Prospects for development of African swine fever virus vaccines.

African swine fever virus is a large DNA virus which can cause an acute haemorrhagic fever in pigs resulting in high mortality. No vaccine is available, limiting options for control. The virus encodes up to 165 genes and virus particles are multi-layered and contain more than 50 proteins. Pigs immunised with natural low virulence isolates or attenuated viruses produced by passage in tissue culture and by targeted gene deletions can be protected against challenge with virulent viruses. CD8+ cells are required for protection induced by attenuated strain OURT88/3. Passive transfer of antibodies from immune to naïve pigs can also induce protection. Knowledge of the genome sequences of attenuated and virulent strains and targeted gene deletions from virulent strains have identified a number of virus genes involved in virulence and immune evasion. This information can be used to produce rationally attenuated vaccine strains. Virus antigens that are targets for neutralising antibodies have been identified and immunisation with these recombinant proteins has been shown to induce partial protection. However knowledge of antigens which encode the dominant protective epitopes recognised by CD8+ T cells is lacking.

A viral mechanism for inhibition of the cellular phosphatase calcineurin.

The transcription factor NFAT (nuclear factor of activated T cells) controls the expression of many immunomodulatory proteins. African swine fever virus inhibits proinflammatory cytokine expression in infected macrophages, and a viral protein A238L was found to display the activity of the immunosuppressive drug cyclosporin A by inhibiting NFAT-regulated gene transcription in vivo. This it does by binding the catalytic subunit of calcineurin and inhibiting calcineurin phosphatase activity.

Survival of African Swine Fever Virus in Excretions from Pigs Experimentally Infected with the Georgia 2007/1 Isolate.

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of swine which can be transmitted through direct contact with infected animals and their excretions or indirect contact with contaminated fomites. The shedding of ASFV by infected pigs and the stability of ASFV in the environment will determine the extent of environmental contamination. The recent outbreaks of ASF in Europe make it essential to develop disease transmission models in order to design effective control strategies to prevent further spread of ASF. In this study, we assessed the shedding and stability of ASFV in faeces, urine and oral fluid from pigs infected with the Georgia 2007/1 ASFV isolate. The half-life of infectious ASFV in faeces was found to range from 0.65 days when stored at 4°C to 0.29 days when stored at 37°C, while in urine it was found to range from 2.19 days (4°C) to 0.41 days (37°C). Based on these half-lives and the estimated dose required for infection, faeces and urine would be estimated to remain infectious for 8.48 and 15.33 days at 4°C and 3.71 and 2.88 days at 37°C, respectively. The half-life of ASFV DNA was 8 to 9 days in faeces and 2 to 3 days in oral fluid at all temperatures. In urine, the half-life of ASFV DNA was found to be 32.54 days at 4°C decreasing to 19.48 days at 37°C. These results indicate that ASFV in excretions may be an important route of ASFV transmission.

Immunization of African Indigenous Pigs with Attenuated Genotype I African Swine Fever Virus OURT88/3 Induces Protection Against Challenge with Virulent Strains of Genotype I.

The attenuated African swine fever virus genotype I strain OURT88/3 has previously been shown to induce protection of European breeds of domestic pigs against challenge with virulent isolates. To determine whether protective immune responses could also be induced in indigenous breeds of pigs from the Kinshassa region in Democratic Republic of Congo, we immunized a group of eight pigs with OURT88/3 strain and challenged the pigs 3 weeks later with virulent genotype I strain OURT88/1. Four of the pigs were protected against challenge. Three of the eight pigs died from African swine fever virus and a fourth from an unknown cause. The remaining four pigs all survived challenge with a recent virulent genotype I strain from the Democratic Republic of Congo, DRC 085/10. Control groups of non-immune pigs challenged with OURT88/1 or DRC 085/10 developed signs of acute ASFV as expected and had high levels of virus genome in blood.

Geographic distribution and epidemiology of peste des petits ruminants virus.

