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Biomedical subjects

R F Ramig

Publications and source records attributed to R F Ramig.

At least 55 records · Page 3Linked to original sources
PubMed1988

The effects of host age, virus dose, and virus strain on heterologous rotavirus infection of suckling mice.

Seven-day-old suckling CD-1 mice, born to seronegative dams, were orally inoculated with a number of animal and human rotaviruses. Simian (SA11), rhesus (RRV), and bovine (B223) rotaviruses were found to replicate and cause severe disease. Canine (K9), bovine (B641), and human (Wa) rotaviruses either replicated minimally and caused minimal disease (K9, B641) or failed to replicate or cause disease (Wa). The features of SA11 infection of mice were examined in greater detail. Suckling mice were susceptible to infection and disease from 1 day of age to 13-15 days of age. Restriction of disease occurred at an earlier age (13 days) than restriction of replication (15 days). Dose-response studies in seven-day-old mice showed that virus replication and disease could be induced with doses as low as 1 x 10(2) pfu/mouse; however, both intestinal virus titers and severity of disease increased in parallel with virus dose. Intestinal virus replication appeared to be restricted in SA11 infections. Only at very low doses (1 x 10(2) pfu/mouse) did virus replication occur to levels above the inoculated dose. While light microscopic examination showed classical features of rotavirus infection in the ileum, electron microscopic examination revealed only the accumulation of large numbers of electron-lucent vacuoles in the ileal enterocytes of infected mice. Structures typical of rotavirus morphogenesis were not detected in enterocytes from mice infected with rotavirus SA11.

Age Factors↗
PubMed1987

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups A, C, F, and G to genome segments.

Crosses were performed between prototype temperature-sensitive (ts) mutants of simian rotavirus SA11 representing reassortment groups A, C, F, and G and ts mutants of rhesus rotavirus RRV that belonged to different reassortment groups. Wild-type (ts+) reassortant progeny were identified by plaque formation at nonpermissive temperature (39 degrees), picked, and grown to high titer. The ts+ phenotype of the resulting progeny clones was verified by titration at 39 degrees and 31 degrees. The electropherotypes of the ts+ clones were determined by electrophoresis, and parental origin of each genome segment was assigned by comparison of segment mobility to parental markers. Analysis of the parental origin of genome segments in the ts+ reassortants derived from SA11 ts X RRV ts crosses revealed the following map locations of the SA11 prototype ts mutants: tsA(778), segment 4; tsC(606), segment 1; tsF(2124), segment 2; and tsG(2130), segment 6. The assignment of tsA was made on the basis of genome segment segregation in two independent crosses with each of two independent RRV ts mutants. The assignment of tsC was made on the basis of segregation in only a single cross with an RRV ts mutant; however, a larger number of progeny clones were examined from this cross. The lesion of tsF was mapped with data from three independent crosses using two different RRV ts mutants. The assignment of tsG was made on the basis of segregation in three independent crosses, two with RRV ts mutants and one with Wa. The assignments of tsA, tsC, and tsF were confirmed in crosses between RRV ts mutants representing those reassortment groups, and SA11 ts mutants in other reassortment groups.

Chromosome Mapping↗
PubMed1987

Mixed infection of Culicoides variipennis with bluetongue virus serotypes 10 and 17: evidence for high frequency reassortment in the vector.

The primary vector species for bluetongue virus (BTV) in the United States, Culicoides variipennis, was orally infected with BTV serotype 10, BTV serotype 17, or a mixture of the two viruses. The recovery of virus from the infected flies was low following a period of extrinsic incubation. Electrophoretic analysis of progeny virus from singly infected flies revealed that only the parental electropherotype could be isolated from those flies. In contrast, electrophoretic analysis of virus from mixedly infected flies revealed that eight of the 11 virus-positive flies produced virus progeny with reassortant electropherotypes. The proportion of reassortant progeny varied from 7 to 78% (mean 42%), depending on the individual fly. Analysis of segregation of the parental origin of genome segments in the reassortant progeny virus suggested that, while reassortment of most segments was random, selection for genome segment 8 from the type 17 parent may have occurred. Analysis of segregation in individual mixedly infected flies showed that each fly yielded a relatively unique set of reassortants, but that specific electropherotypes were isolated repeatedly from individual flies. These data indicated that the vector species C. variipennis was a permissive host for high frequency reassortment of genome segments of BTV.

