Human adenovirus-vectored foot-and-mouth disease vaccines: establishment of a vaccine product profile through in vitro testing.

Next generation, foot-and-mouth disease (FMD) molecular vaccines based on replication deficient human adenovirus serotype 5 viral vectored delivery of FMD capsid genes (AdFMD) are being developed by the United States Dept. of Homeland Security and industry partners. The strategic goal of this program is to develop AdFMD licensed vaccines for the USA National Veterinary Stockpile for use, if needed, as emergency response tools during an FMD outbreak. This vaccine platform provides a unique opportunity to develop a set of in vitro analytical parameters to generate an AdFMD vaccine product profile to replace the current lot release test for traditional, inactivated FMD vaccines that requires FMDV challenge in livestock. The possibility of an indirect FMD vaccine potency test based on a serological alternative was initially investigated for a lead vaccine candidate, Adt.A24. Results show that serum virus neutralization (SVN) based serology testing for Adt.A24 vaccine lot release is not feasible, at least not in the context of vaccine potency assessment at one week post-vaccination. Thus, an in vitro infectious titer assay (tissue culture infectious dose 50, TCID50) which measures FMD infectious (protein expression) titer was established. Pre-validation results show acceptable assay variability and linearity and these data support further studies to validate the TCID50 assay as a potential potency release test. In addition, a quantitative physiochemical assay (HPLC) and three immunochemical assays (Fluorescent Focus-Forming Unit (FFU); tissue culture expression dose 50 (TCED50); Western blot) were developed for potential use as in vitro assays to monitor AdFMD vaccine lot-to-lot consistency and other potential applications. These results demonstrate the feasibility of using a traditional modified-live vaccine virus infectivity assay in combination with a set of physiochemical and immunochemical tests to build a vaccine product profile that will ensure the each AdFMD vaccine lot released is similar to a reference vaccine of proven clinical safety and efficacy.

Pathogenicity and molecular characterization of emerging porcine reproductive and respiratory syndrome virus in Vietnam in 2007.

In 2007, Vietnam experienced swine disease outbreaks causing clinical signs similar to the 'porcine high fever disease' that occurred in China during 2006. Analysis of diagnostic samples from the disease outbreaks in Vietnam identified porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2). Additionally, Escherichia coli and Streptococcus equi subspecies zooepidemicus were cultured from lung and spleen, and Streptococcus suis from one spleen sample. Genetic characterization of the Vietnamese PRRSV isolates revealed that this virus belongs to the North American genotype (type 2) with a high nucleotide identity to the recently reported Chinese strains. Amino acid sequence in the nsp2 region revealed 95.7-99.4% identity to Chinese strain HUN4, 68-69% identity to strain VR-2332 and 58-59% identity to strain MN184. A partial deletion in the nsp2 gene was detected; however, this deletion did not appear to enhance the virus pathogenicity in the inoculated pigs. Animal inoculation studies were conducted to determine the pathogenicity of PRRSV and to identify other possible agents present in the original specimens. Pigs inoculated with PRRSV alone and their contacts showed persistent fever, and two of five pigs developed cough, neurological signs and swollen joints. Necropsy examination showed mild to moderate bronchopneumonia, enlarged lymph nodes, fibrinous pericarditis and polyarthritis. PRRSV was re-isolated from blood and tissues of the inoculated and contact pigs. Pigs inoculated with lung and spleen tissue homogenates from sick pigs from Vietnam developed high fever, septicaemia, and died acutely within 72 h, while their contact pigs showed no clinical signs throughout the experiment. Streptococcus equi subspecies zooepidemicus was cultured, and PRRSV was re-isolated only from the inoculated pigs. Results suggest that the cause of the swine deaths in Vietnam is a multifactorial syndrome with PRRSV as a major factor.

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.

Epizootic vesicular disease in captive California sea lions.

An epizootic of vesicular disease occurred in a group of semi-domesticated California sea lions (Zalophus californianus) during the months of April and May 1997. Ten castrated mature male sea lions, ages 12 to 19 yr, were housed in three adjacent open-ocean net enclosures in San Diego Bay (California, USA). Four animals (40%) developed oral and extremity vesicles, anorexia, and were reluctant to perform learned behaviors. One animal developed vesicles but maintained a normal appetite and behavior. The remaining animals showed no clinical signs of infection. Virus (designated FADDL 7005) was isolated from four of the five animals that developed vesicles. Serum antibody titers to FADDL 7005, a previously untyped calicivirus, were demonstrated in animals that showed any combination of clinical signs and in two animals that did not show any clinical signs. No virus was isolated from five fecal samples collected from four of the group animals. Clinical signs lasted 4 to 20 days in affected animals. All affected animals recovered from infection. An experimental swine was inoculated with FADDL 7005 and developed vesicular disease, which was transmitted to another experimental swine upon contact. It is proposed that FADDL 7005 is a new San Miguel sea lion virus.

Inactivation of viruses by aziridines.

Ethyleneimine (EI) and N-acetylethyleneimine (AEI) have been shown to inactivate viruses belonging to most of the families described by the International Committee for the Taxonomy of Viruses. The mechanism by which they inactivate the viruses has not been established. In this paper, experiments with foot-and-mouth disease virus (FMDV) and poliovirus are described which indicate that the inactivating lesion is on the RNA.

Structural differences between foot-and-mouth disease and poliomyelitis viruses influence their inactivation by aziridines.

Inactivation of foot-and-mouth disease virus (FMDV) and poliovirus by ethyleneimine (EI) and N-acetylethyleneimine (AEI) has been studied at 25 degrees and at 37 degrees C and in different ionic conditions. FMDV is inactivated rapidly in 100 mM Tris pH 7.6 by each reagent at both temperatures. Poliovirus is also inactivated rapidly in 100 mM Tris by EI at both temperatures and by AEI at 37 degrees C. However, it is inactivated much more slowly by AEI at 25 degrees C; but if the virus is first incubated overnight at 2 degrees C with AEI before transferring to 25 degrees C inactivation then proceeds rapidly. Moreover, the rate of inactivation at 25 degrees C is markedly increased if the virus is suspended in 1 mM Tris. We had interpreted these differences as being due to the greater penetrability of poliovirus (i) in 100 mM Tris at 37 degrees C compared with 25 degrees C and (ii) at lower ionic strength. This interpretation has been confirmed by electron microscopy of FMDV and poliovirus particles stained with phosphotungstic acid. At the elevated temperature, poliovirus had an average diameter of 34+/-0. 21 nm and the stain outlined the nucleic acid core and the individual subunits, whereas at 25 degrees C it averaged 28+/-0.13 nm and the stain did not penetrate the particle. This study also showed that the particle diameter alters with changes in buffer concentration, being 28+/-0.13 nm in 100 mM Tris, 31+/-0.16 nm in 10 mM Tris and 34+/-0.21 nm in 1 mM Tris. The changes in poliovirus are reversible as addition of 1/10 volume of 1 M Tris to the virus in 1 mM Tris resulted in the return of the diameter to 28+/-0.13 nm. FMDV, on the other hand, was less sensitive to osmotic differences as its particle diameter only varied by 7% over the 100-fold change in buffer concentration compared with the 22% change observed for poliovirus.

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.

Strategy for producing new foot-and-mouth disease vaccines that display complex epitopes.

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.

Structure and immunogenicity of experimental foot-and-mouth disease and poliomyelitis vaccines.

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.