Aplicação da técnica de RT-PCR para metapneumovírus aviário (aMPV) / Application of RT-PCR technique for avian metapneumovirus (aMPV) / Aplicación de la técnica RT-PCR para metapneumovirus aviario (aMPV)

Os principais hospedeiros do Metapneumovírus aviário (aMPV) são os frangos de corte e perus. O vírus acomete o trato respiratório superior dos perus desencadeando a Rinotraqueíte Viral dos Perus (RVP). O principal objetivo deste trabalho foi padronizar uma técnica de RT-PCR para a detecção do aMPV, por meio do uso do kit AccessQuick™ RT-PCR system (Promega®). Foram utilizados amostras de suabes de traqueia e pulmão de 38 perus comerciais com sintomatologia respiratória e dois suabes oculares de faisão. O RNA viral foi extraído utilizando-se o kit RTP® DNA/RNA Virus Mini Kit (STRATEC Molecular). Em seguida as amostras foram submetidas à RT-PCR One Step, utilizando o kit AccessQuick™ RT-PCR system (Promega®). Todas as 40 amostras testadas por RT-PCR foram negativas, exceto a amostra vacinal que foi utilizada como controle positivo. O aMPV não causa latência em frangos de corte ou perus, logo a excreção viral é limitada. Dessa forma, a ausência da detecção de genoma viral neste estudo pode ser justificada devido à idade que as amostras foram coletadas em perus, com 140 dias no abatedouro, impossibilitando dessa maneira a amplificação do genoma do aMPV. Porém, esse estudo também mostra que a RT-PCR se mostrou eficaz para detectar o genoma viral do aMPV, podendo dessa forma ser utilizado como uma ferramenta de diagnóstico rápido para investigação e estudo de casos de aMPV em rebanho de perus.

The main hosts of Avian metapneumovirus (aMPV) are broilers and turkeys. This virus affects the upper respiratory tract of turkeys, triggering Turkey Rhinotracheitis (TRT). The aim of this study was to optimize a RT-PCR technique in order to detect aMPV using the AccessQuick™ RT-PCR system (Promega®) kit. Tracheal and lung swab samples from 38 commercial turkeys with respiratory symptoms and two ocular swabs from pheasants were analyzed. Viral RNA was extracted using RTP® DNA/RNA Virus Mini Kit (Molecular STRATEC) kit. All 40 samples tested were negative in the RT-PCR. The only positive sample was a vaccine strain, used as the positive control. The aMPV does not cause latency in broilers, chickens or turkeys, thus, the viral excretion is limited. However, the absence of viral genome detection in this study may be justified due to the age the samples were collected, since they were collected in turkeys with about 140 days in the slaughterhouse, thus preventing the amplification of the aMPV genome. This study shows that the RT-PCR is effective to detect aMPV viral genome and may be used as a rapid diagnostic tool for research and for the studying of aMPV cases in turkey flocks in Brazil.

Los principales anfitriones de Metapneumovirus aviario (aMPV) son los pollos de engorde y pavos. El virus afecta el tracto respiratorio superior de los pavos desencadenando la Rinotraqueitis Viral de los pavos (RVP). El principal objetivo de ese estudio fue estandarizar una técnica de RT-PCR para la detección del aMPV, a través del uso del kit AccessQuick™-PCRsystem (Promega®). Se utilizaron muestras de hisopos traqueales y pulmonares de 38 pavos comerciales con síntomas respiratorios y dos hisopos oculares de faisán. El RNA viral se extrajo utilizando el kit DNA RTP® DNA/RNA Virus Mini Kit (STRATEC Molecular). A continuación, las muestras se sometieron a la RT-PCR OneStep utilizando el kit AccessQuick™ RT-PCR (Promega®). Todas las 40 muestras analizadas por RT-PCR fueron negativas, excepto la muestra de vacuna que se utilizó como control positivo. El aMPV no causa latencia en pollos de engorde o pavos, por lo que la excreción viral es limitada. Así, la ausencia de la detección de genoma viral en este estudio puede ser justificada debido a la edad que se recogieron las muestras en los pavos, con 140 días en el matadero, imposibilitando de este modo la amplificación del genoma del aMPV. Sin embargo, ese estudio también muestra que la RT-PCR se ha demostrado eficaz para detectar el genoma viral del aMPV, pudiendo así ser utilizado como una herramienta de diagnóstico rápido para investigación y estudio de casos de aMPV en bandada de pavos.

