[Analysis of Tat protein characteristics in human immunodeficiency virus type 1 sub-subtype A6 (Retroviridae: Orthoretrovirinae: Lentivirus: Human immunodeficiency virus-1)].

INTRODUCTION: Tat protein is a major factor of HIV (human immunodeficiency virus) transcription regulation and has other activities. Tat is characterized by high variability, with some amino acid substitutions, including subtypespecific ones, being able to influence on its functionality. HIV type 1 (HIV-1) sub-subtype A6 is the most widespread in Russia. Previous studies of the polymorphisms in structural regions of the A6 variant have shown numerous characteristic features; however, Tat polymorphism in A6 has not been studied.Goals and tasks. The main goal of the work was to analyze the characteristics of Tat protein in HIV-1 A6 variant, that is, to identify substitutions characteristic for A6 and A1 variants, as well as to compare the frequency of mutations in functionally significant domains in sub-subtype A6 and subtype B. MATERIAL AND METHODS: The nucleotide sequences of HIV-1 sub-subtypes A6, A1, A2, A3, A4, subtype B and the reference nucleotide sequence were obtained from the Los Alamos international database. RESULTS AND DISCUSSION: Q54H and Q60H were identified as characteristic substitutions. Essential differences in natural polymorphisms between sub-subtypes A6 and A1 have been demonstrated. In the CPP-region, there were detected mutations (R53K, Q54H, Q54P, R57G) which were more common in sub-subtype A6 than in subtype B. CONCLUSION: Tat protein of sub-subtype A6 have some characteristics that make it possible to reliably distinguish it from other HIV-1 variants. Mutations identified in the CPP region could potentially alter the activity of Tat. The data obtained could form the basis for the drugs and vaccines development.

Serological diagnosis and prevalence of HIV-1 infection in Russian metropolitan areas.

BACKGROUND: HIV infection is a major health problem in Russia. We aimed to assess HIV prevalence in different population groups and to compare the characteristics of 4th generation immunoassays from Abbott, Bio-Rad, Vector-Best, Diagnostic Systems, and Medical Biological Unit. METHODS: The study included 4452 individuals from the general population (GP), 391 subjects at high risk of HIV infection (HR) and 699 with potentially interfering conditions. HIV positivity was confirmed by immunoblot and by HIV RNA, seroconversion and virus diversity panels were also used. HIV avidity was employed to assess recent infections. RESULTS: The prevalence in GP was 0.40%, higher in males (0.62%) and in people aged < 40 years (0.58%). Patients attending dermo-venereal centers and drug users had a high prevalence (34.1 and 58.8%). Recent infections were diagnosed in 20% of GP and in 4.2% of HR. Assay sensitivity was 100% except for one false negative (99,54%, MBU). Specificity was 99.58-99.89% overall, but as low as 93.26% on HR (Vector-Best). Small differences on early seroconversion were recorded. Only the Abbott assay detected all samples on the viral diversity panel. CONCLUSION: HIV infection rate in the high-risk groups suggests that awareness and screening campaigns should be enhanced. Fourth generation assays are adequate but performance differences must be considered.

[Analysis of HIV-1 (Human immunodeficiency virus-1, Lentivirus, Orthoretrovirinae, Retroviridae) Nef protein polymorphism of variants circulating in the former USSR countries.]

INTRODUCTION: The human immunodeficiency virus (HIV) Nef protein is one of the key factors determining the infectivity and replicative properties of HIV. With the ability to interact with numerous proteins of the host cell, this protein provides the maximum level of virus production and protects it from the immune system. The main activities of Nef are associated with a decrease in the expression of the CD4 receptor and major histocompatibility complex class I molecules (MHC-I), as well as the rearrangement of the cytoskeleton. These properties of the protein are determined by the structure of several motifs in the structure of the nef gene encoding it, which is quite variable. OBJECTIVES: The main goal of the work was to analyze the characteristics of Nef protein of HIV-1 variant A6, which dominates in the countries of the former USSR. The objective of the work was a comparative analysis of natural polymorphisms in the nef gene of HIV-1 sub-subtypes A6 and A1 and subtype B. MATERIAL AND METHODS: The sequences of the HIV-1 genome obtained during the previous work of the laboratory were used, as well as the reference sequence from GenBank. In this work, Sanger sequencing and new generation sequencing methods, as well as bioinformation analysis methods were used. RESULTS AND DISCUSSION: The existence of noticeable differences in the prevalence of Nef natural polymorphisms (A32P, E38D, I43V, A54D, Q104K, H116N, Y120F, Y143F, V168M, H192T, V194R, R35Q, D108E, Y135F, E155K, E182M, R184K and F191L), some of which are characteristic mutations for variant A6, was shown. CONCLUSION: Characteristic substitutions were found in the Nef structure, potentially capable of weakening the replicative properties of HIV-1 variant A6.

Evaluating the accuracy and sensitivity of detecting minority HIV-1 populations by Illumina next-generation sequencing.

