Classical swine fever virus inhibits nitric oxide production in infected macrophages.

Classical swine fever virus (CSFV)-macrophage interactions during infection were analysed by examining macrophage transcriptional responses via microarray. Eleven genes had increased mRNA levels (>2.5-fold, P<0.05) in infected cell cultures, including arginase-1, an inhibitor of nitric oxide production, phosphoinositide 3-kinase, chemokine receptor 4 and interleukin-1beta. Lower levels of nitric oxide and increased arginase activity were found in CSFV-infected macrophages. These changes in gene expression in macrophages suggest viral modulation of host expression to suppress nitric oxide production.

Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection.

Although antibody-mediated immune mechanisms have been shown to be important in immunity to ASF, it remains unclear what role virus neutralizing antibodies play in the protective response. Virus neutralizing epitopes have been identified on three viral proteins, p30, p54, and p72. To evaluate the role(s) of these proteins in protective immunity, pigs were immunized with baculovirus-expressed p30, p54, p72, and p22 from the pathogenic African swine fever virus (ASFV) isolate Pr4. ASFV specific neutralizing antibodies were detected in test group animals. Following immunization, animals were challenged with 10(4) TCID(50) of Pr4 virus. In comparison to the control group, test group animals exhibited a 2-day delay to onset of clinical disease and reduced viremia levels at 2 days postinfection (DPI); however, by 4 DPI, there was no significant difference between the two groups and all animals in both groups died between 7 and 10 DPI. These results indicate that neutralizing antibodies to these ASFV proteins are not sufficient for antibody-mediated protection.

African swine fever virus multigene family 360 genes affect virus replication and generalization of infection in Ornithodoros porcinus ticks.

Recently, we reported that African swine fever virus (ASFV) multigene family (MGF) 360 and 530 genes are significant swine macrophage host range determinants that function by promoting infected-cell survival. To examine the function of these genes in ASFV's arthropod host, Ornithodoros porcinus porcinus, an MGF360/530 gene deletion mutant (Pr4Delta35) was constructed from an ASFV isolate of tick origin, Pr4. Pr4Delta35 exhibited a significant growth defect in ticks. The deletion of six MGF360 and two MGF530 genes from Pr4 markedly reduced viral replication in infected ticks 100- to 1,000-fold. To define the minimal set of MGF360/530 genes required for tick host range, additional gene deletion mutants lacking individual or multiple MGF genes were constructed. The deletion mutant Pr4Delta3-C2, which lacked three MGF360 genes (3HL, 3Il, and 3LL), exhibited reduced viral growth in ticks. Pr4Delta3-C2 virus titers in ticks were significantly reduced 100- to 1,000-fold compared to control values at various times postinfection. In contrast to the parental virus, with which high levels of virus replication were observed in the tissues of infected adults, Pr4Delta3-C2 replication was not detected in the midgut, hemolymph, salivary gland, coxal gland, or reproductive organs at 15 weeks postinfection. These data indicate that ASFV MGF360 genes are significant tick host range determinants and that they are required for efficient virus replication and generalization of infection. The impaired virus replication of Pr4Delta3-C2 in the tick midgut likely accounts for the absence of the generalized infection that is necessary for the natural transmission of virus from ticks to pigs.

African swine fever virus multigene family 360 and 530 genes are novel macrophage host range determinants.

Pathogenic African swine fever virus (ASFV) isolates primarily target cells of the mononuclear-phagocytic system in infected swine and replicate efficiently in primary macrophage cell cultures in vitro. ASFVs can, however, be adapted to grow in monkey cell lines. Characterization of two cell culture-adapted viruses, MS16 and BA71V, revealed that neither virus replicated in macrophage cell cultures. Cell viability experiments and ultrastructural analysis showed that infection with these viruses resulted in early macrophage cell death, which occurred prior to viral progeny production. Genomic cosmid clones from pathogenic ASFV isolate E70 were used in marker rescue experiments to identify sequences capable of restoring MS16 and BA71V growth in macrophage cell cultures. A cosmid clone representing a 38-kbp region at the left terminus of the genome completely restored the growth of both viruses. In subsequent fine-mapping experiments, an 11-kbp subclone from this region was sufficient for complete rescue of BA71V growth. Sequence analysis indicated that both MS16 and BA71V had significant deletions in the region containing members of multigene family 360 (MGF 360) and MGF530. Deletion of this same region from highly pathogenic ASFV isolate Pr4 significantly reduced viral growth in macrophage cell cultures. These findings indicate that ASFV MGF360 and MGF530 genes perform an essential macrophage host range function(s) that involves promotion of infected-cell survival.

