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.

[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.

[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.

[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.

[Epitope mapping of antigenic determinants of E2 glycoprotein from classical swine fever virus using synthetic peptides].

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.

[Preparation and characteristics of monoclonal antibodies to protein E2 (GP55) of classical hog cholera virus].

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.

[Serological monitoring of arbovirus infections in the estuary of the Kuban River (the 2006-2007 data)].

Solid-phase enzyme immunoassay, neutralization test, and the hemagglutination-inhibition test were used to study the sera from human beings (152 samples), agricultural animals (n = 77), hares (n = 3), and wild birds (n = 69), collected in 2006-2007 in the Kuban River estuary (Temryuk District, Krasnodar Territory). There were specific antibodies against viruses of West Nile (WH), tick-borne encephalitis (TBE) (Flaviviridae, Flavivirus), Sindbis (Togaviridae, Alphavirus), the antigenic complex of California, Batai (Bunyaviridae, Orthobunyavirus), Dhori (Orthomyxoviridae, Thogotovirus). The findings suggest the presence of arboviruses from 6 transmitting mosquitoes and ticks in the study area and human infection by the viruses of the antigenic complex of California (20-47%), Batai (3-15%), West Nile (3-12%), Dhori (2%). The index agricultural animals (horses, cattle) were observed to have specific antibodies to the viruses of WN (8-15%), TBE (0-2%), Sindbis (2-9%), the antigenic complex of California (27-54%). Out of the representatives of the wild fauna, virus-neutralizing antibodies to Sindbis virus were found in European hares (Lepus europaeus), California complex virus in gulls (Larus argentatus) and terns (Sterna hirundo), WN and Sindbis viruses in herons (Ardea purpurea), and WN and California complex viruses in bald-coots (Fulica atra).

[Interpretation of the epizootic outbreak among wild and domestic birds in the south of the European part of Russia in December 2007].

The paper presents the results of interpreting the epizootic outbreak etiologically associated with high-virulent influenza virus A/H5N1 among domestic and wild birds in the Zernogradsky and Tselinsky districts of the Rostov Region. Epizooty was characterized by a high infection rate in the synanthropic birds of a ground-based complex. RT-PCT revealed influenza virus A/H5 in 60% of pigeons and crows and in around 20% of starlings, and in 10% of tree sparrows. Fifteen viral strains from chickens (Gallus gallus domesticus), Indian ducks (Cairina moschata), rooks (Corvus frugilegus), rock pigeons (Columba livia), tree sparrows (Passer montanus), common starlings (Sturnus vulgaris), and great white herons (Egretta alba) were isolated and deposited in the State Collection of Viruses of the Russian Federation. Full-sized genomes of 5 strains were sequenced and deposited in the international database GenBank. The isolated strains belong to the Quinhai-Siberian (2.2) genotype, an Iranian-Northern Caucasian subgroup, they are phylogenetically closest to the strain A/chicken/Moscow/2/2007 (inducing epizooty among poultry in the near-Moscow Region in February 2007) and have 13 unique amino acid replacements as the consensus of the Quinhai-Siberian genotypes in the proteins PB2, PA, HA, NP, NA, and M2, by preserving thereby 4 unique replacements first describes for the strain A/chicken/Moscow/2/2007. The findings are indicative of a different mechanism that is responsible for bringing the virus into the northeastern part of the Azov Sea area in September 2007 (during the fall migration of wild birds) and in December 2007 in the south-western Rostov Region where a human factor cannot be excluded. Mass infection of synanthropic birds endangers the further spread of epizooty, including that in the central regions of the Russian Federation in spring after near migrants return after wintering.

[Epizooty caused by high-virulent influenza virus A/H5N1 of genotype 2.2 (Qinghai-Siberian) among wild and domestic birds on the paths of fall migrations to the north-western part of the Azov Sea basin (Krasnodar Territory)].

Isolation, followed by the sequencing the full-size genome of strains of A/chicken/Krasnodarl300/07 and A/Cygnus cygnus/Krasnodar/329/07, has shown that they belong to genotype 2.2 (Qinghai-Siberian). The strains were deposited at the State Virus Collection of the Russian Federation and nucleotide consequences were at the International databank GenBank. The strains contained 10 unique amino acid replacements in reference to the consensus of the Qinghai-Siberian genotype in the PB2, PA, HA, NA, and NS1, which suggests that regional variants may form in different parts of an area.

