Problems of Virology

Hantavirus fevers (HF) are caused by pathogenic orthohantaviruses and are classified into two clinical forms: hemorrhagic fever with renal syndrome (HFRS), reported in Eurasia, and hantavirus pulmonary syndrome (HPS), reported in the Americas.

HF are characterised by an acute course with predominant involvement of internal organs: HFRS is mainly associated with kidney damage, while HPS is characterised by severe lung injury. Both diseases may also affect the central nervous and endocrine systems. In addition, hantavirus fevers are marked by severe complications during the acute phase and a prolonged recovery period.

Currently, there is neither a WHO-approved vaccine nor any effective etiotropic anti-hantavirus therapy. The development of antiviral treatment strategies focuses on preventing orthohantavirus entry into cells or suppressing their intracellular replication. One of the main directions in the search for effective anti-hantavirus therapy is the low-molecular-weight compounds development and testing, many of which have demonstrated pronounced antiviral activity against orthohantaviruses through direct or indirect effects on viral RNA polymerase function.

The review shows that new antiviral nucleoside compounds development and study has promising potential, as well as their combined use for effective etiotropic, pathogenetic and symptomatic treatment of hantavirus fevers.

Introduction. The current state of pig industry in Russia is characterized by the dominance of pig farms of III–IV compartments and a reduction in livestock in private subsidiary and peasant farms against the background of the spread of infectious diseases, mainly caused by viruses of African and classical swine fever (ASFV and CSFV), and porcine reproductive and respiratory syndrome (PRRSV). The classical method of virus cultivation in vitro is still relevant for the diagnosis of diseases, isolation of relevant strains, studying their properties, attenuation, and obtaining vaccine raw materials.

The aim of the study is to select of a universal biological model for in vitro replication of ASFV, CSFV, and PRRSV based on the analysis of permissiveness levels of various cell cultures (primary cell cultures, subcultures, and continuous cell lines).

Materials and methods. The study utilized known virulent, vaccine, and attenuated strains of ASFV, CSFV, and PRRSV. Five primary cell cultures, two subcultures and eleven transferable cell lines / sublines were tested. The degree of tolerance was calculated based on the results of semi-quantitative estimates using the real-time PCR method for five consecutive passages.

Results. For in vitro replication of both virulent and attenuated strains of ASFV, the optimal biological models were primary cell cultures of pBMC, PAM, PL, PS, ST, and PK. For the virulent strain of CSFV, the following were optimal: PAM, PL, ST, PK, PS, RRC, and PK-15 (ATCC). For the vaccine strain of CSFV, the optimal models were synovial membrane subcultures of lamb (MSO/ARRIAH) and kid (MSC/ARRIAH), as well as ST, PAM, PL, and PS. For virulent strains of PRRSV, both American and European types, the optimal model was the primary cell culture PAM. For the attenuated strain of American-type PRRSV, PAM, PL, and MA-104 were suitable, while for the attenuated strain of European-type PRRSV, PAM, PL, and Marc-145 were optimal.

Conclusion. It has been established that some primary cell cultures, such as PAM and PL, with a predominance of mononuclear cells are universal biological models for the in vitro reproduction of virulent and attenuated strains of ASFV, CSFV, and PRRSV.

Introduction. Enteroviruses (genus Enterovirus, family Picornaviridae) are one of the common causes of infectious diseases with neurological and dermatological manifestations in children. Surveillance of these viruses is carried out as part of the program Epidemiological surveillance and prevention of enterovirus (non-polio) infection for 2023–2027.

Objective. To study the type diversity of non-polio enteroviruses (NPEVs) isolated from clinical specimens and wastewater samples collected in Saint Petersburg and the Leningrad Region, and to perform molecular epidemiological characterization of Coxsackievirus B5 isolates.

