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Near full-genome sequence of the Wad Medani Virus (WMV) (strain LEIV-8066Tur) (Orbivirus, Reoviridae) isolated from the ticks Hyalomma asiaticum Schulze et Schlottke, 1929, collected from sheep in Baharly district in Turkmenistan, was determined using next generation sequencing approach. The similarity of the RNA-dependent RNA-polymerase (Pol, VP1) amino acid sequence between WMV and the Kemerovo group orbiviruses (KEMV), as well as of the Baku virus (BAKV), was 64%. The similarity of the conserved structural protein VP3 (T2) of WMV with mosquito-borne and tick-borne orbiviruses reaches 46% and 67%, respectively. For the surface proteins VP2, VP5, and VP7 (T13), which have major antigenic determinants of orbiviruses, the similarity of WMV with tickborne orbiviruses (KEMV and BAKV) is 26-30%, 45% and, 57%, respectively (ID GenBank: KJ425426-35).

The Gissar virus (GSRV) was originally isolated from the ticks Argas reflexus, Fabricius, 1794 collected in a dovecote of Gissar village in Tajikistan (38 degrees 40' N, 68 degrees 40' E). Using electron microscopy, GSRV was classified to Bunyaviridae without referring to genus due to the absence of the antigenic relation with known bunyaviruses. In the present paper genome of GSRV was sequenced (MiSeq, Illumina). Molecular genetics and phylogenetic analysis showed. GSRV has a high level of homology with the Grand Arbaud Virus (GAV) (94% for nucleocapsid protein, 87.5% for RdRp, and 82% for the envelope proteins GnGc) isolated from the ticks A. Reflexus in a dovecote in France. GSRV and GAV have a narrow ecological niche associated with the icks A. Reflexus and birds (predominantly Columbidae). According to the conducted study, GSRV is classified as the topotypic for Central Asia variant of GAV, Uukuniemi group, genuses of the Phlebovirus (Bunyaviridae) (ID GenBank KJ425423, KJ425424, KJ425425).

Complete genome sequence of the Burana virus (BURV) was determined using the next-generation sequencing approach (ID GenBank KF801651). The prototype strain of BURV LEIV-Krg760 was originally isolated from the ticks Haemaphysalis punctata Canestrini et Fanzago, 1877 (Ixodidae, Haemaphysalinae), collected from cows in Tokmak wildlife sanctuary, eastern part of the Chu valley (43 degrees 10' N, 74 degrees 40' E) near Burana village, Kirgizia, in April 1971. Molecular genetics and phylogenetic analyses showed that the BURV belonged to the Nairovirus genus, Bunyaviridae and is related to Tamdy virus (TAMV) that is also associated with the ixodidae ticks of pasture biocenosis in Central Asia. Previous studies showed that TAMV is the prototypic virus of new phylogenetic Tamdy group in the Nairovirus genus. Thus, BURV was classified as a new virus of the Tamdy group, Nairovirus, Bunyaviridae.

The Syr-Darya valley fever virus (SDVFV) was originally isolated from the blood of the patient with fever in the Kyzylorda province, Kazakhstan, in July 1973 and was classified to the Cardiovirus genus (fam. Picornaviridae). Later, SDVFV was isolated from the ticks Hyalomma as. asiaticum Schulze et Schlottke, 1929 (Hyalomminae) (1 strain) and Dermacentor daghestanicus Olenev, 1929 (Rhipicephalinae) (7 strains), collected in the floodplains of the Syr-Darya river and the Ili river. In this paper, complet genome of the SDVFV (strain LEIV-Tur2833) was sequenced using the next-generation sequencing approach (GenBank ID: KJ191558). It was demonstrated that, phylogenetically, the SDVFV is closely related closest to the Theiler's murine encephalomyelitis virus (TMEV) and Vilyuisk human encephalomyelitis virus (VMEV). The similarity of the SDVFV with VHEV and TMEV based on P1 region of the polyprotein-precursor (structural proteins VP1-VP4), reaches 75% and 91% for nucleotide sequences and 80% and 93% for putative amino acid sequences, respectively. For nonstructural proteins regions P2 (2A-2C) and P3 (3A-3D) similarity of SDVFV with TMEV and VHEV is 96%-98%.