An African swine fever virus ERV1-ALR homologue, 9GL, affects virion maturation and viral growth in macrophages and viral virulence in swine.

The African swine fever virus (ASFV) genome contains a gene, 9GL, with similarity to yeast ERV1 and ALR genes. ERV1 has been shown to function in oxidative phosphorylation and in cell growth, while ALR has hepatotrophic activity. 9GL encodes a protein of 119 amino acids and was highly conserved at both nucleotide and amino acid levels among all ASFV field isolates examined. Monospecific rabbit polyclonal antibody produced to a glutathione S-transferase-9GL fusion protein specifically immunoprecipitated a 14-kDa protein from macrophage cell cultures infected with the ASFV isolate Malawi Lil-20/1 (MAL). Time course analysis and viral DNA synthesis inhibitor experiments indicated that p14 was a late viral protein. A 9GL gene deletion mutant of MAL (Delta9GL), exhibited a growth defect in macrophages of approximately 2 log(10) units and had a small-plaque phenotype compared to either a revertant (9GL-R) or the parental virus. 9GL affected normal virion maturation; virions containing acentric nucleoid structures comprised 90 to 99% of all virions observed in Delta9GL-infected macrophages. The Delta9GL virus was markedly attenuated in swine. In contrast to 9GL-R infection, where mortality was 100%, all Delta9GL-infected animals survived infection. With the exception of a transient fever response in some animals, Delta9GL-infected animals remained clinically normal and exhibited significant 100- to 10,000-fold reductions in viremia titers. All pigs previously infected with Delta9GL survived infection when subsequently challenged with a lethal dose of virulent parental MAL. Thus, ASFV 9GL gene deletion mutants may prove useful as live-attenuated ASF vaccines.

African swine fever virus replication in the midgut epithelium is required for infection of Ornithodoros ticks.

Although the Malawi Lil20/1 (MAL) strain of African swine fever virus (ASFV) was isolated from Ornithodoros sp. ticks, our attempts to experimentally infect ticks by feeding them this strain failed. Ten different collections of Ornithodorus porcinus porcinus ticks and one collection of O. porcinus domesticus ticks were orally exposed to a high titer of MAL. At 3 weeks postinoculation (p.i.), <25% of the ticks contained detectable virus, with viral titers of <4 log(10) 50% hemadsorbing doses/ml. Viral titers declined to undetectability in >90% of the ticks by 5 weeks p.i. To further study the growth defect, O. porcinus porcinus ticks were orally exposed to MAL and assayed at regular intervals p.i. Whole-tick viral titers dramatically declined (>1,000-fold) between 2 and 6 days p.i., and by 18 days p.i., viral titers were below the detection limit. In contrast, viral titers of ticks orally exposed to a tick-competent ASFV isolate, Pretoriuskop/96/4/1 (Pr4), increased 10-fold by 10 days p.i. and 50-fold by 14 days p.i. Early viral gene expression, but not extensive late gene expression or viral DNA synthesis, was detected in the midguts of ticks orally exposed to MAL. Ultrastructural analysis demonstrated that progeny virus was rarely present in ticks orally exposed to MAL and, when present, was associated with extensive cytopathology of phagocytic midgut epithelial cells. To determine if viral replication was restricted only in the midgut epithelium, parenteral inoculations into the hemocoel were performed. With inoculation by this route, a persistent infection was established although a delay in generalization of MAL was detected and viral titers in most tissues were typically 10- to 1,000-fold lower than those of ticks injected with Pr4. MAL was detected in both the salivary secretion and coxal fluid following feeding but less frequently and at a lower titer compared to Pr4. Transovarial transmission of MAL was not detected after two gonotrophic cycles. Ultrastructural analysis demonstrated that, when injected, MAL replicated in a number of cell types but failed to replicate in midgut epithelial cells. In contrast, ticks injected with Pr4 had replicating virus in midgut epithelial cells. Together, these results indicate that MAL replication is restricted in midgut epithelial cells. This finding demonstrates the importance of viral replication in the midgut for successful ASFV infection of the arthropod host.

Deletion of a CD2-like gene, 8-DR, from African swine fever virus affects viral infection in domestic swine.

An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (delta8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, delta8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo, 8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant delta8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with delta8-DR. A delay in spread to and/or replication of delta8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for delta8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with delta8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.

African swine fever virus infection in the argasid host, Ornithodoros porcinus porcinus.

