Australia is aiming to end the transmission of HIV and HCV by 2030. National strategies for both viruses highlight the importance of timely high-quality data to improve public health responses. Such data could be obtained through a molecular epidemiology surveillance system, however for successful implementation a robust, time- and cost-effective method for long-range sequencing must be developed and/or verified.
Primers for two novel tiling PCR assays were designed to amplify partial HIV (5kb), or near-full-length HCV (9kb) genomes in overlapping segments of ~1kb. Assays were benchmarked using two panels of 90 infected and 6 uninfected samples from archived plasma as per the WHO HIVResNet Method Validation Framework. Panels included several viral subtypes/genotypes over a range of viral loads (HIV: 1,295-1,301,193 copies/mL, HCV: 121-12,813,253 IU/mL). HIV samples were additionally compared with prior diagnostic sequencing to determine the accuracy of the method.
The novel tiling PCR methods generated viral sequence in 90/90 samples for both viruses. Genome coverage was variable, with a mean sequence length of 5.2kb for HIV, and 7.5kb for HCV. A complete Core-E2 or Reverse Transcriptase was amplified in 74% of HCV and 93% of HIV samples respectively. There was good concordance with prior HIV diagnostic sequencing, with 98% of consensus sequences sharing >99% genetic identity to historical data. For both assays the time from sample to result was <24 hours, at a cost of <$100AUD per sample.
These novel tiling PCRs are an attractive option for routine long-range viral sequencing. However, regional sequencing failures may be a result of underlying viral diversity, and sequence dropout must be monitored. These methods will now undergo pilot deployment to determine their utility in a diagnostic setting. The sequences generated will also be used to assess clustering methods for use in molecular epidemiology surveillance systems for HIV and HCV.