The Artashat virus (ARTSV) was originally isolated fom the Ornithodoros alactagalis Issaakjan, 1936 (Argasidae Koch, 1844), which were collected in the burrow of small five-toed jerboa (Allactaga elater Lichtenstein, 1825) in Armenia in 1972. Later, the ARTSV was isolated from the O. verrucosus Olenev, Sassuchin et Fenuk, 1934 collected in the burrows of Persian gerbil (Meriones persicus Blanford, 1875) in Azerbaijan. Based on the virion morphology, the ARTSV was assigned to the Bunyaviridae viruses. In this work, the ARTSV genome was partially sequenced (GenBank ID: KF801650) and it was shown that the ARTSV is a new member of the Nairovirus genus. ARTSV has from 42% (Issyk-Kul virus) to 58% (Raza virus, Hughes group) similarity with the nairoviruses for nucleotide sequence of part of RNA-dependent RNA-polymerase (RdRp). The similarity on the amino acid level is 65-70%. Low level of homology and the equidistant position of the ARTSV on phylogenetic tree indicate that the ARTSV is a new prototype species of the Nairovirus genus (Bunyaviridae) forming a separate phylogenetic branch.

Full-length genome of the Chim virus (CHIMV) (strain LEIV-858Uz) was sequenced using the next-generation sequencing approach (ID GenBank: KF801656). The CHIMV/LEIV-858Uz was isolated from the Ornithodoros tartakovskyi Olenev, 1931 ticks collected in the great gerbil (Rhombomys opimus Lichtenstein, 1823) burrow in Uzbekistan near Chim town (Kashkadarinsky region) in July of 1971. Later, four more CHIMV strains were isolated from the O. tartakovskyi, O. papillipes Birula, 1895, Rhipicephalus turanicus Pomerantsev, 1936 collected in the great gerbil burrows in Kashkadarinsky, Bukhara, and Syrdarya regions of Uzbekistan, and three strains--from the Hyalomma asiaticum Schulze et Schlottke, 1930 from the great gerbil burrows in Dzheskazgan region of Kazakhstan. The virus is a potential pathogen of humans and camels. The phylogenetic analysis revealed that the CHIMV is a novel member of the Nairovirus genus (Bunyaviridae) and closely related to the Qalyub virus (QYBV), which is prototype for the group of the same name. The amino acid homology between the CHIMV and QYBV is 87% for the RdRp catalytic center (L-segment) that is coincident with both QYBV and CHIMV associated with the Ornithodoros ticks and burrow of rodents as well. The CHIMV homologies with other nairoviruses are 30-40% for the amino acid sequences of precursor polyprotein GnGc (M-segment), whereas 50%--for the nucleocapsid N (S-segment). The data obtained permit to classify the CHIMV as a member of the QYBV group in the genus of Nairovirus (Bunyaviridae).

Full-length genomes of the Sakhalin virus (SAKH) and Paramushir virus (PRMV) (Sakhalin group, Nairovirus, Bunyaviridae) isolated from the ticks Ixodes uriae White 1852 were sequenced using the next-generation sequencing (Genbank ID: KF801659, KF801656). SAKV and PRMV have 81% identity for the part of RNA-dependent RNA-polymerase (RdRp) on the nucleotide level and 98.5% on the amino acid level. Full-length genome comparison shows that SAKV have, in average, from 25% (N-protein, S-segment) to 50% (RdRp, L-segment) similarity with the nairoviruses. The maximum value of the amino acid similarity (50.3% for RdRp) SAKV have with the Crimean-Congo hemorrhagic fever virus (CCHFV) and Dugbe virus (DUGV), which are also associated with the Ixodidae ticks. Another virus studied is Rukutama virus (RUKV) (isolated from ticks I. signatus Birulya, 1895) that recently was classified (based on morphology and antigenic reaction) to the Nairovirus genus, presumably to the Sakhalin group. In this work the genome of the RUKV was sequenced (KF892052-KF892054) and RUKV was classified as a member of the Uukuniemi group (Phlebovirus, Bunyaviridae). RUKV is closely related (93.0-95.5% similarity) with our previously described Komandory virus (KOMV). RUKV and KOMV form separate phylogenetic line neighbor of Manawa virus (MWAV) isolated from the ticks Argas abdussalami Hoogstraal et McCarthy, 1965 in Pakistan. The value of the similarity between RUCV and MWAV is 65-74% on the amino acid level.

