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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 (Δ9GL), 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 Δ9GL-infected macrophages. The Δ9GL virus was markedly attenuated in swine. In contrast to 9GL-R infection, where mortality was 100%, all Δ9GL-infected animals survived infection. With the exception of a transient fever response in some animals, Δ9GL-infected animals remained clinically normal and exhibited significant 100- to 10,000-fold reductions in viremia titers. All pigs previously infected with Δ9GL 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.

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 (Δ8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, Δ8-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 Δ8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with Δ8-DR. A delay in spread to and/or replication of Δ8-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 Δ8-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 Δ8-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.

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.

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.

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.

Infection of naive North American horses with lo4 cell culture infectious doses (CCID,,) of virulence variants of African horsesickness virus (AHSV), designated AHSVMSP, 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. In- creased levels of serum creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and alkaline phos- phatase, 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. African horsesickness (AHS) is an arthropod-borne, noncontagious disease of horses. The causative agent, African horsesickness virus (AHSV), is classified as an orbivirus within the family Reoviridae and is closely related to bluetongue virus (BTV) and epizootic hem- orrhagic disease virus (EHDV).'O Infection of suscep- tible horses with AHSV can result in high morbidity and mortality. In 192 1, Theiler33 described four clin- icopathologic forms of AHS: the acute (pulmonary), subacute (cardiac), mixed, and febrile forms of the dis- ease. The disease in its acute form is characterized by rapid onset of high fever, signs of respiratory distress, pulmonary edema, pleural effusion, hemorrhage, and death. The subacute form is characterized by hydro- pericardium, endocardia1 hemorrhage, marked accu- mulation of edema fluid in the head and neck, partic- ularly in the supraorbital fossae, ligamentum nuchae, and fascia1 planes of the cervical musculature. The mixed form consists of clinical signs and lesions typical of both acute and subacute AHS. The febrile form is characterized by mild, remittent fever that is some- times associated with vague clinical signs, which may escape clinical recognition. Recent evidence indicates that in naive horses, the major determinant of the clin- icopathologic form of disease is the viral virulence phenotype.19

The physico-chemical properties and immunogenicity of experimental vaccines against foot-and-mouth disease (FMD) and poliomyelitis, prepared by treatment of the viruses with N-acetylethyleneimine (AEI), formaldehyde or neutral red, have been studied. None of these reagents affects the rate of sedimentation of the particles or their reaction with antibody against the major immunogenic sites. FMD vaccines prepared by inactivation with AEI or neutral red, behaved like the untreated virus, in that they were disrupted on lowering the pH below 7. The RNA of the AEI-inactivated virus was degraded into slowly sedimenting molecules. Unlike AEI-inactivated virus, from which all the RNA could be extracted with phenol-SDS, the recovery from the neutral red inactivated virus was variable and was sometimes as low as 40%; this RNA gave a heterogenous profile in sucrose gradients. The capsid proteins in the AEI preparation migrated in SDS-PAGE to the same positions as those of untreated virus, but in the neutral red preparation there was evidence of cross-linking. In contrast, the formaldehyde-inactivated vaccine was stable below pH 7 and the RNA could not be released by extraction with phenol-SDS at pH 5, because the capsid proteins had become cross-linked and/or linked to the RNA. As with foot-and-mouth disease virus (FMDV), poliovirus which had been inactivated with formaldehyde did not release its RNA on extraction with phenol-SDS and the capsid proteins were also cross-linked. Surprisingly, although AEI cleaved the viral RNA slowly in situ, the virus was no longer infectious after 6 h. Neutral red did not reduce the infectivity of the virus. All of the preparations gave similar levels of neutralizing antibody after a single inoculation. The high levels obtained with the formaldehyde-inactivated vaccines have implications for the processing of fixed particles by the antigen-presenting cells.

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.

There are three clinicopathologic syndromes associated with African horsesickness (AHS) virus infection in horses. These different forms of AHS (pulmonary, cardiac, and fever forms) vary in the organs affected, the severity of lesions, time of onset of clinical signs and mortality rates. We have studied the effects of infection with three cell culture passaged variants of AHS virus in naive North American horses. One of these viruses, AHS/4SP, consistently caused the pulmonary form of AHS with rapid onset of severe pulmonary edema and 100% mortality. A second variant, AHS/9PI, resulted in signs and lesions typical of the cardiac form of AHS: pericardial effusion, subendocardial hemorrhage and widespread subcutaneous edema. Mortality was approximately 70%. The third variant, AHS/4PI, produced mild to subclinical disease in horses, usually expressed only as transient mild fever. No mortality occurred in horses due to infection with AHS/4PI. All surviving infected animals did, however, seroconvert with both neutralizing and ELISA-reactive antibodies. The results of these studies indicate clearly that in naive horses the form of disease expressed is a property of the AHS virus inoculum.

