Vol 64, No 4 (2019)


Zika fever immunodiagnostics: overview of test systems

Zamarina Т.V., Khrapova N.P., Pimenova E.V., Khanani E.I., Viktorov D.V., Toporkov A.V.


The threat of rapid spread of Zika virus beyond endemic regions has given rise to more research in field of epidemiology and clinic, as well as to the search for Zika fiver new diagnostic and preventive tools. Between 2013 and 2017 in Russia 18 cases of infection transmission by travellers were reported. Fever Zika reference monitoring center in Volgograd Research Anti-Plague Institute (Volgograd, Russian Federation) provides counseling and methodological assistance on laboratory diagnosis and monitoring of Zika fever. In this regard, a literature review of commercial test systems for immunodiagnostics of this infection was performed. Currently, a number of test systems for solid-phase enzyme-linked immunoassay method (ELISA), immunochromatography and indirect immunofluorescent method (IIFT) have been developed for immunodiagnostics of Zika fever. Euroimmun Ltd. remains the only manufacturer that has access to detailed information on validation of the specificity of the produced diagnostic kits. Independent studies confirm that Euroimmun test systems have high specificity and high sensitivity, which is proved by the study of the material from various populations, including Europeans travelling to Zika virus endemic regions and people residing in these regions. A detailed overview of characteristics of Euroimmun test systems for immunodiagnostics of Zika fever allows us to conclude that there is a rationale for the use of these test systems for Russian Federation sanitary protection by identification of antibodies in patients, presumably infected with the Zika virus.

Problems of Virology. 2019;64(4):150-155
pages 150-155 views


Assessment of immunogenic activity of the cloned human rotavirus A WA strain

Latyshev O.E., Eliseeva O.V., Kostina L.V., Alekseev K.P., Khametova K.M., Altaeva E.G., Verkhovsky O.A., Aliper T.I., Grebennikova T.V.


Introduction. Rotovirus infection (RVI) caused by the dsRNA-containing virus from genus Rotavirus, Reoviridae family, belonging to group A (RVA), is the cause of severe diarrhea in human and other mammalian species. Vaccination is the most effective way to reduce the incidence of RVI. At present, the effectiveness of using gnotobiotic piglets as a universal model for reproducing human rotavirus infection and assessing the quality of RVI vaccine preparations has been experimentally proven.

Goals and objectives. Evaluation of immunogenic activity of the cloned RVA Wa strain in the newborn Vietnamese pot-bellied piglets trial.

Material and methods. Development of viral preparations of the cloned human Wa strain PBA, development of human RVA rVP6, ELISA, polymerase chain reaction with reverse transcription, immunization and experimental infection of newborn piglets.

Results. The article presents the results of the experiment on double immunization of newborn piglets with native virus preparations with the infection activity 5.5 lg TCID50/ml, 3 cm3 per dose, HRV with adjuvant 500 pig per dose and mock preparation (control group) followed with experimental inoculation of all animals with virulent virus strain Wa G1P[8] human RVA with infectious activity of 5.5 lg TCID50/ml in 5 cm3 dose. Development of clinical signs of disease and animal death were observed only in control group. RT-PCR system to detect RVA RNA in rectal swabs, samples of small intestine and peripheral lymph nodes was developed. ELISA based on obtained human RVA rVP6 was developed and results on RVA-specific IgG-antibodies in serum samples of experimental piglets are presented.

Conclusion. In the course of the research, a high immunogenic activity of the native and purified virus of the cloned Wa RVA strain Wa was established and the possibility of its use as the main component of the RVI vaccine was confirmed. The possibility of using conventional newborn pigs instead of gnotobiotic piglets as an experimental model was demonstrated.

Problems of Virology. 2019;64(4):156-164
pages 156-164 views

Cytokine-regulating activity of anti-virus preparation CelAgripus in Burkitt's lym-phoma stable B-cell lines

Narovlyansky A.N., Mezentseva M.V., Suetina I.A., Russu L.I., Ivanova A.M., Poloskov V.V., Izmest'eva A.V., Ospelnikova T.P., Sarymsakov A.A., Ershov F.I.


Introduction. Cytokines activated in response to immunosuppressive viral infections can directly or indirectly affect the neoplastic transformation of B cells. In this study, we studied a new substance designed to produce the antiviral drug CelAgrip (CA, CelAgripus), which exhibits interferon (IFN) and cytokine-inducing activity and, apparently, can be used as an activator of antiviral immunity.

Purpose - is to evaluate the cytokine-regulating effect of CA in Burkitt's lymphoma (LB) cell lines latently infected with the Epstein-Barr virus (EBV).

