Qualitative real-time RT-PCR assay for nOPV2 poliovirus detection.

Based on the success of the Sabin2-based vaccine, a next-generation nOPV2 poliovirus vaccine has been developed. For epidemic monitoring and conducting epidemiological investigations, it is necessary to have a diagnostic assay with the ability to differentiate this variant from others. Here we describe such a real-time RT-PCR assay. The region with the cre insertion in the 5'-UTR was chosen as the target, and the limit of detection was 103 copies/mL (2.5×103 copies/mL using Probit analysis) determined using armored RNA particles. Sensitivity and specificity were 86.28 - 100 % and 76.84 - 100 %, respectively (with 95 % CI). Thus, this method can be effectively used when it is necessary to make a differential diagnosis of poliovirus strains.

[Experience of application of IgY-technology for laboratory diagnostics of viral infections.]

INTRODUCTION: The well-known advantages of class Y antibodies (IgY) from egg yolks of immunized hens in comparison with class G antibodies (IgG) of laboratory animals traditionally used in laboratory diagnosis of infectious diseases determine the stable interest of researchers in using IgY for these purposes (IgY technology). Over the past 20 years, the obvious benefits of IgY technology have been demonstrated for a number of viral and bacterial infections. Goals and objectives. Construction of ELISA systems based on specific IgY for laboratory diagnosis of infections caused by tick-borne encephalitis virus, yellow fever virus, poliovirus. MATERIAL AND METHODS: Obtaining yolk preparations of immunized chickens, obtaining highly purified IgY preparations (salting out, affinity chromatography), constructing ELISA systems for determining virus-specific antigens, testing the parameters of ELISA systems. RESULTS AND DISCUSSION: For the first time in laboratory practice, ELISA systems based on the use of specific polyclonal IgY were designed for laboratory diagnosis of topical human viral infections caused by flaviviruses and enteroviruses: determination of antigens of tick-borne encephalitis virus, yellow fever virus, 3 types of poliovirus. It was experimentally shown that these ELISA systems have high sensitivity and specificity, which allows them to be used for the semiquantitative and quantitative determination of antigens of these viruses in various materials (infected cell cultures, vaccines, etc.). CONCLUSION: The ELISA systems developed on the basis of specific IgY for determination of viral antigens can be effectively used for laboratory diagnosis of a number of viral infections, for the validation and control of vaccine preparations.

[Development of inactivated cultural yellow fever vaccine].

INTRODUCTION: The only currently available live vaccine against yellow fever (YF) based on chicken embryos infected with an attenuated 17D strain of the YF virus is one of the most effective vaccine preparations. However, the live vaccine is associated with "viscerotropic syndrome" (approximately 0.4 cases per 100 000 vaccinated). Therefore, the development and introduction of highly purified inactivated vaccine against YF is intended to ensure the maximum safety of vaccination against one of the most common human viral diseases.Goals and objectives. Development and evaluation of immunogenicity of the cultural inactivated vaccine against YF at the laboratory model level. MATERIAL AND METHODS: Adaptation of 17D strain of YF virus to Vero cell culture, cultivation, removal of cellular DNA, inactivation with ß-propiolactone, concentration, chromatographic purification, determination of protein and antigen of YF virus, assessment of immunogenicity in mice in parallel with commercial live vaccine. RESULTS AND DISCUSSION: Immunogenicity: the determination of specific antibodies of class G (IgG) and virus neutralizing antibodies in the sera of immunized mice showed high level of antibodies exceeding that of immunized with commercial live vaccine. The optimal dose of antigen in the vaccine (total protein) was 50 µg/ml (5 µg/0.1 ml -dose and volume per 1 vaccination of mice). Thus, the laboratory version of cultural inactivated vaccine against YF is as effective (and even superior) as the commercial live vaccine. CONCLUSION: Laboratory version of cultural inactivated vaccine against YF, which is not inferior in immunogenicity (in animal model) to commercial live vaccine, has been developed.

[A case of vaccine-associated paralytic poliomyelitis in an infant]. / Sluchai vaktsinoassotsiirovannogo paraliticheskogo poliomielita u rebenka.

AIM: To present the clinical history, vaccination status, features of the clinical picture, composition of the cerebrospinal fluid (CSF), results of laboratory and instrumental examinations of a patient with vaccine-associated paralytic poliomyelitis (VAPP). MATERIAL AND METHODS: In 2017, a child, aged 15 month, mistakenly vaccinated with the first dose of bivalent (types 1 & 3) polioviruses oral vaccine (OPV) was followed up. RESULTS AND CONCLUSION: Clinical parameters of VAPP in the recipient of OPV are considered. Clinical features of disease caused by wild poliovirus and VAPP are compared. The disease was characterized by sudden onset, recurrence, short (2-4 days) period of progression of paresis, persistent residual effects, CSF protein-cell dissociation. It is emphasized that the occurrence of VAPP cases reflects primarily immunization defects.

