Cold‐Adapted Reassortants of Influenza A Virus in MDCK Cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/101846/1/mim03574.pd

Development and characterization of cold-adapted viruses for use as live virus vaccines

Representative viruses from twelve RNA and two DNA virus genera have been successfully adapted to growth at sub-optimal temperature (cold-adapted). In almost every case, there was a correlation between acquisition of the cold-adaptation phenotype and loss of virulence in the normal host whether animal or man. Overall, the best method of cold adaptation to develop a live virus vaccine line appeared to be a stepwise lowering of the growth temperature allowing time for multiple lesions to occur and/or be selected. In addition, the starting virus should be a recent isolate not as yet adapted to a tissue culture host and the cold-adaptation process should then occur in a host heterologous to the virus' normal host. These viruses have been reviewed in the light of their cold-adaptation method and successful production of an attenuated line as virus vaccine candidate. Finally, detailed information is presented for the cold-adaptation process in influenza virus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25479/1/0000019.pd

The development of live attenuated cold-adapted influenza virus vaccine for humans

A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25°C (cold adaptation; ca). Through a series of stepwise passages, two stable mutants were obtained and designated as ‘Master’ strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attentuation, (2) non-transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell-mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups. Copyright © 1999 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35147/1/252_ftp.pd

Characterization of an influenza a host range mutant

A mixed infection of primary chick kidney cells at 38[deg] with A/Ann Arbor/6/60 cold adapted virus and A/Alaska/6/77 wt virus yielded a cold-reassortant virus, CR43-clone 3, which had a host range different from that of either parent. It does not produce detectable virus when grown in Madin-Darby canine kidney cells, while growing normally in primary chick kidney cells at 33[deg]. Both parents, however, grow well in either cell type at 33[deg]. Genotypic analysis of viral RNA electrophoresed in polyacrylamide gels has shown that CR43-clone 3 virus has an aberrant NS gene different from the NS gene of either parent virus. Reassortant viruses made between CR43-clone 3 virus and A/California/ 10/78 (H1N1) virus in primary chick kidney cells at 33[deg] showed the same host range restriction only if the NS gene was derived from the CR43-clone 3 virus. A mixed infection with these same parents, but in Madin-Darby canine kidney cells at 33[deg], produced reassortants that always contained the A/California/10/78 NS gene instead of the CR43clone 3 NS gene. Ferrets inoculated intranasally with the CR43-clone 3 reassortant do not become sick or infected, based on the lack of symptoms: no rhinitis, coryza, or fever; and no detectable virus recovered from nasopharyngeal swabs, turbinate, or lung tissues at 48 hr after infection. Thus, CR43-clone 3 virus contains an aberrant NS gene and manifests a restricted host range phenotype in Madin-Darby canine kidney cells and ferrets.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25081/1/0000512.pd

Genetics of cold-adapted B/Ann Arbor/1/66 influenza virus reassortants: the acidic polymerase (PA) protein gene confers temperature sensitivity and attenuated virulence

The cold-adapted B/Ann Arbor/1/66 influenza virus (ca B/AA/1/66) expresses temperature-sensitive (ts), cold-adapted (ca) and attenuation phenotypes. Reassortants which inherit one or more genes from ca B/AA/1/66 and all other genes from a virulent, wild-type influenza virus, B/Houston/1732/76, were produced and evaluated in order to identify the gene(s) responsible for the ts, ca and attenuation phenotypes. Only reassortants which inherited the PA gene from ca B/AA/1/66 expressed the ts phenotype in MDCK cells at 39 [deg]C. None of the reassortants tested expressed the ca phenotype in embryonated eggs at 25 [deg]C. The virulence of several reassortants was evaluated in ferrets. Inheritance of the PA gene from ca B/AA/1/66 was correlated with significant febrile attentuation and the apparent restriction of viral replication in the lower respiratory tract. Isolation of a virulent, non-ts revertant virus inheriting only the PA gene from ca B/AA/1/66 established a direct relationship between expression of the ts phenotype and attenuated virulence. Evidence for the contribution of at least one other gene from ca B/AA/1/66 to attenuation was observed. Thus, based on the methods used to determine reassortant gene compositions, these results indicate that the PA gene is primarily responsible for attenuation of ca B/AA/1/66 and its reassortants.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26616/1/0000157.pd

Resolution of a common RNA sequencing ambiguity by terminal deoxynucleotidyl transferase

One of the more common ambiguities which arise when using reverse transcriptase and dideoxynucleotide-chain termination to sequence RNA is a radioactive band of cDNA that extends over all four lanes on a sequencing gel. The adjacent sequences both above and below the band are not affected. Assuming then, that these ambiguities are caused by the termination of the DNA polymerase activity of reverse transcriptase for reasons other than the insertion of a dideoxynucleotide in the growing cDNA chain, terminal deoxynucleotidyl transferase should be able to continue to add deoxynucleotides to these products after the sequencing reaction is complete. It does, clearing the improperly terminated cDNA from these pileup sites, revealing the correct sequence. This technique can also be used to identify the template RNA's 5'-terminal base, although far more units of terminal deoxynucleotidyl transferase are required.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26058/1/0000132.pd

The cell receptor level is reduced during persistent infection with influenza C virus

 Persistent influenza C virus infection of MDCK cells perpetuates the viral genome in a cell-associated form. Typically, virus production remains at a low level over extended periods, in the absence of lytic effects of replication. In this study, we demonstrate that persistently infected cells are very restricted in permissiveness for superinfection. By reconstitution experiments, using bovine brain gangliosides as artificial receptors, the degree of super-infection was markedly increased. Analysis of cellular receptor expression revealed reduced concentrations of sialoglycoproteins in general and a limited presentation of the major receptor gp40. Cocultures of persistently infected and uninfected cells (the latter carrying normal receptor levels) initiated a transient rise in virus titers. This kind of induction of virus synthesis appeared to be mainly receptor-linked, since a receptor-deprived subline, MDCK II, did not give rise to a similar effect. Susceptibility of MDCK II cocultures could be partly restored by ganglioside treatment. In accordance to related virus systems, these findings on influenza C virus suggest a role of cell receptor concentrations in the regulation of long-term persistence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42459/1/705-142-6-1155_71421155.pd