The Tyulek virus (TLKV) was isolated from the ticks Argas vulgaris Filippova, 1961 (Argasidae), collected from the burrow biotopes in multispecies birds colony in the Aksu river floodplain near Tyulek village (northern part of Chu Valley, Kyrgyzstan). Recently, the TLKV was assigned to the Quaranfil group (including the Quaranfil virus (QRFV), Johnston Atoll virus (JAV), Lake Chad virus) that is a novel genus of the Quaranjavirus in the Orthomyxoviridae family. In his work, the complete genome (ID GenBank KJ438647-8) sequence of the TLKV was determined using next-generation sequencing (Illumina platform). Comparison of deduced amino acid sequences shows closed relationship of the TLKV with QRFV and JAV (86% and 84% identity for PB1 and about 70% for PB2 and PA, respectively). The identity level of the TLKV and QRFV in outer glycoprotein GP is 72% and 80% for nucleotide and amino acid sequences, respectively. The phylogenetic analysis showed that the TLKV belongs to the genus of the Quaranjavirus in the family Orthomyxoviridae.

Molecular-genetic characteristics of the Okhotskiy virus (OKHV) and Aniva virus (ANIV) were studied (ID GenBank KF981623-32). These viruses are distributed over the shelf and Island areas in the high latitudes in the Okhotsk, Bering, and Barents seas and linked with nesting colonies of Alcidae seabirds and their obligatory parasites, the Ixodes uriae (Ixodidae) ticks. OKHV and ANIV are observed to be independent species within the limits of the Great Island virus (GIV) group of the Orbivirus genus of the Reoviridae family. The majority of the genes of OKHV and ANIV have high homology (VP1 - 96%, T2 - 99%, VP7 (T13) - 98%, NS1 - 94%, NS2 - 98%, NS3 - 72%, VP6 - 93%). Nevertheless, the envelope proteins containing the main specific antigenic determinants (VP2 and VP5) of OKHV and ANIV are sufficiently different (62% and 68% homology for amino acid sequences, respectively).

The prototype strain LEIV-K306 of the Batken virus (BKNV) was isolated from the Ixodidae ticks Hyalomma marginatum Koch, 1844 collected from sheep near town Batken (Kirgizstan) in the April 1970. Later, the BKNV was isolated in Kirgizstan from the mixed pool of the Aedes caspius Pallas, 1771 and Culex hortensis Ficalbi, 1889 mosquitoes. From the very beginning, the BKNV was discussed to be very close to the Dhori virus (DHOV) (Orthomyxoviridae, Thogotovirus) isolated from the Ixodidae ticks Hyalomma dromedarii Koch, 1844 in India. In this work, virtually complete genome sequence (MiSeq, Illumina) of the BKNV was determined (ID GenBank KJ396672-4). Structural and non-structural proteins of the BKNV have a high level of homology with DHOV - 98% (PB1) and 96% (PB2, PA, NP, M). Homology of HA protein between the BKNV and DHOV is 90%, which accounts for antigenic difference between these close relative viruses. Since the differences in the other structural and non-structural proteins are about 96-98%, the BKNV could be suggested as the topotypic DHOV strain for Central Asia, Transcaucasia, and Northern Caspian region. The evolution divergence of the BKNV and DHOV for HA could be explained by local ecologic peculiarities of the BKNV areal.

Complete genome sequencing of three Tamdy (TAMV) virus strains was carried out. The prototype strain TAMV/LEIV-1308Uz was isolated for the very first time from the Hyalomma asiaticum asiaticum Schülce et Schlottke, 1929 (Ixodidae, Hyalomminae) collected in the August 1971 from sheep in the arid area near Namdybulak town (41 degrees 36' N, 64 degrees 39' E) in the Tamdinsky district of the Bukhara region (Uzbekistan). TAMV was revealed to be a prototype member of the new phylogenetic group within the limits of the Nairovirus. The TAMV homology for RdRp (L-segment) amino acid sequence is not less than 40% with Crimea-Congo hemorrhagic fever virus (CCHFV), Hazara virus (HAZV), and Dugbe virus (DUGV), which are also linked with Ixodidae ticks. The TAMV homologies with the Issyk-Kul virus (ISKV) and Caspiy virus (CASV) for RdRp are 37.6% and 37.7%, respectively. These data conformed to the low values of GnGc (M-segment) and nucleocapsid protein N (S-segment) homology. The TAMV homologies with the nairoviruses for GnGc is in average 25%; with the nairoviruses linked with Ixodidae ticks (CCHFV, DUGV, HAZV) - 33%; with Argasidae ticks (ISKV, CASV) - 28%. The TAMV/LEIV-1308Uz, LEIV-6158Ar, and LEIV-10226Az have high level of identity. The TAMV/LEIV-10226Az from Azerbaijan has 99% homology for both nucleotide and amino acid sequences of the prototype TAMV/LEIV-1308Uz RdRp. The TAMV/LEIV-6158Ar from Armenia is more divergent and has 94.2% and 96.3% homologies with the TAMV/LEIV-1308Uz, respectively. The homology between the TAMV/LEIV-1308Uz and TAMV/LEIV-10226Az for GnGc is 93%. The TAMV/LEIV-6158Ar has 90% homology for this protein with the TAMV/LEIV-1308Uz and 93% with the TAMV/LEIV-10226Az, respectively. Differences in nucleocapsid protein between three TAMV strains are 5-7%.

