Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells.

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.     

Bacterially expressed antigenic peptide from foot-and-mouth disease virus capsid elicits variable immunologic responses in animals

A fusion protein consisting of beta-galactosidase (GZ) to which was attached at its N-terminus the amino acid sequence corresponding to residues 142-160 of the immunogenic protein VP1 of foot-and-mouth disease virus (FMDV) has been expressed in E. coli. A chemically synthesized section of DNA corresponding to the amino acid sequence 142-160 was inserted into a vector (pXY410) designed to express fusion proteins with the carboxy terminal 1015 amino acids of GZ. The hybrid protein immunopurified by a GZ-specific monoclonal antibody was soluble, retained full GZ activity, and induced virus-neutralizing antibody in guinea pigs and mice. There were significant differences between the responses of individual mice to the FMDV peptide sequence, although the titers against GZ were uniformly high. This variable pattern did not change after hyperimmunization and was demonstrable in a range of mouse strains of different haplotype. The same results were obtained whether the response was measured by virus neutralization or by RIA against the FMDV peptide sequence. The possible reasons for the variable recognition of the FMDV epitopes by individual mice are discussed.     

Translation and processing of mouse hepatitis virus virion RNA in a cell-free system.

The first event after infection with mouse hepatitis virus strain A59 (MHV-A59) is presumed to be the synthesis of an RNA-dependent RNA polymerase from the input genomic RNA. The synthesis and processing of this putative polymerase protein was studied in a cell-free translation system utilizing 60S RNA from MHV-A59 virions. The polypeptide products of this reaction included two major species of 220 and 28 kilodaltons. Kinetics experiments indicated that both p220 and p28 appeared after 60 min of incubation and that protein p28 was synthesized initially as the N-terminal portion of a larger precursor protein. When the cell-free translation products were labeled with N-formyl[35S]methionyl-tRNAi, p28 was the predominant radioactive product, confirming its N-terminal location within a precursor protein. Translation in the presence of the protease inhibitors leupeptin and ZnCl2 resulted in the disappearance of p28 and p220 and the appearance of a new protein, p250. This product, which approached the maximal size predicted for a protein synthesized from genomic RNA, was not routinely detected in the absence of inhibitors even under conditions which optimized the translation reaction for elongation of proteins. Subsequent chelation of ZnCl2 resulted in the partial cleavage of the precursor protein and the reappearance of p28. One-dimensional peptide mapping with Staphylococcus aureus V-8 protease confirmed the precursor-product relationship of p250 and p28. The results show that MHV virion RNA, like many other viral RNAs, is translated into a large polyprotein, which is cleaved soon after synthesis into smaller, presumably functional proteins. This is in marked contrast to the synthesis of other MHV proteins, in which minimal proteolytic processing occurs.     

Further characterization of a protein kinase from foot-and-mouth disease virus.

Acid disruption of foot-and-mouth disease virus released a protein kinase activity that sedimented at less than 7S. This enzyme was separated into three peaks of activity by ion-exchange and hydroxylapatite chromatography. Analysis of the various enzyme fractions by polyacrylamide gel electrophoresis and silver staining revealed that one of the fractions lacked the major virion structural proteins, but still contained two or three other polypeptides. This enzyme phosphorylated mainly one protein (P17) in an in vitro assay.     

Synthesis of Newcastle disease virus polypeptides in a wheat germ cell-free system.

We have isolated 18S RNA from cytoplasmic extracts of Newcastle disease virus-infected Chinese hamster ovary cells and tested its ability to direct protein synthesis in extracts derived from wheat germ. The products of the cell-free reaction directed by this RNA contain polypeptides that comigrate with NP, M,F, and 47K roteins from virions. In addition, the products contain a polypeptide (67K) that migrates on polyacrylamide gels slightly faster than the HN protein from virions. Tryptic peptide analysis of the cell-free products and proteins from virions confirms their identity.     

Biological activity of ovine IL-23 expressed using a foot-and-mouth disease virus 2A self-cleaving peptide

Hetero-dimeric cytokines often require equi-molar expression of both subunits to achieve biological activity. Previously, we expressed ovine IL-12 p40 and p35 linked using a self-cleaving 2A peptide from foot-and-mouth disease virus (FMDV). We now generated a new improved vector for the expression of hetero-dimeric cytokines and demonstrate the more general applicability of this strategy by cloning and expressing ovine IL-23 using the 2A peptide to link IL-12/IL-23 p40 and p19. The resulting protein was shown to be biologically active when expressed in mammalian COS cells. IL-23 plays a significant role in the differentiation of Th17 cells as well as autoimmunity and the regulation of inflammatory processes. As such this reagents will be invaluable in the unravelling of regulation of the ovine immune system for both veterinary and human animal model applications.     

Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA.

A full-length cDNA plasmid of foot-and-mouth disease virus has been constructed. RNA synthesized in vitro by means of a bacteriophage SP6 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cells. These particles were also found to be highly infectious for primary bovine kidney cells as well as for baby mice. The difficulty in cloning the foot-and-mouth disease virus cytidyl tract in Escherichia coli was circumvented by joining two separate cloned parts, representing the S and L fragments of the genome, and, in a second step, inserting a dC-dG homopolymer. Homopolymeric sequences of up to 25 cytidyl residues did not lead to the production of virus. Replicons containing poly(C) tracts long enough to permit virus replication were first established in yeast cells. One of these constructs could also be maintained in E. coli and was used to produce infectious RNA in vitro. The length of the poly(C) sequence in this cDNA plasmid was 32 nucleotides. However, the poly(C) tracts of two recombinant viruses found in transfected BHK-21 cells were 60 and 80 nucleotides long, respectively. Possible mechanisms leading to the enlargement of the poly(C) tract during virus replication are discussed.     

猪瘟病毒C株共感染口蹄疫病毒影响口蹄疫病毒复制

【背景】猪瘟(Classical Swine Fever)是由猪瘟病毒(Classical Swine Fever Virus,CSFV)引起的猪高度接触性传染病,致死率极高。在临床中存在着CSFV与猪其他病原菌共感染的情况,例如CSFV与口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)的共感染。【目的】利用CSFV与FMDV共感染猪源宿主细胞,研究CSFV与FMDV共感染对FMDV病毒复制的影响。【方法】构建体外共感染细胞模型,在正常PK-15细胞上进行CSFV共感染FMDV实验,通过观察细胞病变效应(Cytopathic Effect,CPE)、实时荧光定量PCR(RT-qPCR)、Western Blot、间接免疫荧光检测CSFV和FMDV共感染及FMDV单独感染情况下FMDV复制水平的差异。利用RT-qPCR筛选鉴定能够影响FMDV复制的CSFV蛋白。【结果】CSFVC株共感染FMDV能够抑制FMDV的复制,而且灭活的CSFV同样抑制FMDV的复制。通过筛选鉴定出CSFV的C蛋白能够抑制FMDV复制。【结论】研究发现CSFV C株共感染FMDV能够抑制FMDV复制,而其C蛋白具有抑制FMDV复制的能力。     

Integrin-alphavbeta6, a putative receptor for foot-and-mouth disease virus, is constitutively expressed in ruminant airways.

Evolved functions of integrin-alpha(v)beta(6) include roles in epithelial cell-extracellular matrix protein interactions and in the binding and activation of latent TGF-beta(1). Integrin-alpha(v)beta(6) is also exploited as a receptor by foot-and-mouth disease virus (FMDV) and may play a significant role in its transmission and pathogenesis. The ovine beta(6) integrin subunit was cloned and sequenced (EMBL accession no. AJ439062). Screening of normal ovine tissues by RT-PCR and immunocytochemistry confirmed that integrin-alphavbeta6 is restricted to sheep epithelial cells. Integrin-alphavbeta6 expression was detected in epithelia of the airways, oral cavity, gastrointestinal tract, kidney, sweat glands, hair follicle sheaths, and the epidermis of pedal coronary band (PB) but not of normal skin. Consistent with FMDV tropism, integrin-alphavbeta6 was detected within the basal layers of the stratified squamous epithelium of the oral mucosa and PB. In addition, integrin-alphavbeta6 appears to be constitutively expressed in the normal airways of both cattle and sheep. The latter finding suggests that ruminant airway epithelium presents a highly accessible target for initiation of infection with FMDV by inhalation.     

