Detection of avian metapneumovirus subtype A from wild birds in the State of São Paulo, Brazil / Detecção de metapneumovirus aviário subtipo A em aves silvestres no estado de São Paulo, Brasil

The present study investigated the circulation of avian metapneumovirus (aMPV) in wild birds in Brazil. To do so, 131 samples from 366 oropharyngeal or cloacal swabs collected from 18 species of birds were tested individually or in pools by RT-PCR. Samples detected by RT-PCR were selected for DNA sequencing. Thirteen (9.9%) samples were detected by the RT-PCR targeting the N gene and four out of 13 samples were sequenced. Sequencing results showed a high identity with the aMPV subtype A. Our results confirm the circulation of the aMPV subtype A in wild birds in Brazil even five years after its last detection.(AU)

O presente estudo investigou a circulação de metapneumovírus aviário em aves silvestres no Brasil. Para tanto, 131 amostras de 366 suabes orofaringeanos ou cloacais coletados de 18 espécies de aves foram testadas individualmente ou na forma de pools por RT-PCR. As amostras detectadas por RT-PCR foram selecionadas para sequenciamento. Treze (9,9%) das amostras foram detectadas por RT-PCR tendo o gene N como alvo; destas, quatro foram sequenciadas com sucesso. Resultados do sequenciamento mostraram alta identidade com o aMPV de subtipo A. Nossos resultados confirmam a circulação de aMPV subtipo A em aves silvestres no Brasil mesmo cinco anos após sua última detecção.(AU)

Brazilian avian metapneumovirus subtypes A and B: experimental infection of broilers and evaluation of vaccine efficacy / Metapneumovirus aviário subtipos A e B brasileiros: infecção experimental em frangos de corte e eficácia vacinal

Avian metapneumovirus (aMPV) is a respiratory pathogen associated with the swollen head syndrome (SHS) in chickens. In Brazil, live aMPV vaccines are currently used, but subtypes A and, mainly subtype B (aMPV/A and aMPV/B) are still circulating. This study was conducted to characterize two Brazilian aMPV isolates (A and B subtypes) of chicken origin. A challenge trial to explore the replication ability of the Brazilian subtypes A and B in chickens was performed. Subsequently, virological protection provided from an aMPV/B vaccine against the same isolates was analyzed. Upon challenge experiment, it was shown by virus isolation and real time PCR that aMPV/B could be detected longer and in higher amounts than aMPV/A. For the protection study, 18 one-day-old chicks were vaccinated and challenged at 21 days of age. Using virus isolation and real time PCR, no aMPV/A was detected in the vaccinated chickens, whereas one vaccinated chicken challenged with the aMPV/B isolate was positive. The results showed that aMPV/B vaccine provided a complete heterologous virological protection, although homologous protection was not complete in one chicken. Although only one aMPV/B positive chicken was detected after homologous vaccination, replication in vaccinated animals might allow the emergence of escape mutants.

O Metapneumovírus aviário (aMPV) é um patógeno respiratório associado à síndrome da cabeça inchada (SHS) em galinhas. Apesar de vacinas vivas contra o aMPV serem utilizadas no Brasil, os subtipos A e B (aMPV/A e aMPV/B) são ainda encontrados no país, com predominância do subtipo B. Este estudo foi conduzido com o intuito de estudar dois isolados brasileiros de aMPV (subtipos A e B) isolados de frango. Para isto, um desafio experimental em frangos foi conduzido com o intuito de explorar a capacidade de replicação dos subtipos A e B Brasileiros. Posteriormente, a protecção virológica conferida por uma vacina do subtipo B em pintos foi realizada com os mesmos isolados. Após o desafio experimental demonstrou-se, por isolamento viral e PCR em tempo real, que o isolado do subtipo B replicou por maior período de tempo e em quantidades maiores, em comparação com o subtipo A. Para o estudo de proteção, 18 pintos de um dia de idade foram vacinados e desafiados aos 21 dias. Usando isolamento viral e PCR em tempo real, em nenhuma ave vacinada e desafiada com aMPV/A foi detectado o vírus, ao passo que uma ave vacinada e desafiada com o aMPV/B foi positiva. Os resultados mostraram que a vacina do subtipo B forneceu protecção heteróloga completa, embora a protecção homóloga não tenha sido conferida em uma ave. Apesar de o aMPV/B ter sido detectado em apenas um frango após vacinação homóloga, a replicação viral em aves vacinadas pode resultar em emergência de mutantes de escape.

