From January 1973, the Commission of the European Communities has given financial support to a second research programme on hog cholera (HC) and African swine fever (ASF). The results obtained during this period on HC have already been discussed. Scientists of the institutes engaged in the programme reported on four aspects of their activities: the virus, pathogenesis and immunity, diagnosis including serological studies, and vaccine and vaccination.
The structure and antigenic composition of HC virus: The main difficulty has been, and remains, that the PK cells which must be used for multiplication of the virus are not an ideal source in many respects, but no better cell culture for virus propagation has been found. Nevertheless, progress has been made in characterizing the HC virus. Virus grown in PK 15 cells has been concentrated and purified by precipitation using polyethyleneglycol or ammonium sulphate, ultra-centrifugation and gradient centrifugation. The buoyant virion density has been found to be in the range of 1.14 to 1.16 g/ml, the isoelectric point has been determined to be 4.8, and particles measuring 40–55 nm in diameter, which may carry fragile surface projections, have been seen in the electron microscope. The virion contains a spherical nucleo-capsid with a diameter of 30 nm. This protects an infectious singlestranded colinear RNA with a molecular weight of about 4 × 106 daltons as shown by 3H-uridine incorporation, enzyme treatment and gradient analysis. The virus particles have been observed in ultra-thin sections. Three major virion structural polypeptides have been suggested by 35S-methionine labelling and the molecular weights of these have been determined as 50,000–55,000, 46,000 and 35,000 daltons, respectively. The 46,000 daltons protein was glycosylated and probably represents the surface projections glycoprotein. Whether the 50,000 to 55,000 protein contains carbohydrates is still under discussion.
A glycoprotein which cross-reacts serologically between HC virus and bovine viral diarrhoea (BVD) virus has been isolated from infected cells by detergent treatment, and it has been shown to protect pigs against an otherwise fatal infection of HC virus. It is not clear whether this component is identical with the virion glycoportein(s) or whether it constitutes a non-structural viral protein. Comparative studies with BVD virus and HC virus support the inclusion of both viruses in the family Togaviridae. For a serological diagnosis of hog cholera, it is most important that components of HC virus be isolated in the near future in order to determine whether any of them reacts negatively to anti-BVD antibodies.
Pathogenesis and immunity: Effective induction of prophylactic immunity and the establishment of eradication programmes demand better knowledge of the properties of the pathogenic agent and better understanding of the mechanisms of immune response and the pathogenesis of the disease. Some projects in the programme under review have therefore focussed interest on basic problems of porcine immunology. It has been demonstrated that IgG, IgM and IgA are synthesized within the small intestine of pigs. In the intestinal secretion, a six-fold excess of IgA over IgM and IgG exists. It has been shown furthermore that after oral vaccination with E. coli, a massive production of antibodies which are mainly of the IgA class took place in the lamina propria of the gut. After intramuscular vaccination with a non-multiplying antigen, a systemic immune response was found, involving principally IgG formed in the local lymph nodes and spleen. However, no antibody was detected in the gut secretions. From studies on the immune response in piglets, where lysozyme was used as the antigen, it can be concluded that parenteral immunization of piglets without maternal antibodies should not be performed during the first four days of life.
Investigations on the pathogenesis of HC have been concerned with intrauterine infection of the foetus by HC virus of low virulence. This is a problem which has implications both for the epizootiology and for the diagnosis of the disease. Infection of pregnant sows with the strain under reference did not result in abortion but there was a relationship between the stage of gestation at which the sows were infected and the number of stillborn or mummified foetuses. Infection during day 65 and 85 resulted in a mortality of about 36 percent, whereas no mortality was observed when infection took place at day 94 and 100. Viral antigen was detected, however, in about 12 percent of these animals. Another group reported on congenital infections with a virus of low virulence resulting in the birth of piglets which carry the virus and excrete it for several months. Such a persistent infection was not accompanied by the synthesis of antibodies. Strains of HC virus were isolated from placentas, foetuses, and newborn piglets in several unvaccinated herds having long-term reproductive failure. These strains had been characterized, showing that they were attenuated for piglets and adults; that they were transmitted from infected piglets to susceptible piglets; that they induced immunity in spite of a low capacity to induce antibody synthesis; and that they are only pathogenic for foetuses in an early stage of gestation.
Work performed on the pathogenesis of hog cholera resulted in the hypothesis that an infection with HC virus causes a disorder of the enzyme system mainly by an increased production of chymotrypsin, leading to direct effects on certain cells and affecting the circulatory system. In this way, the symptoms of HC are produced.
