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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.

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.

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.

Epitope mapping of the major envelope glycoprotein E2 of classical swine fever virus (CSFV) is important for our understanding of E2 and also for development of the CSFV-specific diagnostic assays and epitope- or peptide-based marker vaccines. Previous competitive binding studies showed that monoclonal antibodies raised against E2 protein of CSFV detected 8 individual epitopes. At the present study using a set of synthetic peptides covering the full sequences of E2 glycoprotein five linear non-overlap B-cells epitopes were identified. The identified sequences of 12 strains of the CSFV and 5 other pestiviruses were aligned. The data obtained could be useful for improvement of the CSFV diagnostic systems and studies of amino acid substitutions and their influence on antigenic properties of the CSFV E2 protein.

The epizootic etiologically associated with highly pathogenic avian influenza H5N1 genotype 2.3.2 that is new for Russia among wild and domestic birds in the south of the Primorye Territory during spring migration in April 2008 has been decoded. About 25% of the wild birds of a water complex, which include European teals (Anas crecca), mallard ducks (Anas platyrhynchos), great-crested grebes (Podiceps cristatus), are involved in viral circulation in the area of the Suifun-Khankai plain. Chicken embryos and the cell lines MDCK, SPEV, BHK-21, SW-13 were used to isolate 3 strains from recently deceased hens (A/chicken/Primorje/1/08, A/chicken/Primorje/11/08, and A/chicken/Primorje/12/08) and one strain from a European teal (A/Anas crecca/Primorje/8/08). The strains were deposited in the State Collection of Viruses of the Russian Federation, D. I. Ivanovsky Research Institute of Virology, Russian Academy of Medical Sciences. The nucleotide sequences of the full-sized genomes of A/chicken/Primorje/1/08 and A/Anas crecca/Primorje/8/08 were sent to the International databank GenBank. The strains from domestic and wild birds were shown to be identical. The isolated strains are most close to the strains Alchicken/Viet Nam/10/05, A/chicken/Guangdong/178/04, and A/duck/Viet Nam/12/05. Molecular genetic analysis has indicated that the strains isolated are susceptible to rimantadine and ozeltamivir and less adapted to mammalian cells (particularly, they contain E627 in RV2, which agrees with the biological properties of these strains in vitro). Penetration of the newly isolated virus into the Far East ecosystem provides in the foreseeable future a way for infecting the birds wintering in America and Australia in the nesting places, with further carriage of viral populations there in the period of autumn migrations.

Twenty-eight hybridomas producing monoclonal antibodies (MAbs) to proteins of classical swine virus (CSFV) were obtained by fusion of AS2/0 murine myeloma cells with splenocytes of BALB/c mice. The recombinant E2 glycoprotein of CSFV and the gradient-purified CSFV strain Shimen were used as an antigen for immunization. Twenty-four hybridomas produced MAbs of class IgG and four hybridomas did MAbs of class IgM. All MAbs were specific for E2 protein of CSFV. Competitive enzyme immunoassay showed that MAbs detected 8 epitopes on protein E2.

The porcine reproductive and respiratory syndrome (PRRS) is a contagious viral pathology caused by PRRS virus. There are 2 types of the above virus--the European and American ones. Distribution patterns of the PRRS virus were studied for Russia and Byelorussia. Above 700 porcine sera obtained from 32 households of 21 Russia's administrative regions and from 19 households of 6 Byelorussia's administrative regions were tested for presence of antibodies to the PRRS virus. Simultaneously, the samples were tested for virus presence by polymerase chain reaction (PCR). It was proven serologically that the PRRS virus is widespread in the territories of Russia and Byelorussia. Noteworthily, all field isolates found in Russia and Byelorussia belong to the European type. Not a single viral isolate of the American PRRS type was found. The nucleocapsid (N) recombinant protein was obtained on the basis of the Russian field isolate of the PRRS virus by using the E. coli. expression system. Finally, it was shown as possible to use the recombinant protein in indirect immune enzyme assay for the sake of detecting the antibodies to the PRRS virus.

An effective system for specific prevention of transmissive swine gastroenteritis (TSG) is based on the use of combined vaccine and combined vaccination. Combined vaccine is a lyophilized preparation for intranasal application and emulsion for intramuscular injection. The former is prepared from natural attenuated respiratory variant of TSG virus and the latter from artificially attenuated TSG virus. Combined vaccination consists in parallel double immunization of females on days 70-75 of gestation intramuscularly and intranasally and on days 90-95 of gestation intramuscularly. Vaccinal lactogenic immunity ensures protection of newborn piglets under experimental and field conditions (protection of 70-80 and 90-95% piglets, respectively). The proposed vaccine preparations and protocols of combined immunization of females during gestation by two methods are an effective and safe method for specific prevention of TSG.