The pathogenesis of African swine fever virus (ASFV) infection in Ornithodoros porcinus porcinus was examined in nymphal ticks infected with the ASFV isolate Chiredzi/83/1. At times postinfection (p.i.) ranging from 6 h to 290 days, ticks or dissected tick tissues were titrated for virus and examined ultrastructurally for evidence of virus replication. The ASFV infection rate in ticks was 100% in these experiments, and virus infection was not associated with a significant increase in tick mortality. Initial ASFV replication occurred in phagocytic digestive cells of the midgut epithelium. Subsequent infection and replication of ASFV in undifferentiated midgut cells was observed at 15 days p.i. Generalization of virus infection from midgut to other tick tissues required 2 to 3 weeks and most likely involved virus movement across the basal lamina of the midgut into the hemocoel. Secondary sites of virus replication included hemocytes (type I and II), connective tissue, coxal gland, salivary gland, and reproductive tissue. Virus replication was not observed in the nervous tissue of the synganglion, Malpighian tubules, and muscle. Persistent infection, characterized by active virus replication, was observed for all involved tick tissues. After 91 days p.i., viral titers in salivary gland and reproductive tissue were consistently the highest detected. Successful tick-to-pig transmission of ASFV at 48 days p.i. correlated with high viral titers in salivary and coxal gland tissue and their secretions. A similar pattern of virus infection and persistence in O. porcinus porcinus was observed for three additional ASFV tick isolates in their associated ticks.

Baculovirus expressed 2C of foot-and-mouth disease virus has the potential for differentiating convalescent from vaccinated animals.

Determining whether animals have been infected with foot-and-mouth disease virus or vaccinated is important because infected animals frequently become carriers of the virus, shed it intermittently and thus may be the source of new outbreaks of the disease. We had shown previously that the sera of convalescent animals contain antibodies to 2C, a highly conserved non-structural protein, whereas the sera of vaccinated animals do not. This is explained by observation that 2C is retained on the membranes of cells used for growing the virus for vaccine production. In contrast, the non-structural protein 3D, which is released into the medium, is not removed by centrifugation or filtration during vaccine production and therefore stimulates an immune response in both vaccinated and convalescent cattle. In this study we produced 2C and 3D in insect cells infected with recombinant baculoviruses. As demonstrated by serology and electron microscopy, 2C is also retained on the membranes of the insect cells. Both expressed proteins react with sera of convalescent animals, indicating that they are conformationally similar, but the 2C does not react with sera from vaccinated animals. The baculovirus expressed 2C appears to be a suitable antigen for the development of a reliable diagnostic test.

A BIR motif containing gene of African swine fever virus, 4CL, is nonessential for growth in vitro and viral virulence.

An African swine fever virus (ASFV) gene with similarity to viral and cellular inhibitor of apoptosis genes (iap) has been described in the African isolate Malawi Lil-20/1 (ORF 4CL) and a cell-culture-adapted European virus, BA71V (ORF A224L). The similarity of the ASFV gene to genes involved in inhibiting cellular apoptosis suggested the gene may regulate apoptosis in ASFV-infected cells and thus may function in ASFV virulence and/or host range. Sequence analysis of additional African and European pathogenic isolates demonstrates that this gene is highly conserved among both pig and tick ASFV isolates and that its similarity to iap genes is limited to the presence of a single IAP repeat motif (BIR motif) in the ASFV gene. To study gene function, a 4CL gene deletion mutant, delta 4CL, was constructed from the pathogenic Malawi Lil-20/1 isolate. Growth characteristics of delta 4CL in swine macrophage cell cultures were indistinguishable from those of parental virus. Infected macrophage survival time and the induction and magnitude of apoptosis in virus-infected macrophages were comparable for cells infected with either delta 4CL or parental virus. In infected swine, delta 4CL exhibited an unaltered Malawi Lil-20/1 virulence phenotype. These data indicate that, although highly conserved among ASFV isolates, the 4CL gene is nonessential for growth in macrophage cell cultures in vitro and for pig virulence. Additionally, despite its limited similarity to JAP genes, 4CL exhibits no anti-apoptotic function in infected macrophage cell cultures. The high degree of gene conservation among ASFV isolates, together with the apparent lack of function in the swine host, suggests 4CL may be a host range gene involved in aspects of infection in the arthropod host, ticks of the genus Ornithodoros.

Immunization with VP2 is sufficient for protection against lethal challenge with African horsesickness virus Type 4.

