Genetic factors associated with loss of the temperature-sensitive phenotype of the influenza A/Alaska/77-ts-1A2 recombinant during growth in vivo.
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Child↗
Biomedical subjects
R M Chanock
Publications and source records attributed to R M Chanock.
Rescue of noncultivatable human rotavirus by gene reassortment during mixed infection with ts mutants of a cultivatable bovine rotavirus.
Fastidious human rotaviruses that did not undergo productive infection in tissue culture were rescued by genetic reassortment during mixed infection with a temperature-sensitive (ts) mutant of a cultivatable bovine rotavirus. In this manner, the genes of the fastidious rotavirus that restricted growth in vitro were replaced by the corresponding genes from a tissue culture-adapted rotavirus. We recovered genetically reassorted viruses that grew to high titer and were neutralized specifically by hyperimmune guinea pig type 1 or type 2 human rotavirus antiserum. Preliminary RNA analysis of these clones disclosed that they were indeed viruses with reassorted genes.
Animals↗
Joseph E. Smadel memorial lecture. Strategy for development of respiratory and gastrointestinal tract viral vaccines in the 1980s.
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Acute Disease↗
Evaluation of A/Alaska/6/77 (H3N2) cold-adapted recombinant viruses derived from A/Ann Arbor/6/60 cold-adapted donor virus in adult seronegative volunteers.
The influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) virus was evaluated as a donor of attenuating genes to new variants of influenza A virus. This ca donor virus was mated with the A/Alaska/6/77 (H3N2) wild-type virus, and three A/Alaska/6/77 (H3N2) ca recombinant viruses were produced. The parental origin of the genes in the three ca recombinants had been determined previously (2), and their virulence for adult seronegative volunteers was assessed in the present study to identify the genes present in the ca donor virus that confer attenuation. Each of the recombinants received the hemagglutinin and neuraminidase genes from the A/Alaska/6/77 (H3N2) wild-type parent. One ca recombinant (CR-29) received all six transferable genes from the ca parent and was found to be satisfactorily attenuated in the volunteers. The two other ca recombinants received five of the six transferable genes with a wild-type gene at the M or NS locus. The pattern of infection in humans with these latter two ca recombinants was similar to the CR-29 ca recombinant. These findings demonstrate that inheritance of a gene in ca recombinants at the M or NS locus segregates independently of attenuation and suggest that the M and NS genes present in the ca donor virus are not the major determinants of attenuation conferred by this virus.
Antibodies, Viral↗
Characterization of the temperature sensitive phenotype of the A/Ann Arbor/6/60 cold-adapted virus and its recombinants.
The A/Ann Arbor/6/60 cold-adapted (ca) donor virus had a 38 degrees C shutoff temperature when tested in MDCK tissue culture. ca recombinant viruses bearing all six "internal genes" of the A/Ann Arbor/6/60 ca donor virus and the surface antigens of wild-type virus can manifest a 1,000-fold difference in plaquing efficiency at 38 degrees C. These observations suggest that the A/Ann Arbor/6/60 ca donor genes that specify the temperature sensitive phenotype of the ca recombinants can undergo genetic modification during the production and passage of the recombinants. The NS gene in the ca recombinant virus could be inherited from either parent without influencing the level of temperature sensitivity of the ca recombinant.
Animals↗
Antigenic characterization of human and animal rotaviruses by immune adherence hemagglutination assay (IAHA): evidence for distinctness of IAHA and neutralization antigens.
