Human herpesvirus 8 transfusion transmission in Ghana, an endemic region of West Africa.

BACKGROUND: Human herpesvirus 8 (HHV-8) seroprevalence ranges between less than 5% in Europe and North America and 50% to 70% in sub-Saharan Africa. Evidence of HHV-8 transfusion transmission is only indirect. We conducted a serologic (anti-HHV-8) and molecular (HHV-8 DNA) study of samples from paired donor-immunocompetent recipients transfused with whole blood. STUDY DESIGN AND METHODS: Samples from 252 donor-recipient pairs were tested. Immunoglobulin G to HHV-8 was detected with enzyme immunoassays and confirmed with an in-house immunofluorescence assay. The cellular fraction from seroreactive donors and their recipients was tested for HHV-8 DNA. RESULTS: Anti-HHV-8 was positive (reactive in two or more assays) in 28 (11%) patients and 16 (6%) donors. Of 12 seronegative recipients (at risk of transmission) receiving seropositive blood, one very likely transmission was identified (8.3% confidence interval, 0%-23%). The donor blood contained HHV-8 DNA and his and four other donors' sequences clustered separately from recorded genotypes with a 97% bootstrap constituting a distinct genotype. CONCLUSIONS: HHV-8 is transmitted in Ghana but does not carry clinical consequences since most patients are immunocompetent. The clinical risk will increase with the availability of immunosuppressive drugs in sub-Saharan Africa. We propose that a new genotype (HHV-8-G for Ghana) be added to the current nomenclature.

Rhinovirus genome evolution during experimental human infection.

Human rhinoviruses (HRVs) evolve rapidly due in part to their error-prone RNA polymerase. Knowledge of the diversity of HRV populations emerging during the course of a natural infection is essential and represents a basis for the design of future potential vaccines and antiviral drugs. To evaluate HRV evolution in humans, nasal wash samples were collected daily for five days from 15 immunocompetent volunteers experimentally infected with a reference stock of HRV-39. In parallel, HeLa-OH cells were inoculated to compare HRV evolution in vitro. Nasal wash in vivo assessed by real-time PCR showed a viral load that peaked at 48-72 h. Ultra-deep sequencing was used to compare the low-frequency mutation populations present in the HRV-39 inoculum in two human subjects and one HeLa-OH supernatant collected 5 days post-infection. The analysis revealed hypervariable mutation locations in VP2, VP3, VP1, 2C and 3C genes and conserved regions in VP4, 2A, 2B, 3A, 3B and 3D genes. These results were confirmed by classical sequencing of additional samples, both from inoculated volunteers and independent cell infections, and suggest that HRV inter-host transmission is not associated with a strong bottleneck effect. A specific analysis of the VP1 capsid gene of 15 human cases confirmed the high mutation incidence in this capsid region, but not in the antiviral drug-binding pocket. We could also estimate a mutation frequency in vivo of 3.4x10(-4) mutations/nucleotides and 3.1x10(-4) over the entire ORF and VP1 gene, respectively. In vivo, HRV generate new variants rapidly during the course of an acute infection due to mutations that accumulate in hot spot regions located at the capsid level, as well as in 2C and 3C genes.

New molecular detection tools adapted to emerging rhinoviruses and enteroviruses.

Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5' untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.