Complete genome sequencing of the Kyzylagach virus (KYZV) LEIV-65A strain isolated from the Culex modestus Ficalbi, 1889 (Culicinae), which was collected in the colony of the Ardeidae Leach, 1820 birds on the coast of Caspian sea, Kyzyl-Agach bay, in the southern part of Azerbaijan, was carried out. KYZV has high homology (about 99%) with the Chinese XJ-160 strain of the Sindbis virus (SINV) isolated from Anopheles sp. in Xinjiang Uyghur autonomic region (north-eastern China). Homologies of KYZV and XJ-160 with European SINV strains are 82% and 93% for the nucleotide and amino acid sequences, respectively (GenBank ID: KF981618). The difference between the nucleotide sequences of KYZV and Australian SINV/SW6562 strain is 19%; amino acid sequences, 12%. Since XJ-160 strain is extremely similar to KYZV, the first could be considered as the KYZV strain. The geography of the KYZV and XJ-160 isolation points and their genetic distance from the European viruses allow the KYZV to be suggested as a SINV (genotype IV) topotypic variant typical of Transcaucasia and Central Asia.

The complete genome of Uzun-Agach virus (UZAV), isolated from the liver of Myotis blythii oxygnathus (Monticelli, 1885 (Chiroptera; Vespertilionidae)) bats in Alma-Ata district (Kazakhstan) in 1977 have been sequenced. Based on full-length genome comparison it is shown that UZAV is a new member of the Nairovirus genus (family Bunyaviridae). L-segment and M-segments of UZAV have 69,3% and 64,1% identity with Issyk-Kul virus (ISKV) that also was isolated from bats. S-segment of UZAV have 99,6% identity with the same of ISKV. This allow us to claim that UZAV is a reassortant virus that have S-segment derived from ISKV, and L- and M-segments from another virus that is phylogenetically related to ISKV, but divergent from it. The obtained data that the reassortment between ISKV and UZAV exists in nature suggest that they cocirculated in one ecological niche (bats of the Vespertilionidae family) and the areal of UZAV may coincide with the areal of ISKV.

Genetic research into the Tyuleniy virus (TYUV) (ID GenBank KF815939) isolated in high latitudes from the Ixodes uriae White, 1852, ticks collected in the nesting colonies of the Alcidae (Leach, 1820) birds and Kama virus (KAMV) (ID GenBank KF815940) isolated from the I. lividus ticks collected in the nesting bird colonies in the middle part of the Russian Plane was carried out. Full-genome comparative analysis revealed 70% homology between KAMV and TYUV on the nucleotide level and 74% on the amino acid level. Thus, KAMV is a new member of the TYUV complex belonging to the seabird tick-borne virus group (STBVG) of Flavivirus (Flaviviridae). KAMV is a separate virus and forms separate phylogenetic line together with the TYUV, Meaban virus (MEAV), and Saumarez Reef virus (SREV).

The complete genomes of the three tick-borne flaviviruses (genus Flavivirus, fam. Bunyaviridae) were sequenced: Povassan virus (POWV, strain LEIV-3070Prm, isolated from Haemophysalis logicornis in Primorsky Krai, Russia in 1977), Alma-Arasan virus (AAV, strain LEIV-1380Kaz, isolated from Ixodes persulcatus ticks in Kazakhstan in 1977) and Malyshevo virus (isolated from a pool of Aedes vexans nipponii mosquitoes, in the Khabarovsk Krai, Russia in 1978). It is shown that AAV and Malyshevo virus are the strains of Tick-borne encephalitis virus (TBEV) and belong to Sibirian and Far-Eastern genotypes, respectively (GenBank ID: AAV KJ744033; strain Malyshevo KJ744034). Phylogenetically AAV is closest related (94,6% nt and 98,3% aa identity) to TBEV strains, isolated in Sibiria (Vasilchenko, Aino, Chita-653, Irkutsk-12). Malyshevo virus is closest related (96,4% nt and 98,3% nt identity) to strains of TBEV, isolated in Far Eastern part of Russia (1230, Spassk-72, Primorye-89). POWV LEIV-3070Prm has 99.7% identity with the prototype strain POWV LB, isolated in Canada and 99.5% of isolates with Far-Eastern strains of POWV (Spassk-9 and Nadezdinsk-1991).