An immune adherence hemagglutination assay (IAHA) and a modified enzyme-linked immunosorbent assay for antigenic characterization of human rotaviruses were developed. The designations of type 1 and type 2 were identical to those established previously by specific complement fixation, enzyme-linked immunosorbent assay, and immune electron microscopy. By IAHA (and modified enzyme-linked immunosorbent assay) certain animal rotaviruses were found to be closely related to human rotavirus type 1. The pattern of IAHA reactivity and the cell culture neutralization serotype were found to be distinct properties. The separation of neutralization and IAHA reactivity was apparent when animal rotaviruses which were distinguishable from each other by neutralization assays were found to share IAHA specificity. Further evidence for the dissociation of the neutralization and IAHA specificities was found in studies of human and bovine rotaviruses which underwent genetic reassortment during coinfection. Thus, it appeared that the IAHA and neutralization antigens were coded for by different genes. In view of these findings, we suggest that the term serotype be reversed to identify the antigen that reacts with neutralizing antibodies as is customary for other viruses and that the term subgroup (instead of serotype) be used for the specificity detected by specific complement fixation, enzyme-linked immunosorbent assay, and now IAHA.
Animals↗
Distinctive ribonucleic acid patterns of human rotavirus subgroups 1 and 2.
The ribonucleic acid migration patterns of 7 subgroup 1 and 16 subgroup 2 human rotaviruses recovered from four geographic areas were compared. The subgroup 1 ribonucleic acid patterns had strikingly slower-moving segments 10 and 11, suggesting a correlation between the ribonucleic acid pattern and the subgroup specificity.
Electrophoresis, Polyacrylamide Gel↗
Hemagglutinin-specific enzyme-linked immunosorbent assay for antibodies to influenza A and B viruses.
An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies present in human serum or nasal washes directed against influenza A or B hemagglutinin glycoproteins. The assay was modified to measure the immunoglobulin isotype specificity of the anti-hemagglutinin response in serum and nasal secretions. In the postinfection sera anti-hemagglutinin of the immunoglobulin G isotype was predominant, whereas in nasal secretions the antibody was predominantly immunoglobulin A. The antibody response detected by the ELISA manifested hemagglutinin subgroup specificity. In addition, there was a good correlation between the ELISA antibody titer and the hemagglutination-inhibition or neutralizing antibody titer. The ELISA was more sensitive than the hemagglutination-inhibition assay, and the range of antibody titers measurable by ELISA in human serum was from less than 1:20 for children who had never experienced influenza infection to 1:400,000 for adults convalescing from a secondary infection. With more sensitive tests to detect antibody to the influenza hemagglutinin it should be possible to determine the relative contribution of local and systemic immunity to resistance to influenza virus infection.
Adult↗
Comparison of direct electron microscopy, immune electron microscopy, and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children.
An approximate 10% suspension in water of the first available stool sample from 411 infants and young children with acute gastroenteritis was examined by electron microscopy (EM) after 2 min of negative staining. This procedure enabled the detection of 88% of the 199 rotavirus infections, all of the 22 adenovirus infections, and 47% of the 15 approximately 27-nm virus infections ultimately detected by a combination of techniques, including immune electron microscopy (IEM) and rotavirus enzyme-linked immunosorbent assay (ELISA). Of the 204 infections detected by direct EM of stools, 76% were detected within 2 min of viewing, and 94% were detected within 6 min of viewing. Type 1 and type 2 rotavirus particles were visualized with approximately equal efficiency, although type 2 rotavirus infections were more common. Rectal swab preparations were clearly inferior to stool preparations for the detection of virus infection by direct EM. IEM examination was required for efficient visualization of viruses in rectal swab specimens. ELISA was the most sensitive method for the detection of rotaviruses; with this method, all infections in which rotavirus particles were visualized by EM or IEM were detected. However, 73% of the 1,834 specimens which were presumptively positive for rotavirus by conventional indirect ELISA proved to be falsely positive on the basis of EM, IEM, blocking ELISA, confirmatory ELISA, or a combination of these methods. False-positive rotavirus ELISA reactions apparently were eliminated when fecal specimens were tested in a modified confirmatory ELISA with a lower dilution of rotavirus-negative (pre-immunization) than rotavirus-positive (post-immunization) capture antibody from the same animal.
Acute Disease↗
Proteins of Norwalk virus.
