Search PubMed⌕ Search

Biomedical subjects

R F Ramig

Publications and source records attributed to R F Ramig.

At least 37 records · Page 2Linked to original sources
PubMed1993

Characterization of avian reovirus-induced cell fusion: the role of viral structural proteins.

Cell fusion induced by avian reovirus was analyzed using virus strain FC and Vero cells. One-step growth curves showed that cell fusion was directly associated with viral replication. Cell fusion occurred most efficiently at basic pH (8.0-8.5) and fusion from without could not be demonstrated. Actinomycin D, at low concentrations, increased cell fusion, and cycloheximide prevented cell fusion, indicating that viral protein(s) were responsible for the induction of cell fusion. Immunofluorescence tests indicated that viral proteins were present on the infected cell surface. Radioimmuno-precipitation identified structural proteins mu 2C and sigma 2 as predominant viral protein species present on the infected cell surface. Cell fusion was inhibited by virus-specific antisera, suggesting that mu 2C and/or sigma 2 present on the infected cell surface were involved in the induction of cell fusion. Trypsin and chymotrypsin treatment of purified viruses cleaved both mu 2C and sigma 2 proteins, but generated different cleavage products with each protein. The addition of trypsin to the culture media following infection increased cell fusion, whereas chymotrypsin treatment decreased cell fusion. The opposite effects of trypsin and chymotrypsin on the cell fusion, together with the different specificities of these two proteases in cleavage of mu 2C and sigma 2 proteins, further suggest that the cell surface-associated mu 2C and/or sigma 2 are involved in the syncytium formation.

Alkalies↗
PubMed1993

Rescue of infectivity by sequential in vitro transcapsidation of rotavirus core particles with inner capsid and outer capsid proteins.

We recently developed an in vitro transcapsidation system in which infectivity of single-shelled (ss) rotavirus particles was successfully rescued (Chen and Ramig, Virology [1993]). Here, we report the rescue of infectivity of rotavirus core particles using virus strain B223 (G serotype 10) as the core donor and strain SA11-4F (G3) as the capsid donor. Core particles of B223 were obtained by CaCl2 treatment of B223 ss-particles followed by isopycnic CsCl gradient centrifugation. Inner capsid protein VP6 of SA11-4F was prepared by CaCl2 treatment of SA11-4F ss-particles, followed by removal of core particles by two rounds of centrifugation. Outer capsid proteins VP4 and VP7 of SA11-4F were prepared by EDTA treatment of ds-particles, followed by three rounds of centrifugation to remove ss-particles and minimize residual infectivity. No infectivity (< 3 PFU/ml) was detectable in any of the donor preparations. Transcapsidated ss-particles were obtained by mixing B223 core particles and a 5-fold excess of SA11-4F VP6 at neutral pH. The formation of transcapsidated ss-particles was confirmed by electron microscopy, protein composition analysis, and density determination. Along with the formation of ss-particles by in vitro transcapsidation, some infectivity was also detected and transcriptase activity was reconstituted. Semi-purified transcapsidated ss-particles were then mixed with SA11-4F outer capsid proteins VP4 and VP7 at acidic pH to obtain transcapsidated ds-particles, as described previously. The formation of ds-like particles was also confirmed by electron microscopy, protein composition, and density determination. As the result of formation of transcapsidated ds-like particles, viral infectivity increased significantly (80-fold) relative to that of transcapsidated ss-particles. The infectivity of transcapsidated ds-particles was neutralized by polyclonal anti-SA11 serum, but not by polyclonal anti-B223 serum. The transcapsidated particles formed small plaques like B223 (core donor), and all the progeny plaques contained B223 genomes. These results demonstrate that the infectivity of rotavirus core particles can be rescued by sequential addition of inner and outer capsid proteins in vitro.

Antigens, Viral↗
PubMed1993

Immunodominant neutralizing antigens depend on the virus strain during a primary immune response in calves to bovine rotaviruses.

Sera obtained from gnotobiotic calves (GC antisera) infected with bovine rotavirus strain NCDV or B223 from a previous study (Woode et al., 1987), which have different G (G6 and G10 respectively) and P serotypes, were compared for their neutralization (NT) properties to a number of human and animal rotaviruses (representing G serotype 1-6, 8-10). Two distinct patterns of neutralization were identified from these GC antisera. Of all the serotypes tested, NCDV GC antisera neutralized only B641 to a relatively high titer compared with the homologous titer, implying a narrow pattern of NT response. Analysis with reassortants indicated that the response was primarily to VP4. In contrast, B223 GC antisera neutralized most of the G serotypes tested to titers within 3-7 fold of the homologous titer, demonstrating a broad pattern of NT response. In the earlier study B223 was shown to induce a heterotypic protection against bovine rotavirus B641 (G serotype 6), and the serologic data obtained from this study indicates that a B223 vaccine might provide broad protection against several different serotypes of human and animal rotaviruses.