[VACCINE-ASSOCIATED PARALYTIC POLIOMYELITIS IN RUSSIAN FEDERATION DURING THE PERIOD OF CHANGES IN VACCINATION SCHEDULE (2006-2013 yy.)].

The results of virologic testing of clinical materials and epidemiological analysis of vaccine-associated paralytic poliomyelitis (VAPP) cases obtained in 2006-2013 during AFP surveillance are presented. Among the 2976 cases of AFP 30 cases were VAPP. 15 cases were observed in OPV recipients, whereas 15 cases were observed in non-vaccinated contacts. The age of the patients varied from 4 months to 5.5 years (13.6 ± 12.4 months old). Children younger than 1 year constituted 63.3% of the group; boys were dominant (73.3%); 53.3% of children were vaccinated with OPV; the time period between receipt of OPV and onset of palsy was from 2 to 32 days (18.7 ± 8.2). Lower paraparesis was documented in 48.3% of patients; lower monoparesis in 37.9%; upper monoparesis, in 6.9%; tetraparesis with bulbar syndrome, in 6%. The majority of the patients (85.7%) had an unfavorable premorbid status. The violations of the humoral immunity were found in 73.9% cases: CVID (52.9%), hypogammaglobulinemia (41.2%); selective lgA deflciency (5.9%). In 70.6% cases damage to humoral immunity was combined with poor premorbid status. The most frequently observed (76%, p < 0.05) represented the single type of poliovirus--type 2 (44%) and type 3 (32%). All strains were of the vaccine origin, the divergence from the homotypic Sabin strains fell within the region of the gene encoding VPI protein, which did not exceed 0.5% of nucleotide substitutions except vaccine derived poliovirus type 2--multiple recombinant (type 2/type 3/ type 2/type 1) with the degree of the divergence of 1.44% isolated from 6-month old unvaccinated child (RUS08063034001). The frequency of the VAPP cases was a total of 1 case per 3.4 million doses of distributed OPV in 2006-2013; 2.2 cases per 1 million of newborns were observed. This frequency decreased after the introduction of the sequential scheme of vaccination (IPV, OPV) in 2008-2013 as compared with the period of exclusive use of OPV in 2006-2007: 1 case per 4.9 million doses, 1.4 cases per million newborns and 1 case per 1.9 million doses, 4.9 cases per 1 million newborns, respectively. The study has been financed from Russian Federation budget within the framework of the Program for eradication of poliomyelitis in the Russian Federation, WHO Polio eradication initiative, WHO's European Regional Bureau, Russian Foundation for Basic Research (project No. 15-15-00147).

[Prevalence of hepatitis E markers in children].

AIM: Frequency of detection determination for past and current hepatitis E virus (HEV) infection markers in children with immune suppression, as well as children with normal immune status. MATERIALS AND METHODS: The presence of HEV markers (anti-HEV IgG and IgM, HEV RNA) was studied in 609 sera samples of children with neurologic pathologies, 87 samples--from children with immune deficiencies, as well as 3122 samples from conditionally healthy children of 6 regions of Russia. The children were divided into 5 age groups. Anti-HEV IgG and IgM determination was carried out in EIA, HEV RNA--by RT-PCR. RESULTS: The frequency of detection of anamnestic anti-HEV IgG turned out to be significantly higher among immune-compromised. children compared with healthy children (5.7% against 1.4%, p < 0.05). Anti-HEV IgM, that testify to current or recent infection, were also detected significantly more frequently among children with immune-suppression (1.1-1.6%) compared with healthy children (0.25%, p < 0.05). HEV RNA was detected in 1 child with the absence of anti-HEV IgM and IgG. Nucleotide sequence analysis of HEV confirmed membership of this isolate in genotype 3, that is prevalent in non-endemic territories. CONCLUSION: The data obtained have demonstrated, that HEV-infection is prevalent among children in Russia and its course is, probably, asymptomatic in most cases. Immune suppression is a factor of increased risk of infection of children with HEV.

[Production of the polyclonal enterovirus antibodies of chicken (IgY) and its evaluation as alternative to the rabbit enterovirus neutralizing sera].

