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Biomedical subjects

B R Murphy

Publications and source records attributed to B R Murphy.

At least 253 records · Page 14Linked to original sources
PubMed1980

New enzyme immunoassays for measurement of influenza A/Victoria/3/75 virus in nasal washes.

Nasopharyngeal (NP) washes from 12 patients experimentally infected with influenza A/Victoria/3/75 wild-type virus were tested in 2 newly developed enzyme-immunoassay (EIA) systems. Results were compared with infectivity titres of the NP wash specimens in rhesus monkey kidney-tissue culture. EIA detected A/Victoria/3/75 haemagglutinin antigen for more days than did culture. EIA is also rapid and easy to do in large numbers; hence it may have advantages over culture methods for the diagnosis of influenza infections and the investigation of influenza epidemiology.

Animals↗
PubMed1980

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↗
PubMed1980

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↗
PubMed1980

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↗
PubMed1980

Fluorometric assay for measurement of viral neuraminidase--application to the rapid detection of influenza virus in nasal wash specimens.

A sensitive assay was developed for the measurement of neuraminidase with use of the 4-methylumbelliferkyl-alph-ketoside of N-acetyl-neuraminic acid as a fluorescent substrate. Neuraminidase activity was detected in preparation containing small quantities of cultivated influenza virus as well as in some nasal wash specimens from human volunteers experimentally infected with influenza A/Alaska/78 virus. Activity due to influenza viral neuraminidase could be distinguished from activity due to bacterial or mammalian (fibroblast) neuraminidase by means of enzyme inhibition with ethylenediaminetetraacetate, a chelating agent. The same substrate could be used in a solid-phase assay to determine the antigenic type of the influenza viral neuraminidase. The fluorescent neuraminidase assay takes 2-4 hr to perform and uses equipment available in many laboratories. Thus, it offers promise for the rapid detection of influenza viruses in cell cultures and clinical specimens.

Animals↗
PubMed1980

Evaluation of three strains of influenza A virus in humans and in owl, cebus, and squirrel monkeys.

The virulence of three cloned influenza A viruses was compared in humans and in three readily available species of nonhuman primates (owl, squirrel, and cebus monkeys) in an attempt to identify a species of monkey that could be used to investigate the genetic basis of attenuation of influenza A viruses for humans. Three influenza A viruses from two subtypes, i.e., the A/Udorn/72 (H3N2), A/Alaska/77 (H3H2), and A/Hong Kong/77 (H1H1) viruses, produced febrile influenzal illness in humans. Squirrel monkeys developed mild upper respiratory tract illness in response to each of the three viruses. Illness was accompanied by a high level of virus shedding; each of nine squirrel monkeys that shed equal to or greater than 10(5.0) 50% tissue culture infective doses of virus became ill, whereas those that shed less remained well. In contrast, the cebus and owl monkeys remained clinically well despite infection with each of the three viruses. Thus, squirrel monkeys appear to be moderately permissive primate hosts in which to investigate the genetic basis of virulence of human influenza A viruses.

Animals↗
PubMed1980

Temperature-sensitive mutants of influenza A virus: transfer of the two temperature-sensitive lesions in the Udorn/72-ts-1A2 virus to the A2Hong Kong/123/77 (H1N1) wild-type virus.

The influenza A/Udorn/72-ts-1A2 virus possesses temperature-sensitive mutations in the genes coding for the P1 and P3 polymerase proteins. It is being evaluated as a donor of its attenuating temperature-sensitive genes to produce recombinant live vaccine strains of epidemic variants of influenza A virus. Transfer of the P1 and P3 genes to two viruses within the H3N2 subtype of influenza A virus (i.e., the A/Victoria/3/75 and A/Alaska/6/77 viruses) conferred on each variant the following properties: (i) 37 degrees C shutoff temperature for plaque formation, (ii) almost complete restriction of viral replication in the lungs, (iii) a 100-fold restriction of viral replication in the nasal turbinates, and (iv) genetic stability after replication in hamsters. This study was undertaken to determine whether the transfer of the two ts-1A2 temperature-sensitive genes into a virus belonging to the H1N1 subtype (i.e., the A/Hong Kong/123/77 virus) would result in a restriction of replication in vitro and in vivo comparable to that observed with the previously studied H3N2 recombinant viruses in hamsters. This was found to be the case. In addition, infection of hamsters with the A/Hong Kong/77-ts-1A2 virus induced significant resistance to infection with wild-type A/Hong Kong/77 virus. Thus, the two ts-1A2 temperature-sensitive genes attenuated influenza A viruses belonging to two distinct subtypes to a specific and predictable level. An unexpected genetic interaction was observed between several A/Hong Kong/77-ts-1A2 segregants bearing the group 5 (P1) temperature-sensitive lesion. One interpretation of these results is that intracistronic complementation occurred between these segregants.