Comparative genomics of foot-and-mouth disease virus

Here we present complete genome sequences, including a comparative analysis, of 103 isolates of foot-and-mouth disease virus (FMDV) representing all seven serotypes and including the first complete sequences of the SAT1 and SAT3 genomes. The data reveal novel highly conserved genomic regions, indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Previously undescribed invariant motifs were identified in the 5' and 3' untranslated regions (UTR), as was tolerance for insertions/deletions in the 5' UTR. Fifty-eight percent of the amino acids encoded by FMDV isolates are invariant, suggesting that these residues are critical for virus biology. Novel, conserved sequence motifs with likely functional significance were identified within proteins L(pro), 1B, 1D, and 3C. An analysis of the complete FMDV genomes indicated phylogenetic incongruities between different genomic regions which were suggestive of interserotypic recombination. Additionally, a novel SAT virus lineage containing nonstructural protein-encoding regions distinct from other SAT and Euroasiatic lineages was identified. Insights into viral RNA sequence conservation and variability and genetic diversity in nature will likely impact our understanding of FMDV infections, host range, and transmission.     

Decigram quantities of pure foot-and-mouth disease virus from cell cultures

Baby-hamster kidney (BHK) cell cultures grown in rolling 2-liter Baxter bottles are used for the production of foot-and-mouth disease virus (FMDV) which is subsequently purified. The bottles are held securely in round wire racks (19 per rack) and rotated on a three-tiered roller mill. The use of a strongly buffered growth medium makes changes of the medium unnecessary. A sheet of aluminum foil is used to seal the cultures. It is pressed tightly over all the bottles in a rack by means of a polyurethane foam sheeting bonded to the underside of a rigid snap-on cover. Special equipment eliminates removing the bottles from the racks at any stage in their use. The loaded racks fit directly onto headers of a glassware washer. Spent cell growth media and virus fluids are collected by inverting an entire rack of bottles-Current production is 400 BHK cultures (21 racks) containing 8 × 10⁸ cells each after 6 days growth. About 344 of these cultures (18 racks) are used to grow virus. The purification process yields about 113 mg of pure FMDV per week; the overall recovery based on infectivity is 32%. The projected maximum production of purified virus in the present facilities is approximately 2.5 times greater than this amount.     

Human immunodeficiency virus integration in a cell-free system.

Integration of the viral genome into the nuclear DNA of a host cell plays a pivotal role in the replication of retroviruses. We have developed an in vitro method for studying the biochemistry of human immunodeficiency virus (HIV) integration by using extracts from HIV-infected cells. Analysis of the reaction products showed that HIV integration in vitro accurately reproduces the in vivo process. Integration occurred without apparent specificity for the target sequence, and the integrated provirus was directly flanked by a 5-base-pair duplication of DNA from the target site. HIV integration did not require a high-energy cofactor, and the enzymatic activities required for integration were recovered with the viral DNA when cell extracts were fractionated by gel exclusion chromatography.     

Detection of foot-and-mouth virus antibodies using a purified protein from the high-level expression of codon-optimized, foot-and-mouth disease virus complex epitopes in Escherichia coli

A codon optimized DNA sequence coding for foot-and-mouth disease virus (FMDV) capsid protein complex epitopes of VP1 amino acid residues 21-40, 135-160, and 200-213 was genetically fused to the C-terminal end of a glutathione-S-transferase (GST) gene in pGEX-6P-1 vector with the synonymous codons preferred by Escherichia coli . The gene was synthesized using PCR and subsequently expressed in E. coli producing an intracellular, soluble fusion protein that retained antigenicity associated with FMDV antibodies by western blot analysis. The chimera was purified from bacterial lysates by affinity chromatography and could be used in ELISA tests for antibodies against FMDV.     

Synthesis of encephalomyocarditis virus in a cell-free system: from DNA to RNA virus in one tube

Virus particles are used in vaccination, drug delivery, and material sciences. Here we devised a system where the RNA virus encephalomyocarditis virus (EMCV) is synthesized from DNA templates in vitro. When a plasmid or a PCR product harboring the full-length cDNA of EMCV in the T7 promoter/terminator unit was incubated in a HeLa cell extract supplemented with T7 RNA polymerase, EMCV was produced within 4 h at an efficiency of over 10-fold compared with the system programmed with the EMCV RNA. The EMCV RNA transcribed by the virally encoded RNA-dependent RNA polymerase was predominantly incorporated into the EMCV particle even in the presence of a larger amount of the EMCV RNA transcribed by T7 RNA polymerase from the plasmid.