Dan C. DeBorde

Sequence comparison of wild-type and cold-adapted B/Ann Arbor/1/66 influenza virus genes

Consensus sequences for both wt and ca B/Ann Arbor/1/66 viral PB2, PB1, PA, NP, M, and NS genes were directly determined from vRNA using a combination of chemical and chain-termination sequencing methods. There were 105 sites of difference between the wt and ca sets of these six RNA genes. The differences resulted in 26 amino acid substitutions distributed over the six proteins. The sequence changes were compared to the sequences of other known influenza type B wt viruses to pinpoint those changes that were unique to the ca B/ann Arbor/1/66 virus. Of the 26 amino acid differences, only 11 were unique to the cold-adapted virus. These unique sites were distributed among five of the six genes. The NS protein had no amino acid substitutions. The sequence changes are discussed in terms of their probable mode of origin and selection, and in terms of their importance to the cold-adapted, temperature-sensitive, and attenuation phenotypes of ca B/AA/1/66 virus. The sequence and organization of the PB2 gene and predicted protein are also given. The PB2 gene was 2396 nucleotides long, and it encoded a predicted protein of 770 amino acids with a molecular weight of 88,035 Da for the wt virus and 88,072 Da for the ca virus. Both proteins were predominantly hydrophilic, and each had an overall charge of +24.5 at pH 7.0.

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 degrees C. None of the reassortants tested expressed the ca phenotype in embryonated eggs at 25 degrees 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 attenuation 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.

A mutation in the PA protein gene of cold-adapted B/Ann Arbor/1/66 influenza virus associated with reversion of temperature sensitivity and attenuated virulence

Reassortant SG3 inherits only the acidic polymerase (PA) protein gene from the cold-adapted B/AA/1/66 influenza virus (ca B/AA/1/66) and all remaining genes from a virulent, wild-type virus. This reassortant demonstrates attenuated virulence in ferrets and expresses a ts phenotype characteristic of the ca parent. During virulence evaluation of SG3, a virulent, non-ts revertant virus (designated SG3rFL) was isolated from the lungs of one ferret. In order to determine whether the reversion of SG3 resulted from mutation of the PA gene and/or as the result of extragenic supressor mutations, the revertant PA gene of SG3rFL was transferred to a reassortant (SG3r) inheriting only the revertant PA gene from SG3rFL and all remaining genes from SG3. Reassortant SG3r was non-ts and virulent, indicating that mutation of the PA gene was sufficient for the reversion of the ts and attenuation phenotypes expressed by SG3rFL. The nucleotide and predicted amino acid sequences of the SG3rFL PA gene were determined and compared to those of wt and ca B/AA/1/66. The predicted PA proteins of wt and ca B/AA/1/66 are known to differ by six amino acid substitutions including a valine to methionine substitution at residue 431. The PA proteins of ca B/AA/1/66 and SG3rFL were distinguished by only the single amino acid substitution of methionine to isoluecine also occurring at residue 431. Thus, the methionine residue was implicated in the attenuation of ca B/AA/1/66 and its reassortants. The hydropathic properties of valine, isoleucine, and methionine suggested that reversion involved the restoration of hydrophobic character at this site.

Characterization of an influenza a host range mutant

A mixed infection of primary chick kidney cells at 38 degrees 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 degrees. Both parents, however, grow well in either cell type at 33 degrees C. 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 degrees 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 degrees C, produced reassortants that always contained the A/California/10/78 NS gene instead of the CR43-clone 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.