Animals↗
PubMed1987

Protection between different serotypes of bovine rotavirus in gnotobiotic calves: specificity of serum antibody and coproantibody responses.

In a previous study, different U.S. isolates of bovine rotavirus were studied for their serotypes and cross-protective properties (G. N. Woode, N. E. Kelso, T. F. Simpson, S. K. Gaul, L. E. Evans, and L. Babiuk, J. Clin. Microbiol. 18:358-364, 1983). Three viruses belonging to two different serotype groups were used as vaccines in gnotobiotic calves, which were subsequently challenged with B641 or B223, representing the two bovine serotypes. In the present work, the experiments were repeated with more calves and the specificity of their antibody responses was measured and compared with the results of the protection studies. Protection between different serotypes occurred under both homologous and heterologous conditions but was not directly serotype dependent. B223 virus showed both homologous and heterologous protection against B223 and B641 challenge viruses. This was a one-way reaction, as B641 did not induce protection against B223. Neonatal calf diarrhea virus vaccine produced neither homologous (against B641) nor heterologous (against B223) protection. The plaque reduction neutralization titers of serum antibody and coproantibody did not predict a state of protection against the challenge virus. Calves vaccinated with neonatal calf diarrhea virus or B641 developed neutralizing antibodies to their respective heterologous challenge viruses but were not protected. After challenge, the boosted coproantibody plaque reduction neutralization response to the original vaccine virus was greater than that to the challenge virus.

Animals↗
PubMed1987

Analysis of mixed infection of sheep with bluetongue virus serotypes 10 and 17: evidence for genetic reassortment in the vertebrate host.

Two seronegative sheep were infected intravenously with 10(9) PFU each of bluetongue virus (BTV) serotype 10 and BTV serotype 17. One animal experienced a mild bluetongue-like disease, and both experienced a short-duration viremia and developed neutralizing immune responses to both virus serotypes. Progeny virus was isolated from venous blood from each animal by using conditions in which reassortment could not have occurred during isolation. Electropherotypes were determined for the progeny viruses from the infected sheep, yielding strikingly similar results for the two animals. In both sheep, serotype 10 dominated among the progeny, accounting for 92% of the progeny. Serotype 17 was rarely isolated and accounted for 3% of the progeny analyzed. The remaining 5% of the progeny clones were reassortant and derived genome segments from both serotypes 10 and 17. Analysis of the parental origin of genome segments in the small number of reassortant progeny analyzed suggested that selection of specific genome segments may have occurred in the infected sheep. These data indicate that reassortment of genome segments occurs, at low frequency, in sheep mixedly infected with BTV.

Animals↗
PubMed1986

Analysis of reassortment of genome segments in mice mixedly infected with rotaviruses SA11 and RRV.

Seven-day-old CD-1 mice born to seronegative dams were orally inoculated with a mixture of wild-type simian rotavirus SA11 and wild-type rhesus rotavirus RRV. At various times postinfection, progeny clones were randomly isolated from intestinal homogenates by limiting dilution. Analysis of genome RNAs by polyacrylamide gel electrophoresis was used to identify and genotype reassortant progeny. Reassortment of genome segments was observed in 252 of 662 (38%) clones analyzed from in vivo mixed infections. Kinetic studies indicated that reassortment was an early event in the in vivo infectious cycle; more than 25% of the progeny clones were reassortant by 12 h postinfection. The frequency of reassortant progeny increased to 80 to 100% by 72 to 96 h postinfection. A few reassortants with specific constellations of SA11 and RRV genome segments were repeatedly isolated from different litters or different animals within single litters, suggesting that these genotypes were independently and specifically selected in vivo. Analysis of segregation of individual genome segments among the 252 reassortant progeny revealed that, although most segments segregated randomly, segments 3 and 5 nonrandomly segregated from the SA11 parent. The possible selective pressures active during in vivo reassortment of rotavirus genome segments are discussed.

Animals↗
PubMed1985

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups B and E to genome segments.

