[Adaptation of african swine fever virus (Asfarviridae: Asfivirus)to growth in the continuous culture PPK-66b cells by the method of accelerated passaging].

INTRODUCTION: African swine fever virus (ASF) is a large, enveloped virus with an icosahedral capsid morphology and a double-stranded DNA genome ranging in size from 170 to 190 kb. The replication cycle proceeds in two phases, the early phase lasting 4-6 hours and the late 8-20 hours after infection. The adaptation of the ASF virus to growth in continuous cell lines makes efficient and reliable genetic analysis and more accurate interpretation of its results. OBJECTIVE: Adaptation of a new isolate of the ASF virus to growth in a continuous cell line by the method of accelerated passages and preliminary genetic analysis of the resulting strain. MATERIALS AND METHODS: For virus isolation and passaging of the ASF virus, a porcine leukocyte cell culture (PL) and continuous cell cultures of porcine origin (ST, PK, PPK-66b) were used with Eagle MEM and HLA essential media with 10% porcine or fetal serum. RESULTS: The article presents data on the isolation and analysis of the changes in the reproductive properties of a new African swine fever (ASF) virus isolate in the process of adaptation to growth in a continuous piglet kidney cell culture clone b (PPK-66b). The current state of the problem of cultivation of the ASF virus, the features of its reproduction, and the basis of the genetic differentiation of its isolates are described in detail. Understanding the uniqueness of the nature of the ASF virus determined the approaches to the processes of its cultivation and adaptation. In this regard, the results of studies of cultural properties, and analysis of the nucleotide sequence of 6 genes of the new isolate, as well as phylogenetic analysis of these genes with already known strains and isolates of the ASF virus are presented. CONCLUSION: A new strain obtained in the process of cell adaptation of ASVF/Znaury/PPK-23 ASF virus by the accelerated passaging method reaches a high level of reproduction in 72 hours with an accumulation titer of 7.07 lg HAdE50/cm3. Primary genetic analysis allowed to establish the main phylogenetic relationships of the newly isolated strain with previously known variants of the current ASF panzootic.

[Antigenic and immunogenic activity of virus-like particles based on rabbit hemorrhagic disease virus (Caliciviridae: Lagovirus) genotypes GI1 and GI2 recombinant major capsid proteins].

INTRODUCTION: Rabbit hemorrhagic disease is an acute highly contagious infection associated with two genotypes of pathogenic Lagovirus. Antibodies to major capsid protein (Vp60) are protective. The aim of the work ‒ is an evaluation of antigenic and immunogenic activity of virus-like particles (VLPs) based on recombinant major capsid proteins of both genotypes of rabbit hemorrhagic disease virus (RHDV) (recVP60-GI1 and recVP60-GI2). MATERIALS AND METHODS: Baculovirus-expressed VLPs were evaluated using electron microscopy and administered to clinically healthy 1.53 month old rabbits in a dose of 50 g. Rabbits were challenged with 103 LD50 of virulent strains Voronezhsky-87 and Tula 21 days post immunization. Serum samples were tested for the presence of RHDV-specific antibodies. RESULTS: VLPs with hemagglutination activity forming VLP 3040 nm in size were obtained in Hi-5 cell culture. Specific antibody titers in rabbits measured by ELISA were 1 : 200 to 1 : 800 on 21th day post immunization with VLPs. Immunogenic activity of recVP60-GI1 VLPs was 90 and 40%, while it was 30 and 100% for recVP60-GI2 VLPs after the challenge with RHDV genotypes 1 and 2 respectively. The immunogenicity of two VLPs in mixture reached 100%. DISCUSSION: VLPs possess hemagglutinating, antigenic and immunogenic activity, suggesting their use as components in substances designed for RHDV specific prophylaxis in rabbits. Results of the control challenge experiment demonstrated the need to include the antigens from both RHDV genotypes in the vaccine. CONCLUSION: Recombinant proteins recVP60-GI1 and recVP60-GI2 form VLPs that possess hemagglutinating an antigenic activity, and provide 90100% level of protection for animals challenged with RHDV GI1 and GI2 virulent strains.

[Problems of specific prevention of African swine fever].