Cold-adapted recombinants of influenza a virus in MDCK cells I. Development and characterization of A/Ann Arbor/6/60 × A/Alaska /6/77 recombinant viruses

Abstract Recombinant influenza viruses made at 25 and 33° in Madin-Darby canine kidney (MDCK) cells using the cold-adapted A/Ann Arbor/6/60 virus and the wild-type A/Alaska/6/77 virus were biologically and genetically analyzed. Eight recombinants were separated into two phenotypic groups based on cold-adapted (ca) and temperature-sensitive (ts) markers: ca and ts, ca and non-ts. The ca recombinants showed different degrees of cold adaptibility (DOCA) and different patterns of virus growth at 25°. All recombinants contained at most three genes from the cold variant A/Ann Arbor/6/60 virus (triple-gene recombinant) and most contained two or one gene from the cold variant parent (double-gene and single-gene recombinants, respectively). Further, the same three genes, RNA2, RNA3, and RNA5 (NA) were the only ca A/Ann Arbor/6/60 genes found in the various recombinants. Two clones contained all three A/Ann Arbor/6/60 genes and were both cold-adapted (ca) and temperature-sensitive (ts). All other recombinant clones were ca and non-ts, and contained RNA2 and/or RNA5 (NA). Each set of single-gene ca recombinants correlated with a different, but specific cold-adapted characteristic exhibited by their growth curves at 25°. Single-gene recombinants containing only the RNA2 of A/Ann Arbor/6/60 virus showed rapid growth early in infection and intermediate final virus yield (between the titer of virus yield for the ca A/Ann Arbor/6/60 virus and the wild-type A/Alaska/6/77 virus; while the single-gene recombinant containing only the RNA5 (NA) of A/Ann Arbor/6/60 virus showed slow growth early in infection, but a high final virus yield (equivalent to that of the ca A/Ann Arbor/6/60 parent). The double-gene recombinant containing both these genes showed both rapid growth early in infection and a high final virus yield. Thus, cold adaptation can be transferred to recombinant viruses by at least two independent genes each of which can confer the cold-adaptive property by its own pathway. The genetic basis for temperature sensitivity involves both RNA2 and RNA3, but remains ambiguous in the absence of a single-gene recombinant containing only RNA3 of the cold variant.

Nucleotide sequences of the PA and PB1 genes of B/Ann Arbor/1/66 virus: comparison with genes of B/Lee/40 and type A influenza viruses

The complete sequences of the PA and PB1 genome RNA segments of B/Ann Arbor/1/66 virus have been determined. The PA vRNA is 2308 bases long. Its complementary RNA has a single open reading frame of 2187 bases, capable of encoding a PA protein of 726 amino acids with a molecular weight of 83,175 Da. The predicted PA polypeptide has an overall net charge of -7.5 at pH 7.0. The PB1 vRNA is 2369 bases long. Its complementary RNA has a single open reading frame of 2277 bases, capable of encoding a PB1 protein of 752 amino acids with a molecular weight of 84,332 Da. The predicted PB1 polypeptide has an overall net charge of +18.5 at pH 7.0. Sequence homology comparisons of the PA and PB1 polypeptides from B/Ann Arbor/1/66 virus to the PA and PB1 polypeptides of type A influenza virus reveal respective homologies of approximately 38 and 60%. This high cross-type homology (61%) was previously reported for the PB1 protein of B/Lee/40 virus (Kemdirim et al., 1986). The cross-type homology for the PA protein is similar to that of other non-polymerase proteins, but is substantially lower than that seen for the PB1 protein. Thus, the high cross-type homology that exists for the PB1 gene does not appear to be a characteristic of all polymerase genes.

IN VITRO AND IN VIVO PROPERTIES OF AN INFLUENZA A HOST RANGE VIRUS

Publisher Summary CR43-3 is an influenza A cold-adapted reassortant virus that has a limited host range when compared to either parent virus or to its congenic reassortant, CR31-10. It grows well in primary chick kidney (PCK) cells but very little, if at all, in Madin-Darby canine kidney (MDCK) cells or in ferrets after intranasal inoculation. This host range virus has an aberrant NS gene that migrates differently from the NS gene of either parent on polyacrylamide and agarose gels used for genotyping the reassortants. Reassortants made with experiments using A/Cal/10/78 H1N1 virus and CR43-3 virus showed that the host range property and the aberrant NS gene co-segregated with one another. Sequence studies on this NS gene have demonstrated that the CR43-3 NS gene is a deletion mutant of the NS gene of the A/Alaska/6/77 wt parent. The deletion is 36 bases long and occurs from nucleotides 222–257 of the A/Alaska/6/77 NS gene in the region coding for NS1 protein.