Diagnosis and serological studies: Much of the work of the research programme has been devoted to the improvement of diagnostic procedures, a matter of great importance because diagnosis in the field has become increasingly difficult. As was stressed in the discussion, the disease often runs an atypical course with only a small number of animals becoming sick, mostly young stock around the age of weaning. Clinical symptoms are not severe and may even be intermittent on breeding farms. Outbreaks caused by low virulent strains may therefore pass unnoticed for weeks or months and may give rise to a considerable spread of the virus. Post-mortem lesions often provide little evidence and may be lacking altogether.
Clinical and post-mortem lesions of ASF are becoming less severe than before, and they frequently resemble those of HC, causing an additional diagnostic problem in the countries of the Iberian peninsula.
The direct fluorescent antibody technique, using cryostat sections of suspected tissues, appears to be the common method used for the diagnosis of HC. Evaluation of specimens from positive cases has shown that the tonsils are the organs of choice. Isolation of virus in PK 15 cells is tried if the immunofluorescence test is negative, but some strains of low virulence replicate slowly and require several blind passages under special conditions, e.g. infection of suspended cells. Isolation of low virulent strains is often therefore difficult and time consuming.
A valid answer to the presence or absence of the disease may be obtained more rapidly by serological methods in areas where no vaccination is applied. In recent years these methods have gained considerable ground. In positive herds, a large proportion of the breeding stock is usually found to have antibodies. The percentage of sero-positive animals in a herd obviously depends on several factors, e.g. duration of the infertility problem; housing; management; and possibly the strain of virus. Serological methods have also been used to determine whether or not suspected contacts have taken place.
Most serology has been done so far by a plaque reduction test and a neutralization immunofluorescence test. Both techniques require permanent tissue culture facilities. Other methods such as immuno-electro-osmophoresis (IEOP) and a modified complement fixation test have been adapted to HC and have given promising results. IEOP is suitable for screening a large number of sera and, with the exception of antigen preparation, neither IEOP nor the modified CF test require tissue culture.
A serious drawback for the different serological methods is the presence of antibodies against BVD virus in swine populations. Work in countries in which the national pig herd has not been exposed to either field or to vaccine strains of hog cholera has shown a high incidence of infection. In Ireland, up to 28 percent of the breeding sows from mixed farms (pigs and cattle) had antibodies against BVD virus. Under experimental conditions, BVD virus replicates only to a limited extent in pigs and shows little tendency to spread among pigs. Presumably BVD infections in pigs are chiefly due to contact with cattle and possibly with sheep. More work needs to be done to elucidate the epidemiology of BVD infection in pigs.
Although neutralizing antibodies against BVD may be detected by plaque reduction and by neutralization immunofluorescent tests using hog cholera antigen, titres are normally well below those of the homologous BVD system, provided that for each system a prototype virus strain is used as an antigen. Thus, a herd diagnosis is possible. In this respect it must be mentioned that antigenic variants of both hog cholera and bovine diarrhoea virus have been detected. Furthermore, it was found that pigs infected with BVD virus responded to an infection with hog cholera virus with an anamnestic response.
The IEOP tests and complement fixation tests do not differentiate between antibodies against hog cholera and against BVD virus. These tests, therefore, may be used for screening purposes, but positive results need to be confirmed by neutralization tests.
BVD virus induces some resistance in pigs against infection with hog cholera virus; this partial immunity seems not to be expressed equally against different hog cholera strains; and its influence on the epidemiology of hog cholera in a pig population with a high incidence of BVD deserves more study.
Vaccines and vaccination: Vaccines and vaccination against hog cholera were a main objective of the research programme supported by the EEC. From experience gained in countries applying vaccination and from experimental work, the following conclusions can be drawn:
The modified live virus of choice in most of the countries is at present the Chinese strain. In France, the Thiverval-Grignon strain is also used.
