biphasic febrile response, leucopenia and cell associated viraemia accompanied by virus shedding from the nasopharynx. A second exposure to the virus 18 days later resulted in the isolation of virus from the nasopharynx of one horse. This and a further group of three EHV-1 seropositive horses were subsequently infected with equine herpesvirus type 4 (EHV-4) 147 days after the initial EHV-1 infection and virus was shed from the nasopharynx in the absence of clinical disease. Following the first EHV-1 infection, virus specific immunoglobulin M (IgM) was present by day 5 and remained high until the second exposure at day 18 at which point levels decreased. In contrast, EHV-1 specific IgG, detected at day 6 peaked at day 18, after which time levels remained high. Virus neutralising antibodies and antibodies able to mediate antibody-dependent cellular cytotoxicity were present by day 10. The immune response to EHV-1 is discussed with reference to the disease.

Studies on glycoprotein 13 (gp13) of equid herpesvirus 1 using affinity-purified gp13, glycoprotein-specific monoclonal antibodies and synthetic peptides in a hamster model.

Hamsters were immunized with either an affinity-purified preparation of equid herpesvirus 1 (EHV-1) glycoprotein 13 (gp13) or synthetic peptides representing three sequences within the homologous glycoprotein of EHV-4, resulting in the production of anti-peptide (in the case of peptide-immunized animals) or antivirus antibodies. The sera from gp13-immunized hamsters contained antibodies which showed virus-neutralizing activity and complement-mediated antibody lysis of EHV-1-infected target cells. These hamsters were protected from EHV-1 challenge. The characteristics of a panel of anti-gp13 monoclonal antibodies (P28, P17, 14H7, 16E4 and 16H9) were assessed both in vivo and in vitro. 16E4 and P28 showed high levels of complement-mediated neutralization of virus, complement-mediated lysis of virus-infected target cells and passive protection of hamsters. Furthermore, epitope mapping studies demonstrated that this glycoprotein contains a neutralizing epitope recognized by EHV-1-immune horse serum. The data imply that gp13 has potential as a candidate antigen for a molecular vaccine.

Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype.

Analyses of reassortant and parental strains of BTV serotypes 3 and 10, in serum neutralization tests, confirmed the major role of outer capsid protein VP2 in determination of virus serotype and its involvement in serum neutralization. However, a reassortant BTV strain (R70), containing protein VP5 derived from BTV 3 and VP2 derived from BTV 10, cross-neutralized with both parental virus strains (BTV 3 and BTV 10). It is concluded that VP5 also plays some part in serotype determination of these virus isolates, as analyzed by serum-neutralization, but its role may be less significant than that of VP2.

A reassessment of the dual vaccine against rinderpest and contagious bovine pleuropneumonia.

In the light of the recent outbreaks of rinderpest in Africa a further assessment of the efficacy of the simultaneous inoculation of rinderpest virus vaccine and contagious bovine pleuropneumonia vaccine was undertaken. Groups of cattle were inoculated with a dual preparation of rinderpest vaccine virus and Mycoplasma mycoides subspecies mycoides or M mycoides alone. These groups were then challenged with M mycoides, first unsuccessfully by an in-contact challenge method and then by subcutaneous challenge. All animals were examined clinically after challenge for evidence of contagious bovine pleuropneumonia and serologically for rinderpest virus and M mycoides mycoides antibodies. There was no evidence that the serological response to the dual vaccine was in any way less than that to either agent given alone and no clinical disease was detected in these animals after in-contact challenge. However, after subcutaneous challenge, the dual vaccinated groups reacted similarly to an unvaccinated control group and unlike the group vaccinated only with M mycoides. This would indicate that the rinderpest virus component of the dual vaccine interfered with the ability of the M mycoides component to induce a fully effective immune response. In the pan African rinderpest campaign the use of the dual vaccine in areas where contagious bovine pleuropneumonia occurs should be carefully considered; in areas where the disease does not occur it is contraindicated.

Virulence of bluetongue virus for British sheep.

A South African isolate of bluetongue virus type 3 was inoculated intradermally into three different breeds of British sheep under conditions designed to test its virulence in animals under stress. All animals inoculated developed a pyrexia and viraemia followed by clinical evidence of bluetongue disease. Marked alterations in serum enzyme levels, in particular of creatine phosphokinase, lactate dehydrogenase and aldolase occurred in the more severely affected animals. Nine out of the 12 inoculated animals subsequently died. No major differences in response could be detected in the different breeds of sheep nor in the stressed compared with the unstressed groups. The virulence of this bluetongue virus isolate was thereby confirmed and its potential risk to the British sheep industry. Consequently, stringent import regulations must be maintained to prevent its entry into Britain.

Serial inoculation of sheep with two bluetongue virus types.

Groups of sheep inoculated with bluetongue virus type 4 were challenged at various intervals after inoculation (from seven to 70 days) with bluetongue virus type 3. Examination of the clinical and serological response showed that animals were protected from challenge with a second bluetongue virus for up to 14 days after the inoculation of the first virus type. An adoptive transfer experiment in monozygotic sheep involving both antibody and T lymphocytes was carried out. Only partial protection was observed against heterologous virus challenge, indicating that although the T cell response has a cross-protective component, antibody is not involved. These observations indicate that current vaccination procedures should be reappraised, particularly in terms of revaccination with multiple bluetongue virus type.