Animals↗
PubMed1992

Determinants of rotavirus stability and density during CsCl purification.

The stability of rotavirus infectivity during CsCl gradient purification and subsequent storage was examined using our standard SA11 wild type (SA11-Cl3), the SA11 4F variant (SA11-4F), bovine rotavirus B223, and a panel of bi- and triparental reassortants derived from these parental viruses. Viral stability was determined by the recovery of infectivity at each step during a standard CsCl purification protocol. SA11-4F was the most stable parent (91-93% recovery), SA11-Cl3 had intermediate stability (10-21% recovery), and B223 was least stable (0.5-7% recovery). Among the reassortants, the recovery varied from 0.5 to 88.6% of the initial infectivity and was determined primarily by the parental origin of genome segment 4. The greatest loss of infectivity occurred during Freon extraction, with smaller losses during the CsCl gradient, and the smallest loss during the virus pelleting step. Comparison of the stability of viruses grown in the presence or absence of exogenous trypsin revealed that, in general, viruses grown in the absence of trypsin were more stable during purification. During 4-5 months storage at 4 degrees, the differences in stability of parental and reassortant viruses were not as dramatic as during purification and were not significantly affected by the presence or absence of trypsin during growth. However, survival during storage was as low as 4% and as high as 100% and was also primarily dependent on the parental origin of genome segment 4. It was noted that bovine rotavirus B223 had higher density in CsCl than either SA11-Cl3 or SA11-4F. The observation of heterogeneity in density was investigated using reassortants. These results indicated that all reassortants had intermediate density and suggested that physical interactions among the structural proteins were responsible for the heterogeneity in density. The possible roles of viral structural proteins in rotavirus stability and the relationship between the stability and the density are discussed.

Centrifugation, Density Gradient↗
PubMed1992

Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope.

We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.

Animals↗
PubMed1991

Comparative growth of different rotavirus strains in differentiated cells (MA104, HepG2, and CaCo-2).

The production of viral antigen after infection of MA104, HepG2 (derived from human liver), and CaCo-2 (derived from human colon) cells with various cultivatable human and animal rotavirus strains was compared using immunofluorescence tests. All rotavirus strains examined expressed antigen in CaCo-2 cells and MA104 cells, but only some virus strains, namely, SA11-Cl3 (simian), RRV (simian), CU-1 (canine), and Ty1 (turkey), produced antigen in numbers of infected HepG2 cells comparable to infections in MA104 and CaCo-2 cells. Fl-14 (equine), OSU (porcine), NCDV (bovine), and Ch2 (chicken) strains were found to infect moderate numbers of HepG2 cells. Most human rotaviruses (representing viruses in serotypes 1, 2, 3, 4, 8, and 9), a simian rotavirus variant (SA11-4F), lapine (Ala, C-11 and R-2) viruses and porcine (Gottfried) virus infections resulted either in no detectable antigen or antigen synthesis in a low percentage of HepG2 cells. Human rotavirus isolates obtained from the stool specimens of an immunocompromised child with rotavirus antigen in his liver showed two different patterns of replication in HepG2 cells. Examination of the replication of a subset of viruses in the liver and intestinal tissues of orally infected suckling mice showed the CU-1 and Ty1 strains replicated well, while the OSU and human rotavirus strains did not. These results indicate that growth restriction in HepG2 cells is not serotype-specific, and growth of a virus in HepG2 cells does not necessarily correlate with the hepatotropic potential of a virus strain. Factors that may influence these differences of virus infectivity in HepG2 cells are discussed.

Animals↗
PubMed1991

Rotavirus temperature-sensitive mutants are genetically stable and participate in reassortment during mixed infection of mice.

