Methodological approaches to differential detection of EBV1/EBV2 and HHV6A/HHV6B in saliva

Abstract

Epstein-Barr virus (EBV) and human herpesviruses 6A and 6B (HHV6A and HHV6B) are ubiquitous, infecting all social groups. In Russia, information on the genetic heterogeneity of EBV, even at the level of the main types (EBV1 and EBV2), as well as HHV6A and HHV6B, their prevalence and clinical significance are limited to isolated publications. Plasma and blood leukocytes were mainly investigated. Saliva is the main factor in the transmission and spread of EBV and HHV6A/B infections, an affordable, inexpensive, non-invasive material for detecting viral DNA. The aim of this work is to improve the methodological base for differential detection of HHV6A/HHV6B and the main types of EBV in saliva. The material of the study was the unstimulated mixed saliva of children aged 1-17 years with acute infectious mononucleosis (n=22) and no clinical symptoms of this disease (n=26), as well as conditionally healthy adults (n=9). Samples were collected once and dynamically (daily for 14 days). The quantitative detection of EBV and HHV6A/В DNA was carried out by the method of polymerase chain reaction (PCR) in real time. For differential detection of EBV1/EBV2 and HHV6A/HHV6В, an optimized one-round PCR variant with electrophoretic detection of amplification products in agarose gel was used. As a result, the detection rate of EBV, HHV6A/В and EBV+HHV6A/В DNA in acute infectious mononucleosis was 95%, 91% and 86%, among conventionally healthy children - 69%, 85% and 61.5%, respectively. It was found that among children of the Nizhny Novgorod region, only EBV1 and HHV6B predominate in saliva. According to the results of 14-day dynamic monitoring of saliva virus secretion in healthy virus carriers (adults and children), it was shown that a single EBV DNA study does not allow to reliably assess the infection of individuals or the intensity of EBV secretion. In this case, HHV6A/B is characterized by a more constant and uniform release. The methodological approach optimized in this work makes it possible to separately detect EBV1/EBV2 and HHV6A/HHV6B using a single laboratory protocol, and in combination with an additional stage of saliva sample preparation increases the diagnostic sensitivity of PCR analysis, minimizes the proportion of discordant and false negative results. Such an integrated approach can be applied for diagnostic, epidemiological and research purposes.

Keywords

• ebv1, ebv2, hhv6a, hhv6b, saliva, pcr, genotyping, differential detection.