[The prevalence of the human rhinoviruses and coronaviruses circulating in the Moscow region during 2007-2012].

The rhinoviruses and coronaviruses are the most common causative agents of the acute upper respiratory tract infection in humans. They include several species that vary in the pathogenicity, some causing severe respiratory tract diseases. In this work, the species prevalence of rhinoviruses and coronaviruses was studied in 92 virus-positive clinical patients that were collected at the area of the Moscow region during the period from 2007 to 2012. Using the real-time PCR the virus circulation has been established for all species common in humans, including three rhinoviruses, HRV A, HRV B, and HRV C, and four coronaviruses, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. For eight patients, the identity of the rhinoviruses, including 4 cases of HRV-C, 3 cases of HRV-A, and a single case of HRV-B, was corroborated using partial sequencing of the 5 non-coding regions and phylogenetic analysis. The viruses of HRV-C, HCoV-NL63, and HCoV-OC43 were prevalent in children with severe respiratory diseases.

Quantification of measles, mumps and rubella viruses using real-time quantitative TaqMan-based RT-PCR assay.

In this study, a rapid quantitative method using TaqMan-based real-time reverse transcription-polymerase chain reaction (qPCR-RT) has been developed for estimating the titers of measles, mumps and rubella (MMR) viruses in infected cell culture supernatants. The qPCR-RT assay was demonstrated to be a specific, sensitive, efficient and reproducible method. For MMR viral samples obtained during MMR viral propagations in Vero cells at a different multiplicity of infection, titers determined by the qPCR-RT assay have been compared with estimates of infectious virus obtained by a traditional commonly used method for MMR viruses - 50% cell culture infective dose (CCID(50)) assay, in paired samples. Pearson analysis evidenced a significant correlation between both methods for a certain period after viral inoculation. Furthermore, the established qPCR-RT assay was faster and less-laborious. The developed method could be used as an alternative method or a supplementary tool for the routine titer estimation during MMR vaccine production.

[Evaluation of the efficiency of differential diagnosis of influenza by multiplex real-time polymerase chain reaction].

The paper gives the results of a comparative analysis of the detection of influenza viruses in clinical samples, by using multiplex real-time polymerase chain reaction (RT-PCR) and by virus isolation in MDCK cell cultures. The investigation employed 267 nasopharyngeal swab specimens obtained from patients with influenza symptoms during two epidemic seasons (2008-2009 and 2009-2010). Influenza viruses were found in 104 samples (48 with influenza A virus (IAV) and 56 with influenza B virus (IBV)) by multiplex RT-RCR and in 84 samples (35 with IAV and 49 with IBV) by a cultural technique. The results of detection of influenza viruses by the two methods showed 89.4% agreement. The diagnostic sensitivity of multiplex RT-PCR testing a panel of the clinical samples in question was estimated to be 94.3% for IAV and 95.9% for IBV. The diagnostic sensitivity of multiplex RT-PCR in virus detection was demonstrated to be not only highly competitive with virus isolation, but also superior to the latter.

[Use of multiplex PCR method with real-time detection for differential diagnosis of respiratory viral infections].

Multiplex real-time polymerase chain reaction test-system with fluorescent detection (RT-PCR) for simultaneous identification of main agents of acute respiratory viral infections: influenza A (IAV) and B viruses (IBV), parainfluenza viruses types 1, 2, 3, 4 (PIV 1 - 4), adenoviruses (ADV), respiratory syncitial virus (RSV), rhinoviruses (RV) and enteroviruses (EV), in presence internal positive control (IPC) represented by vaccine strain of rubella virus RA 27/3. Using multiplex RT-PCR method, respiratory viruses were detected in 116 out of 226 clinical samples (nasal swabs) obtained from patients with symptoms of acute respiratory infection: in 68 (58.6%) samples--IBV; in 21 (18.1%)--IAV; in 12 (10.3%) --RV; in 6 (5.2%)--PIV 2; in 4--(3.4%) ADV; in 3 (2.6%)--RSV; in 2 (1.7%)--EV; in 2 (1.7%)--PIV 4; in 1 (0.9%)--PIV 3; in 1 (0.9%)--PIV 1. Mixed infection was observed in 4 (3.4%) patients. PCR assay allowed to reveal various respiratory viruses in 51.3% of samples. At the same time samples were tested for the presence of 12 respiratory viruses--IAV, IBV, PIV 1 - 4, RSV, RV, metapneumoviruses, and coronaviruses NL63, 229E and OC43--in the presence of IPC represented by equine arteritis virus using analogous PCR test-system provided by medical center of Leiden university. Results of tests for detection of IAV, IBV, RSV, PIV 1 - 4, and RV, analyzed by both systems, agreed in 94%. Multiplex format of RT-PCR performing significantly reduces time and cost of the test, which make it suitable and effective instrument of epidemiological studies.