Mixed and single infections of 7-day-old suckling mice with SA11 temperature-sensitive (ts) mutants and RRV wild-type were examined to determine if selection against ts mutations occurred in the suckling mouse model. Single infections with ts mutants indicated that mutant replication was restricted relative to wild-type and that disease was similarly reduced. Revertant (ts+) progeny did not appear to be selected during infection. Mixed infection with ts mutant and RRV wild-type revealed a reduction in the replication of RRV suggesting that the ts mutants displayed an interference phenotype in vivo similar to that observed in vitro. However, reduced replication of the RRV parent in mixed infection did not result in a significant reduction in disease relative to RRV infection. When progeny from the mixed infections were isolated at the permissive temperature both ts and ts+ progeny were observed, and the genome segments of these progeny segregated in a manner consistent with the temperature phenotype of each progeny clone and the location of the ts mutation determined in vitro. Selection of ts+ progeny from mixed infected mice at nonpermissive temperature yielded either the RRV parent or ts+ reassortants. The segregation of genome segments in these ts+ reassortant progeny was consistent with the location of the ts lesion determined previously in vitro. These results indicate the following with respect to infection of suckling mice with ts mutants: (1) ts lesions are genetically stable and are not selected against during in vivo infection, (2) ts mutants cause disease with reduced severity, (3) ts mutants interfere with the replication of wild-type virus in vivo but not with the severity of disease, and (4) mixed infection of suckling mice may be useful in genetic studies with rotaviruses not adapted to growth in cultured cells.

Animals↗
PubMed1990

Superinfecting rotaviruses are not excluded from genetic interactions during asynchronous mixed infections in vitro.

Asynchronous infections of MA104 cells with temperature-sensitive (ts) mutants of simian rotavirus SA11 were performed to determine if the superinfecting ts mutant could contribute to the formation of reassortant progeny. Significant yields of ts+ reassortant progeny were obtained in crosses where infection with the first and second ts mutant was separated by as much as 24 hr, indicating that superinfecting viruses were not excluded from participation in genetic interactions. The practical and theoretical implications of this result are discussed.

Animals↗
PubMed1990

Intracellular RNA synthesis directed by temperature-sensitive mutants of simian rotavirus SA11.

The kinetics of intracellular synthesis of single-stranded (ss) RNA and double-stranded (ds) RNA directed by prototype temperature-sensitive (ts) mutants representing the 10 mutant groups of rotavirus SA11 were examined. Cells were infected with individual mutants or wild type under one-step growth conditions and maintained at permissive temperature (31 degrees) or nonpermissive temperature (39 degrees). At various times postinfection, infected cells were pulse-labeled, ssRNA and dsRNA were purified, RNA species were resolved by electrophoresis and autoradiography, and RNA synthesis was quantitated by computer-assisted densitometry. The mutants representing all groups synthesized significantly less ssRNA and dsRNA at both 31 degrees and 39 degrees, when compared to wild type. When the ratio of synthesis at 39 degrees/31 degrees was determined for ssRNA and dsRNA of each mutant, three RNA synthesis phenotypes were evident. The tsB(339), tsC(606), and tsE(1400) mutants synthesized both ssRNA and dsRNA in a temperature-dependent manner. The group G mutant, tsG(2130), synthesized ssRNA in temperature-independent fashion but was temperature-dependent for the synthesis of dsRNA. The remaining mutants, tsA(778), tsD(975), tsF(2124), tsH(2384), tsI(2403), and tsJ(2131), synthesized both ssRNA and dsRNA in a temperature-independent fashion. The RNA synthesis phenotypes of the ts mutants are discussed in terms of what is known of the function(s) of the protein species to which ts lesions have been assigned.

Animals↗
PubMed1990

Rotavirus genome segment 4 determines viral replication phenotype in cultured liver cells (HepG2).

One-step growth determinations were performed with five strains of rotavirus in HepG2, a cell line derived from human liver. Three virus strains (SA11-C13, SA11-C14, and RRV) replicated in HepG2 cells and attained yields 10- to 100-fold above input titers. Two virus strains (B223 and SA11-4F) failed to replicate above input titer. Analysis of reassortants that segregated the genes of parental virus pairs able and unable to replicate revealed that the HepG2 cell growth phenotype segregated with genome segment 4. Immunofluorescence analysis of infected HepG2 cells showed that the production of detectable antigen correlated with the growth phenotype and also segregated with genome segment 4. Thus, we conclude that (i) some virus strains were capable of replication in cultured liver cells while other strains could not replicate under identical conditions and that (ii) the inability of some virus strains to replicate resulted from a segment 4-associated block in replication before protein synthesis. These results are discussed in terms of what is known of the functions of VP4.

Antigens, Viral↗
PubMed1989

Biological and immunological characterization of a simian rotavirus SA11 variant with an altered genome segment 4.