Experimental data show the usefulness of the Leghorn chicken as a producer of the enterovirus neutralizing antibodies (IgY). The resulting serum is not inferior to the specific activity of the commercial rabbit enterovirus diagnostic sera (EDS) in the neutralization reaction. The IgY have lower backgrounds than mammalian IgG and do not cause toxic effect to cell culture. Compared with the conventional manufacturing method EDS IgY, preparation process is much more effective: the number of serum producers is significantly lower, whereas the yield of the product is higher. Reduction of the volume of the immunogens, immunization cycle, and number of injections is also an advantage of this manufacturing method.

[THE ROLE OF NUTRITION IN THE GENESIS OF-HEPATIC OSTEODYSTROPHY IN PRIMARY BILIARY CIRRHOSIS].

THE PURPOSE: To evaluate the effect of diet on the development of hepatic osteodystrophy in patients with primary biliary cirrhosis. MATERIALS AND METHODS: In 24 women with PBC, including signs of autoimmune hepatitis (PBC/AIH)--9 persons and 19 women in the control group were analyzed the daily diet, body composition, as well as bone mineral density in the lumbar spine and femur by dual-energy X-ray adsorbtsiometrii (DEXA). THE RESULTS: In PBC incidence of osteoporosis and severe osteoporosis was 22.7% and 9.1%, respectively, and osteopenia--59.1%, with a lesion predominantly lumbar spine. Inadequate intake of calcium in patients with PBC is associated with lower density of the femur. The high protein content in the diet has a protective effect on bone mineral density, associating with the development of osteopenia.

The 2010 outbreak of poliomyelitis in Tajikistan: epidemiology and lessons learnt.

A large outbreak of poliomyelitis, with 463 laboratory-confirmed and 47 polio-compatible cases, took place in 2010 in Tajikistan. Phylogenetic analysis of the viral VP1 gene suggested a single importation of wild poliovirus type 1 from India in late 2009, its further circulation in Tajikistan and expansion into neighbouring countries, namely Kazakhstan, Russia, Turkmenistan and Uzbekistan. Whole-genome sequencing of 14 isolates revealed recombination events with enterovirus C with cross-overs within the P2 region. Viruses with one class of recombinant genomes co-circulated with the parental virus, and representatives of both caused paralytic poliomyelitis. Serological analysis of 327 sera from acute flaccid paralysis cases as well as from patients with other diagnoses and from healthy people demonstrated inadequate immunity against polio in the years preceding the outbreak. Evidence was obtained suggesting that vaccination against poliomyelitis, in rare cases, may not prevent the disease. Factors contributing to the peculiarities of this outbreak are discussed. The outbreak emphasises the necessity of continued vaccination against polio and the need, at least in risk areas, of quality control of this vaccination through well planned serological surveillance.

[An ELISA system based on the specific class Y (IgY) antibodies from egg yolks for the quantitative determination of D-antigen in inactivated poliovirus vaccines].

The results of the construction of the first Russian ELISA system for the quantitative determination of D-antigen of 1-3 poliovirus types in the preparations of inactivated poliovirus vaccines are presented. For the first time, this kind of system is based on the use of specific antibodies of class Y (IgY) from egg yolks of immunized hens. It was shown that this ELISA system is specific, sufficiently sensitive, and can be used for quantitative determination of D-antigen of 1-3 poliovirus types in inactivated poliovirus vaccines.

[Seroepidemiology and molecular epidemiology of enterovirus type 71 in the world and the Russian Federation].

A review of recent publications on epidemiology and seroepidemiology of enterovirus type 71 in various regions of the world and authors' own results of study of seroepidemiology and molecular epidemiology of EV71 in Russia are presented.

[Experience of application of multiplex qPCR for differential diagnostics of intestinal viral infections].

AIM: Evaluate the effectiveness of multiplex reverse transcription (RT) and polymerase chain reaction with fluorescence detection in real time mode (qPCR) methods for differential detection of 11 groups of intestine viruses (adenoviruses, enteroviruses, polioviruses, hepatitis A and E viruses, group A and C rotaviruses, orthoreoviruses, noroviruses, sapoviruses and astroviruses) in various biological samples. MATERIALS AND METHODS: Panels of virus isolates and clinical samples characterized by reference methods were used to evaluate sensitivity of detection of various intestine viruses. Nucleic acids were isolated from study samples and multiplex RT and qPCR were carried out. RESULTS: Sensitivity of laboratory reagent kit (LRK) when compared with results obtained from reference methods was 100% for rotavirus A, adenovirus, enterovirus and norovirus, 88.9% for hepatitis E virus and 92.3% for hepatitis A virus, and diagnostic specificity - 99.4%. During analysis of 697 clinical samples from patients with acute intestine infection symptoms nucleic acids of various intestine viruses were isolated in 71.7%. CONCLUSION: Multiplex qRT-PCR was shown as an effective method of etiologic diagnostics of an intestine viral infection. Use of LRK was demonstrated to establish etiology of intestine diseases in 63 - 72% and in children with watery diarrhea - in approximately 90% of cases.