Animals↗
PubMed1980

Use of the enzyme-linked immunosorbent assay to detect serum antibody responses of volunteers who received attenuated influenza A virus vaccines.

Sera from volunteers who received live influenza A wild-type or ts recombinant virus were tested by hemagglutination inhibition (HI) assay, neuraminidase inhibition (NI) assay, and the enzyme-linked immunosorbent assay (ELISA) to determine which assay system was the most sensitive in detecting an immunological response to infection. The ELISA was performed with inactivated whole virus antigen, and the optical density at each of five serial twofold dilutions of pre- and postimmunization sera was measured. The difference in the amount of ELISA antibody in pre- and postinoculation serum specimens was taken to be proportional to the area between the respective titration curves. The ELISA was more sensitive than the HI or NI test in detecting a seroresponse in volunteers infected with A/Hong Kong/123/77 (H1N1), A/New Jersey/8/76 (Hswine N1), or A/Alaska/6/77 (H3N2) ts recombinant virus. These results suggest that the ELISA should be used to determine the frequency of infection with attenuated viruses as well as the 50% human infectious dose of candidate live influenza A vaccine viruses.

Antibodies, Viral↗
PubMed1980

Evaluation of influenza A/Hong Kong/123/77 (H1N1) ts-1A2 and cold-adapted recombinant viruses in seronegative adult volunteers.

Two attenuated influenza A donor viruses, the A/Udorn/72 ts-1A2 and the A/Ann Arbor/6/60 cold-adapted (ca) viruses, are being evaluated for their ability to reproducibly attenuate each new variant of influenza A virus to a specific and desired level by the transfer of one or more attenuating genes. Each of these donor viruses has been able to attenuate influenza A viruses belonging to the H3N2 subtype by the transfer of one or more attenuating genes. To determine whether these two donor viruses could attenuate a wild-type virus that belonged to a different influenza A subtype, ts-1A2 and ca recombinants of a wild-type virus representative of the A/USSR/77 (H1N1) Russian influenza strain were prepared and evaluated in adult doubly seronegative volunteers at several doses. The recombinants derived from both donor viruses were attenuated for the doubly seronegative adults. Less than 5% of infected vaccinees developed a febrile or systemic reaction, whereas five of six recipients of wild-type virus developed such a response. The 50% human infectious dose (HID(50)) for each recombinant was approximately 10(5.0) 50% tissue culture infective doses. The virus shed by the ts-1A2 and ca vaccinees retained the ts or ca phenotype, or both. This occurred despite replication of the recombinant viruses for up to 9 days. No evidence for transmission of the ca or ts-1A2 recombinant virus to controls was observed. A serum hemagglutination inhibition response was detected in less than 50% of the infected vaccinees. However, with the more sensitive enzyme-linked immunosorbent assay, a serological response was detected in 100% of the ca vaccinees given 300 HID(50) and approximately 70% of ca or ts vaccinees who received 10 to 32 HID(50) of virus. These results indicate that the recombinants derived from both donor viruses were satisfactorily attenuated and were stable genetically after replication in doubly seronegative adults although they induced a lower serum hemagglutination inhibition response than that found previously for H3N2 ts and ca recombinants.

Adult↗
PubMed1979

Cell-mediated cytotoxicity following influenza infection and vaccination in humans.