We have studied a variant virus isolated from a stock of SA11 virus (H. G. Pereira, R. S. Azeredo, A. M. Fialho, and M. N. P. Vidal, 1984, J. Gen. Virol. 65, 815-818). This virus, designated 4F, was initially identified by its faster electrophoretic mobility for genome segment 4. The variant was analyzed to determine if the altered electrophoretic mobility of genome segment 4 could be correlated with phenotypic changes. Comparison of our standard laboratory SA11 virus (clone 3) with the 4F variant showed the following: (i) The 4F variant possesses a viral hemagglutinin (VP4) with a higher apparent molecular weight than clone 3. (ii) The 4F variant produces large plaques when assayed in vitro, as compared to clone 3. (iii) The 4F variant produces plaques in the absence of proteolytic enzymes, whereas clone 3 does not. (iv) The 4F variant reacts with serotype-specific neutralizing monoclonal antibodies to VP7, but fails to react with several neutralizing anti-VP4 monoclonal antibodies generated to SA11 clone 3. (v) The 4F variant grows to a higher titer and is more stable than clone 3. (vi) The 4F variant produces a VP4 that appears to be more susceptible to cleavage by trypsin than is the VP4 of clone 3. Further analyses with the 4F variant may lead to an understanding of the molecular basis for these altered phenotypes that appear to be related, at least in part, to the product of genome segment 4.

Antigens, Viral↗
PubMed1989

Phenotypes of rotavirus reassortants depend upon the recipient genetic background.

We have previously characterized the biological and immunological properties of a simian rotavirus SA11 variant (4F) with an altered genome segment 4. The SA11-4F variant formed large plaques in the presence of protease, formed small clear plaques in the absence of protease, and grew to high titer in the presence of protease when compared to our standard wild type (SA11 clone 3). To determine the genome segment of the rotavirus SA11 variant 4F that encoded the unique protease-associated phenotypes of the variant, reassortants were generated that segregated the outer capsid genes of 4F onto a genetic background derived from either the bovine rotavirus B223 or our standard SA11 wild type (clone 3), both of which have contrasting protease-associated phenotypes. The parental and reassortant viruses were examined to determine which genes from the 4F variant encoded the ability (i) to form large plaques in the presence of protease, (ii) to form small clear plaques in the absence of exogenous protease, and (iii) to grow to significantly higher titer in the presence of protease. These phenotypes could be transferred to a clone 3 genetic background by a single genome segment from the 4F variant segment 4. However, in the 4F/B223 reassortants a different and unexpected situation was found. On a B223 genetic background the same phenotypes segregated with a combination of a minimum of two 4F genome segments, segments 4 and 9. These results indicate that the recipient genetic background onto which the genes of a donor rotavirus are reassorted can affect the phenotypes conferred by the presence of the donor segments. Thus, the results of segregation mapping experiments using reassortant viruses should be interpreted with caution.

Cell Line↗
PubMed1989

Studies on rotavirus homologous and heterologous active immunity in infant mice.

Homologous and heterologous active immunity was studied in mice with mammalian group A rotaviruses. One day old mice were vaccinated with one of the following rotaviruses: bovine B641 (serotype 6), bovine B223 (untyped), simian SA11 (serotype 3) and murine EDIM (untyped). At 10 days of age they were challenged with EDIM virulent virus or SA11 virus. All the vaccines induced a serological antibody response in the mice but only the homologous immune response was protective.

Animals↗
PubMed1989

Analysis of reassortment and superinfection during mixed infection of Vero cells with bluetongue virus serotypes 10 and 17.

The reassortment of genome segments during mixed infection of Vero cells with bluetongue virus (BTV) serotypes 10 and 17 was investigated, using non-selective conditions for analysis of the progeny of mixed infections. Reassortment was found to be an early event in the BTV replication cycle, indicating that progeny BTV genomes undergo a single round of reassortment. Non-random segregation of individual genome segments was observed in crosses at equal multiplicity of infection, and was confirmed in crosses performed at unequal multiplicity. Asynchronous infections showed that superinfection exclusion resulted in the failure of the superinfecting virus to contribute genome segments to reassortants if the second virus followed the first by more than 4 h. The significance of these results for the evolution and epidemiology of BTV is discussed.

Animals↗
PubMed1989

Asynchronous mixed infection of Culicoides variipennis with bluetongue virus serotypes 10 and 17.

