Three Strains of European Foot-and-Mouth Disease Virus are Highly Conserved in the 3'-Termini and Highly Variable in the Genes of Two Capsid Proteins

Marquardt, Otfried

doi:10.1099/0022-1317-59-2-283

Cited by 6 publications

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“…These differences might be expected, since there is only a 2 0~ homology in the RNase T1 oligonucleotide maps between the two groups of TMEV (Rozhon et al, 1982;Lorch et al, 1982; our unpublished data), a relationship equivalent to a 1 0~ difference in the R N A sequence (Aaronson et al, 1982). The major differences found between the two groups of TMEV appear to be in the structural polypeptides, which is consistent with the findings from comparison of nucleotide sequences between strains of foot-and-mouth (Marquardt, 1982) and poliovirus types 1 and 3 (Stanway et al, 1983). …”

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Theiler's Virus-specified Polypeptides Made in BHK-21 Cells

Lipton

Rozhon

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1984

Journal of General Virology

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SUMMARYThis study was initiated to determine whether the Theiler's murine encephalomyelitis viruses (TMEV) use the same strategy of gene expression as other picornaviruses. The precursor-product relationships apparent from pulse-chase experiments indicated that the virus-specified polypeptides of the GDVII strain are generated by post-translational cleavages. Pactamycin mapping experiments also showed a gene order for GDVII virus similar to that derived for other picornaviruses by this technique. Finally, six other TMEV induced species of polypeptides similar to those of GDVII virus, but there were minor differences in some of their electrophoretic mobilities.The Theiler's murine encephalomyelitis viruses (TMEV) are members of the picornavirus family and are enteric pathogens of the mouse, their natural host. They are separable into two groups on the basis of the type of central nervous system (CNS) disease they produce in mice after intracerebral (i.c.) inoculation: two of the viruses (GDVII and FA) are highly virulent and produce a rapidly fatal encephalitis (Theiler & Gard, 1940), while other isolates found to date are less virulent and produce a biphasic CNS disease (Theiler, 1937;Lipton, 1975;Lehrich et al., 1976) characterized by poliomyelitis during the first month post-infection (early disease) and, in surviving mice, a persistent CNS infection and a chronic, inflammatory demyelinating disorder weeks to months later (late disease) (Dal Canto & Lipton, 1975).A better understanding of the pathogenesis of the different diseases induced by the TMEV requires further biochemical characterization of these viruses, and more molecular studies of the infection in the mouse. Since the post-translational processing and gene order of TMEV virusspecified polypeptides have not been well defined, we have carried out standard pulse-chase experiments and used the pactamycin mapping technique to determine whether TMEV follow the unique strategy of gene expression found in other picornaviruses (Rueckert et al., 1979).BHK-21 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) as described previously (Rozhon et al., 1982). All the TMEV stocks were high-titred (10 s p.f.u./ml) and plaque-purified at least twice; their origin and passage history except for TO(B15) virus have been described (Lipton, 1978). A suckling mouse brain pool of TO(B 15) virus, kindly provided by Dr B. Mandel (New York City), was passed three times in BHK-21 cells and then plaquepurified twice (Feltz et al., 1953).In the pulse-chase experiments, BHK-2t ceils in 35 mm Petri dishes were infected with a multiplicity of 50 to 100 p.f.u./cell. After virus adsorption for 30 min at 37 °C, the monolayers were washed, and DMEM containing 20 mM-HEPES and 5 ~g/ml actinomycin D was added. At 5 to 6.5 h, the infected ceils were washed once and incubated with methionine-free medium for 30 min. Between 25 and 50 ~tCi L-[35S]methionine (1350 Ci/mmol; Amersham) in 0.4 ml was then added for 4 to 8 min in different experiments. For the chase, the monolayers ...

