Detection of influenza A H1N1 and H3N2 mutations conferring resistance to oseltamivir using rolling circle amplification.

In the event of an influenza pandemic, the use of oseltamivir (OTV) will undoubtedly increase and therefore it is more likely that OTV-resistant influenza strains will also arise. OTV-resistance genotyping using sequence-based testing on viruses isolated in cell culture is time consuming and less likely to detect the low-level presence of drug-resistant virus populations. We have developed a novel rolling circle amplification (RCA) method to achieve the sensitive detection of OTV-resistant viruses from clinical specimens. Using artificially created templates, RCA could detect the presence of OTV-resistant mutations (N2: 119V, 292K, N1: 274Y) even if the population carrying the mutations was <1% of the total. By applying RCA to clinical samples, we identified the emergence of the 274Y mutation in one OTV-treated patient, as well as in seven individuals who were treatment-naïve (confirming community transmission of 274Y-containing resistant influenza A H1N1). These results were further confirmed by neuraminidase region sequencing. In conclusion, RCA technology can provide rapid (<24 h), high-throughput diagnosis of OTV resistance mutations with a high specificity and sensitivity.

Sensitive detection of the K103N non-nucleoside reverse transcriptase inhibitor resistance mutation in treatment-naïve HIV-1 infected individuals by rolling circle amplification.

Primary or transmitted antiretroviral drug resistance mutations pose a significant obstacle for optimizing antiviral treatment. When present at low-levels, resistance mutations are less likely to be detected by standard genotyping assays. This study utilizes a novel rolling circle amplification (RCA) method using padlock probes to achieve the sensitive, specific and low-level detection of the NNRTI resistance K103N from 59 HIV+ treatment-naïve patients from Beijing, China. Using standard genotyping methods, primary drug resistance mutations to either protease or RT inhibitors were found in 25% (15/59) of patients attending hospital clinics in Beijing. Among these 15 patients with antiretroviral (ARV) resistance mutations, standard sequence-based genotyping revealed that most (10/15) had the 103N. Using a highly sensitive RCA assay, 5 more patients among the 59 treatment-naïve cohort were found to have the 103N, but at low-levels, leading to an overall rate of 103N at 25.4% (15/59) in this population. The high prevalence of the 103N suggests that baseline resistance testing should be performed before treatment in this population. Importantly, the new RCA technology allows large-scale, sensitive detection of drug resistance mutations, including detection of minority populations with minimal equipment requirement.

Genetic and functional analysis of R5X4 human immunodeficiency virus type 1 envelope glycoproteins derived from two individuals homozygous for the CCR5delta32 allele.

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) isolated from two HIV-1-infected CCR5delta32 homozygotes. Envs from both subjects used CCR5 and CXCR4 for entry into transfected cells. Most R5X4 Envs were lymphocyte-tropic and used CXCR4 exclusively for entry into peripheral blood mononuclear cells (PBMC), but a subset was dually lymphocyte- and macrophage-tropic and used either CCR5 or CXCR4 for entry into PBMC and monocyte-derived macrophages. The persistence of CCR5-using HIV-1 in two CCR5delta32 homozygotes suggests the conserved CCR5 binding domain of Env is highly stable and provides new mechanistic insights important for HIV-1 transmission and persistence.