Biomedical subjects

S L Ropp

Publications and source records attributed to S L Ropp.

6 recordsLinked to original sources

PubMed2000

Expression of the hepatitis E virus ORF1.

Hepatitis E virus (HEV) is an unclassified, plus-strand RNA virus whose genome contains three open reading frames (ORFs). ORF1, the 5' proximal ORF of HEV, encodes nonstructural proteins involved in RNA replication which share homology with the products of the corresponding ORF of members of the alphavirus-like superfamily of plus-strand RNA viruses. Among animal virus members of this superfamily (the alphavirus and rubivirus genera of the family Togaviridae), the product of this ORF is a nonstructural polyprotein (NSP) that is cleaved by a papain-like cysteine protease (PCP) within the NSP. To determine if the NSP of HEV is similarly processed, ORF1 was introduced into a plasmid vector which allowed for expression both in vitro using a coupled transcription/translation system and in vivo using a vaccinia virus-driven transient expression system. A recombinant vaccinia virus expressing ORF1 was also constructed. Both in vitro and in vivo expression under standard conditions yielded only the full-length 185 kDa polyprotein. Addition of co-factors in vitro, such as divalent cations and microsomes which have been shown to activate other viral proteases, failed to change this expression pattern. However, in vivo following extended incubations (24-36 hours), two potential processing products of 107 kDa and 78 kDa were observed. N- and C-terminus-specific immunoprecipitation and deletion mutagenesis were used to determine that the order of these products within the NSP is NH2-78 kDa-107 kDa-COOH. However, site-specific mutagenesis of Cys483, predicted by computer alignment to be one member of the catalytic dyad of a PCP within the NSP, failed to abolish this cleavage. Additionally, sequence alignment across HEV strains revealed that the other member of the proposed catalytic dyad of this PCP, His590, was not conserved. Thus, the cleavage of the NSP observed following prolonged in vivo expression was not mediated by this protease and it is doubtful that a functional PCP exists within the NSP. Attempts to detect NSP expression and processing in HEV-infected primary monkey hepatocytes were not successful and therefore this proteolytic cleavage could not be authenticated. Overall, the results of this study indicate that either the HEV NSP is not processed or that it is cleaved at one site by a virally-encoded protease novel among alpha-like superfamily viruses or a cellular protease.

Amino Acid Sequence↗

PubMed1998

The rubella virus nonstructural protease requires divalent cations for activity and functions in trans.

The rubella virus (RUB) nonstructural (NS) protease is a papain-like cysteine protease (PCP) located in the NS-protein open reading frame (NSP-ORF) that cleaves the NSP-ORF translation product at a single site to produce two products, P150 (the N-terminal product) and P90 (the C-terminal product). The RUB NS protease was found not to function following translation in vitro in a standard rabbit reticulocyte lysate system, although all of the other viral PCPs do so. However, in the presence of divalent cations such as Zn2+, Cd2+, and Co2+, the RUB NS protease functioned efficiently, indicating that these cations are required either as direct cofactors in catalytic activity or for correct acquisition of three-dimensional conformation of the protease. Since other viral and cell PCPs do not require cations for activity and the RUB NS protease contains a putative zinc binding motif, the latter possibility is more likely. Previous in vivo expression studies of the RUB NS protease failed to demonstrate trans cleavage activity (J.-P. Chen et al., J. Virol. 70:4707-4713, 1996). To study whether trans cleavage could be detected in vitro, a protease catalytic site mutant and a mutant in which the C-terminal 31 amino acids of P90 were deleted were independently introduced into plasmid constructs that express the complete NSP-ORF. Cotranslation of these mutants in vitro yielded both the native and the mutated forms of P90, indicating that the protease present in the mutated construct cleaved the catalytic-site mutant precursor. Thus, RUB NS protease can function in trans.

Cadmium↗

PubMed1997

Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses.