Peste des petits ruminants (PPR) is an important viral disease of goats and sheep prevalent in West Africa and the Middle East. In recent years, PPR has emerged in India, first in the South India and later in North India. To study the genetic relationships between viruses of distinct geographical origin we have sequenced a 322 nucleotide cDNA fragment of the fusion protein gene generated using reverse transcription followed by polymerase chain reaction (PCR) amplification. Viruses from nineteen independent PPR outbreaks were compared; these included the prototype African strain from Senegal and viruses from disease outbreaks which have occurred at different times and locations across Africa, Arabia, the Near East and the Indian subcontinent. Four separate lineages of the virus were identified and the virus isolates from Asia over the past 2 years were all of one lineage which had not previously been identified in Africa or Asia.

Canine distemper virus in Lake Baikal seals (Phoca sibirica).

The virus epizootic which resulted in significant mortality in Siberian seals (Phoca sibirica) in Lake Baikal during 1987/88 was caused by canine distemper virus. Sequence analysis of the virus glycoprotein genes revealed that it was most closely related to recent European field isolates of canine distemper virus. This paper presents evidence that the same virus continued to circulate in seals in Lake Baikal after the initial epizootic. Three out of 45 brain tissue samples collected from seals culled in the spring of 1992 were positive for canine distemper virus-specific nucleic acid by the reverse transcription/polymerase chain reaction and the sequences were closely related to that of the original virus isolated in 1988.

Assay sensitivity and differentiation of monoclonal antibody specificity in ELISA with different coating buffers.

Buffers of different pH and ionic strength were employed as coating buffers for antigen adsorption to microtitre plates. Their efficiency for coating plates with rinderpest virus (RPV) and foot-and-mouth disease virus (FMDV) antigens was studied by ELISA with polyclonal and monoclonal antibody preparations. While the adsorption and detection of RPV antigen with polyclonal antiserum was highly dependent on the ionic strength and pH of coating buffer, adsorption of antigenically active FMDV antigen was relatively unaffected by the buffering conditions. Both antigens were adsorbed optimally in 0.01 M phosphate buffer, pH 8.0. When monoclonal antibodies were used to detect antigen, there was a greater degree of dependence on the coating buffer than that found with polyclonal antisera. Moreover, when they were used to detect antigen adsorbed under several buffering conditions, monoclonal antibodies showed a variety of preferred buffers. The usefulness of this differential reactivity in distinguishing epitope specificity is demonstrated.

Cloning and sequence analysis of the matrix (M) protein gene of rinderpest virus and evidence for another bovine morbillivirus.

We have cloned and sequenced the entire M gene of the vaccine strain of rinderpest virus and that of the virulent Kabete "O" strain from which it was derived. The sequences of these two genes are essentially identical (99% at the nucleotide level), but were very different from a previously published Kabete O M gene sequence (M. Limo and T. Yilma, 1990, Virology 175, 323-327). Inspection of the nucleotide and deduced amino acid sequences of known morbillivirus M genes showed that the earlier sequence was clearly from a morbillivirus, but neither from rinderpest virus nor from peste des petits ruminants virus.

Nucleotide sequence comparisons of the fusion protein gene from virulent and attenuated strains of rinderpest virus.

We have cloned and sequenced the entire fusion (F) protein gene of the RBOK vaccine strain of rinderpest virus and the coding regions for the F genes of two mild field isolates of the virus from Africa. Analysis of the nucleotide and the predicted amino acid sequences showed that the vaccine virus was more than 99% identical in the protein coding region to the virulent Kabete O strain from which it was derived, whereas the field isolates differed by 10 to 12% from each other and from the vaccine strain. No changes were found in the F protein which could explain attenuation of the vaccine; however, each of the mild field isolates had amino acid changes in important functional areas which may be related to their attenuated phenotype.

The genome sequence of the virulent Kabete 'O' strain of rinderpest virus: comparison with the derived vaccine.

We have compared the complete genome sequences of the vaccine strain of rinderpest virus and the virulent strain from which it was derived. Only 87 bases differed between the two genomes (0.55%). Possibly significant differences in amino acid sequence were found in the N, P, F, H and L proteins. A number of differences were also found in the leader region (3' end of the genome), whilst the trailer region appears to be more conserved. In addition, the length of the genome was found in both cases to be 15882, an exact multiple of six, fulfilling predictions made earlier based on work with Sendai and measles viruses.

Dolphin and porpoise morbilliviruses are genetically distinct from phocine distemper virus.