This review presents the current state of the problem of development and application of the specific prevention of African swine fever (ASF) with a brief description of its etiology and pathogenesis. The unique nature of the ASF virus (ASFV) determines some limitations and the complexity of solving the problem of vaccine development. Such situation stimulated the development of highly specific diagnostic methods for rapid and accurate detection of the ASFV. In this regard, results of studies, including our own, concerning the comparative analysis of the genome of vaccine and virulent strains of the ASFV, as well as immunodiagnostic approaches to determine causes of high virulence and low protective activity of the ASFV, are briefly presented. Special attention is given to the issue related to the development of safe and effective vaccines against ASF. In this context disadvantages and possible advantages of live attenuated (LAV) and recombinant (RV) vaccines are considered in details. Results of recent studies on the assessment of the immunogenicity of genetically modified vaccines (GMV) which developed in various laboratories around the world are presented. The obtained data indicate that ASF vaccination is currently the most promising measure to stop the spread of this disease in our country and in the world, however, previous experience with ASF vaccination has revealed some problems in its development and application. The significant contribution of foreign researchers to the study of the basics of virulence of this pathogen and the study of its genes functions are noted. The possible further expansion of ASF in Europe and Asia in bordering Russia territories, as well as the established fact of the persistence of ASFV in wild boar population indicate a constant threat of its re-introduction into our country. In conclusion, the importance of developing a safe effective vaccine against ASF and the assessing of the possible risks of creating the artificial sources of the infection in nature as a result of its use is emphasized.

[Evaluation of the molecularbiological properties of human rotavirus A strain WA.]

BACKGROUND: Rоtaviruses are amоng the leading causes of severe diarrhea in children all over the Wоrld. Vaccination is considered to be the mоst effective way to cоntrоl the disease. Currently available vaccines for prevention of rоtavirus infection are based on live attenuated rotavirus strains human оr animal origin. OBJECTIVES: The aim of this investigation was to study the biological and genetic properties of an actual epidemic human rotavirus A (RVA) strain Wa G1P[8] genotype. METHODS: RVA Wa reproduction in a monolayer continuous cell lines, purification and concentration of RVA antigen, PAAG electrophoresis and Western-Blot, electrophoresis of viral genomic RNA segments, sequencing. RESULTS: Human RVA G1P[8] Wa strain biological and molecular genetic properties were assessed in the process of the adaptation to MARC145 continuous cell line. Cell cultured RVA antigen was purified, concentrated and then characterized by the method of PAAG electrophoresis and immunoblot. To verify RVA Wa genome identity, electrophoresis of viral genomic RNA segments was performed. The lack of accumulation of changes in the RVA Wa genome during adaptation to various cell cultures and during serial passages was demonstrated by sequencing fragments of the viral genome. CONCLUSIONS: RVA Wa strain is stable, it possesses high biological activity: it has been successfully adapted to the MARC145 cell line and RVA Wa virus titer after the adaptation reached 7,5-7,7 lg TCID50/ml. The identity of the cultivated RVA to the original strain Wa G1P[8] was confirmed.

[Assessment of immunogenic activity of the cloned human rotavirus A WA strain.]

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. OBJECTIVES: Evaluation of immunogenic activity of the cloned RVA Wa strain in the new-born Vietnamese potbellied 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 µg 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.

[Molecular and biological characteristics of vaccinary ERA-CB 20M of rabies virus.]

The molecular and biological characteristics of the vaccine against rabies virus strain ERA-CB 20M obtained by the Russian rabiologist, doctor of medical sciences S.V. Gribencha by adapting and cloning the strain ERA and SAD in a transplantable BHK-21 C13 cell culture are presented. The spectrum of the most sensitive strain of rabies ERA-CB 20M cell lines was determined and the level of glycoprotein was quantitatively determined. Primary nucleotide sequences of fragments of the genome of the strain ERA-CB 20M (genes N and G) were obtained and phylogenetic analysis was carried out. Molecular analysis showed that this strain belongs to the group of vaccine strains SAD1. When compared with the reference strain SAD1, 10% of the nucleotide differences were revealed in the gene fragment N; 15%, in the gene fragment G.

Vaccines against avian influenza in poultry.

The review presents the latest data about the types of vaccines against avian influenza that are used in current medical practice or are under development. Inactivated whole virion vaccines, live vector vaccines, as well as experimental vaccines developed using genetic engineering techniques (e.g. subunit vaccines, VLP vaccines, DNA vaccines) were considered. The efficiency of influenza reverse genetic technology for the development of prototype vaccine strains was noted.

Human rotavirus infection. Strategies for the vaccinal prevention.

