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Biomedical subjects

R M Chanock

Publications and source records attributed to R M Chanock.

At least 109 records · Page 6Linked to original sources

Genetic relatedness among human rotaviruses.

The genetic relatedness of 81 clinical rotavirus isolates to the human rotavirus prototype strains Wa (subgroup 2, serotype 1) and DS-1 (subgroup 1, serotype 2) was examined by RNA hybridization techniques. Labeled single-stranded (+) transcripts of Wa or DS-1 virus were incubated with denatured genomic rotaviral RNAs, and the resulting hybrids were subjected to gel electrophoresis and autoradiography. Nineteen of the specimens contained subgroup 1 rotavirus with a "short" RNA migration pattern. These viruses were found to be closely related to the DS-1 strain and were associated with illness of short duration. The remaining 62 isolates belonged to subgroup 2 and exhibited a "long" RNA migration pattern. Fifty-four of these isolates exhibited significant hybridization with the Wa strain probe. Four isolates yielded multiple hybrid bands with the Wa probe but also possessed at least one gene segment homologous to the DS-1 strain. The remaining four subgroup 2 rotaviruses did not exhibit significant homology in the form of labeled hybrid bands when tested with either the Wa or DS-1 probe. These findings suggest that most clinical rotavirus isolates belong to one of two human rotavirus "families" defined as Wa-like or DS-1-like. Our observations also suggest that reassortment occurs in vivo between rotaviruses belonging to the two human rotavirus "families" and that there are one or more additional families of human rotavirus.

Genes, Viral↗

Nucleotide sequence of the structural glycoprotein VP7 gene of Nebraska calf diarrhea virus rotavirus: comparison with homologous genes from four strains of human and animal rotaviruses.

A full-size cloned cDNA copy of the rotavirus gene encoding the structural neutralization glycoprotein (VP7) of Nebraska calf diarrhea virus (NCDV), a strain recently shown to be effective as a vaccine in children, has been sequenced. Comparison of the deduced amino acid sequence of NCDV (serotype 6) VP7 with that of four other rotavirus strains (human WA serotype 1, human HU-5 serotype 2, simian SA-11 serotype 3, and bovine UK serotype 6) indicates that the degree of amino acid homology among VP7 neutralization proteins of these serotypes ranges from 75 to 86%. Four hydrophilic regions at amino acid residues 174-183, 248-256, 287-294, and 310-317 exhibit significant homology and hence may represent common antigenic determinants, while one hydrophilic area at amino acid residues 83-102 exhibits sufficient divergence to suggest it may be involved in serotype specificity.

Amino Acid Sequence↗

Immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in the cotton rat.

Human convalescent antiserum to respiratory syncytial virus (RSV) administered intraperitoneally to cotton rats prior to RSV challenge provided near-complete protection from pulmonary infection. Antiserum given subsequent to viral challenge reduced pulmonary viral titers 100-fold or greater within 24 h. Sandoglobulin, a preparation of purified human IgG with high titer of anti-RSV neutralizing activity, produced the same effects as convalescent antiserum. Sandoglobulin was absorbed rapidly and produced a significant therapeutic reduction in virus titer within 3 h. The level of virus reduction in pulmonary and nasal tissues was directly proportional to the neutralizing antibody titer in the cotton rat serum, and was always greater in the lungs than the nose. Animals treated therapeutically with Sandoglobulin had a depressed primary antibody response to infection, but were completely resistant to reinfection with RSV. Histologic examination of pulmonary tissues from Sandoglobulin-treated animals showed no pathologic changes.

Animals↗

Independent segregation of two antigenic specificities (VP3 and VP7) involved in neutralization of rotavirus infectivity.

Antiserum prepared against the M37 strain of rotavirus, recovered from an asymptomatic newborn infant in Venezuela, neutralized two prototype human rotaviruses that define two separate serotypes: serotype 1 (Wa) and serotype 4 (ST3). Thus, the M37 strain is a naturally occurring intertypic rotavirus. Analysis of reassortant viruses produced during coinfection in vitro indicated that the observed dual serotype specificity of M37 resulted from sharing a related outer capsid protein, VP3, with the ST3 virus and another related outer capsid protein, VP7, with the Wa virus. Analysis of single (VP3)-gene-substitution reassortants indicated that VP3 was as potent an immunogen as VP7. In addition, direct evidence was obtained that the serotype specificity of neutralizing antibody elicited by VP3 can differ from the serotype specificity of neutralizing antibody elicited by VP7, indicating the need for a dual system of rotavirus classification in which the neutralization specificity of both VP3 and VP7 outer capsid proteins are identified.