The association of recombination events in the founding and emergence of subgenogroup evolutionary lineages of human enterovirus 71.

Enterovirus 71 (EV71) is responsible for frequent large-scale outbreaks of hand, foot, and mouth disease worldwide and represent a major etiological agent of severe, sometimes fatal neurological disease. EV71 variants have been classified into three genogroups (GgA, GgB, and GgC), and the latter two are further subdivided into subgenogroups B1 to B5 and C1 to C5. To investigate the dual roles of recombination and evolution in the epidemiology and transmission of EV71 worldwide, we performed a large-scale genetic analysis of isolates (n = 308) collected from 19 countries worldwide over a 40-year period. A series of recombination events occurred over this period, which have been identified through incongruities in sequence grouping between the VP1 and 3Dpol regions. Eleven 3Dpol clades were identified, each specific to EV71 and associated with specific subgenogroups but interspersed phylogenetically with clades of coxsackievirus A16 and other EV species A serotypes. The likelihood of recombination increased with VP1 sequence divergence; mean half-lives for EV71 recombinant forms (RFs) of 6 and 9 years for GgB and GgC overlapped with those observed for the EV-B serotypes, echovirus 9 (E9), E30, and E11, respectively (1.3 to 9.8 years). Furthermore, within genogroups, sporadic recombination events occurred, such as the linkage of two B4 variants to RF-W instead of RF-A and of two C4 variants to RF-H. Intriguingly, recombination events occurred as a founding event of most subgenogroups immediately preceding their lineage expansion and global emergence. The possibility that recombination contributed to their subsequent spread through improved fitness requires further biological and immunological characterization.

[Poliomyelitis and vaccination strategy in Russian Federation in post-certification period].

Immunization schedules implemented in various countries by using poliovirus vaccines are presented. Approaches to prevent development of vaccine associated paralytic poliomyelitis and risk groups for this infection are discussed. In recent years poliomyelitis morbidity situation in the European region has become more complex, with the example of poliomyelitis outbreak in Tajikistan in 2010. The resulting problem of protection of Russian against emergence and spread of poliomyelitis caused by wild type virus is discussed.

[Social and economic significance of enterovirus infection and its role in etiologic structure of infectious diseases in the world].

Human enteroviruses comprised by more than 100 serotypes, they spread everywhere and can cause wide spectrum of diseases as well as significant social and economic loss. Influenza-like illness and mild forms of enterovirus infection (herpangina, exanthema) are widespread and causes of significant number of visits in clinics. Economic cost of mild form of enterovirus infection is not high although great number of cases (10 - 15 mln cases yearly in USA) determines its important economic significance. Single cases and outbreaks of enterovirus aseptic meningitis occur less frequently but lead to significant economic burden due to hospitalization costs. Enteroviruses are also cause up to 30% of sepsis-like disease in newborns and play important role in infant morbidity and mortality. Potential of enteroviruses as a source of new diseases in humans has a special significance for practical healthcare. In XX century enteroviruses became a cause of pandemics of paralytic poliomyelitis, hemorrhagic conjunctivitis, and foot-and-mouth-like disease, which caused vast social and economic loss, and emergence of new forms of enterovirus infection is quite possible in XXI century.

Evolutionary dynamics and temporal/geographical correlates of recombination in the human enterovirus echovirus types 9, 11, and 30.

The relationship between virus evolution and recombination in species B human enteroviruses was investigated through large-scale genetic analysis of echovirus type 9 (E9) and E11 isolates (n = 85 and 116) from 16 European, African, and Asian countries between 1995 and 2008. Cluster 1 E9 isolates and genotype D5 and A E11 isolates showed evidence of frequent recombination between the VP1 and 3Dpol regions, the latter falling into 23 (E9) and 43 (E11) clades interspersed phylogenetically with 46 3Dpol clades of E30 and with those of other species B serotypes. Remarkably, only 2 of the 112 3Dpol clades were shared by more than one serotype (E11 and E30), demonstrating an extremely large and genetically heterogeneous recombination pool of species B nonstructural-region variants. The likelihood of recombination increased with geographical separation and time, and both were correlated with VP1 divergence, whose substitution rates allowed recombination half-lives of 1.3, 9.8, and 3.1 years, respectively, for E9, E11, and E30 to be calculated. These marked differences in recombination dynamics matched epidemiological patterns of periodic epidemic cycles of 2 to 3 (E9) and 5 to 6 (E30) years and the longer-term endemic pattern of E11 infections. Phylotemporal analysis using a Bayesian Markov chain Monte Carlo method, which placed recombination events within the evolutionary reconstruction of VP1, showed a close relationship with VP1 lineage expansion, with defined recombination events that correlated with their epidemiological periodicity. Whether recombination events contribute directly to changes in transmissibility that drive epidemic behavior or occur stochastically during periodic population bottlenecks is an unresolved issue vital to future understanding of enterovirus molecular epidemiology and pathogenesis.