The morbilliviruses recently isolated from two cetacean species in the North and Mediterranean Seas have been shown to differ from phocine distemper virus isolated from European seals using monoclonal antibodies. We have identified a "universal" morbillivirus primer set, based on highly conserved regions of the morbillivirus phosphoprotein (P) gene and used this to amplify a region surrounding the RNA editing site from all known members of the group. Sequence analysis of this region of the gene shows that the dolphin and porpoise viruses are related but quite different from all other members of the group, forming a distinct lineage more closely related to the ruminant morbilliviruses than to the carnivore viruses.

Evidence for different lineages of rinderpest virus reflecting their geographic isolation.

Sequence analysis of part of the fusion protein gene from recent isolates of rinderpest virus revealed that distinct lineages of the virus exist which reflect the geographical location of their isolation in Africa and Asia. Current strains circulating in Kenya and Sudan were most similar, both in terms of nucleotide sequence and pathogenic nature, to viruses isolated in Egypt and in Nigeria in 1983/1984 and they were quite distinct from an East African isolate (RBT-1) from the 1960s. Two older isolates of the virus, the Japanese avianized/lapinized vaccine strain dating from the 1930s and the Old Kabete strain dating from 1911, each differed considerably from the other viruses. The sequence data were derived from the region where the precursor protein is cleaved to yield the biologically active F1/F2 heterodimer; all strains analysed had a highly basic connecting peptide which is required for efficient cleavage by endogenous host cell proteases. No correlation was found between amino acid changes at this site and the rinderpest virus pathogenicity unlike the association reported for Newcastle disease virus.

The CD2v protein of African swine fever virus interacts with the actin-binding adaptor protein SH3P7.

The predicted extracellular domain of the CD2v protein of African swine fever virus (ASFV) shares significant similarity to that of the CD2 protein in T cells but has a unique cytoplasmic domain of unknown function. Here we have shown that CD2v is expressed as a glycoprotein of approximately 105 kDa in ASFV-infected cells. In the absence of an extracellular ligand, the majority of CD2v appears to localize to perinuclear membrane compartments. Furthermore, we have shown using the yeast two-hybrid system and by direct binding studies that the cytoplasmic tail of CD2v binds to the cytoplasmic adaptor protein SH3P7 (mAbp1, HIP55), which has been reported to be involved in diverse cellular functions such as vesicle transport and signal transduction. A cDNA clone encoding a variant form of SH3P7 could also be identified and was found to be expressed in a wide range of porcine tissues. Deletion mutagenesis identified proline-rich repeats of sequence PPPKPC in the ASFV CD2v protein to be necessary and sufficient for binding to the SH3 domain of SH3P7. In ASFV-infected cells, CD2v and SH3P7 co-localized in areas surrounding the perinuclear virus factories. These areas also stained with an antibody that recognizes a Golgi network protein, indicating that they contained membranes derived from the Golgi network. Our data provide a first molecular basis for the understanding of the immunomodulatory functions of CD2v in ASFV-infected animals.

Nuclear and nucleolar localization of an African swine fever virus protein, I14L, that is similar to the herpes simplex virus-encoded virulence factor ICP34.5.

PCR analysis of the genomes of 18 different African swine fever virus (ASFV) isolates showed that the I14L open reading frame (ORF) was present as either a long form or short form in all of the isolates. Sequencing of the ORF from eight isolates confirmed that both forms of the ORF were well conserved. Antisera raised against the I14L protein identified the long form of the protein as a 21 kDa protein expressed late during ASFV infection. Immunofluorescent analysis of transiently expressed haemagglutinin-tagged forms of the I14L protein showed that the long form of the protein localized predominantly to the nucleus and within the nucleoli. In contrast, although the short form of the protein was also present predominantly in the nucleus, it did not localize to the nucleoli. Deletion of the N-terminal 14 amino acids from the long form of the I14L protein, which includes a high proportion of basic Arg/Lys residues, abolished the specific nucleolar localization of the protein, although the protein was still present in the nucleus. Addition of this 14 amino acid sequence to beta-galactosidase or replacement of the N-terminal 14 amino acids of the I14L short form with those from the long form directed both of these modified proteins to the nucleolus. This indicates that this 14 amino acid sequence contains all the signals required for nucleolar localization.