Orthopoxvirus species were identified and differentiated by polymerase chain reaction amplification of genome DNA using a single primer-pair based on sequences coding for the major protein component of the cowpox virus acidophilic-type inclusion body (ATI). DNA available for 6 of 8 Old World (cowpox, variola, monkeypox, camelpox, ectromelia and vaccinia viruses) and 3 New World (skunkpox, volepox, and raccoonpox) resulted in amplicons that ranged in size from 510 to 1673 base pairs depending on the species, except for raccoonpox virus DNA which did not amplify. XbaI digest gel electrophoresis profiles of the amplicons improved resolution of the differences.

Animals↗

PubMed1996

Mousepox outbreak in a laboratory mouse colony.

Mousepox was diagnosed in and eradicated from a laboratory mouse colony at the Naval Medical Research Institute. The outbreak began with increased mortality in a single room; subsequently, small numbers of animals in separate cages in other rooms were involved. Signs of disease were often mild, and overall mortality was low; BALB/cByJ mice were more severely affected, and many of them died spontaneously. Conjunctivitis was the most common clinical sign of disease in addition to occasional small, crusty scabs on sparsely haired or hairless areas of skin. Necropsy findings included conjunctivitis, enlarged spleen, and pale liver. Hemorrhage into the pyloric region of the stomach and proximal portion of the small intestine was observed in experimentally infected animals. In immune competent and immune deficient mice, the most common histologic finding was multifocal to coalescing splenic necrosis; necrosis was seen less frequently in liver, lymph nodes, and Peyer's patches. Necrosis was rarely observed in ovary, vagina, uterus, colon, or lung. Splenic necrosis often involved over 50% of the examined tissue, including white and red pulp. Hepatic necrosis was evident as either large, well-demarcated areas of coagulative necrosis or as multiple, random, interlacing bands of necrosis. Intracytoplasmic eosinophilic inclusion bodies were seen in conjunctival mucosae and haired palpebra. Ectromelia virus was confirmed as the causative agent of the epizootic by electron microscopy, immunohistochemistry, animal inoculations, serologic testing, virus isolation, and polymerase chain reaction. Serologic testing was of little value in the initial stages of the outbreak, although 6 weeks later, orthopoxvirus-specific antibody was detected in colony mice by indirect fluorescent antibody and enzyme-linked immunosorbent assay procedures. The outbreak originated from injection of mice with a contaminated, commercially produced, pooled mouse serum. The most relevant concern may be the unknown location of the source of the virus and the presence of a reservoir for this virus within the United States.

Animals↗

PubMed1995

PCR strategy for identification and differentiation of small pox and other orthopoxviruses.

Rapid identification and differentiation of orthopoxviruses by PCR were achieved with primers based on genome sequences encoding the hemagglutinin (HA) protein, an infected-cell membrane antigen that distinguishes orthopoxviruses from other poxvirus genera. The initial identification step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (raccoonpox, skunkpox, and volepox viruses), and a second pair for amplifying virtually the entire HA open reading frame of the Eurasian-African orthopoxviruses (variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiation, and TaqI digests differentiated the Eurasian-African orthopoxviruses, including vaccinia virus from the vaccinia virus subspecies buffalopox virus. Endonuclease HhaI digest patterns distinguished smallpox variola major viruses from alastrim variola minor viruses. For the Eurasian-African orthopoxviruses, a confirmatory step that used a set of higher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxvirus DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differences within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species.

Animals↗

PubMedDate not supplied

Outbreak of human monkeypox, Democratic Republic of Congo, 1996 to 1997.

Human monkeypox is a zoonotic smallpox-like disease caused by an orthopoxvirus of interhuman transmissibility too low to sustain spread in susceptible populations. In February 1997, 88 cases of febrile pustular rash were identified for the previous 12 months in 12 villages of the Katako-Kombe Health Zone, Democratic Republic of Congo (attack rate = 22 per 1,000; case-fatality rate = 3.7%). Seven were active cases confirmed by virus isolation. Orthopoxvirus- neutralizing antibodies were detected in 54% of 72 patients who provided serum and 25% of 59 wild-caught animals, mainly squirrels. Hemagglutination-inhibition assays and Western blotting detected antibodies in 68% and 73% of patients, respectively. Vaccinia vaccination, which protects against monkeypox, ceased by 1983 after global smallpox eradication, leading to an increase in the proportion of susceptible people.

Adolescent↗