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Biomedical subjects

B R Murphy

Publications and source records attributed to B R Murphy.

At least 235 records · Page 13Linked to original sources
PubMed1983

Evaluation of the A/Seal/Mass/1/80 virus in squirrel monkeys.

An influenza A virus isolated from seals [A/Seal/Mass/1/80 (H7N7)] and an isolate of this virus obtained from a human conjunctiva were evaluated for replication and virulence in squirrel monkeys. When the seal virus was administered intratracheally, it replicated in lungs and nasopharynges and induced illness almost to the same extent that a human influenza A virus [A/Udorn/72 (H3N2)] did. In one monkey that died of pneumonia, the seal virus was recovered from spleen, liver, and muscle as well as lung. After conjunctival administration in monkeys, the seal virus replicated to a peak titer in the conjunctivae 30-fold greater than that attained by the human virus, but this difference was not statistically significant. In contrast, the seal virus replicated less well than the human virus in the tracheae and nasopharynges when administered by the conjunctival route. These results indicate that the seal virus can replicate efficiently in primates, that it can spread systemically, and that it might differ from human virus in being able to replicate slightly better in primate conjunctival tissue.

Animals↗
PubMed1982

Reassortant virus derived from avian and human influenza A viruses is attenuated and immunogenic in monkeys.

An influenza A reassortant virus that contained the hemagglutinin and neuraminidase genes of a virulent human virus, A/Udorn/72 (H3N2), and the six other influenza A virus genome segments from an avirulent avian virus, A/Mallard/New York/6750/78 (H2N2), was evaluated for its level of replication is squirrel monkeys and hamsters. In monkeys, the reassortant virus was as attenuated and as restricted in its level of replication in the upper and lower respiratory tract as its avian influenza virus parent. Nonetheless, infection with the reassortant induced significant resistant to challenge with virulent human influenza virus. In hamsters, the reassortant virus replicated to a level intermediate between that of its parents. These findings suggest that the nonsurface antigen genes of the avian parental virus are the primary determinants of restriction of replication of the reassortant virus in monkeys. Attenuation of the reassortant virus for primates is achieved by inefficient functioning of the avian influenza genes in primate cells, while antigenic specificity of the human influenza virus is provided by the neuraminidase and hemagglutinin genes derived from the human virus. This approach could lead to the development of a live influenza A virus vaccine that is attenuated for man if the avian influenza genes are similarly restricted in human cells.

Animals↗
PubMed1982

Bronchial reactivity in experimental infections with influenza virus.

In an attempt to develop an objective, quantitative test to assess the reactogenicity of candidates for attenuated influenza virus vaccines, the clinical responses and bronchial reactivity to methacholine were assessed in 36 normal volunteers lacking antibody to both hemagglutinin and neuraminidase (doubly seronegative) who were inoculated with either wild-type or temperature-sensitive attenuated influenza A (H1N1) virus by intranasal droplets. Five of six volunteers infected with wild-type virus developed systemic and/or febrile illness that lasted for a mean of five days. Nineteen of 30 persons who received attenuated virus developed infection without febrile or systemic illness. In the six recipients of wild-type virus, bronchial reactivity appeared to increase after inoculation and persist for four weeks. This trend was not observed in infected or noninfected vaccinees. Analysis of variance provided suggestive (but not significant) evidence that the different trends in bronchial reactivity manifested by the wild-type virus and vaccine groups were distinct (P = 0.075).

Airway Resistance↗
PubMed1982

Evaluation of a phenotypic revertant of the A/Alaska/77-ts-1A2 reassortant virus in hamsters and in seronegative adult volunteers: further evidence that the temperature-sensitive phenotype is responsible for attenuation of ts-1A2 reassortant viruses.

In a previous study, a seronegative child to whom attenuated A/Alaska/77-ts-1A2 virus was administered (37 degrees C shutoff temperature for plaque formation) shed virus with an altered temperature-sensitive (ts) phenotype (40 degrees C shutoff temperature) (Murphy et al., Ann. N.Y. Acad. Sci. 354:172-182, 1980; Tolpin et al., Virology 112:505-517, 1981). This ts+ virus (FV1319) was evaluated for its level of replication in hamsters and for its virulence for humans. In hamsters, FV1319 ts+ virus replicated to the same level in the nasal turbinates as that of which the A/Alaska/77 wild-type virus replicated, but its replication in the lungs was reduced 40-fold. In contrast, the A/Alaska/77-ts-1A2 reassortant achieved a titer in hamster nasal turbinates that was significantly lower (P less than 0.005) than those achieved by the wild-type and the FV1319 viruses; the A/Alaska/77-ts-1A2 reassortant was not recoverable from the lungs. In seronegative adult volunteers, the pattern of replication of the FV1319 virus was similar to that of the A/Alaska/77 wild-type virus. The illness induced by the FV1319 ts+ virus was also similar to that caused by the wild-type virus. In contrast, the A/Alaska/77-ts-1A2 reassortant was satisfactorily attenuated in adult volunteers. These results suggest that attenuation of the A/Alaska/77-ts-1A2 reassortant virus in humans is a function of the ts phenotype: loss of this phenotype restored virulence. The ability of the A/Alaska/77-ts-1A2 reassortant to lose its ts phenotype and regain virulence during growth in a permissive host limits the usefulness of the ts-1A2 reassortants as vaccine viruses for humans.