Materials and methods. Stool samples were collected from resident children with clinical symptoms, healthy migrants under 5 years of age; wastewater samples were also included in the study. Enterovirus isolation was performed by inoculation of specimens onto RD, HEp-2, and L20B cell cultures. Molecular genetic methods included PCR, Sanger sequencing, nucleotide sequence analysis and phylogenetic analysis using the R programming language. Estimation of tMRCA and construction of nucleotide identity heatmaps were performed using TreeTime and SDTv 1.3 software, respectively.

Results. A total of 25 NPEV types were identified. At the species level, Enterovirus betacoxsackie predominated – 53/71 (74.6%; 95% CI: 62.9–84.2%), followed by Enterovirus alphacoxsackie – 17/71 (23.9%; 95% CI: 14.6–35.5%), and Enterovirus coxsackiepol – 1/71 (1.4%; 95% CI: 0.04–7.6%). In Saint Petersburg and the Leningrad Region, viruses belonging to Enterovirus betacoxsackie were most frequently detected: Coxsackievirus B5 (12.7%), ECHO 11 (11.3%), ECHO 13 (8.5%), ECHO 14 (8.5%), and ECHO 6 (8.5%). A notable prevalence of ECHO 25 (5.6%) and ECHO 29 (5.6%) was also observed. A comprehensive analysis of the phylogenetic tree, nucleotide identity heatmap, and tMRCA estimates showed that the CVB5 strains isolated from resident children in 2018 and from migrant children in 2021 belonged to two distinct genetic lineages, indicating multiple independent introductions of the virus into the Russian Federation.

Conclusion. The frequent detection of Enterovirus betacoxsackie in the studied collection of isolates and the identification of two genetic lineages of Coxsackievirus B5 confirm the complexity of its epidemiological dynamics and highlight the necessity of continuous surveillance for the timely detection of new genetic variants.

Introduction. Overcoming the immunosuppressive microenvironment of pancreatic cancer (PC) using oncolytic viruses appears to be a promising therapeutic approach. However, tumor cells are characterized by significant heterogeneity, including populations of resistant cells with high basal JAK/STAT signaling activity. One potential approach to overcoming this resistance is the use of JAK1/2 kinase inhibitors, such as ruxolitinib, the efficacy of which has already been demonstrated in several studies.

Objective. To study the effect of ruxolitinib (a JAK1/2 kinase inhibitor) on the replicative activity of certain oncolytic flaviviruses (YFV 17D) and alphaviruses (SINV K1487) in human pancreatic adenocarcinoma cell lines AsPC-1, BxPC-3, and Capan-2, which are characterized by high resistance to viral oncolysis.

Materials and methods. Human pancreatic adenocarcinoma cell lines: AsPC-1, BxPC-3, and Capan-2 from the collection of Laboratory of Tick-Borne Encephalitis Chumakov FSC R&D IBP RAS (Institute of Poliomyelitis). Viruses: yellow fever virus strain 17D and Sindbis virus strain K1487 (GenBank AC № PZ244961.1). Virus quantification was performed using the plaque assay on Vero cell culture and real-time PCR.

Results. In this study, we investigated the effect of ruxolitinib (a JAK1/2 kinase inhibitor) on the replicative activity of certain oncolytic flaviviruses (YFV 17D) and alphaviruses (SINV K1487) in virus-resistant human pancreatic adenocarcinoma cell lines AsPC-1, BxPC-3, and Capan-2. A significant dose-dependent increase in infectious titers (by 2–3 orders of magnitude) of YFV 17D and SINV K1487 was demonstrated in the studied cultures in the presence of various concentrations of ruxolitinib (0.1 μM, 0.5 μM, 1.0 μM, and 5.0 μM).

Conclusion. Enhanced replication of oncolytic flaviviruses and alphaviruses in the presence of ruxolitinib could significantly improve the efficacy of therapy for resistant and highly immunosuppressive pancreatic cancer. This combined virotherapy approach can activate powerful immunomediated cell death mechanisms even without an overt cytopathic effect in culture.