We have improved the methods for the standard competitive growth assay of human immunodeficiency virus type 1 (HIV‐1). The cloning step for the mixed viral population and subsequent genotype analysis for arbitrary numbers of clones were excluded from procedures. Instead, a single nucleotide polymorphism (SNP)‐detection step was devised for the determination of viral populations. The quantitative SNP‐detection method can rapidly estimate the proportion of wild‐type and mutant populations with high reproducibility. Consequently, this method allows manipulation of many samples within a short period. Using this new competitive growth assay, replicative fitness of drug‐resistant HIV‐1 containing an M46I amino acid mutation in the protease was assessed in the presence or absence of indinavir. Without indinavir, replicative fitness of wild‐type HIV‐1 surpassed that of M46I‐mutated HIV‐1, and the fraction of mutated virus was reduced to about 10% at passage #9. In contrast, the fraction of M46I‐mutated virus increased to >90% at passage #5 in the presence of 26.4 nM indinavir. Almost identical results were obtained for L90M‐mutated HIV‐1 with or without saquinavir. HIV‐1 can survive under indinavir pressure by acquiring M46I mutation, as with acquisition of the L90M mutation under saquinavir pressure. However, these mutations damage viral replicative fitness under natural conditions without any drugs. Subtle differences between wild‐type and mutant viruses are thus easily detected using the improved method.

To monitor active HIV-1 transmission in Nagoya, Japan, we have been determining the subtypes of HIV-1 infecting therapy-naive individuals who have newly visited the Nagoya Medical Center since 1997. The subtypes were determined by phylogenetic analyses using the base sequences in three regions of the HIV-1 genes including gag p17, pol protease (PR) and reverse transcriptase (RT), and env C2V3. Almost all HIV-1 subtypes from 1997 to 2007 and 93% of all HIV-1 isolates in 2007 were subtype B. HIV-1 subtypes A, C, D, and F have been detected sporadically since 1997, almost all in Africans and South Americans. The first detected circulating recombinant form (CRF ) was CRF01_AE (11-year average annual detection rate, 7.7%). Only two cases of CRF02_AG were detected in 2006. A unique recombinant form (URF ) was first detected in 1998 and the total number of URFs reached 25 by year 2007 (average annual detection rate, 4.7%). Eleven of these 25 were detected from 2000 to 2005 and had subtypes AE/B/AE as determined by base sequencing of the gag p17, pol PR and RT, and env C2V3 genes (average annual detection rate, 3.7%). Unique subtype B has been detected in six cases since 2006. All 17 of these patients were Japanese. Other recombinant HIV-1s have been detected intermittently in eight cases since 1998. During the 11-year surveillance, most HIV-1s in Nagoya, Japan were of subtype B. We expect that subtype B HIV-1 will continue to predominate for the next several years. Active recombination between subtype B and CRF01_AE HIV-1 and its transmission were also shown.

We studied the emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) with major amino acid mutations in 402 therapy-naive patients at Nagoya Medical Center, Japan, between 1999 and 2006. The mean prevalence of drug-resistant HIV-1 was 6.7% (range, 2.3-10.0%; n = 27). HIV-1 variants with protease inhibitor (PI)-resistant mutations alone were most frequently found (3.5%, n = 14), followed by those with nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutations alone (1.7%, n = 7). Variants with nucleoside reverse transcriptase inhibitor (NRTI)-resistant mutations alone were sporadically found (1.0%, n = 4). A variant possessing both NRTI- and PI-resistant mutations was detected in one patient (0.2%) and a variant possessing both NNRTI- and PI-resistant mutations was identified in another patient (0.2%). In addition, another 17 variants (4.2%, n = 17) with only 215-revertant mutations (T215C/D/G/L/S) that can easily reconvert to the nucleoside analogue-associated mutation of T215Y/F were found. The 402 viruses were phylogenetically analyzed, revealing three independent clusters comprising PI-resistant variants with the M46I or L90M mutation, NNRTI-resistant variants with the K103N mutation, and 215-revertant variants. The PI-resistant and 215-revertant strains have been spreading since 2000, and the NNRTI-resistant strain has started spreading since 2003. The nature of the epidemic and information for successfully blocking the spread of drug-resistant HIV-1 were clarified in this study.