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Background Hepatitis E virus (HEV) is a major causative agent of acute hepatitis worldwide, prompting continuous HEV vaccine efforts. Vaccine development is hampered by the lack of convenient animal models susceptible to infection with different HEV genotypes. We produced recombinant open reading frame 2 protein (pORF2; p551) of HEV genotype (GT) 3 and assessed its immunogenicity and protectivity against HEV challenge in common marmosets (Callithrix jacchus, CM). Methods p551 with consensus sequence corresponding to amino acid residues 110–660 of HEV GT3 pORF2 was expressed in E. coli and purified by affinity chromatography. CMs were immunized intramuscularly with 20 μg of p551 VLPs with alum adjuvant (n = 4) or adjuvant alone (n = 2) at weeks 0, 3, 7 and 19. At week 27, p551-immunized and control animals were challenged with HEV GT1 or GT3 and thereafter longitudinally screened for markers of liver function, anti-HEV IgG and HEV RNA in feces and sera. Results Purified p551 formed VLPs with particle size of 27.71 ± 2.42 nm. Two immunizations with p551 induced anti-HEV IgG mean titer of 1:1810. Immunized CMs challenged with homologous and heterologous HEV genotype did not develop HEV infection during the follow-up. Control CMs infected with both HEV GT1 and GT3 demonstrated signs of HEV infection with virus shedding and elevation of the levels of liver enzymes. High levels of anti-HEV IgG persisted in vaccinated CMs and control CMs that resolved HEV infection, for up to two years post challenge. Conclusions CMs are shown to be a convenient laboratory animal model susceptible to infection with HEV GT1 and GT3. Immunization with HEV GT3 ORF2/p551 triggers potent anti-HEV antibody response protecting CMs from homologous and heterologous HEV challenge. This advances p551 in VLPs as a prototype vaccine against HEV.

The factors influencing hepatitis E virus (HEV) circulation remain largely unexplored. We investigated HEV seroprevalence in humans and the prevalence of infection in farm pigs and rabbits in different regions of the Russian Federation, as well as the genetic diversity and population dynamics of HEV. Anti-HEV IgG antibody detection rates in the general population increase significantly with age, from 1.5% in children and adolescents under 20 years old to 4.8% in adults aged between 20 and 59 years old, to 16.7% in people aged 60 years and older. HEV seroprevalence varies between regions, with the highest rate observed in Belgorod Region (16.4% compared with the national average of 4.6%), which also has the country’s highest pig population. When compared with the archival data, both increases and declines in HEV seroprevalence have been observed within the last 10 years, depending on the study region. Virus shedding has been detected in 19 out of the 21 pig farms surveyed. On one farm, circulation of the same viral strain for five years was documented. All human and animal strains belonged to the HEV-3 genotype, with its clade 2 sequences being predominant in pigs. Sequences from patients, pigs, and sewage from pig farms clustered together, suggesting a zoonotic infection in humans and possible environmental contamination. The HEV-3 population size predicted using SkyGrid reconstruction demonstrated exponential growth in the 1970s–1990s, with a subsequent decline followed by a short rise around the year 2010, the pattern being similar to the dynamics of the pig population in the country. The HEV-3 reproduction number (Re) predicted using Birth-Death Skyline analysis has fluctuated around 1 over the past 20 years in Russia, but is 10 times higher in Belgorod Region. In conclusion, HEV-3 circulation varies both geographically and temporally, even within a single country. The possible factors contributing to this variability are largely related to the circulation of the virus among farm pigs.