[Molecular genetic characteristics of the strain A/chicken/Moscow/2/2007 (H5N1) strain from a epizootic focus of highly pathogenic influenza A among agricultural birds in the neear-Moscow region (February 2007)].

Among agricultural birds in the near-Moscow Region (February 2007), local epizootics caused by the highly pathogenic avian influenza A/H5N1 virus seem to be of unintended manual origin. Such a situation may be considered to be model when the source of inoculation is elucidated in cases of potentially possible acts of bioterrorism. Molecular genetic analysis of isolated A/chicken/Moscow/2/2007 strain established its genetic similarity with the highly pathogenic strains detected in the Black-and-Caspian Sea region in 2006. At the same time, comparison of nucleotide sequences of the strain A/chicken/Moscow/2/2007 with the strains of Qinghai-Siberian genotype (CSG) for which the sequences of full-sized genomes are known in the international databases revealed a significant distinction of the near-Moscow strain from the earlier known analogues. The uniqueness of the primary structure of the PB1 gene is shown. The paper discusses the functional value of amino acid substitutions in the proteins of the strain A/chicken/Moscow/2/2007 and in other variants of CSG of the subtype H5N1.

[Complex environmental and virological monitoring in the Primorye Territory in 2003-2006].

The paper presents the results of monitoring of viruses of Western Nile (WN), Japanese encephalitis (JE), tick-borne encephalitis (TBE), Geta, Influenza A, as well as avian paramicroviruses type I (virus of Newcastle disease (ND)) and type 6 (APMV-6) in the Primorye Territory in 2003-2006. Totally throughout the period, specific antibodies to the viruses were detected by neutralization test in wild birds (7.3%, WN; 8.0%, Geta; 0.7% Batai; 2.8%, Alpine hare (Lepus timidus); by hemagglutination-inhibition test in cattle (11.4% WN; 5.9%, JE; j 3.0%, TBE; 11.6%, Geta), horses (6.1, 6.8, 0, and 25.3%, respectively), and pigs (5.4, 1.5, 0, and 5.9%, respectively) by enzyme immunoassay (IgG) in human beings (0.8, 0.5, 6.8, and 3.2%, respectively. Reverse-transcription polymerase chain reaction (RT-PCR) was used to reveal RNA of the NP segment of influenza A virus in 57.9 and 65% of the cloacal swabs from wild and domestic birds, respectively; and the HA-segment of subtype HH was not detected in 2005. HA/H5 RNA was recorded in 5.5 and 6.7% of the swabs from wild and domestic birds, respectively; 6% of the specimens from domestic birds were M-segment positive in 2006. RNA of influenza A virus NA/H7 and RNA was not detected throughout the years. In 2004, the cloacal swabs 8 isolated influenza A strains: two H3N8 and two H4N8 strains from European teals (Anas crecca), two (H3N8 and H6N2) strains from Baikal teals (A. formosa), one (H10N4) strain from shovelers (A. clypeata), and one (H4N8) from garganeys (A. querquedula). In 2004, one ND virus strain was isolated from the cloacal swabs from European teals (A. crecca). RT-PCR revealed RNA of this virus in some 8 more cloacal swabs from black ducks (A. poecilorhyncha) (3 positive specimens), pheasants (Phasianus colchicus) (n = 2), garganeys (A. querquedula) (n = 1), gadwalls (A. strepera) (n = 1), and geese (Anser anser domesticus) (n = 1). Sequencing of the 374-member fragment of the ND virus F gene, which included a proteolytic cleavage site, could assign two samples to the weakly pathogenetic variants of genotype 1, one sample to highly pathogenic variants of genotype 3a, five to highly pathogenic ones of genotype 5b. Isolation of APMV-6 (2003) from common egrets (Egretta alba) and geese (Ans. anser domesticus) is first described.

[Newcastle disease virus in the populations of wild birds in the south of the Primorye Territory in the period of autumn migrations in 2001-2004].

