Simultaneous detection of infectious bronchitis virus and avian metapneumovirus genotypes A, B, and C by multiplex RT-qPCR assay in chicken tracheal samples in Ecuador.

Respiratory RNA viruses such as Infectious bronchitis virus (IBV) and Avian metapneumovirus (aMPV), which are characterized by generating both respiratory damage and adverse effects on reproductive organs, affect poultry production economically due to high mortality rate and decrease in egg production and quality. Particularly, aMPV has three genotypes that have been reported with greater frequency in chickens: aMPV-A, aMPV-B, and aMPV-C. The present study proposes the design of a multiplex RT-qPCR assay for the simultaneous diagnosis of the 3 genotypes of interest of aMPV and IBV, followed by testing of 200 tracheal samples of vaccinated chickens with respiratory symptoms and finally a phylogenetic analysis of the sequences found. The assay detected up to 1 copy of each viral genome. The standard curves showed an efficiency between 90 and 100% in the multiplex assay and inter- and intra-assay coefficients of variation of 0.363 and 0.459, respectively and inter- and intra-assay coefficients of variation of 0.363 and 0.459, respectively. 69.5% of samples were found positive alone or in coinfection. 114 samples were positive for IBV, 13 for aMPV-A and 25 for aMPV-B. RNA of aMPV-C was no detected. The most commonly found combination was aMPV-B and IBV within 6 samples, and the least common was aMPV-A and aMPV-B in coinfection in 2 samples. The assay was specific for amplification of the genomes of the studied respiratory viruses (IBV, aMPV-A, aMPV-B, aMPV-C) as no amplification was shown from other viral genomes (ChPV, CAstV, ANV, and FAdV) or from the negative controls. Partial genomic Sanger sequencing enabled to identify circulating vaccine-derived and wild-type strains of IBV and vaccine and vaccine-derived strains of aMPV-B. In conclusion, this newly developed multiplex RT-qPCR was shown to be able to detect individual infections as well as co-infections among the respiratory viruses investigated. It was demonstrated to be a reliable and efficient tool for rapidly and safely diagnosing these infections. Furthermore, this study represents the first report of aMPV strains in Ecuadorian poultry and demonstrates the circulation of aMPV-A, aMPV-B, and GI-13 IBV strains in unvaccinated chicken populations in the country. Thus, it highlights the importance of simultaneously identifying these pathogens in greater detail and on a regular basis in Ecuador.

Detection and Genome Sequence Analysis of Avian Metapneumovirus Subtype A Viruses Circulating in Commercial Chicken Flocks in Mexico.

Avian metapneumoviruses (aMPV subtypes A-D) are respiratory and reproductive pathogens of poultry. Since aMPV-A was initially reported in Mexico in 2014, there have been no additional reports of its detection in the country. Using nontargeted next-generation sequencing (NGS) of FTA card-spotted respiratory samples from commercial chickens in Mexico, seven full genome sequences of aMPV-A (lengths of 13,288-13,381 nucleotides) were de novo assembled. Additionally, complete coding sequences of genes N (n = 2), P and M (n = 7 each), F and L (n = 1 each), M2 (n = 6), SH (n = 5) and G (n = 2) were reference-based assembled from another seven samples. The Mexican isolates phylogenetically group with, but in a distinct clade separate from, other aMPV-A strains. The genome and G-gene nt sequences of the Mexican aMPVs are closest to strain UK/8544/06 (97.22-97.47% and 95.07-95.83%, respectively). Various amino acid variations distinguish the Mexican isolates from each other, and other aMPV-A strains, most of which are in the G (n = 38), F (n = 12), and L (n = 19) proteins. Using our sequence data and publicly available aMPV-A data, we revised a previously published rRT-PCR test, which resulted in different cycling and amplification conditions for aMPV-A to make it more compatible with other commonly used rRT-PCR diagnostic cycling conditions. This is the first comprehensive sequence analysis of aMPVs in Mexico and demonstrates the value of nontargeted NGS to identify pathogens where targeted virus surveillance is likely not routinely performed.

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)

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)

Detection of infectious bronchitis virus and Avian metapneumovirus by a duplex semi-nested reversetranscription–polymerase chain reaction assay / Detecção do vírus da bronquite infecciosa das galinhas e do metapneumovírus aviário utilizando uma reação de transcrição reversa com reação em cadeia pela polimerase duplex

Duplex RT-PCR assay is reported for the simultaneous detection of avian infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV), the causative agents of major diseases in poultry. The duplex RT-PCR assay optimized showed a detection limit of 10-3 (101 EID50/50m L for IBV and 100.5 EID50/50m L for aMPV, respectively when two viruses were mixed and 10-1 for each one separated (103 EID50/50m L for IBV and 102.5 EID50/50m L for aMPV, respectively. It was specific, sensitive and applicable for the rapid detection of these viruses in clinical samples.

Descreve-se um ensaio de duplex RT-PCR assay para a detecção simultânea do vírus da bronquite infecciosa das galinhas (IBV) e do metapneumovirus aviário (aMPV), agentes etiológicos de doenças de elevada importância em avicultura. A duplex RT-PCR otimizada mostrou um limiar de detecção de 10-3 (101 EID50/50m L para IBV e 100.5 EID50/50m L para aMPV, respectivamente, quando da combinação dos dois vírus e 10-1 para cada um dos vírus em separado(103 EID50/50m L para IBV e 102.5 EID50/50m L para aMPV, respectivamente. O ensaio foi demonstrado como específico, sensível e aplicável à rápida detecção destes vírus em amostras clínicas.

Detection of infectious bronchitis virus and Avian metapneumovirus by a duplex semi-nested reversetranscription–polymerase chain reaction assay / Detecção do vírus da bronquite infecciosa das galinhas e do metapneumovírus aviário utilizando uma reação de transcrição reversa com reação em cadeia pela polimerase duplex

duplex RT-PCR assay is reported for the simultaneous detection of avian infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV), the causative agents of major diseases in poultry. The duplex RT-PCR assay optimized showed a detection limit of 10-3 (101 EID50/50m L for IBV and 100.5 EID50/50m L for aMPV, respectively when two viruses were mixed and 10-1 for each one separated (103 EID50/50m L for IBV and 102.5 EID50/50m L for aMPV, respectively. It was specific, sensitive and applicable for the rapid detection of these viruses in clinical samples.(AU)

Descreve-se um ensaio de duplex RT-PCR assay para a detecção simultânea do vírus da bronquite infecciosa das galinhas (IBV) e do metapneumovirus aviário (aMPV), agentes etiológicos de doenças de elevada importância em avicultura. A duplex RT-PCR otimizada mostrou um limiar de detecção de 10-3 (101 EID50/50m L para IBV e 100.5 EID50/50m L para aMPV, respectivamente, quando da combinação dos dois vírus e 10-1 para cada um dos vírus em separado(103 EID50/50m L para IBV e 102.5 EID50/50m L para aMPV, respectivamente. O ensaio foi demonstrado como específico, sensível e aplicável à rápida detecção destes vírus em amostras clínicas.(AU)