Cell-mediated cytotoxic activity in circulating mononuclear cells from 31 volunteers challenged with live influenza A/Victoria virus, and 22 volunteers vaccinated with inactivated influenza vaccine, was examined employing target cells infected with several viruses by means of a 51Cr release assay. Effectors from infected volunteers, and from volunteers who manifested four-fold rises in serum HAI antibody after vaccination, demonstrated significantly elevated levels of cytotoxicity against targets infected with the homologous virus. Elevated cytotoxicity was seen by days 3 and 4 after challenge or vaccination and returned to baseline levels by day 9 to 10. In infected volunteers, cytotoxic activity was broadly directed, rising against targets infected with an antigenically distinct virus within the same influenza type (A), against targets infected with a serologically unrelated virus of a different influenza type (B), and also against cells infected with Newcastle disease virus, a paramyxovirus from another species. However, elevated levels of cytotoxicity were not observed when targets infected with herpes simplex virus, a member of an entirely different virus group, or when uninfected target cells were employed. In vaccinated volunteers, the rise in cytotoxicity was more restricted than after infection, since elevated cytotoxic activity was seen only against cells infected with the homologous virus and not against inflenza B-infected cells. Fractionation of mononuclear cell populations indicated that effector cell activity is associated with T-cell depleted fractions and can only partially be reduced by depletion of adherent cells. The rapid development, short duration, and broadly directed specificity of this cytotoxic response suggest that it may be involved in early events following acute influenza infection in humans.

Adolescent↗
PubMed1979

Temperature-sensitive mutants of influenza A virus: evaluation of the A/Victoria/75-ts-1A2 temperature-sensitive recombinant virus in seronegative adult volunteers.

An influenza A virus recombinant bearing the surface antigens of the A/Victoria/3/75 (H3N2) strain and the two ts genes of the A/Udorn/72-ts-1A2 virus was evaluated for attenuation, antigenicity, and protective effect in 42 doubly seronegative adult volunteers (i.e., individuals who lacked detectable serum antibodies for the hemagglutinin and neuraminidase antigens). This 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 90% of the volunteers. Of the volunteers, 5% developed mild coryza or rhinitis but other signs or symptoms were not observed, indicating that the A/Victoria/75-ts-1A2 recombinant was more attenuated than the A/Victoria/75-ts-1[E] recombinant. Vaccinees shed virus for a shorter interval and at a lower titer than did the A/Victoria/75-ts-1[E] vaccinees. Each ts-1A2 isolate retained the ts phenotype indicating that the recombinant was stable genetically in doubly seronegative adults. Finally, the ts-1A2 recombinant induced significant resistance to subsequent challenge with A/Victoria/75 wild-type virus.

Adult↗
PubMed1979

Cold-adapted variants of influenza A virus: evaluation in adult seronegative volunteers of A/Scotland/840/74 and A/Victoria/3/75 cold-adapted recombinants derived from the cold-adapted A/Ann Arbor/6/60 strain.

Influenza A/Scotland/74 (H3N2) and A/Victoria/75 (H3N2) cold-adapted (ca) recombinant viruses, prepared by mating the A/Ann Arbor/6/60 (H2N2) ca donor virus and influenza A wild-type virus, were evaluated in adult seronegative volunteers (serum hemagglutination-inhibiting antibody titer, </=1:8) for level of attenuation, antigenicity, and genetic stability of the temperature-sensitive and ca phenotypes. At 10(7.0) to 10(7.5) 50% tissue culture infective doses the A/Scotland/74 and A/Victoria/75 ca recombinant viruses were clearly attenuated and antigenic. However, one of eight vaccinees infected with 10(7.5) 50% tissue culture infective doses of the A/Scotland/74 ca recombinant had a febrile reaction (39 degrees C). At a 10-fold higher dose (10(8.5) 50% tissue culture infective doses), 4 of 12 A/Scotland/74 vaccinees had a febrile and/or systemic reaction. Febrile reactions were not observed in volunteers who received the A/Victoria/75 ca recombinant virus, whereas 3 of the 12 vaccinees had mild upper respiratory tract symptoms, in one instance associated with mild systemic manifestations. Significantly, the serum hemagglutination- and neuraminidase-inhibiting antibody responses were comparable to those induced by wild-type virus. Both ca recombinant viruses were shed in low titer for a short period of time. Each isolate retained the temperature-sensitive phenotype. However, there was evidence of genetic instability of the ca marker in that 7 of 24 isolates exhibited some loss of the ca property, and one isolate completely lost the capacity to produce plaques at 25 degrees C. The retention of a low level of residual reactogenicity in the A/Scotland/74 ca recombinant suggests that acquisition of the ca and temperature-sensitive phenotypes by a ca recombinant virus may not always bring about a satisfactory level of attenuation for individuals lacking hemagglutinin immunity.