The accuracy and sensitivity of deep sequencing were assessed using viral standards (pNL4-3 and pLAI.2) of both DNA and RNA. The sequencing accuracy did not depend on the type of nucleic acid, but critically depended on the number of reads and threshold of sensitivity to minor viral populations. With coverage of more than 236 reads, the accuracy of viral RNA sequencing was equal to or exceeded 99.9%, with a sensitivity threshold to minor nucleotides of 20%. When the sensitivity threshold was below 1%, reduced accuracy dynamics were clearly visible even when the coverage was massive (more than 9.000 reads). It was found that the floating sensitivity threshold allowed the sequencing accuracy to be maintained at an acceptable level in cases of low coverage (less than 1.500-2.000) of reads. These results indicate the quality that can be expected with a specific number of reads and sensitivity threshold. Deep sequencing is a very powerful tool that can significantly improve the value of study results, but despite its superior performance, it should be used with caution regarding its sensitivity to minor populations below 1%.

[Molecular epidemiological analysis of HIV-1 variants circulating in Russia in 1987-2015]. / Molekuliarno-épidemiologicheskii analiz variantov VICh-1, tsirkulirovavshikh v Rossii v 1987-2015 gg.

AIM: To simultaneously analyze HIV-1 samples from all Russian regions to characterize the epidemiology of HIV infection in the country as a whole. SUBJECTS AND METHODS: The most extensive study was conducted to examine nucleotide sequences of the pol gene of HIV-1 samples isolated from HIV-positive persons in different regions of Russia, with the diagnosis date being fixed during 1987-2015. The nucleotide sequences of the HIV-1 genome were analyzed using computer programs and on-line applications to identify a virus subtype and new recombinant forms. RESULTS: The nucleotide sequences of the pol gene were analyzed in 1697 HIV-1 samples and the findings were that the genetic variant subtype A1 (IDU-A) was dominant throughout the entire territory of Russia (in more than 80% of all infection cases). Other virus variants circulating in Russia were analyzed; the phenomenon of the higher distribution of the recombinant form CRF63/02A in Siberia, which had been previously described in the literature, was also confirmed. Four new recombinant forms generated by the virus subtype A1 (IDU-A) and B and two AG recombinant forms were found. There was a larger genetic distance between the viruses of IDU-A variant circulating among the injecting drug users and those infected through heterosexual contact, as well as a change in the viruses of subtype G that caused the outbreak in the south of the country over time in 1988-1989. CONCLUSION: The findings demonstrate continuous HIV-1 genetic variability and recombination over time in Russia, as well as increased genetic diversity with higher HIV infection rates in the population.

[The first experience of application of standardized genotype technique of identification of HIV-tropism].

The antagonists of co-receptor CCR5 are an ultimately new class of preparations to treat HIV-infection. The mechanism of action of the preparations of this class is in the selective binding with co-receptor CCR5. This process results in the prevention of penetration of HIV into cell. Before prescribing the CCR5 antagonists the detection of viral tropism has to be done. Recently, in Russia the genotype technique of tropism detection was registered which can be used in clinical practice. The present article describes first experience of application of the given technique to clinical samples. The high correlation was established while comparing with the results of reference laboratory.

[The first break-through of the genotype 2.3.2 of high-virulence influenza A virus A/HSN1, which is new for Russia, in the Far East].

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.

[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.

[Isolation of influenza virus A (Orthomyxoviridae, Influenza A virus), Dhori virus (Orthomyxoviridae, Thogotovirus), and Newcastle's disease virus (Paromyxoviridae, Avulavirus) on the Malyi Zhemchuzhnyi Island in the north-western area of the Caspian Sea].

The paper presents the results of the 2003 and 2006 environmental virological monitoring surveys on the Malyi Zhemchuzhnyi Island where a large breeding colony of sea gull (Laridae) is located. In the past several years, expansion of cormorants (Phalacrocorax carbo) has enhanced the intensity of populational interactions. The investigators isolated 13 strains of influenza A virus (Orthomyxoviridae, Influenza A virus) subtype H13N1 (from sea gulls (n = 4), cormorants (n = 9) 1 strain of Dhori virus (Orthomyxoviridae, Thogotovirus) from a cormorantwith clinical symptoms of the disease, 3 strains of Newcastle disease virus (Paramyxoviridae, Avulavirus) from cormorants. RT-PCR revealed influenza A virus subtype H5 in 3.1% of the cloacal lavages from cormorants. Neutralization test indicated that sera from cormorants contained specific antibodies against West Nile (Flaviviridae, Flavivirus) (15.0%), Sindbis (Togaviridae, Alphavirus) (5.0%), Dhori (10.0%), and Tahini (Bunyaviridae, Orthobunyavirus) (5.0%); sera from herring gulls had antibodies against Dhori virus (16.7%); there were no specific antibodies to Inco (Bunyaviridae, Orthobunyavirus) and mountain hare (Lepus timidus) (Bunyaviridae, Orthobunyavirus) virus.

[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.

[Development of polymerase chain reaction-based test systems for influenza A virus isolation and typing].

Influenza viruses are spread worldwide and cause the disease in birds and mammals, including human beings. Moreover, birds are the natural reservoir of influenza A viruses. Early detection of newly emerging influenza viruses requires permanent monitoring. Traditional viral shedding methods using cell cultures and chicken embryos are time-consuming and laborious analysis when a large number of samples are examined. The paper describes the use of polymerase chain reaction-based test systems to detect influenza A virus and to differentiate its two subtypes: H5 and H7. The developed test systems were evaluated on 19 reference influenza A virus strains. Thereafter, more than 1500 samples from wild and domestic birds, collected in the Russian Federation in 2004-2006, were studied. The data of these test systems were compared with the techniques of viral shedding in the cell cultures and chicken embryos.

[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.

[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.