Comparison of broth microdilution and agar dilution for susceptibility testing of Neisseria gonorrhoeae.

Broth microdilution was compared with agar dilution to test the susceptibilities of 170 clinical isolates of Neisseria gonorrhoeae to several antimicrobial agents. Percents agreement between the two methods within 1 doubling dilution were 98.8% for enoxacin, 94.3% for penicillin G, 93.5% for spectinomycin, and 98.4% for ceftriaxone.

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.

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.

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.