Recombinant major surface glycoprotein E2 from virulent Shimen strain of classical swine fever virus (CSFV) has been tested for immunogenicity in animal immunization experiments. Immunization of 3-month-old piglets with 200 micrograms of recombinant protein protected the animals from lethal challenge with virulent CSFV strain. CSFV-specific antibody detection test based on competitive ELISA has been developed using the recombinant E2 protein. The test can evaluate specific antibody levels after subunit vaccination with recombinant E2 after immunization with live vaccine based on attenuated CSFV strain.

Recombinant E2 protein from vaccine strain of classical swine fever virus (CSFV) and from SCFV virulent strain Shimen was synthesized in SF-21 and High-Five cell culture with baculovirus as the expressing vector. For secretion, hydrophobic C-terminal transmembrane domain was removed and N-terminal signal polypeptide of 38 amino acids was added. Maximum accumulation of recombinant products in SF-21 cells was observed after 48 h and in medium 96 h after infection with recombinant baculovirus. In High-Five cells and in culture medium the maximum accumulation of E2 was observed after 96 h. The level of E2 expression is 5-10 micrograms/106 cells. The products of expression were purified by affinity chromatography and their specificity confirmed in immunochemical tests with a series of reference monoclonal antibodies. The product can be used for detecting antibodies to SCFV by competitive enzyme immunoassay.

The CS vaccine strain of Classical Swine Fever Virus is a derivative from the LK parental strain that has been used in Russia for more than 30 years. A 10697 nucleotide fragment of the CS strain's genome has been sequenced. Sixteen unique restriction markers have been found in the CS genome comparing to the following strains: Alfort187, Alfort Tubingen, Brescia, CAP, Glentorf, ALD, GPE-, Chinese, C-strain, Riems, P97. Fourteen of these sites (AflII, AvaI, CfoI, Eco47II, HaeII, KpnI, MunI, NspI, PstI, ScaI, SmaI, SpeI, StyI, VspI) are only present in the CS strain genome. The 2 sites (BgII, NdeI) are present in all other genomes except for the genome of vaccine strain. A PCR/restriction test has been developed based on these findings in order to distinguish the vaccine strain from field isolates. Two pairs of nested primers and a criteria of analysis have been designed for each restriction marker site. The tests have been conducted first on the reference strains resulting in predicted restriction patterns. Finally, the tests have been applied to a number of field isolates obtained at different locations in Russia in different years. These results give further evidence that PCR/restriction tests can identify the LK and CS vaccines helping to avoid confusion with field strains.

Primary structure of a genome fragment of attenuated strain CS of hog cholera virus (HCV) coding for three surface glycoproteins Erns, E1, and E2 (fragment size 2379 nucleotides) is analyzed. By the nucleotide sequence the homology between strain CS and ten other virulent and attenuated HCV strains in this area is 84.9-94.6%, 87.2-94.6% in gene Erns, 84.6-96.9% in gene E1, and 83.3-94.3% in gene E2. By amino acid sequence the homology is 90.9-94.3%, 92.9-95.0%, 92.3-95.6%, and 88.9-94.1%, respectively. Computer analysis demonstrated philogenetic ratios between these strains and other HCV strains and the areas of potential antigenic differences between CS strain and other HCV strains. The data indicate that strain CS used as live vaccine protecting from HCV contains unique nucleotide and amino acid positions and its evolution history is different from that of analyzed reference strains. The data will be further used for detecting the fine antigenic structure of strain CS surface glycoproteins with the aim of disclosing unique antigenic markers.

Numerous monoclonal antibodies (MAb) to hog cholera virus are a highly specific and effective instrument for studies of this agent. Panels of MAb for differential diagnosis of Pestiviruses are characterized. International reference panel of 30 MAbs is a result of cooperation of European scientists; it was approved as the official reference for assessing all available and new diagnostic agents. MAb permit intraspecies differentiation between hog cholera virus strains and, which is particularly important, between vaccine and field strains. Study of antigenic structure and functional characteristics of surface proteins of the virus with the use of MAb panels helped single out and map the functions of four antigenic sites of surface glycoprotein E2 and detect a relationship between RNAse activity and structural component of another surface glycoprotein E0.

The review emphasizes the significance of laboratory methods for the diagnosis of hog cholera virus. In addition to virus isolation and immunofluorescent method, enzyme immunoassay (EIA) of virus-specific antigen and antibodies are recommended. Commercial EIA kits for laboratory diagnosis of hog cholera virus and test-systems whose development is in progress now are characterized.