Available evidence indicates that the modified live viruses are genetically stable, i.e. passages in pigs do not increase their virulence for pigs; they do not produce disease or otherwise affect the health of vaccinated pigs of different ages; and it has been demonstrated with the Chinese strain that application of this virus to sows two weeks before or two weeks after service is harmless. Vaccination with the Chinese strain under field conditions did not reveal any untoward effects on the sows or their litters. Inoculation of the Thiverval-Grignon strain into sows at day 30 and 60 of pregnancy also had no unfavourable effect on pregnancy or on the litters. Vaccination of piglets from non-vaccinated sows also proved harmless when one to five week old pigs were vaccinated, and it has been shown that piglets can be vaccinated within the first two weeks of life with the Thiverval-Grignon strain. The modified live viruses induce a solid immunity in a large proportion of vaccinated pigs which lasts under laboratory conditions more than a year in most cases. The immunity has been reported to persist for as long as three years after vaccination with the Thiverval-Grignon strain. It is, however, necessary to bear in mind that under field conditions more than half of the pigs which had been vaccinated with the Chinese strain were sero-negative one year after vaccination. This means that to obtain optimal results revaccination has to be considered in breeding farms. Furthermore, it has been demonstrated that some of the pigs vaccinated either with the Chinese strain or with the Thiverval-Grignon strain excrete the virus which is transmitted to nonvaccinated contact pigs.
Immunization of pregnant sows with the Chinese or the Thiverval-Grignon strain can result in transmission of the vaccine virus to the foetuses. As already mentioned, this seems to have no harmful effect, but the possibility that the vaccine virus persists in the new generation must be considered.
In prednisolon-treated pigs, virus of the Chinese strain could be detected in tonsils and kidneys up to 15 days post-vaccination. There may therefore be an effect of a recent vaccination on the diagnosis of HC by the immunofluorescence technique.
It was demonstrated that pigs immunized with a relatively small quantity of virus of the Chinese strain were immune but became virus carriers after exposure to virulent virus 14 days after vaccination. A minimal potency of 100 ID50 units per dose is therefore proposed as a requirement for a HC vaccine containing the China strain.
Vaccinated sows will transmit antibodies with the colostrum to their young. This has some implications. Firstly, there is protection of the piglets against virulent virus, as shown in an experiment in which pigs born from vaccinated sows survived exposure to a virulent virus when five to seven weeks old. Secondly, maternal antibodies in young animals prevent a response to vaccination. This has been demonstrated and it has been shown that maternal antibodies decline at the age of six weeks to a level which allows a successful vaccination. Thirdly, there may be the possibility that modified live virus administered to pigs with maternal antibodies may persist in these animals.
In one instance promising results have been obtained in the development of an inactivated HC vaccine. Hog cholera virus grown in PK 15 cells has been concentrated, inactivated with glycidaldehyde and then mixed with an oil adjuvant; and an inoculation of 5 ml of this vaccine has induced a solid and long lasting immunity.
The results of the research programme under review have broadened the basis on which decisions on the control and eradication of hog cholera can be taken. Moreover, the research programme has offered a means of close cooperation between laboratories of European countries.
The EEC-sponsored programme on African swine fever (ASF) has covered:
Virus morphology and morphogenesis.
Immunology and pathogenesis.
Diagnosis and serological studies.
Because of security risks only a small number of laboratories have been involved, but some participation has been possible by workers from non-affected EEC countries [Pirbright (United Kingdom) and Lindholm (Denmark)].
Virus morphology and morphogenesis: Significant progress has been made in a study of the detailed structure of ASF virus, utilizing thin sections of infected cells and negatively-stained virus particles. Nunes et al have shown that virions usually consist of an outer “shell” made up of a unit membrane with an external dense coat and an inner core containing a dense nucleoid. The external coat, in negatively stained particles, appears to consist of subunits with a triangular appearance, which are bound to the membrane. The cores, after sonication or Nonidet P-40 treatment, appear spherical with a net-like periphery, suggesting the appearance of subunits.
Three groups of workers had reported on the physicochemical-chemical characterization of virus purified by essentially similar methods. Two of these (Black and Brown and Vigario et al) found five major polypeptides, of which at least two were glycoproteins according to Vigario et al. Minor polypeptides have been described, in numbers varying up to 19 (Tabares) in intracellular virus preparations.
Preliminary results of controlled disruption experiments, using NP-40, suggested that empty capsid shells contained two polypeptides of MW 76,000 (VP2) and 50,000 (VP3). The membrane included VP1 (MW 125,000) and VP4 (MW 44,000). VP5 may be associated with the DNA (MW 39,000).
Comparative studies have been made of the immunofluorescence and immunoperoxidase techniques for studying the morphogenesis of virions (Nunes et al).
The cross-immunoelectrophoresis technique was used to separate five virus-specific antigens derived from infected cells. Monospecific antisera were prepared against three of these by inoculation into pigs. These reagents should be useful in relating structure and function of virion components.
