HIV subtype distribution varies in the United States depending on the population screened. Generally, the frequency of non-B subtypes has remained low in high-risk groups such as MSM and IDUs, as well as in non-IDU heterosexuals and blood donors [20
]. The frequency of non-B subtype infections was greater in populations enriched for immigrants from nonclade B epidemic countries or military personnel who became infected overseas [24
]. The frequency of non-B subtype in blood donors appears to be increasing at only a modest rate over the last 2 decades. Studies from the 1980s of seropositive donors and recipients of blood products found no nonclade B infections [23
], whereas studies of seropositive donors from the 1990s identified approximately 1% nonclade B infections [7
]; more recent studies of infected donors identified since 2000 reported rates of nonclade B infection in the 2%–5% range [3
], similar to the 2.5% rate documented here.
Because the samples analyzed here were from asymptomatic blood donors who denied knowledge of their HIV infection, their drug resistance mutations are likely attributed to resistant virus acquired from their sources of infection who are presumed to have been on antiviral therapies. The frequency of HIV drug resistance mutations among blood donors trended but was not significantly higher in incident vs prevalent infections (P
= .06). A stable frequency of drug resistance mutations also applied when resistance to the more recently introduced protease and the longer used RT inhibitors were analyzed separately. The frequency of transmitted drug resistance mutations appears to be stable among blood donors based on comparisons of rates among incident and prevalent infections in this study and in prior studies of HIV in US blood donors [3
], an observation in keeping with reported rates of transmitted drug resistance mutations in high-risk untreated groups [29
There are currently 7 HCV genotypes that are further subdivided into 83 subtypes (http://hcv.lanl.gov/content/sequence/HCV/classification/genotable.html
] that can vary widely in their geographic distribution (http://hcv.lanl.gov/components/sequence/HCV/geo/geo.comp
). In high-risk groups in the United States, subtypes 1a and 1b predominate, whereas in most other countries the majority of HCV infections belong to other subtypes. Because HCV transmissions in the United States occur mainly among young IDUs [34
] and reinfections can displace the original resident strain [35
], the distribution of HCV genotypes may rapidly change. Eight HCV subtypes were identified here with 1a (55%) and 1b (15%) predominating. The subtype distribution in prevalent cases was nearly identical to that reported for HCV-seropositive samples collected in 1988–1994 from a population reflecting that of the US [37
], which supports the validity of our sampling strategy. In this study, we document a higher frequency of subtype 3a (21% vs 9%) and a lower frequency of 1b (8% vs 18%) in incident vs prevalent donors in keeping with a recent analysis showing decreasing genotype 1 frequencies in younger vs older IDUs [38
HBV genotypes also vary greatly in their geographic distribution. Currently there are 8 genotypes that can be further subdivided into at least 24 subgenotypes defined as having >4% nucleotide difference [39
]. Twenty of these 24 subgenotypes were identified among the 193 sequenced HBV strains. When the frequencies of the subgenotypes were compared, A2 occurred more frequently in incident cases (67% vs 27%) while A1, B2, and B4 frequencies were higher in prevalent cases (8%–14% vs 0%–4%).
No HBV antiviral drug resistance mutations were observed. Drug-resistant HBV variants may be inefficient at transmission and/or establishment of a chronic infection or may be underrepresented in the pool of HBV being actively transmitted by sexual or parenteral routes. Neutralization escape mutations in the HBV envelope protein were heavily overrepresented in prevalent vs incident HBV infections (22% vs 6%). This observation is consistent with these mutations having been more strongly selected for in long-term infected donors in whom a strong antibody response develops than in very recent, anti-HBc–negative incident cases [40
This study of viral diversity has several limitations. First, the analysis was restricted to infections detected as NAT or HBsAg positive by current blood supply screening assays, most of which were also confirmed antibody positive. Consequently, infections by highly divergent variants that would not be detected by these assays would not be identified. Given efforts of test manufacturers and regulators to ensure that blood donor screening and confirmatory tests are sensitive to viral variants, we believe that this issue has limited impact on our findings. Second, a moderate proportion of donations selected for molecular analysis were not able to be characterized due to failure of long-amplicon PCR. These results were largely explained by absence of detectable nucleic acid or lower viral load in the PCR-refractory samples. It is also well recognized that all donor screening assays have low but significant rates of false positivity, especially if the classification is made only upon the routine testing results and does not include further testing of an independent sample such as the retrieved frozen plasma unit or follow-up donor sample. This is a particular problem with possible NAT yield samples (ie, seronegative and reactive by a single NAT assay), as evidenced by the high rate of incident cases with negative PCR results in this study (), many of which are likely due to false- NAT results. Third, we performed bulk sequencing of PCR products and therefore may not have detected cases of dual infection or minor populations of drug resistance or immune escape variants represented in viral quasi-species.
Overall, our analysis indicates that the HIV epidemic is relatively stable in terms of subtypes and transmitted drug resistance mutations. The HCV data provide evidence of differences in subtypes between incident and prevalent cases that may be stochastically driven by random founder effects and/or result from immigration of infected individuals to the United States. The HBV subgenotypes also showed evidence of change, possibly driven by similar epidemiological factors. The relative frequencies of different viral genetic clades and resistance patterns observed in our study population showed general concordance with those in populations with admitted high-risk behavior [44
]. Molecular characterization of recently transmitted blood-borne viruses detected through the large-scale routine NAT and antibody screening of blood donors is therefore a good complement to studies in highly exposed populations. As predominant viral strains change over time, sequence data generated by such blood donor molecular surveillance studies may be of use to adjust primers used in nucleic acid detection methods [46
], as well as the specificities of antibodies and antigens used in serologic assays [48
] in order to maintain the high sensitivity of blood donation screening assays.