Ancient variants of the Epstein–Barr virus (Herpesviridae, Lymphocryptovirus, HHV-4): hypotheses and facts

Introduction. People living with human immunodeficiency virus (HIV) are more likely to experience Epstein-Barr virus (EBV) reactivation and develop EBV-associated diseases. In Russia, the clinical significance of EBV genetic diversity in HIV-infected patients has not been assessed. The aim was to analyze a relationship between the major EBV types and LMP-1 genovariants with clinical and laboratory parameters in HIV-infected subjects. Materials and methods. Peripheral blood leukocytes were collected from 138 HIV(+) individuals aged 20-69 years. Association between EBV types, LMP-1 variants and subvariants with clinical and laboratory parameters (CD4+ T-lymphocyte count, HIV and EBV viral load, use and adherence to antiretroviral therapy (ART)), was performed using the principal component analysis method and the Mann-Whitney U test. Results. It has been shown that detectable HIV viral load increases in patients with low CD4+ T-lymphocyte counts, high EBV viral load, and low or no ART adherence. In general, infection with EBV-2 or the LMP-1 B95-8 alone resulted in lower EBV and HIV viral loads compared with other variants. Significant EBV-1 LMP-1 subvariants were identified, the biological potential of which was enabled in immunodeficiency state (CD4+ T-lymphocyte count ≤200 cells/μl). In “naive” patients, EBV-1/LMP-1(S309N)+HIV co-infection occurred with a higher, and EBV-1/LMP-1(E328Q)+HIV with the lowest HIV viral load. The highest EBV DNA concentrations were observed with EBV-1/LMP-1(Q334R)+HIV. In “experienced” patients, the level of EBV DNA was significantly lower when infected with EBV-1/LMP-1(E328Q)+HIV and, conversely, higher in case of detected EBV-1/LMP-1(H358P)+HIV. Conclusion. The features of clinical and laboratory parameters EBV+HIV co-infection caused by different EBV-1 LMP-1 subvariants (at the level of amino acid substitutions S309N, E328Q, Q334R, H358P) have been identified. It is necessary to study the functional role of such mutations in vitro and in vivo. In the context of assessing a clinical significance of EBV molecular genetic diversity, it is advisable to conduct larger-scale studies in different territories of Russia.

Assessment of the relationship between Epstein–Barr virus major types and genovariants as well as clinical and laboratory parameters in HIV-infected adults

Popkova,

Filatova,

Minaeva

et al. 2024

Molecular and genetic characteristics of Nizhny Novgorod Regionepstein–Barr virus isolates in children with infectious mononucleosis and healthy virus carriers

Попкова¹,

Уткин²,

Bryzgalova³

et al. 2023

Numerous foreign studies evidence about a pronounced heterogeneity of the Epstein-Barr virus (EBV) populationcirculating throughout the world. Various EBV classifications have been proposed. The attention of Russian researchers has focused on the study of the structural and functional polymorphism of the EBV LMP-1 oncogene in the context of oncological diseases in adulthood. The aim of the work was to assess EBV molecular genetic diversity in children with EBV infection in the Nizhny Novgorod region. There were analyzed blood leukocyte and saliva specimens from children aged 117 years with EBV-infectious mononucleosis (n = 69) and sex- and age-matched healthy virus carriers of (n = 32). A total of 178 EBV isolates were studied. For differential detection of EBV-1/EBV-2, we used an optimized one-round PCR variant with electrophoretic detection of amplification products in agarose gel. Nucleotide sequences of the LMP-1gene C-terminal fragment were determined by Sanger sequencing. Bioinformatics data analysis was performed using MEGA X software. As a result, during EBV-infectious mononucleosis, only the EBV-1 type was detected in all children, among healthy virus carriers EBV-1 (93.84.3%) and EBV-2 (6.24.3%). Based on the EBV classification according to R.H. Edwards et al. the strain affiliation of EBV isolates was determined. A total of five variants of LMP-1 were identified, namely B95-8, China 1, Med, NC and Alaskan, among which B95-8 dominated. The LMP-1 Med+, China 2, and China 3 variants were not found in any of the studied samples. It has been shown that the region of tandem repeats makes a significant contribution to the genetic diversity of the EBV population. A total of 100 amino acid substitutions were identified, of which the most common in the Nizhny Novgorod region EBV isolates are G212S, S366T, E328Q and S309N. A comparative analysis showed that strains, deletions, repeats, amino acid substitutions in EBV isolates from biological samples in children with infectious mononucleosis had common characteristics with a group of healthy virus carriers. In the active form of EBV infection, the appearance of structurally heterogeneous EBV sequences isolated from blood leukocytes and saliva from a single source was noted. Thus, for the first time, the molecular genetic diversity of EBV in children with various forms of EBV infection was assessed, which is the basis for the prospective development of clinical and epidemiological studies of EBV infection at a new methodological level.

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

Попкова

Уткин

Bryzgalova

et al. 2022

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.