Takato Odagiri

Effect of Specific Immunity to Viral Neuraminidase on Subsequent Influenza Virus Infection in Man

It is now well established that influenza A virus has two distinct glycoprotein spikes, hemagglutinin and neuraminidase, on its surface (4). The specific antibody to hemagglutinin is known as the neutralizing antibody and immunity to influenza has long been correlated with the presence of hemagglutination-inhibition (HAI) antibody directed against hemagglutinin (2). In contrast, the specific antibody to viral neuraminidase is non-neutralizing, except when present in relatively high concentrations. However, it has also been demonstrated that anti-neuraminidase antibody (ANAB) partially inhibits the virus yield by its effect on the release of influenza virus from host cells in vitro (3, 7, 8). Also, although anti-hemagglutinin antibody showed a greater protective effect, it was emphasized that the presence of ANAB produced by infection or artificial immunization resulted in a marked decrease in pulmonary virus titers and lung lesions in mice and chickens (1, 6). Subsequently, Murphy et al (5) reported that ANAB reduced the severity of the clinical expression of influenza infection and contributed to the prevention of the spread of epidemics in man. Our results in this paper also confirm the effectiveness of anti-neuraminidase antibody on influenza virus infection in man. Forty-two paired sera were selected at random from 403 first grade students at the Daiichi Junior High School, Sendai, in December, 1977 (pre-epidemic phase) and March, 1978 (post-epidemic phase). Between those two dates, a Hong Kong influenza, A/Texas/1/77 (H3N2)-like virus outbreak occurred among these students. Serological diagnosis of each serum was performed by hemagglutination-inhibition (HAI) and neuraminidase-inhibition (NAI) tests on the A/Texas/1/77 strain as previously described (9). In the NAI test, the A/Texas/1/77 strain was solubilized with 0.1% Nonidet P 40 to prevent steric hinderance by any HAI antibodies that might also have been present in the test serum specimens (10). From the results of the HAI and NAI tests, 42 persons were classifiable, 15 as infection cases and 27 as non-infection cases. The 15 infection cases showed significant antibody rise to both hemagglutinin (H3) and neuraminidase (N2) (a fourfold or greater rise in the HAI titer and a threefold or greater rise in the NAI titer). In addition, evidence of infec-

Cold-Adapted Reassortants of Influenza A Virus in MDCK Cells

Attenuation of influenza viruses for the development of live vaccines can be accomplished by transferring attenuated genes from an attenuated donor virus to the new epidemic or pandemic strain by genetic reassortment (4). One donor virus, an influenza A/Ann Arbor/6/60 cold-adapted variant, was produced by serial passage in primary chick kidney (PCK) cells by gradually lowering the incubation temperature to 25 C which restricts the growth of wild type influenza viruses (1). During adaptation at the lower temperature, the A/Ann Arbor/6/60 virus acquired two phenotypic mutations, a cold-adapted (ca) property and a temperature-sensitive (ts) property at a shutoff temperature of 37-38 C (10). In studies of reassortant viruses derived from the A/Ann Arbor/6/60 donor strain, reassortants possessing the ca and ts properties lost their virulence for animals and humans (2, 3). However, A/Ann Arbor/6/60 donor virus genes which confer both the ca and ts properties and attenuation have not been unequivocally identified since most of the reassortants available for examination have multiple genes from the A/Ann Arbor/6/60 virus and possess both ca and ts properties as a linked phenotype. Recently, we isolated some limited gene reassortants of A/Ann Arbor/6/60 X A/Alaska/6/77 viruses and demostrated that the ca property can be segregated from the ts property at either 25 or 33 C, which is a permissive temperature for growth of influenza virus (7). Correlations between the reassortants gene constellations and their phenotypic markers, ca and ts, were also assessed. The present study explores the functions of both the ca and ts properties in the ca reassortants with respect to their virulence or attenuation in mice. Influenza A viruses used in this study were the A/Ann Arbor/6/60 (H2N2) ca variant as an attenuated donor strain for mice, the A/Alaska/6/77 (H3N2) wild type, and four ca reassortant viruses. The reassortants were produced in Madin-Darby canine kidney (MDCK) cells by mixed infection with the A/Ann Arbor/6/60 ca donor and the A/Alaska/6/77 viruses at either 25 or 33 C (7). Table 1 presents a summary of the gene constellations of the ca reassortants and their biological phenotypes as expressed in MDCK cells. Two (T66-1-1 and T 66-2-1) of the four reassortants exhibited ca and ts phenotypes while the others (T325-1-l