Complete genome sequences were obtained for the LEIV-13841Ka (ID GenBank KF767463-65) and LEIV-279Az (ID GenBank KF767460-62) virus strains, which were classified as different strains of the Zaliv Terpeniya virus (ZTV). LEIV-13841Ka was isolated from the ticks Ixodes (Ceratixodes) uriae White, 1852 collected on Ariy Kamen (Commander Islands) in 1986. LEIV-279Az was isolated from the mosquitoes Culex modestus Ficalbi, 1889, collected in heron colony (Ardea Linnaeus, 1758) in Azerbaijan (1969) and was initially identified as Uukuniemi virus (UUKV). According to the results obtained LEIV-279Az is ZTV strain as well. LEIV-13841Ka and LEIV-279Az RdRp sequences have high level of homology (99%) with previously sequenced ZTV/LEIV-271Ka. The L-segment nucleotide sequences are homological with ZTV/LEIV-271Ka on the level of 94% and 98% for LEIV-13841Ka and LEIV-279Az, respectively; M-segment--89% and 88%, respectively. Such homologies for the amino acid sequences of Gn/Gc polyprotein are 98.3% and 97.7%. NP proteins of ZTV/LEIV-13841Ka and LEIV-279Az have 88.7% and 84.6% homologies with ZTV/LEIV-271Ka both for amino acid and nucleotide sequences, respectively. Thus, for the very first time we demonstrated ZTV strain isolated from mosquitoes in subtropical Transcaucasia zone. Obtained results permit to expand suggested areal of ZTV and to fill up data upon the ecology of the Uukuniemi virus group.

The full-length genome of the unclassified Geran virus (GERV, strain LEIV-10899Az) isolated from the ticks (Ornithodoros verrucosus Olenev, Zasukhin and Fenyuk, 1934 (Argasidae, Ornithodorinae)) collected in the burrow of the red-tailed gerbils (Meriones (Cricedidae) erythrourus Grey, 1842) near the Geran station (Azerbaijan) was sequenced using the next-generation approach (GenBank ID: KF801649). It was shown that the GERV is a new representative of the Nairovirus genus (family Bunyaviridae). The comparative analysis of the full-length genome sequences of the GERV with other nairoviruses showed that the highest level of homology (55.6% for N protein (S-segment) of 54.2% for the polyprotein Gn/Gc (M-segment) and 74.8% for the RNA-dependent RNA polymerase (L-segment)) GERV had with the Chim virus (CHIMV) that is also associated with the shelters biocenoses (rodent burrows) in Central Asia and was previously assigned to the Qalyub virus group (QYBV). Comparing the GERV with the QYBV sequences (partial sequence 413 n.o. of RdRp gene) revealed a high level of homology: 74.3 and 97.4% for the nucleotide and amino acid sequences, respectively. The data obtained in this work provided an opportunity to classify the GERV to the QYBV group; the Nairovirus genus, to the family Bunyaviridae.

Complete genome sequencing of the Sokuluk virus (SOKV) isolated in Kyrgyzstan from bats Vespertilio pipistrellus and their obligatory parasites--Argasidae Koch, 1844, ticks was carried out. SOKV was classified as attributed to the Flaviviridae family, Flavivirus genus. The maximum homology (71% for nucleotide and 79% for amino acid sequences) was detected with respect to the Entebbe bat virus (ENTV). ENTV and SOKV form a group joining to the yellow fever virus (YFV) within the limits of the mosquito flavivirus branch. Close relation of SOKV with bat covers and human housings permits to assume SOKV potentially patogenic to human health.

Full-genome sequencing of the Caspiy virus (CASV - Caspiy virus) (ID GenBank KF801658) revealed its attribution to the Nairovirus genus of the Bunyaviridae family as a separate species. CASV forms separate line, which is the most close to the Hughes virus (HUGV) and Sakhalin virus (SAKV) groups containing viruses linked with seabirds and ticks parasitizing on them and distributed over the shelf and island ecosystems in the Northern Eurasia, as well as the North and South America.