Animals↗
PubMed1982

Secretory and systemic immunological response in children infected with live attenuated influenza A virus vaccines.

An enzyme-linked immunosorbent assay was used to measure isotype-specific antibody to purified hemagglutinin (HA) of influenza A virus, using serum and nasal-wash specimens from young children undergoing primary infection with live cold-adapted influenza A/Alaska/77 (H3N2) or A/Hong Kong/77 (H1N1) candidate vaccine virus. The serum antibody response followed the pattern expected for a primary viral infection. Each of 17 vaccinated children had a serum immunoglobulin G (IgG) HA antibody response, 16 had an IgM antibody response, and 13 had an IgA antibody response. Nasal-wash HA antibody was detected in the IgA, IgM, and IgG isotypes. Of the 17 vaccinated children, 14 had an IgA response, 13 had an IgM response, and 9 had an IgG response. Most of the IgA and IgM HA antibody was actively secreted locally, whereas only some of the IgG HA antibody could be shown to be actively secreted into the respiratory tract. There was a good correlation between the level of nasal-wash antibodies measured by the HA-specific IgA enzyme-linked immunosorbent assay and by a plaque neutralization assay. These data indicate that intranasal vaccination of susceptible children with live, attenuated, cold-adapted influenza A viruses efficiently stimulates both systemic and local antibody responses.

Antibody Formation↗
PubMed1982

Production and level of genetic stability of an influenza A virus temperature-sensitive mutant containing two genes with ts mutations.

Temperature-sensitive (ts) reassortant vaccine strains derived from the A/Udorn/72 ts-1A2 donor virus were not sufficiently stable genetically in humans. We therefore sought to produce a new, more stable donor virus. We had previously identified a stable ts virus with a ts P3 gene and in the current study identified another relatively stable single-lesion ts virus with a ts mutation in the NP gene. A new ts reassortant virus was constructed by mating these two single mutants and by isolating three reassortant progeny, clones 20, 53, and 55, that contained both a ts P3 and a ts NP gene. These reassortant progeny possessed a 37 to 38 degrees C shutoff temperature and were as restricted in their replication in hamster lungs as the A/Udorn/72 ts-1A2 virus. All isolates from the lungs and nasal turbinates of hamsters were temperature sensitive. An in vitro stress test was used to determine whether the new ts P3 ts NP reassortant virus would undergo loss of its ts phenotype after replication at semipermissive temperature. Clone 20 and 55 reassortants underwent progressive loss of their ts phenotype in vitro, although at a rate slightly less than that of the A/Udorn/72 ts-1A2 virus. The level of genetic stability after replication in vivo was assessed in cyclophosphamide-treated hamsters in which virus replication continued for up to 15 days. Again, both the A/Udorn/72 ts-1A2 and the new ts P3 ts NP reassortant clone 55 manifested a progressive loss of temperature sensitivity after 7 days of replication. Clone 55 virus lost temperature sensitivity significantly less rapidly than the A/Udorn/72 ts-1A2 virus. These results indicated that, although the new ts P3 ts NP reassortant virus was more stable than the A/Udorn/72 ts-1A2 virus, it nevertheless underwent progressive loss of temperature sensitivity after replication in vitro and in vivo. Therefore, it does not appear to be a satisfactory donor virus. This experience plus that gained earlier with other ts mutants of influenza A virus suggest that influenza A virus mutants that rely solely upon their ts phenotype for attenuation are unlikely to exhibit the phenotypic stability required of a vaccine virus. Other genetic techniques are needed to produce more stable influenza A virus strains.

Animals↗
PubMed1982

Virulence of avian influenza A viruses for squirrel monkeys.

Ten serologically distinct avian influenza A viruses were administered to squirrel monkeys and hamsters to compare their replication and virulence with those of human influenza A virus, A/Udorn/307/72 (H3N2). In squirrel monkeys, the 10 avian influenza A viruses exhibited a spectrum of replication and virulence. The levels of virus replication and clinical response were closely correlated. Two viruses, A/Mallard/NY/6874/78 (H3N2) and A/Pintail/Alb/121/79 (H7N8), resembled the human virus in their level and duration of replication and in their virulence. At the other end of the spectrum, five avian viruses were restricted by 100- to 10,000-fold in replication in the upper and lower respiratory tract and were clearly attenuated compared with the human influenza virus. In hamsters, the 10 viruses exhibited a spectrum of replication in the nasal turbinates, ranging from viruses that replicated as efficiently as the human virus to those that were 8,000- fold restricted. Since several avian viruses were closely related serologically to human influenza viruses, studies were done to confirm the avian nature of these isolates. Each of the avian viruses plaqued efficiently at 42 degrees C, a restrictive temperature for replication of human influenza A viruses. Avian strains that had replicated either very efficiently or very poorly in squirrel monkeys still grew to high titer in the intestinal tracts of ducks, a tropism characteristic of avian, but not mammalian, influenza viruses. These observations indicate that some avian influenza A viruses grow well and cause disease in a primate host, whereas other avian viruses are very restricted in this host. These findings also provide a basis for determining the gene or genes involved in the restriction of replication that is observed with the attenuated avian viruses. Application of such information may allow the preparation of reassortant viruses derived from a virulent human influenza virus and an attenuated avian virus for possible use in a live attenuated vaccine for prevention of influenza in humans.