Animals↗

Antigenic analysis of a putative new strain of respiratory syncytial virus.

A recent isolate (9320) of respiratory syncytial virus (RSV) that had been reported to differ from all previous strains was compared with RSV strains isolated over a period of 23 years by using the techniques of in vitro neutralization by specific antiserum and in vivo resistance to infection in cotton rats. The 9320 strain did not appear to be a novel antigenic variant and exhibited greater homology to several prototype strains than did an earlier isolate. The antigenic differences detected by in vitro neutralization disappear when virus strains are compared in vivo. Thus, it appears that concerns that antigenic instability might compromise efforts at RSV prophylaxis are unfounded.

Animals↗

Nucleoprotein and membrane protein genes are associated with restriction of replication of influenza A/Mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract.

An avian influenza A virus, A/Mallard/NY/6750/78(H2N2), was restricted in in replication in the respiratory tract of squirrel monkeys. Avian-human influenza A reassortant viruses possessing the six RNA segments coding for nonsurface proteins (i.e., internal genes) of this avian virus were as restricted in replication in squirrel monkeys as their avian influenza parent. These findings indicated that restriction of replication of the avian influenza virus is a function of one or more of its internal genes. For an investigation of which of the avian influenza genes was responsible for restricted replication in the respiratory tract of primates, reassortant viruses were produced that contained human influenza virus surface antigens from the A/Udorn/72(H3N2) virus and one or more of the internal genes derived from the avian influenza virus parent. Avian-human reassortant influenza A viruses containing only the nucleoprotein or matrix protein RNA segment from the avian influenza virus parent were as restricted in their growth as an avian-human influenza reassortant virus containing each of the six avian influenza internal genes. In addition, an avian-human influenza reassortant virus possessing only the avian RNA 1 and nonstructural genes (which by themselves do not specify restricted replication) manifested a significant reduction of virus replication in squirrel monkey tracheas. Thus, the avian nucleoprotein and matrix genes appear to play a major role in the host range restriction exhibited by the A/Mallard/78 virus and its reassortants, but the combination of RNA 1 and nonstructural genes also contributes to restriction of replication.

Animals↗

Reassortant rotaviruses as potential live rotavirus vaccine candidates.

A series of reassortants was isolated from coinfection of cell cultures with a wild-type animal rotavirus and a "noncultivatable" human rotavirus. Wild-type bovine rotavirus (UK strain) was reassorted with human rotavirus strains D, DS-1, and P; wild-type rhesus rotavirus was reassorted with human rotavirus strains D and DS-1. The D, DS-1, and P strains represent human rotavirus serotypes 1, 2, and 3, respectively. Monospecific antiserum (to bovine rotavirus, NCDV strain) or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7 (of the rhesus rotavirus), was used to select for reassortants with human rotavirus neutralization specificity. This selection technique yielded many reassortants which received only the gene segment coding for the major neutralization protein from the human rotavirus parent, whereas the remaining genes were derived from the animal rotavirus parent. Single human rotavirus gene substitution reassortants of this sort represent potential live vaccine strains.

Animals↗

Quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats.

The amount of passively acquired serum respiratory syncytial virus (RSV)-neutralizing antibodies required to protect the respiratory tract of cotton rats against infection was studied. Infant cotton rats were inoculated intraperitoneally with various dilutions of a single pool of sera derived from cotton rats convalescent from RSV infection. After 24 h, these animals were inoculated with RSV intranasally. Virus replication in the respiratory tract was suppressed in cotton rats which had a serum neutralizing antibody titer of 1:100 or greater. Resistance was greater in the lungs than in the nose. Complete or almost complete resistance in the lungs was observed in cotton rats with a serum neutralizing antibody titer of 1:380 or greater. The level of serum RSV-neutralizing antibodies required to confer significant resistance to infection in the cotton rat was similar to the level of maternally derived serum antibodies possessed by human infants less than 2 months of age, who as a group exhibit relative resistance to RSV disease compared with infants 2 to 6 months of age.

Animals↗

Avian-human reassortant influenza A viruses derived by mating avian and human influenza A viruses.

