Human immunodeficiency virus type 1 (HIV-1) is classified into different phylogenetic subtypes, with subtype C representing more than half of the novel infections globally. However, there are relatively few subtype C envelopes available for study. We amplified 18 unique env genes from 13 patients who were infected with subtype C HIV-1 in six African countries and in Scotland to create replication-competent viruses. These envelopes are phylogenetically diverse across the subtype C spectrum, and have on average more N-linked glycosylation sites and slightly longer variable loops than previously described C envelopes. We found that CCR3 coreceptor usage is less prevalent in subtype C than in subtype B viruses, and these envelopes have varied sensitivity to neutralization. The subtype C chimeric viruses generated in this study will be useful for evaluating the breadth of neutralizing antibodies and other entry inhibitors.Subtype C human immunodeficiency virus type 1 (HIV-1) accounts for .50 % of all new infections worldwide, and is the most prevalent subtype in sub-Saharan Africa, South Asia and Brazil (UNAIDS/WHO, 2009). Although useful panels of subtype C primary isolates (Brown et al., 2005;Bures et al., 2002;Fernandez-Garcia et al., 2009) and pseudoviruses (Gray et al., 2006;Li et al., 2006b) have been developed, more need to be characterized due to the diversity of the envelope gene, env, within this subtype. For instance, the standard reference panel of subtype C envelopes (Envs) included isolates from only two African countries, Zambia and South Africa (Li et al., 2006b). Here, we analysed Envs from infections acquired in six African countries and from Scotland, UK. Our replicationcompetent viruses with new Envs represent a diverse range of contemporary subtype C isolates for which we have determined coreceptor usage and sensitivity to neutralizing antibodies.Plasma samples were obtained from 13 HIV-1 subtype C infected, treatment naive individuals diagnosed in Scotland through the National Surveillance System (Yirrell et al., 2004). The undisclosed patients' origins and viral loads are presented in Supplementary Table S1 (available in JGV Online). Nine samples were derived from individuals infected in Africa and four from individuals infected in Scotland. The subtype was determined by gag sequence typing. Viral envs (gp160 and gp120) were amplified directly from plasma viral RNA extracted with QIAamp Viral RNA Mini kit (Qiagen) using primers shown in Supplementary Table S2 (available in JGV Online) and Titan One Tube RT-PCR kit (Roche Applied Sciences).Env genes were cloned into pHXB2Denv (gp120) or the C2 cassette (gp160), which enables production of replicationcompetent chimeric viruses (McKeating et al., 1996;Zheng & Daniels, 2001). Therefore, unlike the previous panels of subtype C envs, those in this panel are uniquely able to spread infection. Viruses were generated by transfection of 293T cells and clones that yielded infectious progeny [¢1000 focus forming units (f.f.u.) ml 21 on NP2/CD4/ CCR5 or NP2...