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

Abstract

EpsteinBarr virus (EBV) and human herpesviruses 6A and 6B (HHV6A and HHV6B) are ubiquitous, infecting representatives of all social groups, starting from early childhood. Currently, 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 mainly by foreign data. In Russia, there are not so many publications devoted to this issue. In this case, the objects of study are mainly plasma and leukocytes of peripheral blood, scrapings or swabs from the oropharynx are used much less often. Saliva is the main factor in the transmission and spread of EBV and HHV6A/B infections. Saliva testing is an affordable, inexpensive, and non-invasive method for detecting viral DNA. The purpose 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 for the study was unstimulated mixed saliva of children aged 117 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 detection and quantification of EBV DNA and HHV6A/B DNA was performed using real-time PCR. For the differential determination of EBV1/EBV2 and HHV6A/HHV6B, an optimized one-round PCR variant with electrophoretic detection of amplification products in an agarose gel was used. Statistical data processing was carried out using the R programming language and the RStudio environment. According to the results of our own research, the frequency of detection of EBV, HHV6A/B and EBV+HHV6A/B DNA in acute infectious mononucleosis was 95, 91 and 86%, and among conventionally healthy children 69, 85 and 61.5%, respectively. It was found that among the examined children of the Nizhny Novgorod Region, EBV1 and HHV6B prevail in the viral population, which is consistent with existing ideas about their geographical distribution in the adjacent territories. EBV2 and HHV6A were not detected in any of the examined saliva samples. 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 according to 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