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confidence: 85%

Theiler's Virus-specified Polypeptides Made in BHK-21 Cells

Lipton

Rozhon

Black

1984

Journal of General Virology

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confidence: 99%

“…The Y-proximal RNA sequences of aphthoviruses are highly homologous, an observation providing evidence for the close relationship of the serotypes. The Y-proximal sequences coding for the capsid proteins VP1 and VP3 are less homologous (Marquardt, 1982); this may explain the antigenic variation between the serotypes noticed some time ago (Galloway et al, 1948;Wild et al, 1969).The specific distribution of sequence homology among aphthoviruses made us consider whether sequence homologies exist between distinct genome areas of aphthoviruses and those of other picornaviruses. The availability of bacterial clones which contain foot-and-mouth disease virus (FMDV) O~ K-specific complementary DNA (K/ipper et al, 1981) facilitated such studies.…”

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“…Qualitative results were obtained in the form of autoradiographs of the RNase A-treated and cleaned filters (experimental details are given in Marquardt, 1982) and are shown in Fig. 2.…”

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“…By comparing the capacity of the same DNA fragments to hybridize with heterologous RNA under stringent conditions, the degree of homology of both RNA species can be estimated (Marquardt, 1982). The homologies shown in Fig.…”

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Multiple Homologies of Oligonucleotide Size Exist between Nucleic Acids of Picornaviruses

Marquardt

Bachmann

1983

Journal of General Virology

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SUMMARYA semi-quantitative analysis of hybrid formation between restriction enzymegenerated subgenomic fragments of cloned cDNA prepared from RNA of foot-andmouth disease virus (FMDV) strain O1K and radiolabelled RNA from bovine enterovirus, bovine rhinovirus or Mengo virus indicated that the hybrids were of oligonucleotide size. They were located in those parts of the FMDV O1K genome that code for the two capsid proteins VP3 and VP1 and the precursor protein P52 as well as at the Tend. No hybridization was observed with poliovirus type 1 RNA.Picornaviruses (for review, see Cooper et al., 1978) consist of a single-stranded RNA molecule 7000 to 8000 bases long of (+) polarity which is encapsidated by 60 copies of each of the four capsid proteins. The primary product of protein synthesis is a precursor polyprotein from which the mature proteins are released after extensive processing. The aphthoviruses represent one of the four genera grouped in this virus family. They can be distinguished from the others (enteroviruses, rhinoviruses and cardioviruses) by their different buoyant densities in caesium chloride, stabilities in the pH range 3 to 7 or ribonucleotide compositions. The Y-proximal RNA sequences of aphthoviruses are highly homologous, an observation providing evidence for the close relationship of the serotypes. The Y-proximal sequences coding for the capsid proteins VP1 and VP3 are less homologous (Marquardt, 1982); this may explain the antigenic variation between the serotypes noticed some time ago (Galloway et al., 1948;Wild et al., 1969).The specific distribution of sequence homology among aphthoviruses made us consider whether sequence homologies exist between distinct genome areas of aphthoviruses and those of other picornaviruses. The availability of bacterial clones which contain foot-and-mouth disease virus (FMDV) O~ K-specific complementary DNA (K/ipper et al., 1981) facilitated such studies. The experimental strategy is based on the correlation of the cloned cDNA to the map of FMDVinduced polypeptides (Fig. 1). It takes advantage of restriction enzymes which cleave FMDVspecific double-stranded DNA into defined subgenomic fragments. These fragments are analysed individually as to whether or not they hybridize with radiolabelled RNA by use of the technique of Southern (1975). Since RNA that did not hybridize is removed by RNase A digestion, the remaining RNA/DNA hybrids consist of true double-strands. They require a minimum size between more than 6 and about 30 base pairs (bp) to be detectable.Such a technique which detects homologies of oligonucleotide size is required to reveal common picornavirus sequences, because a study comparing the RNase T1 digestion products of the RNAs of representative members of three genera (Frisby et al., 1976) did not find large common oligonucleotides. However, a pentanucleotide resistant to the RNases T1 and A was shown to be shared by at least four RNAs. This could form the core of a common oligonucleotide consisting of more than six bases. The only homology thu...

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