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


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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.

About the authors

Mariia Igorevna Popkova

Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology

Author for correspondence.
Email: popmarig@mail.ru
ORCID iD: 0000-0001-5864-5862

PhD (Мedicine), Leading Researcher of Laboratory of Molecular Biology and Biotechnology

SPIN: 4485-2459

Russian Federation

Oleg Vladimirovich Utkin

Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology

Email: utkino2004@mail.ru
ORCID iD: 0000-0002-7571-525X

PhD (Biology), Head of Laboratory of Molecular Biology and Biotechnology

SPIN: 1174-6500

Russian Federation

Daria Alekseevna Bryzgalova

Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology

Email: moskvinadara7@gmail.com
ORCID iD: 0000-0002-6663-8440

Junior Researcher of Laboratory of Molecular Biology and Biotechnology

SPIN: 5626-3491

Russian Federation

Anna Olegovna Senatskaia

Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology

Email: seanna@bk.ru
ORCID iD: 0000-0003-2024-4283

Junior Researcher of Laboratory of Molecular Biology and Biotechnology

SPIN-код: 6891-2140

Russian Federation

Evgeniya Andreevna Soboleva

Privolzhsky Research Medical University

Email: Fullofcarrot@pimunn.ru
ORCID iD: 0000-0003-3591-9618

MD. Assistant of the Infectious Diseases department

 

Russian Federation

Nikolai Aleksandrovich Sakharnov

Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology

Email: saharnov@nniiem.ru
ORCID iD: 0000-0003-3965-2033

Researcher of Laboratory of Molecular Biology and Biotechnology

SPIN:8457-3501

Russian Federation

Elena Nikolaevna Filatova

Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology

Email: filatova@nniiem.ru
ORCID iD: 0000-0002-6683-7191

PhD (Biology), Leading Researcher of Laboratory of Molecular Biology and Biotechnology

SPIN: 1986-8147

Russian Federation

Ekaterina Aleksandrovna Kulova

“Tonus Krokha i Semeynaya STOMATOLOGIYA” (Nizhniy Novgorod)

Email: dr_kulova@mail.ru
ORCID iD: 0000-0002-5207-1164

PhD (Medicine), Infectious Disease Physician, Allergologist and Immunologist 

SPIN: 4331-6958

Russian Federation

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Copyright (c) 2022 Popkova M.I., Utkin O.V., Bryzgalova D.A., Senatskaia A.O., Soboleva E.A., Sakharnov N.A., Filatova E.N., Kulova E.A.

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