The Baku virus (BAKV) was originally isolated from the ticks Ornithodoros capensis Neumann, 1901 (Acari: Argasidae) collected from the seagull (Larus argentatus) seating nests on the islands of the Baku archipelago, the Caspian sea. BAKV was assigned to Kemerovo group (KEMV) (Orbivirus, Reoviridae). The BAKV was frequently isolated from the ticks O. coniceps Canestrini, 1980, collected from L. argentatus and tern (Sterna hirundo) nests in Turkmenia and pigeon (Columba livia neglecta) nests in Uzbekistan. In this work, the genome of the BAKV was sequenced using the next-generation sequencing technology. The BAKV Pol protein has 48.6% identity level with the viruses of the Great Island Virus group and at average 41% with non-tick orbiviruses. The BAKV T2 protein level identity with the orbiviruses ranges from 23.7% to 64.8%. The maximum identity level of the T2 protein (64.8%) is observed for the tick-borne viruses of the GIV (KEMV) group. According to the conducted molecular-genetic and phylogenetic analysis, the BAKV is a novel species of the genus Orbivirus. It forms a phylogenetic group distinctly related to the GIV group.

Unclassified virus named Komandory virus (KOMV) isolated in Komandorski islands from the ticks Ixodes uriae in 1986 was partially sequenced. The KOMV nucleocapsid (N) protein sequence shows 30-40% identity level with the mosquito-borne phleboviruses and 43% with the Uukuniemi virus (UUKV) group (Phlebovirus, Bunyaviridae). The maximum identity (65%) for the KOMV N protein is observed for the Manawa virus. The KOMV glycoprotein identity with the UUKV group viruses ranges from 45% to 59%. The KOMV RdRp identity with the Manawa virus reaches 74%, while showing 63% level at average with the other UUKV group viruses. According to the results of molecular-genetic and phylogenetic analysis, the KOMV is a new member of the UUKV group (Phlebovirus, Bunyaviridae).

The Issyk-Kul virus (etiological agent of the Issyk-Kul fever) was originally isolated from bats (Nyctalus noctula Schreber, 1774 (Chiroptera: Vespertilionidae)) and their parasites ticks (Argas (Carios) vespertilionis Latreille, 1796 (Parasitiformes: Argasidae)) in Kirghizia. Sporadic cases and epidemics of the Issyk-Kul fever are observed in Central Asia since 1979. The ISKV genome was de novo sequenced using the next-generation sequencing technology. According to the molecular-genetic and phylogenetic analysis, the ISKV is a member of a novel group in the genus Nairovirus (Bunyaviridae). Based on the data obtained, molecular-genetic methods can be used for ISKV detection (PCR) for the Issyk-Kul fever monitoring and diagnosis in the endemic areas.

The Khasan virus (KHAV) was originally isolated in Khasansky District and Maritime Territory in 1971 from the ticks Haemophysalis longicornis (Neumann, 1901) collected from the deers Cervus nippon (Temmink, 1838). Based on the biological properties and virion morphology, KHAV was identified as an unclassified member of the Bunyaviridae family. In order to elucidate the KHAV taxonomy in more detail, viral genome was partially sequenced using the next-generation sequencing technology. According to the phylogenetic analysis conducted for partial sequences of the three genome segments, KHAV was attributed to the genus Phlebovirus. KHAV is phylogenetically mostly related to the viruses of the Uukuniemi group and has up to 62% identity with them. The maximum identity level is observed for sequences of the RNA-dependent-RNA-polymerase (RdRp) gene. The KHAV homology level with the tick-borne Uukuniemi group viruses is 50 to 62%; however, for the mosquito-borne phleboviruses it does not exceed 30%.

Unidentified Khurdun virus (KHURV) was isolated in 2001 from coot (Fulica atra, Linnaeus, 1758) in the Volga River delta (Astrakhan Region, Russian Federation). Here we report that the KHUV genome was de novo sequenced (on Illumina platform) and the KHURV was classified as a novel prototypic bunyavirus. The KHURV genome comprises three negative-sense RNA segments (L, M, and S); its terminal nucleotide sequences are canonical for the Orthobunyavirus genus. Based on the results of the molecular-genetic and phylogenetic analysis we suggest that the KHURV belongs to the genus Orthobunyavirus (Bunyaviridae).