Isolation and characterization of avian metapneumovirus from chickens in Korea

Avian metapneumovirus (aMPV) causes upper respiratory tract infections in chickens and turkeys. Although the swollen head syndrome (SHS) associated with aMPV in chickens has been reported in Korea since 1992, this is the study isolating aMPV from chickens in this country. We examined 780 oropharyngeal swab or nasal turbinate samples collected from 130 chicken flocks to investigate the prevalence of aMPV and to isolate aMPV from chickens from 2004-2008. Twelve aMPV subtype A and 13 subtype B strains were detected from clinical samples by the aMPV subtype A and B multiplex real-time reverse transcription polymerase chain reaction (RRT-PCR). Partial sequence analysis of the G glycoprotein gene confirmed that the detected aMPVs belonged to subtypes A and B. Two aMPVs subtype A out of the 25 detected aMPVs were isolated by Vero cell passage. In animal experiments with an aMPV isolate, viral RNA was detected in nasal discharge, although no clinical signs of SHS were observed in chickens. In contrast to chickens, turkeys showed severe nasal discharge and a relatively higher titer of viral excretion than chickens. Here, we reveal the co-circulation of aMPV subtypes A and B, and isolate aMPVs from chicken flocks in Korea.

Development of Competitive ELISA for Detection of Avian Metapneumovirus Antibodies in Chicken

Avian metapneumovirus (aMPV) causes an acute and highly contagious upper respiratory tract infection in turkeys and chickens. In this study, a competitive ELISA (C-ELISA) was developed for the detection of antibodies to aMPV in chicken sera and/or their egg yolks. This assay is based on the competitive binding of monoclonal antibody with serum antibodies to recombinant aMPV N protein expressed by a recombinant baculovirus. The C-ELISA showed specificity and sensitivity of 100% and 98.0%, respectively, when compared to the virus neutralization test. In specific pathogen-free chickens experimentally infected with aMPV SC1509 strain, the C-ELISA started to detect antibodies to aMPV as early as 5 days post infection from birds infected with aMPV, while a commercial ELISA kit detected first 10 days post infection. The C-ELISA was similar or superior to a commercial ELISA kit when serum and egg yolk samples collected from chickens on six outbreak farms were tested for diagnosis. The C-ELISA developed in the present work provides a short turnaround time and can be a useful diagnostic and screening tool for aMPV infection in the field.

Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A / Comparação entre as técnicas de RT-PCR convencional e RT-PCR em tempo real para a detecção do metapneumovírus aviários subtipo A

Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an established test for the attachment (G) gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3) to 10Õ TCID50 mL-1). The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.

O metapneumovírus aviário (AMPV) pertence ao gênero Metapneumovirus, família Paramyxoviridae. Isolamento viral, sorologia e detecção do RNA genômico são atualmente as técnicas utilizadas para o diagnóstico desse agente. O objetivo do presente estudo foi comparar a detecção de RNA viral de seis isolados de AMPV, subtipo A (AMPV/A), utilizando diferentes métodos de RT-PCR convencional e real time RT-PCR (RRT-PCR). Duas novas técnicas de RT-PCR convencional e duas técnicas de RRT-PCR, ambas para a detecção dos genes da nucleoproteína (N) e da proteína de fusão (F), foram comparadas com um RT-PCR previamente estabelecido para a detecção do AMPV (gene da glicoproteína -G). Todos esses métodos foram capazes de detectar os isolados AMPV/A. As técnicas RRT-PCR (genes F e N) mostraram os menores limites de detecção (10(0.3) to 10Õ TCID50 mL-1). Os resultados sugerem que as técnicas RT-PCR convencional (gene F) e as técnicas de RRT-PCR (gene F e N) desenvolvidas no presente estudo podem ser utilizadas com sucesso para a detecção do AMPV/A. Além disso, o RRT-PCR gera resultados rápidos e sensíveis, o que o torna uma ferramenta alternativa para o isolamento viral.

Isolation and Characterization of Avian Metapneumovirus from Broiler Breeder Chickens in Korea

Avian metapneumovirus (AMPV) is an emerging pathogen causing respiratory and reproductive illness in poultry worldwide. To demonstrate the presence of AMPV in domestic chickens in Korea, we attempted to isolate AMPV from affected chickens. A cytopathic agent was isolated using chicken tracheal ring culture from dead chickens from a broiler breeder farm with reduced egg production in Korea. This agent, termed SC1509 strain, subsequently passed in Vero cells with distinct cytopathic effects. The SC1509 strain was confirmed as avian metapneumovirus (AMPV) using both RT-PCR test and monoclonal antibody-based immunofluorescence assay. Sequence analysis based on the G glycoprotein revealed that the SC1509 strain had 22.5 to 96.0% nucleotide sequence identity and 11.1 to 92.7% predicted amino acid sequence identity with previously published AMPV strains, particularly with the highest sequence homology (95.8 to 96% for nucleotides and 92.2 to 92.7% for amino acids) to European strains belonging to genotype B. The SC1509 strain was phylogenetically clustered with genotype B viruses, confirming that the SC1509 strain belongs to genotype B. This is the first report of genotype B avian metapneumovirus from chickens in Korea.