[Testing of sewage water in orphanage for younger children as a way of surveillance for circulation of polioviruses].

Ability to test sewage water for surveillance on circulation of polioviruses was assessed. Stool samples from children living in orphanage for younger children were collected monthly. Simultaneously, samples of sewage water from orphanage's collector and community collector, in which sewage from neighborhood dwellings is dumped, were collected by snap sample and sorption methods. Rate of isolation of polio--and nonpolioenteroviruses (NPEV) from stool samples for 6 months was 44%; rate of isolation from sewage water for the same period was 79% for sorption method and 50% for snap sample method. Between viruses circulating in orphanage, NPEV of different serotypes predominated (99 isolates out of 170). Domination of polioviruses in isolates from sewage samples obtained by sorption method (23 strains out of 32) can be associated with properties of the sorbent. Number of poliovirus strains and NPEV isolated by snap sample method was equal. Season fluctuations in proportion of stool and sewage samples containing viruses coincided. Comparison of efficacy of the methods during total study period (14 samplings) did not reveal significant difference in rate of virus isolation (in orphanage's collector--72% and 50% for sorption and snap sample collection methods respectively; in community collector--31% for both methods). Detection of type 1 poliovirus with changed antigenic properties in one stool sample and one sewage water sample argue for possibility to detect in sewage minor quantity of virus excreting by one person. Thus testing of sewage water provides information on viruses circulating in the community of children. Study of stool samples revealed high rate of poliovirus isolation (up to 32%) including nonvaccinated children.

Transmission networks and population turnover of echovirus 30.

Globally, echovirus 30 (E30) is one of the most frequently identified enteroviruses and a major cause of meningitis. Despite its wide distribution, little is known about its transmission networks or the dynamics of its recombination and geographical spread. To address this, we have conducted an extensive molecular epidemiology and evolutionary study of E30 isolates collected over 8 years from a geographically wide sample base (11 European countries, Asia, and Australia). 3Dpol sequences fell into several distinct phylogenetic groups, interspersed with other species B serotypes, enabling E30 isolates to be classified into 38 recombinant forms (RFs). Substitutions in VP1 and 3Dpol regions occurred predominantly at synonymous sites (ratio of nonsynonymous to synonymous substitutions, 0.05) with VP1 showing a rapid substitution rate of 8.3 x 10(-3) substitutions per site per year. Recombination frequency was tightly correlated with VP1 divergence; viruses differing by evolutionary distances of >0.1 (or 6 years divergent evolution) almost invariably (>97%) had different 3Dpol groups. Frequencies of shared 3Dpol groups additionally correlated with geographical distances, with Europe and South Asia showing turnover of entirely distinct virus populations. Population turnover of E30 was characterized by repeated cycles of emergence, dominance, and disappearance of individual RFs over periods of 3 to 5 years, although the existence and nature of evolutionary selection underlying these population replacements remain unclear. The occurrence of frequent "sporadic" recombinants embedded within VP1 groupings of other RFs and the much greater number of 3Dpol groups than separately identifiable VP1 lineages suggest frequent recombination with an external diverse reservoir of non-E30 viruses.

[Molecular epidemiology of ECHO 6 virus, the causative agent of the 2006 outbreak of serous meningitis in Khabarovsk].

A total of 3194 cases of enterovirus meningitis were notified in the Russian Federation in 2005, of them there were 1434 cases in the Khabarovsk Territory. Enteroviruses were isolated from 1020 out of the virologically studied 1362 patients from the Khabarovsk Territory. Viruses E6 and E30 were isolated in 80 and 14.7% of cases, respectively. E1, E3, E7, E33, Coxsackie virus B1, B4, B5, and A10 were sporadically detected. The E6 strains isolated in Komsomolsk-on-Amur were identical while E6 strains isolated in Khabarovsk belonged to two different genotypes and greatly differed from those isolated in Konsomolsk-on-Amur. The virus E30 strains isolated in Khabarovsk and Komsomolsk-on-Amur had a 1% difference in VP1 genome nucleotide sequence and belonged to E30 subtype that circulated in Russia and Kazakhstan in 2004-2005.