The paper presents the results of molecular virological monitoring of Newcastle disease virus (NDV) by reverse-polymerase chain reaction (followed by sequence of F-gene fragment 374 p.n.) and chick embryo isolation of samples from the avian cloacal swabs collected in the south of the Primorye Territory in September-October 2001-2004. It shows that before 2004, there were only slightly pathogenic variants of NDV of genotype 1 in this region and in 2004 they were added by highly pathogenic variants of subtypes 3a and 5b. The impact of landscaping features of the south of the Primorye Territory on the environment of NDV is discussed.

[Highly pathogenic influenza A/H5N1 virus-caused epizooty among mute swans (Cygnus olor) in the lower estuary of the Volga River (November 2005)].

Molecular virological studies of the field material collected in the epicenter of epizooty with high mortality among mute swans (Cygnus olor) in the area of the lower estuary of the Volga River (November 2005) could establish the etiological role of highly pathogenic influenza A (HPAI) virus of the subtype H5N1. Ten HPAI/H5N1 strains deposited at the State Collection of Viruses of the Russian Federation with the priority dated December 1, 2005 were isolated from the cloacal/tracheal swabs and viscera of sick and freshly died mute swans. Complete nucleotide sequences of all fragments of the genome of 6 strains have been deposited in the Gene Bank. The paper discusses the molecular genetic characteristics of isolated strains.

[Isolation of influenza A/H5N1 virus strains from poultry and wild birds in West Siberia during epizooty (July 2005) and their depositing to the state collection of viruses (August 8, 2005)].

Six strains of influenza AH5N1 virus were isolated, by using PS and MDCK continuous cell lines from poultry and wild birds, which were collected on July 28, 2005 in the samples taken from 5 examines species of wild birds in the Novosibirsk region during the epizootic outbreak with a high mortality. The strains were identified by means of HIT, RT-PCR, and microchip-based techniques. Two strains, A/Grebe/Novosibirsk/29/05 (H5N1) and A/Duck/Novosibirsk/56/05 (H5N1), were deposited to the Russian State Collection of Viruses (Registration Nos. 2372 and 2371, respectively) with the priority date 08.08.2005. Positive results in RT-PCR for influenza A/H5N1 virus detec- tion were obtained in 100% of the samples from dead and sick poultry; 93% from the clinically healthy poultry kept together with sick one; positive results ranged from 0 to 36%. Sequencing established the identity of genetic characteristics of strains isolated for wild birds and poultry as well as their affiliation to high pathogenic avian influenza (HPAI). Phylogenetic analysis revealed a high homology of hemagglutining of West-Siberian strains and strains isoolated from bar-headed gooses (Eulabeia indica) on the Qinghai Lake (Western China) in the 2005 spring.

[Isolation of highly pathogenic avian influenza (HPAI) A/5H5N1 strains from wild birds in the epizootic outbreak on the Ubsu-Nur Lake (June 2006) and their incorporation to the Russian Federation State Collection of viruses (July 3, 2006)].

Virological and molecular genetic studies of the field material collected in the epicenter of epizooty with high mortality rates among the wild birds on the coast of the Ubsu-Nur lake (Republic of Tyva, 51 degrees NL, 93 degrees EL, June 2006) revealed the etiological role of highly pathogenic avian influenza (HPAI) H5N1. Seven HPAI/H5N1 strains were isolated from the tracheal/cloacal swabs of clinically healthy, ill and recently dead great-crested grebes (Podiceps cristatus), cormorants (Phalacrocorax carbo), balt-coots (Fulica atra), and common terns (Sterna hirundo) collected on June 24, 2006, and incorporated to the RF State Collection of Viruses (with the July 3, 2006 priority). Full-length genome nucleotide sequences were incorporated to the GenBank (with the July 23, 2006 priority) (DQ852600-DQ852607). Comparative analysis of molecular genetic characteristics showed their belonging to the Qinghai-Siberian genotype. The strains were sensitive to rimantadine.

[West Nile virus infection of agricultural animals in the Astrakhan region, as evidenced by the 2001-2004 serological surveys].