The three SAT serotype viruses, endemic in Africa, are well known for their difficulty to adapt to cell culture. The viral mechanism involved in foot-and-mouth disease virus (FMDV) tissue tropism and cellentry is not well understood. A recombinant, small plaque-forming virus (vSAT1tc), derived from a tissue culture-adapted SAT1 virus (SAR/9/81tc), revealed four amino acid substitutions (VP3 Asp192 → Tyr; VP3 Ser217 → Ile; VP1 Ala69 → Gly and VP1 Asn110 → Lys) in the capsid, compared to the SAR/9/81wt isolate collected from infected impala epithelium. One substitution added a positively charged lysine residue to the short F-G loop of VP1. Furthermore, vSAT1tc displayed a high affinity for CHO-K1 cells possibly via interaction with negatively charged sulphated polysaccharides while SAT1 impala strain relied strongly on V6 integrin receptors for cell entry. The cell culture adaptation and small plaque phenotype of vSAT1tc was accompanied by differences in particle aggregation and significant differences in acid stability. Based on limited cross neutralization data, the antigenic features seem to be unchanged. Thus, acquisition of positively charged residues in the virion may be beneficial for adaptation of SAT type field strains to cell culture.

Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus within the Picornaviridae family. Infection of cells with positive-strand RNA viruses results in a rearrangement of intracellular membranes into viral replication complexes. The origin of these membranes remains unknown; however induction of the cellular process of autophagy is beneficial for the replication of poliovirus, suggesting that it might be advantageous for other picornaviruses. By using confocal microscopy we showed in FMDV-infected cells co-localization of non-structural viral proteins 2B, 2C and 3A with LC3 (an autophagosome marker) and viral structural protein VP1 with Atg5 (autophagy-related protein), and LC3 with LAMP-1. Importantly, treatment of FMDV-infected cell with autophagy inducer rapamycin, increased viral yield, and inhibition of autophagosomal pathway by 3-methyladenine or small-interfering RNAs, decreased viral replication. Altogether, these studies strongly suggest that autophagy may play an important role during the replication of FMDV.

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.

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, Pβ. 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.

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 (Pr4Δ35) was constructed from an ASFV isolate of tick origin, Pr4. Pr4Δ35 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 Pr4Δ3-C2, which lacked three MGF360 genes ( 3HL , 3Il , and 3LL ), exhibited reduced viral growth in ticks. Pr4Δ3-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, Pr4Δ3-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 Pr4Δ3-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.

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.

African horsesickness (AHS) is an arboviral disease of horses, donkeys and mules. The causative agent, classified in the Reoviridae, genus Orbivirus, is transmitted primarily by biting midges of the genus Culicoides. African horsesick­ ness virus (AHSV) infection results in severe pulmonary ede­ ma and pleural and pericardial effusion in naive horses.>' The mechanism(s) by which these alterations in fluid balance occur has been the subject of limited research. The results of one recent study of the morphologic basis for the devel­ opment of pulmonary edema in AHS revealed no ultrastruc­ tural evidence for endothelial damage or infection by AHS virus .. The present report describes the ultrastructural evi­ dence for infection of pulmonary microvascular endothelial cells of horses experimentally infected with AHSV. Two groups of four horses each were used. Animals in group NO.1 were infected by intravenous injection with 10 2

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.), 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.

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.

Widely used inactivated vaccines for foot-and-mouth disease (FMD) induce protective immunity, but vaccine production plants and residual virus in the vaccine itself have been implicated in disease outbreaks. The structure of the FMD virion has been determined, and although much of the surface of the viral particle is produced by complex folding of the three surface-exposed capsid proteins (VP1-3), some surface regions representing important linear epitopes can be mimicked by recombinant proteins or synthetic peptides. Vaccine candidates based on these products stimulate immune responses to foot-and-mouth virus (FMDV), but do not always protect livestock from disease. The basis of protective immunity to FMDV has been explored using genetic engineering to produce antigenic chimeras of the virus. Studies with these chimeras have shown that a strong and protective immune response can be generated in livestock to epitopes outside the sequential epitopes incorporated into previous subunit vaccine candidates. Genetic engineering of the virus has also been used to demonstrate that changes within the sequence encoding an arginine-glycine-aspartic acid (RGD) sequence in VP1 abrogate virus binding to cells in culture, confirming the role of RGD as the receptor binding site. Based on this information, genetically stable viruses which cannot bind to cells have been created by deleting the nucleotides coding the RGD sequence. The receptor binding site-deleted viruses have been shown to be non-infectious in tissue culture, mice, and swine. Cattle vaccinated with these viruses are protected from disease when challenged with virulent FMDV, demonstrating that they could serve as the basis for safer FMD vaccines.

Here we describe an African swine fever virus (ASFV) protein encoded by the open reading frame 5-AR that shares structural and functional similarities with the family of bacterial histone-like proteins which include histone-like DNA binding proteins, integration host factor, and Bacillus phage SPO1 transcription factor, TF1. The ASFV 5-AR gene was cloned by PCR and expressed in E. coli. Monospecific antiserum prepared to the 5-AR bacterial expression product specifically immunoprecipitated a protein of approximately 11.6 kDa from ASFV infected swine macrophages at late times post infection. Additionally, the 5-AR expression product was strongly recognized by ASFV convalescent pig serum, indicating its antigenicity during natural infection. Cloned p11.6 bound both double and single stranded DNA-cellulose columns. Consistent with a DNA binding function, immunoelectronmicroscopy localized p11.6 to the virion nucleoid, To our knowledge, p11.6 is the first bacterial histone-like DNA-binding protein found in an animal virus or eukaryotic cell system.