The proteins of the Norwalk virus were studied by polyacrylamide gel electrophoresis. Highly purified specifically immunoprecipitated virions appeared to contain a single primary structural protein with a molecular weight of 59,000. In addition, a soluble Norwalk viral protein with a molecular weight of 30,000 was identified in fecal specimens containing Norwalk virus. The protein structure of the virion is similar to that of the Calciviridae family.
Caliciviridae↗
Immunoenzymatic examination of autoptic tissues from the outbreak of fatal infantile disease in Naples, in 1978-1979.
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Antigens, Viral↗
Respiratory syncytial virus antibodies in nonhuman primates and domestic animals.
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Animals↗
ts P1 and P3 genes are responsible for satisfactory level of attenuation of ts-1A2 recombinants bearing H1N1 or H3N2 surface antigens of influenza A virus.
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Adult↗
Genetic approaches to attenuation of influenza A viruses for man.
The explosion of new information concerning the influenza A viral genome provides a basis for deliberate manipulation of its genes with the intent of introducing specific mutations that render influenza virus attenuated and useful for prevention of disease. Currently there is considerable effort to develop a defined set of mutant genes that confer a specific and desired level of attenuation upon any viral recombinant into which they are transferred. In this manner new antigenic variants of influenza A virus may be satisfactorily attenuated after transfer of the mutant genes. The mutant genes must be readily identifiable by simple in-vitro techniques, thus enabling the genetic basis of attenuation to be monitored directly during all phases of vaccine development, manufacture and utilization in man. We describe our experience with two sets of ts mutant genes which affect viral RNA transcription or synthesis and which effect a reproducible level of attenuation in wild-type influenza A virus.
Animals↗
Human rotavirus type 2: cultivation in vitro.
A strain of type 2 human rotavirus (Wa) was grown to relatively high titer through 14 passages in primary cultures of African green monkey kidney (AGMK) cells. This passage series was initiated with virus that had been passaged 11 times serially in newborn gnotobiotic piglets. In contrast, virus present in the stool of patient Wa as well as virus from the first, second, or third passage in piglets could not be propagated successfully in African green monkey kidney cells. Prior to each passage in cell culture, the virus was treated with trypsin and the inoculated cultures were centrifuged at low speed. Cultivation of a type 2 human rotavirus should aid attempts to characterize this virus and to develop a means of immunoprophylaxis for a serious diarrheal disease of human infants.
Animals↗
Rotavirus gastroenteritis in the Washington, DC, area: incidence of cases resulting in admission to the hospital.
The incidence of rotavirus gastroenteritis in infants and children that required admission to the hospital was estimated for a defined population of approximately 105,000 individuals, including 29,000 children aged 15 years or younger whose primary health care was provided by Group Health Association, Inc, a health maintenance organization in the Washington, DC, area. From January 1977 through March 1979, almost all infants and children in this age group who required hospitalization for gastroenteritis were studied for evidence of infection with human rotavirus (HRV) and other agents. On the average, one in 272 (3.7/1,000) infants less than 12 months old and one in 451 (2.2/1,000) children aged 13 through 24 months were hospitalized for HRV disease each winter. The incidence of rotavirus gastroenteritis requiring hospitalization declined precipitously in children after the second birthday and such illness was not detected in children aged 5 years or older. The role of other agents in acute gastroenteritis requring hospitalization was minimal, compared with that of rotavirus.
Adolescent↗
Temperature-sensitive mutants of influenza A virus: evaluation of the Alaska/77-ts-1A2 temperature-sensitive recombinant virus in seronegative adult volunteers.