Horses were immunized by inoculation with a vaccinia construct containing a full-length cDNA corresponding to the L2 gene segment of African horsesickness virus type 4(AHSV-4). All immunized horses developed serum neutralizing antibodies prior to challenge with virulent AHSV-4. No ELISA-reactive antibodies were present prior to challenge. A group of four seronegative control horses died after developing clinical signs and lesions typical of the pulmonary form of African horsesickness while the immunized horses were clinically normal. Increases in serum neutralizing and ELISA-reactive antibody titers following challenge indicate that at least some replication of challenge virus occurred in immunized horses. These results demonstrate that AHSV VP2 alone is sufficient to induce a protective immune response in horses and indicate the usefulness of ELISA-reactive antibodies for differentiation of vaccinated and naturally exposed horses.

Absence of protein 2C from clarified foot-and-mouth disease virus vaccines provides the basis for distinguishing convalescent from vaccinated animals.

We have recently reported that cattle and pigs which have been vaccinated against foot-and-mouth disease can be distinguished from convalescent animals by the absence of antibodies to viral non-structural protein 2C (Lubroth and Brown, Res. Vet. Sci., 1995, 59, 70-78(1)). In this study, we show that the absence of 2C antibodies from the sera of vaccinated animals can be explained by the association of this viral protein with cellular debris which is separated from the virus harvest prior to inactivation of the supernatant for vaccine production. This serological marker can be of great value in countries where the disease occurs or in the veterinary regulatory arena when livestock are transported across borders, since it can be used to identify convalescent, persistently infected animals and vaccinates exposed to wild-type virus variants which have infected the vaccinated animals.

Clinical pathology and hemostatic abnormalities in experimental African horsesickness.

Infection of naive North American horses with 10(4) cell culture infectious doses (CCID50) of virulence variants of African horsesickness virus (AHSV), designated AHSV/4SP, AHSV/9PI, and AHSV/4PI, reproduced three classical forms of African horsesickness: acute (pulmonary), subacute (cardiac), and febrile, respectively. Distinct clinicopathologic and hemostatic abnormalities were associated with each form of disease. Hemostatic abnormalities included increased concentration of fibrin degradation products and prolongation of prothrombin, activated partial thromboplastin, and thrombin clotting times. Hemostatic findings indicated activation of the coagulation and fibrinolytic systems with clotting factor consumption in acute and subacute cases of African horsesickness. Hematologic abnormalities in acute and subacute cases of African horsesickness included leukopenia, decreased platelet counts, elevated hematocrit, and increased erythrocyte counts and hemoglobin concentration. Leukopenia was characterized by lymphopenia, neutropenia, and a left shift. Increased levels of serum creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and alkaline phosphatase, hypocalcemia, hypoalbuminemia, hypoproteinemia, and elevated creatinine, phosphorus, and total bilirubin levels were present in some but not all horses. Metabolic acidosis, indicated by decreased total bicarbonate and increased lactate and anion gap, was present in horses with the acute form of disease. Mild thrombocytopenia and leukopenia were occasionally associated with the febrile form of disease. These results suggest a role for intravascular coagulation in the pathogenesis of African horsesickness.

Characterization of an acid-resistant mutant of foot-and-mouth disease virus.

A foot-and-mouth disease virus mutant which is stable at pH 6.4 has been isolated from a virus of serotype A. In contrast to the parent (P) virus, which gave a mixture of large and small plaques in BHK21 cells and in a bovine kidney cell line, the acid-resistant (AR) virus gave small plaques which did not increase markedly in size after 24 hr. The infectivity titer of the acid-resistant virus was about 100-fold lower in suckling mice than in BHK21 cells, whether the inoculation was made intraperitoneally or intracerebrally, whereas the parent virus gave similar titers in both systems. Furthermore, in mice the AR virus reached its end point two to three times more slowly. The diameter of the AR virus was almost 20% less than that of the P virus and it had a more distinct topography, but the two viruses cosedimented in sucrose gradients. However, the buoyant density in CsCl of the AR virus was slightly lower (1.42 compared with 1.43 g/cc) in coruns. The RNAs and capsid proteins of the two viruses gave similar profiles in sucrose gradients and by SDS-PAGE, respectively. However, isoelectric focusing of the capsid proteins revealed considerable differences between the two viruses. Whereas the P virus gave four protein bands, corresponding to VP1-VP4, the AR virus gave one band for VP4, two for VP3, two for VP2, and four for VP1. Sequence analysis of the genes coding for the capsid protein regions of the two viruses showed four changes (one silent), resulting in an Ala-3-->Ser substitution in VP1 and Glu-131-->Lys and Asp-133-->Ser substitutions in VP2.

African horsesickness: pathogenesis and immunity.