The gene composition of the viral population obtained via mixed infection of embryonated chick eggs with influenza viruses at a high multiplicity of infection was analyzed. In mixed infection caused by influenza A/WSN/33 (H1N1) and A/Duck/Czechoslovakia/56 (H4N6) viruses, the population showed a preponderance of the reassortants whose content of genomic segments originating from either of the parent virus deviated strongly from the random pattern: the hemagglutinin (HA) gene of A/WSN/33 (H1N1) virus and the NP gene of A/Duck/Czechoslovakia/56 (H4N6) virus were prevalent in the gene composition of the reassortants. The mixed infection produced by influenza A/Udorn/72 (H3N2) virus and the reassortant R8 containing the HA gene of A/Duck/Ho Chi Minh/014/78 (H5N3) virus, the population of reassortants contained mainly the HA gene of A/Udorn/72 (H3N2) virus and the NP gene of the reassortant R8. The findings are discussed due to the problem of specific recognition of gene segments when incorporated into the viral particles.

Avian influenza A viruses belonging to hemagglutinin (HA) subtypes H5 and H6 were studied in the infectivity neutralization test and radioimmunoprecipitation assay (RIPA) with monoclonal antibody MAb C179. This MAb recognizes a conformational antigenic epitope in the stem region of HA formed by two regions (amino acid positions 318-322 in HA1 subunit and 47-58 in HA2), conserved in all H1 and H2 influenza viruses. MAb C179 reacts with HA of H5 viruses in RIPA and neutralizes these strains as efficiently as H2 viruses. C179 precipitates H6 subtype HA but does not neutralize the infectivity of these viruses. Comparison of amino acid sequences of H2, H5, and H6 strains showed identical epitope recognized by MAb C179 in H5 and H6 HAs, which differs from epitopes of H1 and H2 by two amino acids in the HA2 subunit. Causes of disagreement between immunoprecipitation of H6 HA by MAb C179 and neutralization of this serosubtype by this MAb are discussed.

Avian influenza A virus with H2 hemagglutinin has been adapted to mice for the first time. Alterations in the hemagglutinin of adapted variants of the virus as a result of adaptation to a new host are described. Hemagglutinin of a highly virulent adapted variant differed from the parental avirulent strain by antigenic structure, electrophoretic mobility, and receptor activity during interactions with murine red cells.

In the cytoplasms of chick embryo fibroblast and Ehrlich ascitic carcinoma cells infected with influenza virus (fowl plague virus), in addition to fragmented virus nucleocapsid larger nucleocapsid structures were found which sedimented in the region of 90 -120S. The structures were detected upon short 3H-uridine label of the cells. Their buoyant density in cesium chloride was higher than that of the fragmented nucleocapsid (1.34 -1.39 g/cm3). In electron microscope, the structures were visualized as thin nonhelical filaments 3.5 nm in diameter, their morphology being no different from that of a similar rapidly sedimenting structure isolated from the nucleoplasms of the same cells. To determine the possibility of transfer of the rapidly sedimenting structure from the nucleus into the cytoplasm, a cell-free system was used containing nuclei from influenza virus-infected cells labeled with 3H-uridine for 5 min, as well as the cytoplasm from uninfected and unlabeled cells. The presence of a labeled rapidly sedimenting structure in the cytoplasm of the cell-free system suggests that the structure is synthesized in the nucleus and then transported into the cytoplasm. The relation of this structure to the fragmented nucleocapsid is unknown. It may be assumed to be its intracellular precursor.

The sedimentation and density properties of ribonucleoproteins were studied with different strains of influenza type A virus: A/turkey/Wisconsin/66, A/swine/Iowa/15/31, A/horse/Prague/56, antigenic (A/H3Neq1) and biological (MRC-11) recombinants of the A/Port-Chalmers/1/73 strain. In all the viruses under study, ribonucleoprotein (RNP) was found to be heterogeneous and to sediment in a zone of 30-70 S. Variability in the distribution of fragments in different strains was revealed. In A/turkey/Wisconsin/66 and MPC-11 strains the highest radioactivity was found in the zone of "heavy" fragments (60-70 S), in A/horse/Prague/56 and A/swine/Iowa/15/31 strains it was prevalent in the zone of 40-50 S. In recombinant A(H3Neq1) the distribution of radioactivity varied in different tests. The buoyant density in cesium chloride in all RNP compenents was found to be similar, 1.34-1.36 g/ml.

Radioimmunoassay were carried out with influenza A/Cambridge/46, A/FM/1/47, A/England/1/51, A/Denwer/1/57, and A/USSR/90/79 of the H1N1 subtype to determine the antigenic determinants of influenza A/USSR/90/77 virus hemagglutinin. The test system included 125I-labeled hemagglutinin of A/USSR/90/77 virus and homologous antiserum to this virus. The most marked antigenic similarity was found between A/USSR/90/77 and A/USSR/90/79 viruses. The A/Cambridge/46 and A/Denwer/1/57 viruses showed clear-cut differences in the antigenic determinants from the A/USSR/90/77 virus.