An influenza A virus recombinant bearing the surface antigens of the A/Alaska/6/77 (H3N2) wild type virus ands the two ts genes of the A/Udorn/72-ts-1A2 (H3N2) virus was evaluated for attenuation, antigenicity, and transmissibility in 28 adult volunteers all of whom possessed a preinoculation serum hemagglutination-inhibiting (HAI) antibody titer of less than or equal to 1:8 and 18 of whom also possessed a serum neuraminidase-inhibiting (NI) antibody titer of less than or equal to 1:4. The Alaska/77-ts-1A2 recombinant, which had a 37 degrees C shutoff temperature for plaque formation and ts mutations on the genes thought to code for the P1 and P3 polymerase proteins, infected 71 percent of the vaccinees when administered at a dose of 10(6.5) TCID50. Only 3 percent of the vaccinees developed symptoms in contrast to 50 percent of volunteers who received 10(4.2) TCID50 of wild type virus. Vaccinees shed virus for a shorter interval and at a lower titer than the volunteers who received wild type virus. Each ts-1A2 isolate retained the ts phenotype indicating that the recombinant was stable genetically in seronegative adults. An immunological response, as measured by a rise in serum HAI and/or NI antibody, was detected in 71 percent of the vaccinees and 87 percent of the recipients of wild type virus. Transmission of vaccine virus to susceptible contacts was not observed. The two ts-1A2 ts genes have now been transferred to two variants within the H3N2 subtype, the Vic/75 and Alaska/77 viruses, and have rendered the viruses satisfactorily attenuated for adults. The level of infectivity of the Alaska/77-ts-1A2 virus appeared to be low, however.
Adult↗
Temperature-sensitive mutants of influenza A virus. Transfer of the two ts-1A2 ts lesions present in the Udorn/72-ts-1A2 donor virus to the influenza A/Alaska/6/77 (H3N2) wild type virus.
The Udorn/72-ts-1A2 temperature-sensitive influenza A virus has a 37 degrees C shutoff temperature and a ts mutation on the genes coding for the P1 and P3 proteins. This ts donor virus was produced with the expectation that the transfer of its two ts genes would regularly and predictably attenuate each new variant of influenza A virus. It had previously been mated with the A/Victoria/75 (H3N2) virus and five Vic/75-ts-1A2 rcombinants were isolated that had both ts-1A2 ts genes and in vitro and in vivo genetic and biological properties similar to their Udorn/72-ts-1A2 parent. The present study was designed to determine if the acquisition of the two ts-1A2 ts genes would also confer a specific level of attenuation on the influenza A/Alaska/6/77 (H3N2) wild type virus. Fifteen recombinant Alaska/77-ts-1A2 viruses were isolated and characterized genetically for the number and location of ts mutations. These clones were also studied for their level of replication and genetic stability in hamsters. Four recombinants possessed both of the ts-1A2 mutations and had a 37 degrees C shutoff temperature for plaque formation. Two recombinants possessed only a ts P1 gene and had either a 38 degrees C or 39 degrees C shutoff temperature. The remaining nine clones had the ts P3 gene and a shutoff temperature of 37 degrees C, 38 degrees C or 39 degrees C. Each of the four 37 degrees C shutoff temperature recombinants that possessed both ts P1 and P3 genes were restricted at least 10,000-fold in replication in the hamster's lung and approximately 100-fold in the nasal turbinates compared to the level of replication of wild type virus in these sites. All isolates from these animals retained the ts phenotype. The level of replication in vivo of the ts P1 and P3 segregants was related to their shutoff temperature of plaque formation in vitro, e.g. the 38 degrees C ts P3 segregant was less restricted in replication in the lungs than a 37 degrees C ts P3 segregant. All isolates from animals infected with the ts P3 segregants were ts, whereas a low level of genetic instability was detected for one of the ts P1 segregants. Since ten independent ts-1A2 recombinants (one Udorn/72, 5 Victoria/75 and 4 Alaska/77) with both ts-1A2 mutations exhibited the same genetic and biologic properties, it is likely that these ts P1 and P3 genes were the prime determinants of attenuation and could effect a similar level of attenuation in other influenza A viruses within the H3N2 subtype.
Animals↗