Rotavirus was first isolated in 1973 in Australia from children with diarrhea. Hundreds of thousands of children die annually in developing countries from this virus with the mortality peaks in the most impoverished among them. According to wHo, rotavirus infection claims about 440 thousands children lives each year, being third in the mortality rate after pneumonia and malaria. Rotavirus is widely spread throughout the world and by the age of five years almost every child encountered this pathogen at least once. Rotavirus has a high genetic and antigenic diversity. The most important for humans is the group A rotavirus, and the most common by far genotypes are G1P [8], G2P [4], G3P [8], G4P [8], G9P [8], and to a lesser extent G12P [8]. There are three gene constellations described in rotavirus designated Wa, Ds-1, and Au-1. It is believed that they originated from rotaviruses of pigs, cattle, dogs, and cats, respectively. Cases of rotavirus interspecies transmission from animal to humans were reported. The first vaccines against rotavirus infection were based on naturally attenuated virus of the animal origin. Their efficiency, especially in developing countries, was inadequate, but today China and India use vaccines based on animal rotaviruses. Using the method of gene reassortation with the cattle rotavirus WC3 as a backbone, pentavalent vaccine against most common human rotavirus serotypes was developed and now successfully used as RotaTeq. The ability of rotavirus to protect against heterologous isolates was taken into account in the development of other vaccine, Rotarix, created on the basis of rotavirus genotype G1P1A [8]. The efficacy of these vaccines in developing countries is significantly reduced (51%), the cost of a dose is high, and so the search for more effective, safe, and inexpensive vaccines against rotavirus continues around the world.

Monitoring of rabies in wild animals in the Kirov region after oral immunization.

This work presents the results of the molecular genetic research on genomes of field isolates of the rabies virus circulating in the territory of the Kirov region in order to analyze the phylogenetic relationship between the wild isolate genomes and to determine the possible reversion of the vaccine strain of the rabies virus used in the oral vaccine to virulent variant. We studied 24 brain samples from wild carnivores shot after oral immunization of the area with Rabivak-O/333. A bait with the vaccine provided by the Veterinary Service of the Kirov was also studied. All samples were found to be positive for the presence of the rabies virus as established by FAT and RT-PCR techniques. Phylogenetic analysis of N genome fragments of the rabies virus showed that the field isolates from the Kirov regions were genetically close to the field isolates from Buryatia 2012. Analysis of G genome fragments showed that the Kirov field isolates were close to the isolates from Lipetsk (2011), as well as to the Ukrainian isolates (2006 and 2010). Molecular genetic analysis of the gene fragments N and G for the field isolates and fragments of the genome of the rabies virus vaccine did not reveal any reversion to the virulent vaccine strain.

[Two cases of hydrophobia in the Republic of Tatarstan: in vivo and postmortem laboratory diagnosis].

The results of rabies in vivo and postmortem laboratory detection in two cases registered in the Republic of Tatarstan are reported: a victim bitten by a wolf in 2002 and another one bitten by a stray dog on Goa Island, India, in 2013. In the patient bitten by a wolf cornea imprints studies using the method of fluorescent antibodies (MFA) showed rabies-positive result 6 days before the patient's death. The results were confirmed by postmortem examination of different parts of the brain and salivary glands using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. In the patient bitten by a stray dog the rabies virus specific antigen was detected by eye cornea studies using the MFA method and saliva studies using the ELISA. The rabies virus genome was also isolated from saliva and tear fluid using nested reverse-transcription polymerase chain reaction (RT-PCR) 9 days before the patient's death. The in viva studies results were consistent with the postmortem study of different parts of the brain using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. All the infection-positive results of both in viva and postmortem studies were consistent with the clinical studies, i.e. rabies diagnosis was confirmed. The analysis of the rabies virus gene G fragment nucleotide sequence of 238 nd length showed a slight difference between the studied isolates (2 rabies) and the RABV AY9563I9 (1.68%), difference by 10.5% from the Vnukovo-32 vaccine strains and by 10.9% from the SAD B19 rabies strain, respectively (rabies viruses of 1st genotype). It was also significantly different from the lissaviruses of 2,4,5, and 6 genotypes (21 .0-32.7%). The obtained results indicate phylogenetic closeness of the studied isolates (2 rabies) with the RABV AY956319 rabies virus strain belonging to the 1st genotype.

[Engineering by reverse genetics and characterization of the new reassortant influenza virus strain H5N1].

Reverse genetics was applied to engineering of the reassortantvaccine candidate strain against highly pathogenic avian influenza viruses (HPAIVs) of the H5 subtype. The new strain recPR8-H5N1 contains the HA gene from the Russian HPAIV A/Kurgan/05/2005 (H5N1), the NA and internal genes from A/Puerto Rico/8/34 (H1N1). The strain recPR8-H5N1 demonstrated the antigenic specificity (H5), high proliferation rate in 12 days chicken embryos, and was lethal for the embryos in 36 hours. An inactivated emulsified vaccine based on the strain recPR8-H5N1 elicited high antibody titers and protected 6-week-old chickens from lethal challenge with the HPAIV A/Kurgan/05/2005 (H5N1) on day 21 after single immunization. Infection of non-vaccinated birds with the strain recPR8-H5N1 did not cause any pathology, and the virus was not detected using PCR in blood and cloacal swabs on day 7 p.i. Specific weak seroconversion caused by infection with the strain recPR8-H5N1 was detected on day 14 p.i. As a result, a new influenza virus strain was obtained with modified properties.