Culicoides variipennis (Diptera: Ceratopogonidae) the primary vector of bluetongue virus (BTV) in the U.S.A. were asynchronously mixedly infected with two BTV serotypes (BTV-10 and BTV-17); flies first ingested a blood meal that contained BTV-17 and 1, 3, 5, 7, and 9 days later selected flies ingested a second blood meal that contained BTV-10. Control flies ingested each parental virus separately, or both viruses simultaneously, in a single blood meal. Electrophoretic analysis of progeny virus clones indicated that superinfection with BTV-10 occurred when the flies ingested the second virus 1, 3 and 5 days post-initial infection. Parental BTV-17 and reassortant virus clones were isolated from these flies, but parental BTV-10 virus was not isolated from any flies. Reassortant clone frequencies were 67%, 71% and 17% when superinfection occurred on days 1, 3 and 5 after initial infection, respectively, as compared to 48% for simultaneously infected flies. Only parental BTV-17 clones were isolated from flies that ingested the second virus on days 7 and 9 after initial BTV-17 infection. The results indicated that interference to superinfection occurred in C. variipennis by 5 days and flies were refractory to superinfection by 7 days post-initial infection. Analysis of segregation of the parental origin of genome segments in the reassortant clones indicated selection against most segments of BTV-10 parental origin. This occurred both in individual flies and in individual groups. The fact that C. variipennis readily fed on a second blood meal and their ability to produce new viral genotypes suggested that these vectors are highly permissive hosts for evolution of BTV by genome reassortment.

Animals↗
PubMed1989

Comparative studies of the antigenic polypeptide species VP4, VP6, and VP7 of three strains of bovine rotavirus.

Three bovine rotavirus strains belonging to two distinct serotype groups, serotype 6 (NCDV and B641) and B223, distinct from the other six mammalian rotavirus serotypes but not yet assigned to a serotype group, were compared with each other and with canine rotavirus (K9, serotype 3) by studying the properties of their cognate polypeptide species VP4, VP6, and VP7. The three viruses showed distinct differences in the polyacrylamide gel electrophoretic migration rates of protein species VP4 and VP7, with minor differences in VP6. Differences were also observed among the migration patterns of genome segments 4, 6, and the 7-8-9 triplet, which encode VP4, VP6, and VP7, respectively. Monoclonal antibodies (MAbs) to B223, which were directed against VP4 or VP7, showed homologous specificity for neutralization and immunofluorescence (IF), although one MAb reactive with VP4 also reacted by IF and by immunoprecipitation (IP) with all four viruses and weakly neutralized B641 and K9. This MAb may react with the epitope responsible for the B223-induced one-way neutralizing and protection response of calves against B641 observed in earlier studies. MAbs reactive with VP6 by IP showed enzyme-linked immunosorbent assay and IF reactivity with all three bovine viruses and the canine virus. The two serotype 6 viruses could be distinguished by the two B641 MAbs, B641-N2b reacting by neutralization and IF with both viruses and B641-N1 reacting with B641 and the serotype 3 canine rotavirus but not with NCDV. One nonneutralizing B641 MAb reacted by IP and IF with VP7 of all four rotaviruses examined, and one B223 MAb neutralized B223 and, to low titer, B641 and K9 although reacting by IP and IF with all four viruses. Three MAb-resistant mutants were selected by passage of B223 in the presence of one of three selected B223 MAbs at concentrations which only neutralized approximately 90% of the infectious virions. The resulting mutants were 100% resistant to neutralization with their respective MAb but remained neutralizable by the same selection of MAbs as the parent B223 virus.

Animals↗
PubMed1989

Passive immunity modulates genetic reassortment between rotaviruses in mixedly infected mice.

Genetic reassortment between simian rotavirus SA11 and rhesus rotavirus (RRV) occurs with high frequency following mixed infection of nonimmune suckling mice (J. L. Gombold and R. F. Ramig, J. Virol. 57:110-116, 1986). We examined the effects of passively acquired homotypic or heterotypic immunity on reassortment in vivo. Passively immune suckling mice obtained from dams immune to either serotype 3 simian rotavirus (SA11) or serotype 6 bovine rotavirus (NCDV) were infected orally with either SA11 or RRV or a mixture of SA11 and RRV (both serotype 3 viruses). At various times postinfection, signs of disease were noted and the intestines of individual mice were removed and homogenized for titration of infectious virus and isolation of progeny plaques. Electrophoresis of genomic RNA was used to identify reassortants among the viral progeny isolated from infected animals. No reassortants (less than 0.45%) were detected among 224 clones examined from mixedly infected, homotypically immune mice. Twenty-nine reassortants (10.66%) were identified among 272 progeny clones from mixedly infected, heterotypically immune mice. Thus, reassortment was reduced more than 50-fold by homotypic immunity and approximately threefold by heterotypic immunity compared with prior data obtained from mixed infections of nonimmune mice. In addition, reassortment between SA11 and RRV in nonimmune mice was shown to be dependent on the virus dose. Taken together, these results suggest that immune responses may modulate the frequency of reassortment by reducing the effective multiplicity of infection (by neutralization or other immune mechanisms), thereby preventing efficient mixed infection of enterocytes.

Animals↗