Reassortant influenza A viruses were produced by mating an avian virus (A/Mallard/NY/78, A/Mallard/Alberta/78, or A/Pintail/Alberta/79) with a wild-type human influenza A virus. From each mating a reassortant virus was obtained that contained the genes coding for the hemagglutinin and neuraminidase surface antigens of the human influenza A wild-type virus and the six other RNA segments ("internal genes") of the avian influenza A virus parent. The avian-human reassortant influenza viruses produced resembled their avian virus parent in that they produced plaques on MDCK monolayers at 42 C, a temperature restrictive for the human influenza viruses. In the trachea of squirrel monkeys, each avian-human reassortant influenza virus was as restricted in its replication as was its avian influenza virus parent. Thus, one or more of the six internal genes of each avian parent virus was responsible for restriction of the reassortant virus in monkeys. The A/Washington/80 X A/Mallard/NY/78 reassortant virus retained its phenotype of restricted replication in monkeys after five serial passages in vivo. It also failed to transmit to cagemates or induce resistance to wild-type virus challenge, and it did not initiate a systemic or enteric infection. These findings form the basis for evaluation of these attenuated avian-human reassortant influenza A viruses as live attenuated vaccines for humans.

Animals↗

A dot hybridisation assay for detection of rotavirus.

A "dot" hybridisation technique for the detection of rotavirus in stools and other biological materials is described. The assay is based on the in-situ hybridisation of labelled single-stranded RNA probes, obtained by in-vitro transcription of rotavirus particles, to heat-denatured rotavirus RNA immobilised on nitrocellulose membranes. The method is highly specific and allows for the detection of as little as 8 pg of viral RNA. Its use for the detection of rotavirus in stool suspensions and rectal swabs obtained from children with diarrhoea may facilitate epidemiological studies of rotavirus gastroenteritis.

Child↗

Temperature-sensitive mutants of influenza A/Udorn/72 (H3N2) virus. III. Genetic analysis of temperature-dependent host range mutants.

One hundred thirty-three ts mutants of influenza A/Udorn/72 virus were arranged into eight complementation groups, A-H, on Madin-Darby canine kidney (MDCK) monolayer cultures at the restrictive temperature of 40 degrees. The eight complementation groups, A-H, on MDCK cells corresponded to the eight recombination groups, A-H, on rhesus monkey kidney (RMK) cells, respectively, and this suggested that each MDCK complementation group represented one of the eight influenza A RNA gene segments. These ts viruses were used to identify the locus of the ts mutation in temperature-dependent host range (td-hr) mutants of the A/Udorn/72 virus. Sixteen of the 133 ts mutants exhibited distinct host (MDCK)-dependent restriction of plaque formation at 40 degrees but not at 34 degrees and were referred to as td-hr mutants. These 16 td-hr mutants were ts+ (not ts) on RMK cells but ts on MDCK cells. The td-hr mutants did not share a common lesion and the ts lesions were distributed among the eight complementation groups, A-H, when tested on MDCK cells. An analysis of one of the td-hr mutants indicated that an extrageneic RMK-dependent suppressor mutation did not account for the td-hr phenotype. These data suggested that a host-dependent ts mutation was responsible for the td-hr restriction of this mutant. Representation of td-hr mutations in each of the eight complementation groups indicates that the influenza A virus genome can undergo mutation leading to an altered host range in any of its eight RNA segments.

Animals↗

Construction and characterization of cDNA clones for four respiratory syncytial viral genes.

Cytoplasmic poly(A)-containing RNA from respiratory syncytial virus-infected cells was used as a template to synthesize oligo(dT)-primed cDNAs. Discrete size classes of single-stranded cDNAs, resolved by alkali agarose gel electrophoresis, were used separately to construct double-stranded cDNAs that were subsequently inserted into the plasmid vector pBR322 at the Pst I site by means of oligo(dG)oligo(dC) tailing. After transfection of Escherichia coli, recombinant plasmids were screened mostly by serial rounds of hybrid selection of mRNAs from virus-infected cells and subsequent in vitro translation of the selected mRNAs. Comparative peptide mapping of the translation products with those of authentic virion proteins served to establish the viral origin of the cDNA recombinants. In this manner, four distinct classes of recombinant plasmids were identified. These encode sequences corresponding to those of respiratory syncytial virus nucleocapsid protein, matrix protein, phosphoprotein, and a nonstructural protein.

DNA↗

Oral administration of human rotavirus to volunteers: induction of illness and correlates of resistance.

Four of 18 volunteers challenged orally with human rotavirus strain D (subgroup 2, serotype Wa) developed a diarrheal illness two to four days after inoculation. Viral shedding was detected in five of the 18 volunteers, whereas 12 (67%) developed serologic evidence of infection. Two volunteers who developed diarrheal illness after the initial inoculation were given the same inoculum 19 months later; neither developed diarrhea, although one developed constitutional and gastrointestinal symptoms. The presence of preinoculation serum immunofluorescent antibody to rotavirus strain D or high levels of neutralizing antibody to Wa or reassortant DS-1 human rotavirus correlated with resistance to diarrheal illness. Although prechallenge serum antibody correlated with resistance to diarrhea and/or shedding of rotavirus, the relationship of preexisting local neutralizing activity in intestinal fluid was less clear-cut.