[Real-time PCR kits for the detection of the African Swine Fever virus].

The results obtained using the diagnostic kit based on real-time polymerase chain reaction to detect the DNA of the African Swine Fever in the pathological material, as well as in the culture fluid, are presented. A high sensitivity and specificity for detection of the DNA in the organs and tissues of animals was shown to be useful for detection in the European Union referentiality reagent kits for DNA detection by real time PCR of ASFV. More rapid and effective method of DNA extraction using columns mini spin Quick gDNA(TM) MiniPrep was suggested and compared to the method of DNA isolation on the inorganic sorbent. High correlation of the results of the DNA detection of ASFV by real-time PCR and antigen detection results ASFV by competitive ELISA obtained with the ELISA SEROTEST/INGEZIM COMRAC PPA was demonstrated. The kit can be used in the veterinary services for effective monitoring of ASFV to contain, eliminate and prevent further spread of the disease.

[Molecular-genetic analysis of the genomes of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 circulating in the area of Russian Federation].

The molecular genetic analysis of the genomes of the virus of porcine reproductive respiratory syndrome (VPRRS) and porcine circovirus type 2 (PCV-2) circulating in the area of the Russian Federation was discussed. The results of this work showed the circulation of the strains of the European genotype VPRRS similar to those found in France and Denmark from 1998 to 2001. The homology of the fragment of one of the genes between the Russian isolates and the vaccine strain Porcilis PRRS (Intervet) was found. It requires further study. The strains representing the North American genotype VPRRS were not found. The PCV-2 genomes fall into three separate goups. One (genotype 2b) is formed by isolates in Malaysia, Brazil, Switzerland, China, Slovakia, UK, USA, isolated during the period from 2004 to the present time. The second group consists of sequences of the viruses isolated in 2000-2012 in Canada, the U.S., China, and South Korea (genotype 2a). The third group is formed by highly pathogenic isolates in 2013 from China (highly pathogenic genotype 2c). The circulation of all three known genotypes of PCV-2: 2a, 2b, and 2c in Russian Federation was demonstrated.

[Production of the monoclonal antibodies to the rabies virus nucleoprotein].

Five hybridomas secreting monoclonal antibodies (MAbs) for the nucleocapsid protein of the rabies virus were obtained through the fusion of the SP2/0 murine myeloma cells with splenocytes of BALB/c mice immunized with fixed rabies virus (CVS strain). All hybridomas secret MAbs of the IgG class that display different specificity to the nucleocapsids of rabies and rabies-related viruses. MAbs 2ell showed the specificity for the prevalent in Russia rabies viruses that are similar to commercially available anti-rabies conjugate.

[African swine fever in Russian Federation].

African swine fever (ASF) is an infectious viral disease that causes high economic losses due to the necessity of depopulation of pigs in affected areas, sanitary measures, trade restrictions, etc. The virus (ASFV) is relatively stable in the unprocessed meat products and environment. Thus, large areas are at risk due to free movement of people and products. The ASFV does not affect people and animals, except the wild and domestic pigs. Some ticks can become infected and carry the virus for years. Adaptation of the virus by changing into the less virulent form would mean the threat of an endemic situation to the area. The disease is endemic in domestic and wild pigs in most of sub-Saharan Africa and Sardinia, Italy. There is no treatment for ASF, and no vaccine has been developed. In case of infection with less virulent ASFV strains, the recovered pigs could spread the virus as long as their live. In terms of clinical symptoms, ASF is very similar to Classical Swine Fever. The methods of laboratory diagnostics are well developed and efficient for identification of ASFV and virus-specific antibodies. Experience of eradication of ASF in Spain suggests the importance of serological monitoring of pigs. In the spring of 2007, the ASF was detected in the Caucasus region. Same virus was detected in Georgia, Armenia, Azerbaijan, and Russia. The ASFV circulating in the Caucasus and the Russian Federation is a highly virulent virus. No reduction of the virulence was observed since the first outbreak in Georgia. In the last years, the ASF remained in the Caucasus, southern parts of Russia and appeared occasionally as far as St. Petersburg and St. Petersburg region, and in the area of Nizhny Novgorod. Domestic pigs play an important role in the ASFV spread; they transfer the virus to the wild boars. The virus circulates in the population of wild boars depending on their density in the area. Occasionally, the disease is spread from wild to domestic pigs. There is no evidence of ticks being involved in the process. Thus, the human activity in raising pigs is largely responsible for continuous spread of the disease. Despite vigorous monitoring and sanitary measures, the disease has not been stopped. The control strategy for ASF should consider International (especially Spanish) experience and local situation. The strategy is based on the number of important steps including rapid localization of the disease by trained specialists, setting up buffer zones, constant serologic monitoring of swine population and farms, improvement of diagnostic facilities, training of veterinary personnel, development of the system of information and international collaboration.