Recombinant (reassortant) viruses were selected from crosses between temperature-sensitive (ts) mutants of simian rotavirus SA11 and wild-type human rotavirus Wa. The double-stranded genome RNAs of the reassortants were examined by electrophoresis in Tris-glycine-buffered polyacrylamide gels and by dot hybridization with a cloned DNA probe for genome segment 2. Analysis of replacements of genome segments in the reassortants allowed construction of a map correlating genome segments providing functions interchangeable between SA11 and Wa. The reassortants revealed a functional correspondence in order of increasing electrophoretic mobility of genome segments. Analysis of the parental origin of genome segments in ts+ SA11/Wa reassortants derived from the crosses SA11 tsB(339) X Wa and SA11 tsE(1400) X Wa revealed that the group B lesion of tsB(339) was located on genome segment 3 and the group E lesion of tsE(1400) was on segment 8.

Animals↗
PubMed1985

Genome RNAS of virulent and attenuated strains of bluetongue virus serotypes 10, 11, 13 and 17.

An improved system was developed for the electrophoretic resolution of bluetongue virus (BTV) genome segments. This procedure was used to compare the genome segments of wild type (W) and vaccine (V) strains of BTV serotypes 10, 11, 13 and 17. Numerous alterations in the migration of individual genome segments could be detected between W and V strains of each serotype, suggesting that many mutations had occurred during attenuation of the V strains. In addition, electrophoretic polymorphisms not previously detected, were found between W strains of the 4 serotypes.

Bluetongue virus↗
PubMed1985

Growth characteristics of virulent and attenuated strains of bluetongue virus serotypes 10, 11, 13 and 17.

The growth properties of wild type (WT) and live, attenuated vaccine (V) strains of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were compared. All BTV strains produced maximal yields when grown at 34 C and harvested 24-36 hr after infection. The 4 V strains were temperature-sensitive (ts) for growth at 39 C as compared to the corresponding WT strains. The 4 V strains were ts for plaque formation when 37 C was used as the nonpermissive temperature. One-step growth curves showed that only the V strain of type 13 had significantly altered growth kinetics as compared to the corresponding WT. All BTV strains were strongly cell associated throughout the growth cycle.

Animals↗
PubMed1984

Extragenic suppression of temperature-sensitive phenotype in reovirus: mapping suppressor mutations.

Independently isolated, spontaneous pseudorevertants of temperature-sensitive (ts) mutants of reovirus type 3 have previously been genetically characterized (R. F. Ramig and B. N. Fields, 1979, Virology 92, 155-167). Eighteen of these pseudorevertants were backcrossed to wild-type reovirus type 1 and reassortant progeny expressing the parental ts phenotype were selected. Analysis of segregation of genome segments in the reassortant, parental ts, progeny clones allowed the determination of the genome segment bearing the suppressor mutation of four pseudorevertants. The suppressor of tsA(201) phenotype mapped to segment S4 in the pseudorevertants RtsA(201)101 and RtsA(201)121 and to segment L3 in pseudorevertant RtsA(201)122. The suppressor of tsB(352) phenotype mapped to segment S1 in the pseudorevertant RtsB(352)b. In two other pseudorevertants the suppressor could not be mapped to a single genome segment due to the small number of progeny clones examined. These genetic results indirectly support the "compensating protein interactions" hypothesis for the mechanism of suppression.

Animals↗
PubMed1984

Characterization of temperature-sensitive mutants of simian rotavirus SA11: protein synthesis and morphogenesis.

The synthesis of viral polypeptides, distribution of viral antigens, and morphogenesis of viral structures have been examined in cells infected with temperature-sensitive (ts) mutants of SA11 representing 10 recombination groups. At the permissive temperature (31 degrees C) the synthesis of viral polypeptides and the distribution of viral antigens did not differ significantly from those of the wild type. At the nonpermissive temperature (39 degrees C) some mutants (tsB, -C, -E, -F, and -G) synthesized significantly smaller amounts of viral polypeptides and had a very diffuse distribution of viral antigen. Several of the mutants synthesized one or more electrophoretically aberrant polypeptide species at both 31 and 39 degrees C. All of the mutants, except tsF, assembled morphogenetic intermediates at 39 degrees C. Aberrant intermediates were assembled in all mutants at 31 and 39 degrees C. No specific morphogenic defect could be associated with any of the ts mutants.

Animals↗
PubMed1983

Isolation and genetic characterization of temperature-sensitive mutants that define five additional recombination groups in simian rotavirus SA11.