Chronic HCV infection and associated liver cancer impose a heavy burden on the healthcare system. Direct acting antivirals eliminate HCV, unless it is drug resistant, and partially reverse liver disease, but they cannot cure HCV-related cancer. Possible remedy could be a multi-component immunotherapeutic vaccine targeting both HCV-infected and malignant cells, also those not infected with HCV. To meet this need we developed a two-component DNA vaccine based on the highly conserved core protein of HCV to target HCV-infected cells, and a renowned tumor associated antigen telomerase reverse transcriptase (TERT) based on the rat TERT, to target malignant cells. Their synthetic genes were expression-optimized, and HCV core was truncated after aa 152 (Core152opt) to delete the domain interfering with immunogenicity. Core152opt and TERT DNA were highly immunogenic in BALB/c mice, inducing IFN-γ/IL-2/TNF-α response of CD4+ and CD8+ T cells. Also, DNA-immunization with TERT enhanced cellular immune response against luciferase encoded by a co-delivered plasmid (Luc DNA). However, DNA-immunization with Core152opt and TERT mix resulted in abrogation of immune response against both components. A loss of bioluminescent signal after co-delivery of TERT and Luc DNA into mice indicated that TERT affects the in vivo expression of luciferase directed by the immediate early cytomegalovirus and interferon-β promoters. Panel of mutant TERT variants was created and tested for their expression effects. TERT with deleted N-terminal nucleoli localization signal and mutations abrogating telomerase activity still suppressed the IFN-β driven Luc expression, while the inactivated reverse transcriptase domain of TERT and its analogue, enzymatically active HIV-1 reverse transcriptase, exerted only weak suppressive effects, implying that suppression relied on the presence of the full-length/nearly full-length TERT, but not its enzymatic activity. The effect(s) could be due to interference of the ectopically expressed xenogeneic rat TERT with biogenesis of mRNA, ribosomes and protein translation in murine cells, affecting the expression of immunogens. HCV core can aggravate this effect, leading to early apoptosis of co-expressing cells, preventing the induction of immune response.

Therapeutic DNA-vaccination against drug-resistant HIV-1 may hinder emergence and spread of drug-resistant HIV-1, allowing for longer successful antiretroviral treatment (ART) up-to relief of ART. We designed DNA-vaccines against drug-resistant HIV-1 based on consensus clade A integrase (IN) resistant to raltegravir: IN_in_r1 (L74M/E92Q/V151I/N155H/G163R) or IN_in_r2 (E138K/G140S/Q148K) carrying D64V abrogating IN activity. INs, overexpressed in mammalian cells from synthetic genes, were assessed for stability, route of proteolytic degradation, and ability to induce oxidative stress. Both were found safe in immunotoxicity tests in mice, with no inherent carcinogenicity: their expression did not enhance tumorigenic or metastatic potential of adenocarcinoma 4T1 cells. DNA-immunization of mice with INs induced potent multicytokine T-cell response mainly against aa 209–239, and moderate IgG response cross-recognizing diverse IN variants. DNA-immunization with IN_in_r1 protected 60% of mice from challenge with 4Tlluc2 cells expressing non-mutated IN, while DNA-immunization with IN_in_r2 protected only 20% of mice, although tumor cells expressed IN matching the immunogen. Tumor size inversely correlated with IN-specific IFN-γ/IL-2 T-cell response. IN-expressing tumors displayed compromised metastatic activity restricted to lungs with reduced metastases size. Protective potential of IN immunogens relied on their immunogenicity for CD8+ T-cells, dependent on proteasomal processing and low level of oxidative stress.

Highlights • There were multiple importations and several patterns of spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Russia. • There are no signs of direct virus importations from China. • SARS-CoV-2 variants circulating in Russia mostly contain seven common mutations., Objectives The outbreak of coronavirus disease 2019 (COVID-19) started in December 2019 in China and then spread worldwide over the following months, involving 188 countries. The objective of this study was to determine the molecular epidemiology of the COVID-19 outbreak in Russia. Methods In this study, two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains were isolated and genetically characterized. A phylogenetic analysis of all available Russian sequences was then performed and these were compared to the epidemiological data on COVID-19 incidence to evaluate the molecular epidemiology and pattern of virus spread in the territory of Russia. Results and conclusions Whole genome analysis of the isolates obtained in this study and 216 others isolated in Russia revealed a set of seven common mutations when compared to the original Wuhan virus, including amino acid substitutions in spike protein S and nucleoprotein N, possibly affecting their properties. Phylogenetic analysis of all Russian sequences and 8717 sequences from other countries showed multiple importations of the virus into Russia, local circulation, and several patterns of virus spread.

Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence in patients with chronic HBV infection. Understanding the mechanisms underlying stability and persistence of HBV cccDNA in hepatocytes is critical for developing novel therapeutics and managing chronic hepatitis B. In this study, we observed an unexpected increase in HBV cccDNA levels upon suppression of transcription by de novo DNA methyltransferase DNMT3A and uncovered additional mechanisms potentially involved in HBV cccDNA maintenance. Methods: HBV-expressing cell lines were transfected with a DNMT3A-expressing plasmid. Real-time PCR and HBsAg assays were used to assess the HBV replication rate. Cell cycling was analyzed by fluorescent cell sorting. CRISPR/Cas9 was utilized to abrogate expression of APOBEC3A and APOBEC3B. Alterations in the expression of target genes were measured by real-time PCR. Results: Similar to previous studies, HBV replication induced DNMT3A expression, which in turn, led to reduced HBV transcription but elevated HBV cccDNA levels (4- to 6-fold increase). Increased levels of HBV cccDNA were not related to cell cycling, as DNMT3A accelerated proliferation of infected cells and could not contribute to HBV cccDNA expansion by arresting cells in a quiescent state. At the same time, DNMT3A suppressed transcription of innate immunity factors including cytidine deaminases APOBEC3A and APOBEC3B. CRISPR/Cas9-mediated silencing of APOBEC3A and APOBEC3B transcription had minor effects on HBV transcription, but significantly increased HBV cccDNA levels, similar to DNMT3A. In an attempt to further analyze the detrimental effects of HBV and DNMT3A on infected cells, we visualized &gamma, H2AX foci and demonstrated that HBV inflicts and DNMT3A aggravates DNA damage, possibly by downregulating DNA damage response factors. Additionally, suppression of HBV replication by DNMT3A may be related to reduced ATM/ATR expression. Conclusion: Formation and maintenance of HBV cccDNA pools may be partially suppressed by the baseline expression of host inhibitory factors including APOBEC3A and APOBEC3B. HBV inflicts DNA damage both directly and by inducing DNMT3A expression.

Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contrast, transcriptional activation of ATM or ATR using CRISPRa significantly increased HBV replication. We conclude that ATM and ATR are essential for HBV replication. Furthermore, DNA damage leading to the activation of ATM and ATR transcription, results in the reactivation of HBV replication.

The hepatitis C virus (HCV) has remarkable genetic diversity and exists as eight genotypes (1 to 8) with distinct geographic distributions. No complete genome sequence of HCV subtype 2b (HCV-2b) is available from Latin American countries, and the factors underlying its emergence and spread within the continent remain unknown. The present study was conducted to determine the first full-length genomic sequences of HCV-2b isolates from Latin America and reconstruct the spatial and temporal diversification of this subtype in Brazil. Nearly complete HCV-2b genomes isolated from two Brazilian patients were obtained by direct sequencing of long PCR fragments and analyzed together with reference sequences using the Bayesian coalescent and phylogeographic framework approaches. The two HCV-2b genomes were 9318 nucleotides (nt) in length (nt 37&ndash, 9354). Interestingly, the long RT-PCR technique was able to detect co-circulation of viral variants that contained an in-frame deletion of 2022 nt encompassing E1, E2, and p7 proteins. Spatiotemporal reconstruction analyses suggest that HCV-2b had a single introduction in Brazil during the early 1980s, displaying an epidemic history characterized by a low and virtually constant population size until the present time. These results coincide with epidemiological data in Brazil and may explain the low national prevalence of this subtype.

Wild ducks are known to be able to carry avian influenza viruses over long distances and infect domestic ducks, which in their turn infect domestic chickens. Therefore, prevention of virus transmission between ducks and chickens is important to control the spread of avian influenza. Here we used a low pathogenic wild aquatic bird virus A/duck/Moscow/4182/2010 (H5N3) for prevention of highly pathogenic avian influenza virus (HPAIV) transmission between ducks and chickens. We first confirmed that the ducks orally infected with H5N1 HPAIV A/chicken/Kurgan/3/2005 excreted the virus in feces. All chickens that were in contact with the infected ducks became sick, excreted the virus, and died. However, the ducks orally inoculated with 104 50% tissue culture infective doses of A/duck/Moscow/4182/2010 and challenged 14 to 90 days later with H5N1 HPAIV did not excrete the challenge virus. All contact chickens survived and did not excrete the virus. Our results suggest that low pathogenic virus of wild aquatic birds can be used for prevention of transmission of H5N1 viruses between ducks and chickens.