[Molecular-genetic analysis of the field isolates of avian leucosis viruses in the Russian Federation].

Results of monitoring of different subtypes of avian leukosis virus (ALV) from commercial poultry farms in 14 regions of Russian Federation were discussed. Only three regions were found to be negative. ALV was detected in other 11 regions in 46-64% cases (for different regions). The phylogenetic analysis of the genomes for the 12 field isolates of ALV was carried out in different regions of Russian Federation. The isolates belong to different subtypes of the virus and form two large groups. The genomic differences between Russian and foreign isolates within each group range from 5% to 10%.

[High-yield reassortant virus containing hemagglutinin and neuraminidase genes of pandemic influenza A/Moscowl/01/2009 (H1N1) virus].

The crossing of influenza A/Moscow/01/2009 (H1N1) virus and reassortant strain X31 (H3N2) containing the genes of internal and non-structural proteins of A/Puerto Rico/8/34 (H1N1) strain gave rise to reassortant virus ReM8. The reassortant contained hemagglutinin (HA) and neuraminidase (NA) genes of pandemic 2009 influenza virus and 6 genes of high-yield A/Puerto Rico/8/34 (H1N1) strain. The reassortant ReM8 produced higher yields in the embryonated chicken eggs than the parent pandemic virus, as suggested by infectivity and HA activity titration as well as by ELISA and the measurement of HA protein content by scanning electrophoresis in polyacrylamide gel slabs. High immunogenicity of ReM8 reassortant was demonstrated by immune protection studies in mice. The reassortant virus ReM8 is suitable as a candidate strain for the production of inactivated and subunit influenza vaccines.

[Molecular diagnostics of influenza].

The review is concerned with molecular diagnostic tools for influenza A viruses. Test systems based on PCR, real-time PCR, IEA, sequenation, and microchip-based methods are discussed as applied at this Institute for comprehensive monitoring influenza A viruses.

[The 24 May, 2009 isolation of the first A/IIV-Moscow/01/2009 (H1N1)swl strain similar to swine A(H1N1) influenza virus from the first Moscow case detected on May 21, 2009, and its deposit in the state collection of viruses (SCV No. 2452 dated May 24, 2009)].

The paper presents the results of the first isolation of the new influenza virus in Moscow and the Russian Federation, which was similar to the swine A/IIV-Moscow/01/2009(H1N1)swl strain isolated on May 24, 2009 from a Russian arrived in Moscow from the USA on May 19, 2009. The antigenic, biological, and molecular genetic properties of this virus were studied. The virus was isolated on MDCK and chick embryos, the hemagglutination titers being 1:8-1:16 AE; the infectious titers being 6.51g of the tissue cytopathogenic infective dose (TCID50) and 7.01g of the common infective dose (CID50). The virus was sensitive to arbidol, ribavirin, oseltamivir, and resistant to rimantadine. The complete virus genome was sequenced; the data were accepted to the Gen Bank on May 28, 2009 under GQ219584-GQ219590 and GQ202724. The significant gene substitution of neuraminidase Asp for Gly in position 451, which has been undetectable in any other strain published in the Gen Bank by the present time is unique only to A/IIV-Moscow/01/2009 (H1N1)swl. The virus has been deposited in the State Collection of Viruses, D. I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, under No. 2452 dated May 24, 2009.

[Isolation of noncytopathogenic genotype 2 bovine viral diarrhea virus from the cattle mucosa in the Russian Federation].

The paper presents the results of genotyping and phylogenetic analysis of three noncytopathogenic isolates of bovine viral diarrhea virus from the mucosae of the cattle with different clinical presentations of the disease. On the basis of the phylogenetic analysis of high-conserved and variable virus genome regions (5'-UTR, N(pro), and E2), the authors referred two isolates to as genotype 1, subgenotypes 1b and 1d, and the third isolate to as genotype 2. This is the first communication about the isolation of genotype 2 virus in Russia.