African horsesickness (AHS) is a serious, non-contagious disease of horses and other solipeds caused by an arthropod-borne orbivirus of the family Reoviridae. In horses, AHS causes three distinct clinicopathologic syndromes, the pulmonary, cardiac and fever forms of the disease. Recent work has shown that the primary determinant of the form of disease expressed by naive horses is the virulence of the virus inoculum. Horses which recover from AHS exhibit solid humoral immunity against homologous challenge. Protective antibodies appear to be directed towards neutralizing epitopes on AHS virus VP2. The relationship of neutralization to protection and vaccination is discussed.

Foot-and-mouth disease virus particles contain replicase protein 3D.

An antibody against the Escherichia coli-expressed RNA polymerase of foot-and-mouth disease virus (FMDV) reacts with the virus in ELISA and radioimmunoprecipitation experiments and with a protein of the disrupted virus particle in an immunoblot analysis. Treatment of the virus with trypsin, which cleaves capsid protein VP1 and a 56-kDa polypeptide present in trace amount in the particles, reduces the level of the reaction in ELISA and radioimmunoprecipitation and eliminates the immunoblot reaction. Electron microscopy showed that only approximately 20% of the virus particles reacted with the anti-polymerase antibody, whereas most reacted with an antibody against the immunodominant G-H loop of the virus. In the presence of ammonium ions, the expressed polymerase degrades the RNA of the virus into molecules sedimenting at approximately 12 S, indicating that it can act as a hydrolytic as well as a polymerizing enzyme. Moreover, the RNA in trypsin-treated virus particles is degraded when incubated at 37 degrees C, suggesting that the cleaved 56-kDa protein still possesses hydrolytic activity. In addition, the anti-polymerase antibody, which inhibits the polymerase activity of the E. coli-expressed protein, also partially inhibits the hydrolytic activity of the previously described endonuclease of the virus particle, suggesting that this enzyme is identical with the polymerase or forms part of it.

Neutralizing epitopes of African horsesickness virus serotype 4 are located on VP2.

A panel of monoclonal antibodies (MAbs) was generated against African horsesickness virus serotype 4 (AHSV/4). Three of the MAbs (SA6, OH3, and ME11) strongly neutralized the homologous virus and a heterologous type 4 isolate. The MAbs did not cross-neutralize AHS serotypes 1-3 or 5-9. The MAbs immunoprecipitated a viral protein of 108 kDa which co-migrated with VP2. Pretreatment with SA6 prevented mortality of 71% of day-old mice after intracranial injection of 100 LD50 of AHSV/4, while OH3 and ME11 significantly increased the average survival time of challenged animals. This study demonstrates that neutralizing epitope(s) for AHS are located on VP2 and that antibodies to these epitope(s) are protective in a neonatal mouse model.

Immunolocalization of Alzheimer beta-amyloid peptide precursor to cellular membranes in baculovirus expression system.

One characteristic of Alzheimer's disease (A beta disease) is the accumulation of amyloid deposits within the extracellular space of the brain and meninges. A 40 amino acid peptide called beta-peptide or A4 protein is the subunit of the amyloid fibrils found in these deposits. The sequence of beta-peptide is contained within those of a family of larger proteins called the Alzheimer beta-amyloid peptide precursor (APP). These APPs contain, in addition to a signal sequence, a hydrophobic sequence that is believed to span cell membranes. Although biochemical studies indicate that some APPs have properties of integral membrane proteins, morphological confirmation of this has not been reported. We recently described an expression system in which human APP751 cDNA was placed under the transcriptional regulation of the polyhedrin gene promoter in the baculovirus Autographica californica infecting a Spodoptera frugiperda cell line (Ramakrishna et al., Biochem Biophys Res Commun 174:983-989, 1991). As part of a larger biochemical and molecular biological study of APP, we have carried out an immunocytochemical study using antibodies directed against several epitopes within APP to reveal, at both the light and the electron microscopic levels, the cellular localization of APP in the baculovirus expression system. These studies demonstrate that APP751 is abundantly synthesized and inserted into certain of the membrane compartments of the cell. As early as 24 hr postinfection, APP751 is found associated with all membrane compartments excepting mitochondrial membranes. The patterns of immunolabeling are consistent with our biochemical findings that the protein is processed in these cells so as to release the extracellular domain and to retain a transmembrane and intracellular segment. These data provide the first morphological demonstration of the membrane location of APP751, its posttranslational processing to a secreted fragment, and its exclusion from the mitochondrial membranes. This system is especially valuable for identifying conditions under which antibodies raised against APP or appropriate synthetic peptides will react with native APP.