Population Sequence Analysis of a Simian Immunodeficiency Virus (32H Reisolate of SIV_mac251): A Virus Stock Used for International Vaccine Studies

Abstract

The virus structural genes gag and env (both gp120 and gp41 regions) of the 32H isolate of SIVmac251 were amplified using the polymerase chain reaction (PCR). The proviral template used in the PCR was DNA isolated from cells used to prepare several experimental SIV vaccines, which have been tested in simians, and a standard challenge stock of virus, which has been used in international collaborative studies. The PCR products were cloned and the nucleotide sequences of several clones were determined for each gene. From a comparison of the sequences obtained the predominant amino acid sequences of gag and env were predicted and the degree of sequence heterogeneity was determined. Conserved and more variable regions of each protein were identified. The gp120 region of env was more heterogeneous than gag or the transmembrane protein of env (gp41). Within gp120, sequence variability was concentrated to specific regions equivalent to the V1, V2, and, to a lesser extent, the C1 regions identified for human immunodeficiency virus type 1 (HIV-1). In contrast the region equivalent to the hypervariable "V3-loop" of HIV-1 was highly conserved. The implications of the data is discussed in relation to the ability of this virus stock to prepare effective vaccines against SIV.

Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants

“…It therefore appears that the SIVmac32H(pJ5) molecular clone represents a dominant virus within the 11/88 pool. The V3 region is identical to the consensus sequence and this region does not seem to be variable within SIV (Almond et al, 1992;Burns & Desrosiers, 1991). The V4 region is identical to that of SIVmac251 but surprisingly differs significantly from the 11/88 consensus.…”

“…The gag and env sequence variants in this uncloned pool have been examined by Almond et al (1992). Comparison of the env gene sequence of SIVmac32H(pJ5) to those of the variants in the 11/88 pool shows that the V1/V2 region is essentially identical to the consensus sequence (Almond et al, 1992) and identical to five of the six sequences derived from a macaque infected with the 11/88 pool (E. G. J. Hulskotte et al, personal communication) and quite different in this region from the other SIVmac clones (SIVmac251, SIVmac239, SIVmaclAll and SIVmac142). It therefore appears that the SIVmac32H(pJ5) molecular clone represents a dominant virus within the 11/88 pool.…”

Resistance to superinfection by a vigorously replicating, uncloned stock of simian immunodeficiency virus (SIVmac251) stimulates replication of a live attenuated virus vaccine (SIVmacC8)

“…The vaccine virus, SIVmacC8, is a virus clone with an attenuated phenotype in vivo Rud et al, 1994) due to a 12 bp in-frame deletion in nef and two further conservative amino acid changes. Macaques were inoculated intravenously with 5000 TCID 50 SIVmacC8 (9/90 pool; Almond et al, 1992), with an end-point titre of 10 4 TCID 50 ml 21 on C8166 cells . Wild-type challenge virus, SIVmac32H/L28, was prepared by inoculation of a cynomolgus macaque (L28) with 10 MID 50 11/88 stock of SIVmac251/32H reisolate (Stott et al, 1990;Cranage et al, 1992); the macaque was killed humanely after 9 weeks.…”

“…The serum dilution representing 75 % inhibition of p27 antigen production was recorded as the titre (Kent et al, 1994). The 11/88 virus stock of SIVmac251/ 32H (Almond et al, 1992) represented the challenge virus and SIVmacJ5 represented the vaccine virus (Stebbings et al, 2002). Virus isolation from Ficoll-purified PBMC was determined by co-culture with C8166 indicator cells up to 28 days, by syncytium formation and p27 antigen detection Almond et al, 1990).…”

Characterisation of a human homologue of a yeast cell division cycle gene, MCM6, located adjacent to the 5′ end of the lactase gene on chromosome 2q21

“…Taq polymerase was obtained from Advanced Biotechnologies or Promega and amplification performed in a Hybaid Omnigene apparatus. Products were cleaned using a 4B Sepharose column (Pharmacia) and the resulting column fraction was used directly as sequencing template [13].…”