Sera sampled from 2,884 farming animals in the Astrakhan region in 2001 to 2004 were investigated by the hemagglutination inhibition test (HIT) in order to indicate specific antibodies to West Nile virus (WNV). HIT-positive samples were investigated by the neutralization test (NT). WNV antibodies were detected in all the examined species of animals: horses (the proportion of positive tests throughout the observation averaged 9.8%; the agreement with NT results was 94.1%), cattle (6,4 and 72.%), camels (5.2 and 41.7%), pigs (3.1 and 75%), and sheep (2.2 and 57.1). Relationships between the environmental features of WNV in different natural zones, the infection rate, and the conditions of keeping farming animals in the Astrakhan region are analyzed.

[Ecology and evolution of influenza viruses in Russia (1979-2002)]. / Ekologiia i évoliutsiia virusov grippa v Rossii (1979-2002 gg.).

The research results on ecology and evolution of influenza A viruses, which has been conducted by the Center of Ecology and Evolution of influenza Viruses of Ivanovsky's Institute of Virology, Russian Academy of Medical Sciences, for more than 30 years, are summarized in the paper. A gene pool of influenza A viruses circulating in Russia's territory was defined. Foci of influenza A viruses were detected in natural biocenosis. Issues conditioned by the population interrelations of influenza viruses, i.e. between the populations of wild and home animals and the populations of people, are also under discussion.

[Analysis of genome determinants of virulence and setting-up of a library of full-size genome copies of the attenuated virus strain of the porcine reproductive and respiratory syndrome virus North American type]. / Analiz genomnykh determinant virulentnosti i sozdanie biblioteki polnorazmernykh infektsionnykh kopii genoma attenuirovannogo shtamma virusa reproduktivno-repriratornogo sindroma svinei severoamerikanskogo tipa.

Primary genome structures of 3 variants of the NADC-8 North American virulent strain of porcine reproductive and respiratory syndrome virus (PRRSV) were compared for the purpose of detecting any potential genetic virulence determinants of genus Arterivirus. Apart from the virulent variant, we also investigated the attenuated variant, obtained after 251 passages in cell culture, and the intermediate variant isolated from a pig after a partial reversion of the attenuated virus. The attenuated variant genome acquired a 3-nucleotide deletion and 50 mutations versus its virulent precursor. A comparison of the attenuated and intermediary virus variants denoted 8 nucleotide mutations entailing substitutions of 6 amino acids in 3 open reading frames (ORF1a, ORF1b and ORF6). A 32-clone library was constructed in the pACYC177 plasmid vector, which comprised full-size copies of the genome of the NADC-8 attenuated variant strain (251), virus PPCC, for the purpose of experimentally verifying the functional role of the obtained mutations. Full-size analogues ((+)-chain of RNA) of the viral genome, comprising the CAP-structures and polyadenylated ones were obtained in vitro on the basis of the cloned DNA. Seven of the 8 analyzed clones of the viral genome were infected and their insertion into the MARC-145 cell resulted in obtaining of infectious PRRSVs. Four of the constructed recombinant viruses had delayed growth parameters, and 3 of them were similar to the parental strain. The described technology (inverse genetics) would make it possible to introduce changes into the viral genome in applied and fundamental research of Arteriviruses.

[Genetic variability of the nucleocapsid protein of the virus of the porcine reproductive and respiratory syndrome]. / Variabel'nost' gena belka nukleokapsida virusa reproduktivnogo i respiratornogo sindroma svinei (RRSS).

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.

[Synthesis of recombinant proteins from the American and European viruses of the porcine reproductive and respiratory syndrome in baculovirus expression system and their antigenic properties]. / Sintez v bakulovirusnoi sisteme rekombinantnykh belkov amerikanskogo i evropeiskogo tipov virusa reproduktivnogo i respiratornogo sindroma svinei i ikh antigennye svoistva.

The baculovirus expression system was made use of to derive the recombinant nucleocapsid (N) proteins of the American and European virus types and of the porcine reproductive and respiratory syndrome (PRRS). The obtained products were purified by metal-affine chromatography and their specificity was confirmed in immunechemical reactions with reference monoclonal antibodies. The antigenic activity of recombinant proteins was studied by indirect immune enzyme assay (IEA) with porcine serum, which had been in advance characterized by the "HerdCheck" kit (IDEXX Co.). It was shown as possible to apply the derived recombinant antigens in determining, by indirect IEA, antibodies to the PRRS virus.