Imported cases of dengue fever in Russia during 2010-2013.

OBJECTIVE: To confirm dengue infection among Russian tourists returned from Southeast and Mexico in 2010-2013 with clinical signs of infection. METHODS: Blood and serum samples from patients were collected. NS1 antigen and human IgM/IgG antibodies to dengue virus were identified using commercial tests manufactured by "Standard Diagnostics, INC.", Korea. ELISA test was used for the quantitative analyses of human IgM/IgG antibodies to dengue virus ("Orgenics Ltd.", Israel). Viral RNA was detected using commercial real-time PCR tests manufactured by "Genome Diagnostics Pvt. Ltd.", India and "Vector", Russia. Genotypes of revealed dengue viruses were determined employing nucleotide sequencing and phylogenetic analysis of 5'-UTR of the viral genome. RESULTS: A total of 98 collected blood samples were analyzed. Fifty samples were positive for at least one of four markers of dengue infection. IgM to dengue virus were revealed in 38 samples, in 25 samples IgM were combined with IgG. NS1 antigen was detected in 43 samples. 22 serum samples were positive for dengue virus RNA. The majority of samples (12 patients) from tourists returned from Thailand were positive for genotype 1 of dengue virus, 2nd and 4th genotype were identified each in 1 patient. CONCLUSIONS: Due to laboratory confirmed cases of imported dengue fever in Russia, the differential diagnosis of dengue is strictly recommended for tourists returning from endemic areas.

Infection of chickens caused by avian influenza virus A/H5N1 delivered by aerosol and other routes.

This study presents results of the study of infectivity of avian influenza virus (AIV) A subtype H5N1 strains isolated from agricultural birds across the territory of the Russian Federation and CIS countries. The results of the susceptibility of chickens to the AIV isolates delivered by the aerosol route and the dissemination of the virus in the organs of infected birds are presented. As was observed, the sensitivity of birds to AIV by the aerosol route of infection is 30 times higher than by intranasal route, 500 times higher than by the oral route and 10000 times higher than by the intragastric route of infection, which is indicative of higher permissivity of respiratory organs to AIV. The highest titres of AIV A subtype H5N1(A/Chicken/Kurgan/05/2005 strain) in aerosol-infected chickens were found in nasal cavity mucosa, lungs, cloaca, serum and kidney, where viable virus accumulation was detected by 18h post-infection (p.i.). The highest virus titres were observed 54h p.i. in lungs, serum and kidney, reaching the value of 8.16 lg EID50 /g(ml) in the lungs. The results showed that birds infected by the aerosol route developed higher titres of virus than those infected by other routes.

Development and verification of real-time PCR assay for identification of viral agents causing acute respiratory infections in human beings.

A multiplex polymerase chain reaction (PCR) for identification of four viruses causing acute respiratory diseases in human beings was developed. The analytical sensitivity of developed RT-PCR for identification of adenovirus, respiratory-syncytial virus, flu viruses types A and B, and actual subtypes of type A flu virus (seasonal and pandemic variants H1N1, seasonal H3N2, and viruses of bird flu that are pathogenic to human beings H5 and H7) was 1 × 103 genome equivalents per milliliter. Diagnostic sensitivity for flu virus type A and B, and also subtypes H1 (seasonal H1N1, pandemic variant of H1N1 of year 2009), H3, H5 was 1 × 103-104 viral particles per milliliter. The method developed has high specificity and does not have positive signal in experiments with DNA/cDNA of human beings and viral DNA. We have studied 50 samples using the developed set. Etiology was defined in 33 samples.

[Development and validation of the real-time PCR assay for the identification of viral agents causing acute respiratory infections in humans].

The real time PCR assay targeting influenza A and B virus, 5 subtypes of influenza A virus (seasonal H1N1, pandemic H1N1 (2009), seasonal H3N2, pathogenic for human subtypes of avian influenza H5 and H7), respiratory syncytial virus, and adenovirus was developed. The analytical sensitivity of the developed assay was 1 x 10(3) genome equivalents per ml. The diagnostic sensitivity of the method was 1 x l0(3)-10(4) viral particles per ml. Experiments with human DNA/cDNA and viral cDNA showed a markedly high diagnostic specificity of the developed PCR assay. In the assay of the developed PCR test, 50 nasopharyngeal swab specimens were tested. The etiology was identified in 33 samples.

[Outbreak of acute enterovirus intestinal infection in Sakhalin region in August 2010].

The investigation of cases of acute intestinal infections in the Sakhalin region of Russia in August, 2010 is described. Epidemiological and molecular biological studies were conducted. After initial PCR screening and determining the nucleotide sequences of the positive samples the following enteroviruses were found: Coxsackie A2 - 42 samples (45%), Coxsackie A4--31 sample (34%), Enterovirus 71--6 samples (6,5%), Coxsackievirus B5--6 samples (6,5%), Coxsackie B3--4 samples (4%) and Coxsackie B1--4 samples (4%). The phylogenetic analysis of sequences showed that the closest analogues for the nucleotide sequences of these genotypes were previously identified in Japan, Korea and China in 2000-2010.

[Studies of properties of pandemic influenza virus A/H1N1 circulated in Russian Federation in 2009 - 2010].

AIM: Studies of cultural, virologic, antigenic properties of 89 samples of pandemic influenza A(H1N1) 2009 virus isolated in Russian Federation from May 2009 to March 2010. MATERIALS AND METHODS: Properties of isolated samples were compared with those of the reference strain A/ California/04/2009 (H1N1). RESULTS: Studies of biological properties and analysis of genome nucleotide sequences of the isolated samples showed that those strains are closely related to the reference strain. CONCLUSION: Monitoring of genetic, virologic and antigenic properties of pandemic influenza A(H1N1) 2009 virus isolates carried out from May 2009 to March 2010 did not reveal significant changes in the abovementioned properties of the virus or emergence of mutations that can lead to such changes.

[Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009].

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.

[In vivo efficacy of Ingavirin against pandemic A (H1N1/09)v influenza virus].

Ingavirin was shown to be efficient in inhibition of the pandemic influenza virus strains A/California/04/2009 (H1N1)v, A/California/07/2009 (H1N1)v, A/Moscow/225/2009 (H1N1)v and A/Moscow/226/2009 (H1N1)v. as well as the influenza virus strain A/Aichi/2/68 (H3N2) in the lungs of the infected mice. After oral administration of Ingavirin the titers of the influenza virus strains in the lung homogenates lowered.

[Investigation of therapeutic and protective effect of Reaferon-ES Lipint on mice infected with avian influenza virus (subtype H5N1)].

The efficacy of the therapeutic, prophylactic and urgent prophylactic schemes for the use of Reaferon-ES lipint, a liposomal human recombinant alpha-interferon for oral use, was studied on mice infected with the avian influenza virus. Strain A/Chicken/Kurgan /05/2005 (subtype H5N1) of the avian influenza virus showed high virulence with respect to mice ICR. Theoretically-based calculations allowed to design an optimal therapeutic and prophylactic dose of the drug for the mice (1000 units/animal). It was observed that only after prophylactic use of Reaferon-ES lipint it was effective in protection of the mice infected with 10 LD50 of the avian influenza virus (A/Chicken/Kurgan/05/2005, H5N1). The protection coefficient was 0.35. Under such conditions the drug efficacy was comparable with that of Tamiflu. Therefore, Reaferon-ES lipint could be recommended for prophylaxis of the infection due not only to the season strains of the influenza virus, but also to the strains of the avian influenza virus.