Objectives: to study the CA-induced expression of the cytokine genes IL-ip, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IFN-a, IFN -Y, IFN-p, IFN-A1, IFN-A2, IFN-A3, TNF-a in normal and EBV transformed LB cells.

Material and methods. Cell line: the human embryo fibroblasts (HEF), Namalva, Daudi, Raji, P3HR-1. Preparations: CA, gossypol-acetic acid (GAA), sodium carboxymethyl cellulose (Na-CMC). Methods: RT-PCR and methods for assessing cytotoxicity (MTT and Scepter 2.0 Merck cell counter).

Results. The effect of the CA preparation on the expression of IFN-Л, IL-1p, IL-6, IL-8 and IL-10 genes was revealed. Discussion. We observed the activation of gene expression of IFN-A, IL-1P, IL-6, IL-8 and suppression of IL-10 gene activity when treatment CA of LB cells.

Conclusion. The substance CA has new effects on the activation of the expression of a number of key cytokine genes in stable Burkitt lymphoma cell lines.

Problems of Virology. 2019;64(4):165-172
pages 165-172 views

Detection of specific antibodies of classes G and M to bovine leukemia virus in the blood serum

Gulyukin M.I., Kapustina O.V., Ezdakova I.Y., Valtsiferova S.V., Stepanova T.V., Anoyatbekov M.


Introduction. Bovine leukemia is a widespread infection worldwide, the causative agent of which is the bovine leukemia virus (BLV) in structural structure and functional features similar to human T-cell leukemia virus (HTLV-1 and HTLV-2) and It is considered as an actual medical and social problem. The study of the immune response in experimentally infected calves at an early stage of the disease development, synthesis of specific antibodies of classes G and M (IgG and IgM), diagnostic informativeness of detection of IgM in cattle leukemia is relevant and determines the purpose of this study.

Material and methods. Samples of blood and serum of cattle: animals experimentally infected with VLCRS, patients with cattle leukemia; control negative; specific to heterologous pathogens of cattle diseases. Indirect and sandwich variant enzyme-linked immunosorbent assay (ELISA); commercial ELISA kits (IDEXX, USA; Hema LLC, FKP Kursk Biofactory Firm BIOK, Russia) for the detection of specific IgG and IgM for BLV in the agar gel immunodiffusion reaction (RID).

Results. The humoral immune response develops shortly after infection — by 1-8 weeks. IgM are detected starting from the 3rd day, and IgG from the 7th day after infection. Up to 97% of coincidence of positive results in RID and indirect variant of TF ELISA based on monoclonal antibodies to cattle IgM (IgMbovine ) were found.

Discussion. The dynamics of the synthesis of antibodies of classes M and G to the glycoprotein gp 51 BLV has a dose- dependent wave-like character, is consistent with the levels of increase / decrease in the absolute and relative number of leukocytes / blood lymphocytes of infected calves.

Findings. Serum specific IgM was detected starting 3 days after infection with BLV. Early detection of IgM in serum of cattle can be used as an additional test for the detection of sick animals.

Problems of Virology. 2019;64(4):173-177
pages 173-177 views

Detection of bovine herpesvirus 4 DNA in cattle by realtime PCR

Nefedchenko A.V., Yuzhakov A.G., Koteneva S.V., Glotova T.I., Glotov A.G., Zaberezhny A.D.


Introduction. BoHV-4 is poorly understood. Data on the circulation of the virus among animals and its role in infectious diseases insufficient.

Aimes and goals. Development of real-time PCR for detecting the BoHV-4 and studying the frequency of its presence in samples from sick animals.

Material and methods. The nucleotide sequences of the glycoprotein L gene served as a target for amplification. The sequences of reference strains published in GenBank were used to analyze and design the primers. Studies were conducted in 3 regions of Western Siberia on 5 large dairy farms.

Results. 27.7% of samples contained the virus. The virus was present as a monoagent in nasal cavity of calves (80.0%), lungs (46.2%) and bronchial lymph nodes (38.5%) in pneumonia. In the cases of diarrhea the virus was detected in 20%, and in cows with gynecological pathology in 10.0%. In respiratory diseases of calves the virus was detected in association with BoHV-1 (21.6%) and BoCV (20.3%), and in gynecological pathology of cows with BVDV1 (6%).

Discussion. According to the phylogenetic analysis of 5 identified virus isolates, four belonged to the American branch and one to the European branch. The circulation of American strains occurred in the territory of the Republic of Kazakhstan (1), Tyumen (1) and Novosibirsk (2) regions, and the European - in the Novosibirsk region.