Competitive radioimmunoassay in the homologous system using 125I-labeled neuraminidase (NA) of A/Japan/305/57 virus and antibodies to it showed that influenza viruses NA of subtypes H2N2 (A/Singapore/1/57 and A/Tokyo/3/67) and H3N2 (A/Aichi/2/68, A/England/42/72, A/Port Chalmers/1/73, and A/Texas/1/77) had undergone clear antigenic change despite the presence of Japan strain NA determinant. The use of a heterologous system (125I-NA of the Japan strain--antibodies to NA of England/42/72 strain) for the study of the intratype determinant showed the presence of a common determinant in all the strains under study, this determinant being represented dissimilarly, however. Neuraminidase of the Texas strain has a determinant differing from the intratype one, common for the Japan and England viruses.

The intracellular influenza virus-containing structures involved in RNA synthesis in the cytoplasm and in the nucleoplasm of infected chicken fibroblasts were studied. Two approaches were used: (1) short pulse labeling of infected cell with [3H]uridine; (2) determination in vitro of polymerase activity of intracellular virus-specific structures. Both methods revealed functionally active virus-specific structures in the nucleoplasm and showed that a functionally active virus-specific structure was localized in the nucleoplasm of infected cells. This structure contained proteins of the viral ribonucleoprotein, but sedimented somewhat faster (at 60--90S in velocity sucrose and glycerol gradients). Meanwhile, polymerase-containing structures in the cytoplasm of infected cells sedimented in the position of viral ribonucleoproteins (25--60S).

Non-infectious virus particles produced by influenza virus (classical fowl plague)-infected Ehrlich ascitic carcinoma cells have the same morphology, size and sedimentation rate as the standard virions. Their main difference from the allantoic virus is their extreme fragility. They remain intact upon a short-term centrifugation in sucrose solutions but desintegrate upon prolonged centrifugation. In isopicnic fractionation they are detected in two forms, with a density of 1.23 g/ml retaining the hemagglutinating but not the neuraminidase activity and with a density of 1.27 g/ml deprived of both hemagglutinating and neuraminidase activity. In the electron microscopic examination the 1.23 g/ml structure appears as virus particles with defective areas in the surface spikes layer whereas the 1.27 g/ml structure has no spike layer at all. The protein analysis by polyacryl amide gel electrophoresis revealed a sharply reduced amount of the membrane protein in the ascitic virus. A hypothesis is suggested according to which the reduced amount of the membrane protein is the cause of the unusual fragility of ascitic virus particle membranes as a result of which they readily lose glycoproteins which, in its turn, leads to their reduced infectious activity.

Influenza virus hemagglutinin of H3 type was isolated using the proteolytic enzyme bromlaine. The isolated hemagglutinin was shown to have no neuraminidase activity and to contain no admixtures of extraneous proteins. This antigen was used to immunize rabbits and the resulting antiserum had high antibody titers in HI tests. The method of precipitation with the serum of labeled viral proteins showed the serum to be monospecific and to contain antibody to the hemagglutinin alone.

A test system for the detection and quantitation of influenza virus hemagglutinin (HA) in biological specimens by solid-phase enzyme immunoassay (SPEIA) is described. The test system implies sequential adsorption on polystyrene base of anti-HA guinea pig gammaglobulins, detectable HA-containing antigen, monospecific anti-HA rabbit serum, and peroxidase antirabbit conjugate. Adsorption of the HA-containing antigen is run in the presence of a high concentration of nonionic detergent. The developed method is highly sensitive (0.3-0.5 ng in a specimen) and permits the detection and quantitation of HA in whole influenza virions, preparations of surface glycoproteins and in preparations of hemagglutinin recovered from virus particles. The analysis (after pre-sensitization of the polystyrene adsorbent) takes from 2 to 21/2 hours. The possibility of using the developed test system for control and standardization of inactivated influenza vaccines (whole-virion, split, and subunit) is discussed.

In the course of classical fowl plague virus reproduction in Ehrlich ascites carcinoma cells both hemagglutinins and S-antigen accumulate and titers of the infectious activity increase. However virus reproduction does not terminate in formation of virus, and subviral structures are found in the liquid fraction of the infected cells. Analysis of these structures has shown them to have a sedimentation coefficient of 350-370S and buovant density 1.29 g/ml. The rapidly sedimenting structure has complement-fixing hemagglutinating activity but bow infectivity.