Animals↗
PubMed1982

Live temperature-sensitive equine influenza virus vaccine: generation of the virus and efficacy in hamsters.

Temperature-sensitive (ts) reassortants of an equine influenza virus, subtype A-1, were produced by mating a human influenza ts donor virus with an equine influenza A/Cornell/16/74 wild-type virus and by isolating a ts reassortant virus possessing the equine hemagglutinin and neuraminidase surface antigens. Two equine its reassortant clones, 8B1 and 71A1, were produced which had an in vitro shutoff temperature for plaque formation of 38 and 37 C, respectively. The human ts donor virus had ts mutation(s) on the polymerase 3 (P3) and nucleoprotein genes so that a ts equine reassortant virus could have either or both of these ts genes. It was found by complementation analysis that reassortant clone 8B1 had a ts lesion on the P3 gene and clone 71A1 had ts lesions on the nucleoprotein and P3 genes. An analysis of the parental origin of the genes in each ts equine reassortant virus indicated that clone 8B1 received 6 of its 8 genes and clone 71A1, 3 of its 8 from the equine parent virus, the remainder genes being from the human ts donor virus. The growth of both clones was restricted in the lungs of hamsters, but similar to that of the equine wild-type virus in the nasal turbinates. Each virus isolate obtained from the hamster's lungs or nasal turbinates retained the ts phenotype. These findings form the basis for further evaluation of the equine ts reassortant viruses for their level of attenuation and immunogenicity in horses.

Animals↗
PubMed1981

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↗
PubMed1981

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↗
PubMed1981

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↗
PubMed1981

Specific anti-influenza virus antibody production in vitro by human peripheral blood mononuclear cells.

Cultures of human peripheral blood mononuclear cells stimulated in vitro with influenza virus were found to produce anti-influenza virus antibody. An enzyme-linked immunosorbent assay was used to measure cumulative antibody secretion into culture supernatants. Optimal antibody production was obtained with 2 x 10(6) peripheral blood mononuclear cells cultured in flat-bottomed wells for 12 days in 2 ml of RPMI 1640 medium supplemented with 10% fetal calf serum, antibiotics, and 4 mM L-glutamine. Antibody could be detected in cultures stimulated with live A/Hong Kong/68 (H3N2), purified formalin-inactivated A/Aichi/68 (H3N2), or live B/Hong Kong/68 influenza virus. Antibody of both IgG and IgM isotypes was produced. Under optimum conditions, the secreted IgG antibody was found to be directed preferentially at antigens on the stimulating virus. Antibody production was shown to require the cooperative interaction of B and T cells. Poisson analysis of B cell precursor frequency showed that approximately 1 in 40,000 B cells could be induced to produce IgG anti-influenza virus antibody in virus-stimulated cultures. Further investigation of this in vitro response should allow dissection of the cellular mechanisms involved in the regulation of antibody production in man.

Antibodies, Viral↗
PubMed1981

In vitro production of anti-influenza virus antibody after intranasal inoculation with cold-adapted influenza virus.

We have studied the production of anti-influenza virus antibody in vitro by peripheral blood mononuclear cells (PBMC) obtained from 7 normal volunteers at various times after intranasal inoculation with cold-adapted A/Alaska/6/77 [H3N2] influenza virus. Antibody released into culture supernatants was assayed by a 2-step enzyme-linked immunosorbent assay (ELISA). Cells obtained 6 days after intranasal inoculation spontaneously released both IgG and IgA anti-influenza antibody; this antibody production occurred within 24 hr, was specific for the virus used to inoculate the volunteers, and was inhibitable by cycloheximide. When day 6 cells were cultured in vitro for 12 days with the polyclonal activator pokeweed mitogen (PWM), no increase in the amount of antibody above that released in the absence of PWM was seen. In contrast, cells obtained 27 days after inoculation made no spontaneous antibody. In addition, cultures of these day 27 cells with PWM resulted in the production of large amounts of IgG antibody but relatively little IgA anti-virus antibody. Thus, after a mucosal influenza virus infection, several subpopulations of functionally different cells sequentially appear in the peripheral circulation: an initial population of cells secreting IgG and IgA antibody spontaneously, followed by a 2nd population of cells secreting IgG antibody when stimulated with PWM.

Administration, Intranasal↗