Immunology and pathogenesis: Nunes, Petisca and Gonzalves described the histopathology of acute ASF in pigs, noting a marked increase in lymphocytes and reticulum cells during the first 4 days after inoculation, followed by degenerative changes in vessel walls and reticulum. The proliferating lymphocytic cells were pyroninophilic and considered to be blast cells of the T series, resulting from stimulation by virus antigens. Plasmablasts (cells of the B series) were rare. The thymus was spared, but extensive destruction was seen in the bone marrow.
Wilkinson at Pirbright began to investigate resistance factors and demonstrated that UV-inactivated virus did not interfere with the production of ASF virus in cultures. Live ASF virus also did not induce interferon production in PK or PBM cultures and interferon produced in PK 15 cells failed to inhibit production of ASF virus in pig kidney cells or PBM cells.
Diagnostic methods and serological studies: Carnero et al reported on the virological and serological procedures adopted to confirm the outbreak of ASF in France in 1974. This appeared in the Pyreneean Atlantic Department, 30 Km from the frontier, and was thought to have been derived from a recrudescence of the disease in northern Spain. The strain of virus appeared to have a small capacity to spread and was clinically mild, accompanied by anorexia, cough, haemorrhages on ears, loss of weight, with restricted lesions at autopsy.
Twenty-five animals from one property were examined at the laboratory. Four spleens were positive on HA but 10 of 22 blood samples required a single subinoculation to reveal HA. Five animals were negative on 3 subcultures but 3 of these had antibody (IIF) and the other 2 were serologically negative. No HA I antibodies were detected but 18 of 25 sera were positive in IIF tests at a dilution of 1:10 and 11 at 1:100.
Carnero et al investigated the conditions for haemadsorption by infected leucocytes. The phenomenon occurred without modification at temperatures between 20–40°C and between pH 4 and pH 8. It was not influenced by prior treatment with trypsin (4 mg/m). No elution or haemagglutination was demonstrable.
The only virus which was found to interfere seriously with the Malmquist test for virus isolation was that of Aujeszky's disease which produced a cytopathic effect in leucocyte cultures. FMD virus, enteroviruses, SVD virus, adenovirus type 5 and TGE virus did not interfere.
A preliminary study (by French workers) of the 3-step immunoperoxidase technique showed it to be less easy to interpret than the IIF method for serum and tissue antibody.
Ordas and Sanchez-Botija have encountered increasing numbers of cases of Aujeszky's disease in young pigs in Spain and reported that inoculation of leucocyte cultures would reveal both viruses. If field materials did not produce HA in the first passage, they examined inoculated cultures by direct immunofluorescence and then stained for cytological changes, taking account of the fact that nuclear changes are characteristic for the herpesvirus.
Dr. Botija described development of a laboratory diagnostic technique which depends on the detection of antibodies to ASF virus in tissue extracts, using IIF tests. The importance of this procedure lies in the fact that 2–3 years ago 80 percent of positive cases could be confirmed by direct IF on tissue smears or sections; this figure is now around 30 percent. The tissue antibody technique using spleen, lung and liver for preference now detects 81 percent. In combination with direct IF, 92 percent of the total cases can be confirmed.
The immuno-electro-osmophoresis (IEOP) technique was investigated in Madrid for the detection of chronic cases and virus carriers. It had a sensitivity similar to that of IIF (c. 90 percent), the 2 tests combined detecting 95 percent of all positives. IEOP tests have the advantage that they can be readily applied to large numbers of sera for survey purposes and will probably be very useful in future.
Dr. Vigario and his colleagues in Lisbon have attempted to show the relationship of virus structural and non-structural antigens to some serological tests. They inoculated into rabbits semi-purified virus before and after treatment with Tween 80/ether or Triton X100. The resulting sera reacted with ASF virus in immunodiffusion and IIF tests, while antisera to semipurified virus (in 2 instances only) showed low levels of neutralizing activity. Antisera to extracts of infected cells inhibited HA in one instance only and it was suggested that the antigen response was not a structural component of the virion.
Attempts by the same workers to obtain monospecific antisera to structural polypeptides isolated in polyacrylamide gels were not successful, probably due to inadequate amounts of protein.
In summary, it is evident from the material presented that while many highly effective techniques have been established for large scale diagnostic applications much remains to be done on the structural components of the virus and virus-induced products involved in diagnostic serological reactions. Studies of the pathogenesis of the disease and of the immunological reactions involved are still in a very early stage and should be continued if an understanding is to be gained of the progressive loss of virulence of the virus strains in circulation at present, and the accompanying increase in very mild clinical cases or inapparent carriers. Methods of transmission also require further investigation.