Nineteen independent temperature-sensitive (ts) mutants were isolated from SA11 following mutagenesis with proflavin or 5-azacytidine. Fourteen of the ts mutants fell into one or another of five mutant groups previously defined by recombination. Five of the ts mutants defined five recombination groups (F, G, H, I, and J) that had not been previously identified. Thus, 10 of the 11 expected mutant groups have been identified in SA11. The prototype mutants of the 10 mutant groups were tested for recombination at nonpermissive temperature to determine if any group had a lesion affecting recombination. Most mutant pairs recombined efficiently; however, the tsH mutant was restricted in its recombination with the tsB and tsI mutants and the tsG and tsJ mutants failed to recombine at detectable levels at nonpermissive temperature. The mutants of groups F-J did not complement, or did so inefficiently, and interfered with the growth of wild type at both permissive and nonpermissive temperatures. The growth properties of the mutants of groups F-J are described.

Animals↗
PubMed1983

A genetic map of reovirus: assignment of the newly defined mutant groups H, I, and J to genome segments.

Mutants representing three previously undefined reovirus type 3 mutant groups have been isolated following backcross of suppressed pseudorevertants to wild type (R.F. Ramig and B.N. Fields, 1979, Virology 92, 155-167; R. Ahmed, P.R. Chakraborty, A.F. Graham, R.F. Ramig, and B.N. Fields, 1980, J. Virol. 34, 383-389). The prototype mutant of each of the three new mutant groups was mapped by analysis of genome segment segregation in intertypic recombinants derived from crosses between the type 3 ts mutants and ts mutants of type 1 or type 2. Segregation analysis revealed the location of the group H prototype mutant tsH(26/8) to be genome segment M1, that of the group I prototype mutant tsI(138) to be segment L3, and that of the group J prototype mutant tsJ(128) to be segment S1. Mapping of the group I and J lesions required the identification of suppressed ts lesions in some of the intertypic rcombinant clones.

Animals↗
PubMed1983

Factors that affect genetic interaction during mixed infection with temperature-sensitive mutants of simian rotavirus SA11.

A number of factors that affect genetic interaction during mixed infection with temperature-sensitive mutants of simian rotavirus SA11 have been examined. (1) Statistical analyses of recombination frequency (RF) indicated that (a) the variability noted in RF was not related to variations in experimental conditions and (b) a linear map of the mutations could not be drawn. (2) The wild phenotype of recombinant progeny was stable on passage. (3) Aggregates of progeny virus or heterozygous progeny virus particles did not contribute significantly to the observed RF. (4) RF increased in parallel with multiplicity of infection. (5) A maximal, or near maximal, RF was obtained at the earliest time significant recombinants could be detected. (6) Recombination was efficient at nonpermissive temperature. (7) Complementation did not occur or was inefficient. (8) Mutants from all recombination groups interfered with the growth of wild-type virus at both permissive and nonpermissive temperatures.

Genetic Complementation Test↗
PubMed1980

Genetic variation during persistent reovirus infection: presence of extragenically suppressed temperature-sensitive lesions in wild-type virus isolated from persistently infected L cells.

Persistent reovirus infection of L cells was established with a serially passaged stock of temperature-sensitive (ts) mutant C(447) containing greater than 90% defective interfering particles. Within a month after establishment of the carrier culture, the ts mutant was replaced by virus that expressed the wild-type (ts(+)) temperature phenotype (R. Ahmed and A. F. Graham, J. Virol. 23:250-262, 1977). To determine whether the ts(+) phenotype of the virus was due to intragenic reversion or to the presence of an extragenic mutation suppressing the original ts defect, several clones were backcrossed to wild-type reovirus, and the progeny of each cross were screened for temperature sensitivity. The results indicated that the original tsC lesion had reverted. However, in two of the seven clones examined, new ts lesions were found. These new ts lesions appeared phenotypically as ts(+) due to the presence of extragenic suppressor mutations. Temperature-sensitive mutants representing three different groups were rescued from one suppressed clone, indicating that this ts(+) clone contained multiple ts lesions. Among the ts mutants rescued were the initial isolates of a new recombination group which we have designated H. Some of the ts mutants rescued from the suppressed clones are capable of interfering with the growth of wild-type reovirus and may play a role in maintaining the carrier state. The results of this study show that persistently infected L cells contain a genetically heterogeneous population of reovirus even though all virus clones express the ts(+) phenotype. It is thus critical to distinguish between genotype and phenotype when analyzing viruses that emerge during persistent infection.

Animals↗