Adaptation, Physiological↗
PubMed1979

Live Victoria/75-ts-1[E] influenza A virus vaccines in adult volunteers: role of hemagglutinin immunity in protection against illness and infection caused by influenza A virus.

To explore the relationship between neuraminidase immunity and the degree of attenuatíon of live influenza A virus vaccines, a comparative evaluation of three Victoria/75-ts-1[E] (Vic/75-ts-1[E]) recombinant viruses in serum hemagglutination-inhibiting-negative (titer, </=1:8) adult volunteers was performed. These three ts-1[E] viruses had a similar restriction of replication at 38 degrees C in vitro, and each possessed the two attenuating genes of the ts-1[E] donor strain (13). However, Vic/75-ts-1[E] recombinants 81 and 113 possessed both Vic/75 hemagglutinin (H3(75)) and Vic/75 neuraminidase (N2(75)), whereas Vic/75-ts-1[E] recombinant 67 had Vic/75 hemagglutinin but the N2(65) neuraminidase. Vic/75-ts-1[E] recombinant 67 was significantly more attenuated than Vic/75-ts-1[E] recombinants 81 and 113 in that fewer local and systemic signs and symptoms of illness were observed in those volunteers who received clone 67. These findings were consistent with our previous observations which suggested that the following two factors contribute to the attenuation of ts-1[E] vaccine strains in adults: (i) the attenuating effect of the two ts-1[E] genes and (ii) the neuraminidase immunity of the host. Vic/75-ts-1[E] recombinant clone 67 vaccinees developed an immunological response to the H3(75) hemagglutinin in the absence of a response to the N2(75) neuraminidase. To assess the role that anti-hemagglutinin immunity induced by an attenuated live virus vaccine plays in resistance to influenza A virus, vaccinees who received recombinant 67 were challenged with Vic/75 wild-type virus, and their responses were compared with those of Vic/75-ts-1[E] vaccinees who received recombinant 81 or 113. Each of the three groups of ts-1[E] vaccinees was significantly protected against illness induced by wild-type virus infection, although resistance was not complete. However, the clone 67 vaccinees were protected less against infection. The infection-permissive resistance induced by clone 67 resembled that previously described for inactivated neuraminidase-specific vaccines. These results suggested that a ts-1[E] recombinant that possessed the hemagglutinin of a new pandemic variant, the neuraminidase of the preceding subtype, and the two ts-1[E] ts genes would be satisfactorily attenuated for children and adults with neuraminidase immunity and could induce resistance to illness caused by the new pandemic wild-type influenza A virus.

Adolescent↗
PubMed1979

Relationship of genotype of recombinants of influenza A/Hong Kong/68-ts-1[E]virus used as live virus vaccines to virulence in humans.

Influenza A/Hong Kong/68-ts-1[E] virus is a temperature-sensitive mutant developed for use as a live virus vaccine (B. R. Murphy, E. G. Chalhub, S. R. Nusinoff, J. Kasel, and R. M. Chanock, J. Infect. Dis. 128:479--487, 1973). This virus and temperature-sensitive recombinants derived by mating it with A/Udorn/72, A/Georgia/74, or A/Victoria/75 wild-type virus have been administered to volunteers in clinical trials on the assumption that the ts-1[E] temperature-sensitive genetic lesions on a polymerase gene (P3) and on the nucleoprotein gene (NP) would determine a satisfactory and reproducible level of attentuation regardless of the genetic constitution of ts-1[E] recombinants at other loci (B. R. Murphy, D. D. Richman, S. B. Spring, and R. M. Chanock, Postgrad. Med. 52:381--388, 1976). In this paper, the parental origin of genes in the ts-1[E] recombinants was determined by using the technique of polyacrylamide gel electrophoresis of virion ribonucleic acid segments in the presence of a denaturing agent (urea). When tested in individuals who lacked immunity to hemagglutinin antigen, attenuation of the ts-1[E] recombinants appeared to correlate with inheritance of the ts-1[E] temperature-sensitive genes at the P3 and NP loci and with the level of preinfection neuraminidase immunity. There was no evidence that other genes from the ts-1[E] donor virus played a role in attenuation.

Genes, Viral↗