The highest prevalence of human metapneumovirus in Ahwaz children accompanied by acute respiratory infections.

PURPOSE: The newly described human metapneumovirus (hMPV) has been recently discovered as an etiological agent of acute respiratory infections (ARTI) in infants and children. The aim of this study was to determine the prevalence of hMPV and its potential role as causative agent of ARTI in Ahwaz children. METHODS: In the present study, we examined 124 nasal swabs from infants affected by ARTI for the presence of hMPV by RT-PCR technique. RESULTS: Sixty-eight out of 124 (54.4%) cases were positive for hMPV which is the highest incidence of hMPV ever reported in the world, 94.1% of positive cases belonged to genotype A; whereas no B genotype was detected. Our positive hMPV children were affected by upper (URTI) as well as lower respiratory tract infection (LRTI); however, LARTIs had higher prevalence. CONCLUSIONS: We suggest a probable role of F protein alteration as the causative agent for the highest prevalence of hMPV infection among Ahvaz children.

Evaluation of Seeplex(TM) RV Detection Kit for Detecting Rhinovirus, Human Metapneumovirus, and Coronavirus / 대한진단검사의학회지

BACKGROUND: Direct antigen test (DAT) and culture are primary tests to diagnose infections by respiratory viruses, but are mainly available for the traditional viral pathogens such as respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), and adenovirus in clinical laboratories. The objective of this study was to evaluate a multiplex reverse transcriptase-PCR method using Seeplex(TM) RV Detection kit (Seegene, Korea) for the detection of rhinovirus, coronavirus, and human metapneumovirus (hMPV). METHODS: From January to May 2007, nasopharyngeal aspirates (NPAs) from pediatric patients negative for culture and DAT of traditional viral pathogens were tested with Seeplex(TM). All the amplicons were directly sequenced and homology of the sequences was searched in the National Center for Biotechnology Information (NCBI) database. Patients' medical records were reviewed for clinical and demographic features. RESULTS: Forty-seven (26.4%) of 178 NPAs were positive: 18 rhinovirus, 15 hMPV, 4 RSV A, 3 coronavirus OC43, 3 influenza virus A, 2 adenovirus, 1 coronavirus NL63, and 1 RSV B. Based on maximum identity, each of the sequences indicating rhinovirus, hMPV, and coronavirus OC43 matched to the corresponding viruses with homology of 94-98%, 96-99%, and 98-100%, respectively. Seeplex(TM) positive patients were 0-11 yr old with a male:female ratio of 1.5:1. Clinical diagnoses included 9 pneumonia, 6 bronchiolitis, 2 cold, 1 asthma exacerbation for rhinovirus; 10 pneumonia, 4 bronchiolitis, and 1 clinical sepsis for hPMV; and 1 pneumonia, 2 croup, and 1 cold for coronavirus. CONCLUSIONS: Multiplex reverse transcriptase-PCR method using Seeplex(TM) RV Detection kit is a reliable test to detect rhinovirus, hMPV, and coronavirus. It may improve the diagnostic sensitivity for RSV, influenza virus, PIV, and adenovirus.

Human Metapneumovirus Infection in Hospitalized Children with Acute Respiratory Disease in Korea

Human metapneumovirus (hMPV) is a recently isolated virus, mostly associated with acute lower respiratory infection in children, of which symptoms are similar to those of respiratory syncytial virus (RSV) infection. The aim of our study was to determine the frequency of hMPV in hospitalized children with acute respiratory tract disease in Korea. Nasal aspirates from hospitalized children with respiratory infections under 15 yr old between December 2003 and February 2005 were included in the study. Each sample was analyzed for RSV, adenovirus, influenza virus A and B, and parainfluenza virus by indirect fluorescent assay (IFA). F-gene sequences were used for PCR for the detection and sequencing of hMPV. In total 381 samples, negative samples in which any viral pathogen could not be identified by IFA were 231 cases. hMPV was detected using reverse transcriptase-PCR (RT-PCR) in 28 of 231 (12.1%) children who were not infected with another respiratory viruses. The hMPV-infected children were diagnosed as having pneumonia, bronchiolitis, bronchial asthma exacerbation, croup, and upper respiratory tract infection. Most of the RT-PCR positive samples for hMPV were collected in winter season. These results suggest that hMPV may be a responsible pathogen causing acute respiratory tract infection in Korean children.