We have previously described a monoclonal antibody (mAb 135D4) to an unidentified 70- to 72-kDa African swine fever virus (ASFV) protein that exhibited high levels of neutralizing activity against various virulent ASFV isolates. Here, we identify the reactive ASFV protein as the major virus structural protein p72. In vitro -translated products of the p72 protein gene were specifically immunoprecipitated by mAb 135D4. Immunoprecipitation of a nested set of truncated p72 in vitro translation products defined the region between amino acid residues 400 and 404 as necessary for mAb 135D4 reactivity. Five partially overlapping peptides (15mers) covering residues 388-446 failed to react with mAb 135D4, suggesting the conformational dependence of the epitope. Supporting this interpretation, larger in vitro translation products representing residues 56282, 159-361, 360-508, and 507-646 also failed to react with mAb 135D4. Consistent with its involvement in virus neutralization, immunoelectromicroscopy, using a rabbit antiserum against mAb 135D4-purified p72, located the protein on the surface of unenveloped virus particles.

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.

INTRODUCTION. In the swine host ASFV infects cells of the mononuclear-phagocytic system including fixed tissue macrophages (1). Cytopathological changes consistent with apoptotic cell death, including karyorrhexis and chromatin condensation, have been observed in mononuclear cells in tissues of pigs infected with highly virulent ASFV isolates (2). ASFV infection induced apoptosis in primary swine macrophages in vitro at late time postinfection, a time at which viral replication had already occurred in these cells (3). Thus, transient modulation of infected macrophage survival may be necessary for productive viral replication to occur in this cell type.

The growth, stability and seroprevalence in laboratory rodents of the two known strains of mouse adenovirus were compared. The FL strain of mouse adenovirus grew in both L 929 murine fibroblasts and in CMT-93 murine rectal carcinoma cells, whereas the K 87 strain grew only in CMT-93 cells. The bulk of the FL progeny virus was released from the host cells. K 87 virus was largely cell-associated. Both virus strains were stable at 37 degrees C in liquid medium. The K 87 strain was completely inactivated after 5-15 minutes at 56 degrees C, whereas FL infectivity was still detected after two hours at this temperature. Both virus strains were stable in the dessicated state for 14 days, although FL viability was more dependent on the presence of protein in the virus diluent. Seroepidemiologic data suggest that viruses antigenically related to mouse adenovirus are more prevalent among laboratory rats than among laboratory mice and that the virus(es) infecting rats differ from those infecting mice. Results of retrospective serologic testing suggest an association between mouse adenovirus and an outbreak of disease in a mouse breeding colony.

Cardiomyoblasts in the myocardium of embryonic lobsters at 3-4 weeks and 6 months of development were examined with the transmission electron microscope in order to describe the events in the formation of sarcomeres in a neurogenic cardiac system. Thick and thin myofilaments appear first in the cell periphery near the sarcolemma. They align in parallel in a sequential fashion to form consecutive sarcomeric units. Well-defined A and I bands appear before any semblance of a Z line is present. The initial sarcomere is anchored to the sarcolemma by the insertion of thin myofilaments into a region of electron dense material associated intimately with the sarcolemma. Myofibrils grow outward in several planes away from the electron-dense regions of membrane that serve as focal points for myofibril formation.

The cellular organization of the embryonic heart of the lobster Homarus americanus was examined in 6-week and 6-month-old animals. The heart wall consists of an outer adventitial layer of fibroblast cells and an inner layer of transversely striated myocardial cells. Present in close association with the myocardium are cardiac neurons, hemocytes and so-called storage cells.

Characterization of specific host cell receptors for enveloped viruses is a difficult problem because many enveloped viruses bind to a variety of substrates which are not obviously related to tissue tropisms in the intact host. Viruses with a limited cellular tropism in infected animals present useful models for studying the mechanisms by which virus attachment regulates the disease process. Rabies virus is a rhabdovirus which exhibits a marked neuronotropism in infected animals. Limited data suggest that spread occurs by transsynaptic transfer of virus. The results of recent experiments at Yale suggest that viral antigen is localized very soon after injection at neuromuscular junctions, the motor nerve endings on muscle tissue. On cultured muscle cells, similar co-localization with the acetylcholine receptor is seen both before and after virus multiplication. Pretreatment of these cells with some ligands of the acetylcholine receptor results in reduced viral infection. These findings suggest that a neurotransmitter receptor or a closely associated molecule may serve as a specific host cell receptor for rabies virus and thus may be responsible for the tissue tropism exhibited by this virus. In addition to clarifying aspects of rabies virus pathogenesis, these studies have broad implications regarding the mechanism by which other viruses or viral immunizations might mediate autoimmune diseases such as myasthenia gravis. Images FIG. 1 FIG. 2