Biological characteristics of a cold-adapted influenza A virus mutation residing on a polymerase gene

SummaryThe biological function of a cold-adapted (ca) mutation residing on the PB2 gene of an influenza A/Ann Arbor/6/60 (A/AA/6/60) ca variant virus in the viral replication cycle at 25° C was studied. The viral polypeptide synthesis of A/AA/6/60 ca variant at 25° C was evident approximately 6 hours earlier than the wild type (wt) virus and yielded twice as many products. The quantitative analysis of viral complementary RNA (cRNA), synthesized in the presence of cycloheximide, revealed that A/AA/6/60 ca variant and a single gene reassortant that contains only the PB2 gene of the ca variant with remaining genes of the wt virus produced equal amount of cRNA at 25° and 33° C, which was an amount approximately four fold greater than the wt virus cRNA synthesized at 25° C. These results strongly suggest that the ca mutation residing on the PB2 gene of A/AA/6/60 ca variant affects the messenger RNA synthesis at 25° C in the primary transcription.

Serological evaluation of an influenza A virus cold-adapted reassortant live vaccine, CR-37 (H1N1), in Japanese adult volunteers.

A cold-adapted influenza A virus, CR-37 (H1N1), derived from genetic reassortment between A/Ann Arbor/6/60 (H2N2) cold-adapted variant virus and A/California/10/78 (H1N1) wild-type virus, was tested in Japanese adult volunteer. The CR-37 live virus preparation induced only low-grade clinical reactions in volunteers for the first 3-4 days after inoculation. Two vaccinees who did not show any antibody changes became febrile (over 38.0 degrees C). Skin tests using the vaccine preparation and uninfected allantoic fluid were performed, and indicated that one of these two vaccines was positive for the CR-37 vaccine preparation. A high proportion of the vaccinees whose sera had a haemagglutination-inhibition (HI) antibody titre against the vaccine strain of less than or equal to 64 before inoculation, seroconverted in both HI and neuraminidase-inhibition (NAI) antibody titrations, and only a few seroconverted in the titration of antibody against type-specific internal antigens. The serological examinations against heterotypic H1N1 variants indicated that the cold-adapted live influenza virus vaccine could induce a broad spectrum of HI antibody reactivity and immunity of long duration.

Antibody responses after repeated influenza A virus immunizations among schoolchildren in Japan

Antibody responses to influenza virus immunizations were examined among junior high school students. The students received two doses of a commercial split-product vaccine containing influenza A H1N1 during a 2-year period following the first appearance of H1N1 virus in the winter of 1977-78. In haemagglutination-inhibition (HI) tests, the students who had been infected with H1N1 virus in 1977-78 showed a better response and wider cross-reactivity to the drift strain than the students who had not experienced earlier H1N1 influenza infection. Neuraminidase-inhibition (NAI) antibody titres after immunization depended upon a history of natural infection with H1N1 virus, since students not previously infected showed no significant NAI antibody rise after immunization.

Antigenic and genetic characterization of H1 influenza viruses isolated from feral ducks and swine in Japan.

SummaryThe strains of H1N4 influenza A virus isolated from feral ducks in Japan in 1977–78 were compared to swine-origin H1N1 viruses antigenically and genetically. Homologous characteristics were found among the H1N4 isolates from feral ducks in hemagglutination-inhibition (HI) tests, viral RNA patterns on polyacrylamide gel electrophoresis and oligonucleotide mapping. Although the hemagglutinins of duck-origin viruses employed in this study were identified as H1, the viruses were distinguishable from A/New Jersey/8/76 (H1N1), A/duck/Alberta/35/76 (H1N1) and the virus isolated from swine in Japan in the cross HI test. Also, the viral RNA patterns of the duck- and swine-origin H1 viruses were found to be quite different, indicating that genetic reassortment of HA genes between them is unlikely. After H1N4 virus of duck-origin was intranasally inoculated into pigs, a brief period of virus recovery with no serological response was observed; whereas swine-origin H1N1 virus produced seroconversion in the pigs inoculated.