The Complete Nucleotide Sequence and Construction of an Infectious cDNA Clone of a Highly Virulent Encephalomyocarditis Virus

Zimmermann, Albert; Nelsen-Salz, Birgit; Kruppenbacher, J. P.; Eggers, Hans J.

doi:10.1006/viro.1994.1495

Cited by 23 publications

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“…EMCV Rueckert (EMCV R/45), the prototype strain, was isolated from a 5-year-old chimpanzee that suffered acute fatal myocarditis in 1945 (15), whereas EMC-M/58 virus was isolated from a naturally infected domestic pig suffering from severe myocarditis (29). A number of EMC-M/58 variants, including EMC-D/58, EMC-B/58, PV2/58, and PV21/58, were generated in laboratories and used to study the pathogenesis of viral diabetes (6,7,18,25,46,49). Mengo virus, another cardiovirus that shares the same serotype as EMCV, was isolated from the cerebrospinal fluid of a paralyzed rhesus macaque in 1948 (8).…”

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“…Mengo virus, another cardiovirus that shares the same serotype as EMCV, was isolated from the cerebrospinal fluid of a paralyzed rhesus macaque in 1948 (8). EMCV 30/87 and 2887A/91 are more recent EMCV isolates obtained from aborted swine fetuses in the United States and Belgium, respectively, following natural EMCV outbreaks in domestic pigs (19,20).Nucleotide sequencing of most EMCV strains and Mengo virus has identified a poly(C) tract, consisting of 61 to 146 cytosine residues (C 61 to C 146 ), occasionally interrupted by 1 to 3 uridine residues, located at the 5Ј nontranslated region (NTR) of the positive-sense RNA genome (1,9,10,19,31,49). Studies to determine the role of the poly(C) tract in virus replication, virulence, and host range have produced conflicting findings.…”

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“…Nucleotide sequencing of most EMCV strains and Mengo virus has identified a poly(C) tract, consisting of 61 to 146 cytosine residues (C 61 to C 146 ), occasionally interrupted by 1 to 3 uridine residues, located at the 5Ј nontranslated region (NTR) of the positive-sense RNA genome (1,9,10,19,31,49). Studies to determine the role of the poly(C) tract in virus replication, virulence, and host range have produced conflicting findings.…”

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A Wild-Type Porcine Encephalomyocarditis Virus Containing a Short Poly(C) Tract Is Pathogenic to Mice, Pigs, and Cynomolgus Macaques

LaRue

Myers

Brewer

et al. 2003

J Virol

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Previous studies using wild-type Encephalomyocarditis virus (EMCV) and Mengo virus, which have long poly(C) tracts (61 to 146 C's) at the 5 nontranslated region of the genome, and variants of these viruses genetically engineered to truncate or substitute the poly(C) tracts have produced conflicting data on the role of the poly(C) tract in the virulence of these viruses. Analysis of the nucleotide sequence of an EMCV strain isolated from an aborted swine fetus (EMCV 30/87) revealed that the virus had a poly(C) tract that was 7-to 10-fold shorter than the poly(C) tracts of other EMCV strains and 4-fold shorter than that of Mengo virus. Subsequently, we investigated the virulence and pathogenesis of this naturally occurring short-poly(C)-tractcontaining virus in rodents, pigs, and nonhuman primates. Infection of C57BL/6 mice, pigs, and cynomolgus macaques resulted in similar EMCV 30/87 pathogenesis, with the heart and brain as the primary sites of infections in all three animals, but with different disease phenotypes. Sixteen percent of EMCV 30/87-infected pigs developed acute fatal cardiac failure, whereas the rest of the pigs were overtly asymptomatic for as long as 90 days postinfection (p.i.), despite extensive myocardial and central nervous system (CNS) pathological changes. In contrast, mice infected with >4 PFU of EMCV 30/87 developed acute encephalitis that resulted in the death of all animals (n ‫؍‬ 25) between days 2 and 7 p.i. EMCV 30/87-infected macaques remained overtly asymptomatic for 45 days, despite extensive myocardial and CNS pathological changes and viral persistence in more than 50% of the animals. The short poly(C) tract in EMCV 30/87 (CUC 5 UC 8 ) was comparable to that of strain 2887A/91 (C 10 UCUC 3 UC 10 ), another recent porcine isolate.Encephalomyocarditis virus (EMCV) is a picornavirus belonging to the Cardiovirus genus that infects many animal species including pigs (17), rodents (45), cattle (41), elephants (13), raccoons (47), marsupials (38), baboons, macaques, chimpanzees, and humans (2,16,21,38,44,45). Rats and mice are the natural hosts of the virus, but pigs are the most commonly and severely infected domestic animals (5, 36). EMCV strains have been isolated from primates, pigs, and rodents (8,15,20,22,24,29). EMCV Rueckert (EMCV R/45), the prototype strain, was isolated from a 5-year-old chimpanzee that suffered acute fatal myocarditis in 1945 (15), whereas EMC-M/58 virus was isolated from a naturally infected domestic pig suffering from severe myocarditis (29). A number of EMC-M/58 variants, including EMC-D/58, EMC-B/58, PV2/58, and PV21/58, were generated in laboratories and used to study the pathogenesis of viral diabetes (6,7,18,25,46,49). Mengo virus, another cardiovirus that shares the same serotype as EMCV, was isolated from the cerebrospinal fluid of a paralyzed rhesus macaque in 1948 (8). EMCV 30/87 and 2887A/91 are more recent EMCV isolates obtained from aborted swine fetuses in the United States and Belgium, respectively, following natural EMCV outbreaks in domestic ...