Administration, Oral↗

Dose response of A/Alaska/6/77 (H3N2) cold-adapted reassortant vaccine virus in adult volunteers: role of local antibody in resistance to infection with vaccine virus.

An attenuated influenza A candidate vaccine virus, derived from the A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) donor virus and the A/Alaska/6/77 (H3N2) wild-type virus, was evaluated in adult seronegative volunteers (serum hemagglutination-inhibiting antibody titer, less than or equal to 1:8) for level of attenuation, infectivity, antigenicity, and genetic stability. Four groups with similar preinoculation mean titers of serum and nasal wash antibodies were inoculated intranasally with 10(4.5), 10(5.5), 10(6.5), or 10(7.5) 50% tissue culture infectious doses (TCID50) of the ca reassortant virus, and eight other seronegative adult volunteers received the wild-type virus. Only 2 of 66 vaccinees developed fever or mild and brief systemic or upper respiratory tract illness or both. Both volunteers with vaccine-related reactions received the highest dose (10(7.5) TCID50) of ca virus, which indicates that the vaccine retains some mild reactogenicity at a high dosage. In contrast, four of eight volunteers infected with the wild-type virus became ill. Each of the 54 isolates tested retained the temperature-sensitive phenotype of the vaccine virus. Thus, the ca reassortant was genetically stable and attenuated at 10(4.5) to 10(7.5) TCID50 for seronegative adults. The 50% human infective dose of ca virus was approximately 10(5.3) TCID50. Ten and one hundred 50% human infectious doses infected 73 and 83% of vaccinees, respectively, and approximately 75% developed an immunological response at these doses. The failure of the vaccine virus to infect some volunteers was correlated with the presence of pre-inoculation nasal wash immunoglobulin A hemagglutinin antibody.

Adult↗

Mechanisms of immunity to respiratory syncytial virus in cotton rats.

Active immunity and maternally transmitted passive immunity to respiratory syncytial virus (RSV) were studied in cotton rats. Animals infected with respiratory syncytial virus developed complete resistance to pulmonary reinfection, which lasted at least 18 months. Nasal resistance was of shorter duration and began to diminish in 8 months. Pulmonary resistance was transferred by parabiosis, but nasal resistance was not. Adoptive transfer studies with fractionated convalescent blood showed that serum antibody, but not circulating lymphocytes, conferred pulmonary resistance. Immune females conferred antibody to their young prenatally and postnatally, with most of the antibody being transferred via colostrum and milk. Maternally transmitted immunity was more effective in the lungs than in the nose and was transient in both organs. Foster nursing experiments showed colostrum and milk to be the most important routes of immune transfer. Although resistance in infants generally correlated with serum neutralizing antibody levels, several exceptions to this correlation suggested that immune factors other than neutralizing antibody may also play an important role in maternal passive immunity.

Animals↗

Pediatric viral gastroenteritis during eight years of study.

During the period January 1974 through July 1982, fecal samples from 1,537 pediatric inpatients with gastroenteritis were tested for enteric viruses by electron microscopic and rotavirus enzyme-linked immunosorbent assay techniques. Rotaviruses were detected in 34.5% of these patients, enteric adenoviruses were detected in 4.7%, approximately 27-nm viruses were detected in 1.6%, and at least one of these agents was found in 40.1% of the study subjects. Three infections were by an apparently new agent which morphologically is a rotavirus, but which failed to react in the rotavirus enzyme-linked immunosorbent assay. During the first 8 calendar years of study, rotaviruses were detected in 39.0% of 577 patients in the even-numbered years and 30.3% of 702 patients in the odd-numbered years. Adenoviruses were found in all calendar months. Rotaviruses were found in inpatients in November through July, whereas approximately 27-nm viruses were found in October through June. The percentage of patients who had a demonstrated viral infection rose steadily from 7.4% in September to 72.0% in January and then steadily declined to 2.9% in August. Viral infection was especially common in study subjects who were 7 through 24 months of age; 61% of such children had one or more enteric viruses. Rotavirus-infected patients tended to be younger during the months of greatest rotavirus activity than at the beginning and end of the rotavirus season, presumably because of a greater exposure to virus at the height of the rotavirus outbreak.

Adenoviridae Infections↗