Novel pH sensing semiconductor for point-of-care detection of HIV-1 viremia.

The timely detection of viremia in HIV-infected patients receiving antiviral treatment is key to ensuring effective therapy and preventing the emergence of drug resistance. In high HIV burden settings, the cost and complexity of diagnostics limit their availability. We have developed a novel complementary metal-oxide semiconductor (CMOS) chip based, pH-mediated, point-of-care HIV-1 viral load monitoring assay that simultaneously amplifies and detects HIV-1 RNA. A novel low-buffer HIV-1 pH-LAMP (loop-mediated isothermal amplification) assay was optimised and incorporated into a pH sensitive CMOS chip. Screening of 991 clinical samples (164 on the chip) yielded a sensitivity of 95% (in vitro) and 88.8% (on-chip) at >1000 RNA copies/reaction across a broad spectrum of HIV-1 viral clades. Median time to detection was 20.8 minutes in samples with >1000 copies RNA. The sensitivity, specificity and reproducibility are close to that required to produce a point-of-care device which would be of benefit in resource poor regions, and could be performed on an USB stick or similar low power device.

Detection of elephant endotheliotropic herpesvirus type 1 in asymptomatic elephants using TaqMan real-time PCR.

This study assessed the feasibility of identifying asymptomatic viral shedders using a novel TaqMan real-time PCR on trunk washes and swabs from the conjunctiva, palate and vulva of elephants. Six elephants from a UK collection were sampled weekly over a period of 11 weeks for this study. The herd prevalence of elephant endotheliotropic herpesvirus-1 (EEHV-1) was 100 per cent by PCR. The virus DNA was detected in all the sampling sites; however, the prevalence of virus DNA in the conjunctiva swabs was higher. In addition, Asian elephants from two continental European collections were sampled once and one animal tested positive on a trunk wash. The virus from this animal was phylogenetically typed as EEHV-1A based on 231 nucleotides of the terminase gene.

Experimental infection of common warthogs (Phacochoerus africanus) and bushpigs (Potamochoerus larvatus) with classical swine fever virus. I: Susceptibility and transmission.

An incursion of classical swine fever virus (CSFV) into the domestic pig population in South Africa, identified in 2005, raised the concern that infection might spread to wildlife species and be maintained in these hosts. This study sought to determine whether two wildlife Suidae species present in South Africa, the bushpig (Potamochoerus larvatus) and the common warthog (Phacochoerus africanus), could support productive CSFV infection. Both species could be infected with CSFV and transmitted infection to in-contact animals of the same species. Viral antigen and RNA genome were detected in blood/serum and animals that survived initial infection seroconverted approximately 10-14 days post-inoculation. Viral RNA remained detectable in nasal and saliva secretions for prolonged periods until monitoring ended at 42-44 days after initial challenge. These data suggest that both Suidae species could serve to spread circulating CSFV within wild populations, with implications for disease control.

Development of a DNA microarray for simultaneous detection and genotyping of lyssaviruses.

The lyssavirus genus of the Rhabdoviridae family of viruses includes 7 genotypes and several non-assigned isolates. The source of lyssavirus infections is diverse with numerous reservoirs in a wide geographical area. In many parts of the world reservoir hosts can potentially be carrying one of several lyssavirus strains and possibly new divergent isolates await discovery. Accordingly, generic detection methods are required to be able to detect and discriminate all lyssaviruses and identify new divergent isolates. Here we have allied a sequence-independent amplification method to microarray to enable simultaneous detection and identification of all lyssavirus genotypes. To do so, lyssavirus RNA was converted to cDNA and amplified in a random PCR, labelled and hybridized to probes on the microarray chip before being statistically analysed. The probes were to a 405 bp region of the relatively conserved N gene. Here we demonstrate a microarray capable of detecting each of the seven lyssavirus genotypes. The random amplification of lyssavirus RNA and the numerous oligonucleotide probes on the microarray chip also offer the potential to detect novel lyssaviruses.