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A Wild-Type Porcine Encephalomyocarditis Virus Containing a Short Poly(C) Tract Is Pathogenic to Mice, Pigs, and Cynomolgus Macaques

LaRue

Myers

Brewer

et al. 2003

J Virol

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“…Since then, several infectious clones of picornaviruses have been constructed. These include Hepatovirus [39] , Enterovirus [40,41] , Rhinoviruses [42] and Cardioviruses [30,43,44] , and the first infectious clone of FMDV (Aphthovirus) O1K strain was recovered by Zibert et al [16] . In 1993/2004, infectious clones of FMDV A12 and ZIM/7/83 (SATⅡ serotype) were constructed by Rieder et al [15] and van Rensburg et al [17] , respectively.…”

Section: Discussionmentioning

confidence: 99%

Engineering infectious foot-and-mouth disease virus in vivo from a full-length genomic cDNA clone of the A/AKT/58 strain

Bai

Cao

et al. 2009

SCI CHINA SER C

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Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10(-7.5)). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10(-6)), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA transcripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.

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“…To further elucidate the function of the poly(C) region for the EMCV life cycle, construction of cDNA clones with modified poly(C) tracts was intended. Since long dC ⅐ dG tracts are neither strictly reproduced from template-dependent polymerases nor precisely maintained in the bacterial host, several approaches have been used to overcome this problem (1,7,16,19,22). To begin with, we constructed and sequenced a full-length cDNA clone of PV2 from overlapping fragments (17).…”

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The poly(C) region affects progression of encephalomyocarditis virus infection in Langerhans' islets but not in the myocardium

Zimmermann

Botta

Arnold

et al. 1997

J Virol

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The diabetogenic variant PV2 of encephalomyocarditis virus was cloned, and three recombinants differing in their 5 poly(C) tracts were analyzed. It is shown that the poly(C) region is not essential for infectivity in mice but does influence the virus load and degree of pathological lesions within the Langerhans' islets but not in the myocardium.Variant PV2 of encephalomyocarditis virus (EMCV) induces an insulin-dependent diabetes mellitus-like syndrome in certain inbred mouse strains by destruction of insulin-producing pancreatic beta cells (14,17). To analyze further the molecular basis of the pathogenic properties, PV2 was cloned and sequenced (17).A feature of murine cardioviruses (EMCV and mengovirus) and bovine aphthoviruses (FMDV) is the homopolymeric cytosine tract (60 to 350 C's) within the 5Ј noncoding region. The length of this region and the distance from the 5Ј end of the RNA depends on virus strain and isolate (3, 4). The task of the poly(C) region in the viral replication cycle is not yet clear; however, it is not likely that it plays a direct role in viral RNA replication or protein synthesis, since cardioviruses with shortened or deleted poly(C) tracts replicate efficiently in cell culture (2,7,10,15). On the other hand, at least in the case of mengovirus the length of the poly(C) tract seems to influence viral pathogenesis upon inoculation into mice, i.e., the fewer the C's, the more attenuated appears the corresponding virus (6,15,18). In contrast to mengoviruses, a full-length poly(C) region seems not to be a prerequisite for infectivity of EMCV applied intracerebrally (10). To further elucidate the function of the poly(C) region for the EMCV life cycle, construction of cDNA clones with modified poly(C) tracts was intended. Since long dC ⅐ dG tracts are neither strictly reproduced from template-dependent polymerases nor precisely maintained in the bacterial host, several approaches have been used to overcome this problem (1,7,16,19,22). To begin with, we constructed and sequenced a full-length cDNA clone of PV2 from overlapping fragments (17). Although the original poly(C) region of EMCV PV2 is composed of 117 or 118 cytosines, after reverse transcription and further cloning procedures the longest poly(C) tract amounted to only 20 cytosines (rPV2/C20 in Fig. 1A). To elongate the 5Ј noncoding region, the EcoRV-HindIII fragment of the clone rPV2/C20 was replaced by the corresponding fragments of pSPORT/EMC-ran or pSORT-EMC-dT, leading to rPV2/ran and rPV2/dT, respectively ( Fig.

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The Complete Nucleotide Sequence and Construction of an Infectious cDNA Clone of a Highly Virulent Encephalomyocarditis Virus

Cited by 23 publications

References 0 publications

A Wild-Type Porcine Encephalomyocarditis Virus Containing a Short Poly(C) Tract Is Pathogenic to Mice, Pigs, and Cynomolgus Macaques

A Wild-Type Porcine Encephalomyocarditis Virus Containing a Short Poly(C) Tract Is Pathogenic to Mice, Pigs, and Cynomolgus Macaques

Engineering infectious foot-and-mouth disease virus in vivo from a full-length genomic cDNA clone of the A/AKT/58 strain

The poly(C) region affects progression of encephalomyocarditis virus infection in Langerhans' islets but not in the myocardium

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