Conclusion. The search for viruses involved to the infectious pathology, as well as studying the genetic diversity of viruses circulating on a particular farm including imported from other countries, is relevant.

Problems of Virology. 2019;64(4):178-184
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Molecular-genetic characterization of Avian avulavirus 20 strains isolated from wild birds

Karamendin K.O., Sayatov M.K., Kydyrmanov A.I., Kasymbekov E.T., Asanova S.E., Daulbayeva K.D., Khan E.Y.


Introduction. Previously unknown paramyxovirus strains were isolated from wild birds in 2013-2014 in Kazakhstan and subsequently identified as representatives of the novel Avian avulavirus 20 species. The aims and tasks were molecular genetic characterization of novel avulaviruses and investigation of their phylogenetic relationships.

Material and methods. Embryonated chicken eggs were inoculated with cloacal and tracheal swabs from wild birds with subsequent virus isolation. The complete nucleotide sequences of viral genomes were obtained by massive parallel sequencing with subsequent bioinformatics processing.

Results. By initial infection of chicken embryos with samples from 179 wild birds belonging to the Anatidae, Laridae, Scolopacidae and Charadriidae families, 19 hemagglutinating agents were isolated, and five of them were identified as representatives of new viral species. The study of their sequenced genomes revealed their similarity in size, but there was a significant genetic variability within the species. 2,640 nucleotide substitutions were identified and 273 of them were non-synonymous, influencing the protein structure of viruses. It was shown that isolates Avian avulavirus 20/black-headed gull/ Balkhash/5844/2013 and Avian avulavirus 20 /great black-headed gull/Atyrau/5541/2013 were 86% and 95% respectively identical to the previously described reference strain, indicating a significant evolutionary divergence within species.

Discussion. The authors suggest the existence of two independent lineages - the Caspian, represented by the reference strain Aktau/5976 and Atyrau/5541, as well as the second, geographically significantly distant Balkhash lineage.

Conclusion. The study confirms the role of the birds of the Laridae family as the main reservoir of Avian avulavirus 20 in the avifauna that plays a key role in maintaining viruses of the genus Avulavirus in the biosphere and is a potential natural source for the emergence of new viral variants. Continuous surveillance of them in the wild is one of the most important tasks in ensuring the safety of the poultry industry.

Problems of Virology. 2019;64(4):185-192
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ASF virus replication features in the presence of recombinant proteins CD2v, pX69R and pE248R

Mazloum A., Zhukov I.U., Aronova E.B., Igolkin A.S., Vlasova N.N.


Introduction. African swine fever (ASF), sever hemorrhagic disease of swine caused by a large DNA virus of the Asfaviridae family.

Since there are no effective and safe vaccines against ASF yet, it is urgent to study the functions of its proteins, which is applicable by analyzing the features of ASF virus replication in the presence of recombinant proteins in vitro.

Purpose. To study the effect of ASFV recombinant proteins CD2v, pE248R and pX69R on the speed and level of reproduction of ASF virus in vitro. Thus, obtain the necessary knowledge to develop approaches for creating a vaccine against ASF.

Materials and methods. ASFV isolate Krasnodar 07/17 and strain ASF/ARRIAH/CV-1 were used. Cloning of X69R, EP402R, and E248R genes was performed in the pJET1.2 / blunt vector and pCI-neo in E. coli JM-109 cells, according to the manufacturer's manual. Localization of recombinant proteins in CV-1 cell line carried out by direct immunofluorescence reaction (DIF) using polyclonal antibodies conjugated to FITC.

The ASF virus reproduction level was assessed by hemadsorption reaction and qPCR kit (Central Research Institute of Epidemiology).

Results. Recombinant plasmids pCI-neo / E248R, pCI-neo / EP402R and pCI-neo / X69R were constructed.

The localization and the specificity of the obtained recombinant proteins CD2v, pE248R and pX69R was confirmed. It was established that these recombinant proteins induce the level of ASF virus reproduction on days 3-5 of the experiment by ~ 1.2-1.5 lgHADU50/cm3 in comparison with the negative control.

Discussion. The data obtained demonstrate the important role of CD2v, pX69R and pE248R proteins in the reproduction of the virus, since they significantly affect its level. The exact function of pX69R protein was not determined, however, in the experiments its positive effect on ASF virus reproduction was established, manifested in an increase in its reproduction level.

Conclusion. This methodology allows us to study the nature of the effect of proteins with unknown function on ASF virus replication.

Problems of Virology. 2019;64(4):193-200
pages 193-200 views

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