Identification of human herpesvirus 6 variants A and B by primer-specific real-time PCR may help to revisit their respective role in pathology.
BACKGROUND
Human herpesvirus 6 (HHV-6) isolates are classified into two variants, termed HHV-6A and HHV-6B, on the basis of distinct genetic, antigenic and biological characteristics, but the specific pathogenicity of each variant remains poorly understood.


OBJECTIVE
To design a rapid, sensitive and specific real-time variant-specific PCR (VS-PCR) method to differentiate both variants in biological specimens.


METHODS
The VS-PCR was adapted from a real-time PCR assay, based on TaqMan technology, previously developed for the genome quantitation of both HHV-6 variants [Gautheret-Dejean A, Manichanh C, Thien-Ah-Koon F, Fillet AM, Mangeney N, Vidaud M, et al. Development of a real-time polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients. J Virol Meth 2002;100:27-35], a consensual reverse primer (Taq2) being changed into two variant-specific primers named H6A and H6B. This method was applied to a large set of biological specimens obtained in different pathological contexts.


RESULTS
The sensitivity threshold was about 10 copies/well for HHV-6A-specific PCR (PCR-A) and 1 copy/well for HHV-6B-specific PCR (PCR-B). Both assays showed a linear dynamic range from 10 to 100,000 copies of HHV-6 DNA. Regarding the specificity and the capacity of discrimination of each assay, one variant could be detected and identified in the presence of more than 1000 times higher concentrations of the other variant in virus mixtures. The comparison of the results obtained with this VS-PCR with those previously obtained with a classic PCR method allowed us to validate our new technique on a wide panel of biological samples, including numerous patients with severe HHV-6-related symptoms. The high prevalence of HHV-6B was confirmed in healthy individuals and immunocompromised patients. HHV-6A was identified in distinct samples from several patients exhibiting neurological disorders.


CONCLUSIONS
We developed a new VS-PCR assay, able to differentiate HHV-6A and HHV-6B in biological samples, even in the case of mixed infections. Our study confirms the wide prevalence of HHV-6B and highlights the potential greater neuropathogenic role of HHV-6A in immunocompromised patients and young infants.
Boutolleau
David
D
Laboratoire de Virologie, UPRES EA 2387, Groupe Hospitalier Pitié-Salpêtrière, Paris, France. david.boutolleau@psl.ap-hop-paris.fr
Duros
Caroline
C
Bonnafous
Pascale
P
Caïola
Delphine
D
Karras
Alexandre
A
Castro
Nathalie De
ND
Ouachée
Marie
M
Narcy
Philippe
P
Gueudin
Marie
M
Agut
Henri
H
Gautheret-Dejean
Agnès
A
D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005260:Female; D015654:Herpesvirus 6, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D006801:Humans; D007223:Infant; D007413:Intestinal Mucosa / Q000821:virology; D007962:Leukocytes / Q000821:virology; D008297:Male; D008875:Middle Aged; D009061:Mouth Mucosa / Q000821:virology; D016133:Polymerase Chain Reaction; D019349:Roseolovirus Infections / Q000473:pathology / Q000821:virology*; D012463:Saliva / Q000821:virology; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral