Simultaneous detection and partial molecular characterization of five RNA viruses associated with enteric disease in chickens: chicken astrovirus, avian nephritis virus, infectious bronchitis virus, avian rotavirus a and avian orthoreovirus, via multiplex RT–qPCR

Figures

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  Figure 1. Test for the formulation of the standard curve in multiplex and single-plex assays. (A) Calibration curve of the multiplex assay. (B) Amplification plot of the multiplex assay [IBV in red color, ANV in green color, CAstV un blue color, AvRVA in purple color and ARV in brown color]. (C) Calibration curve of the single-plex assay for IBV. (D) Amplification plot of the single-plex assay for IBV. (E) Calibration curve of the single-plex assay for ARV. (F) Amplification plot of the single-plex assay for ARV. (G) Calibration curve of the single-plex assay for CAstV. (H) Amplification plot of the single-plex assay for CAstV. (I) Calibration curve of the single-plex assay for AvRVA. (J) Amplification plot of the single-plex assay for AvRVA. (K) Calibration curve of the single-plex assay for ANV. (L) Amplification plot of the single-plex assay for ANV.

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  Figure 2. Map of the percentage of positive samples for each virus across the provinces of Ecuador. Provinces not sampled are shown in gray.

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  Figure 3. Phylogenetic relationships between the sequences of CAstV obtained here and other sequences of CAstV from Brazil, India, Canada, the United States, China, Tanzania, Poland, Malaysia, the Netherlands, Belgium, Iran, Iraq, Italy, and Croatia based on part of the ORF 1b gene nucleotide sequence. Sequences were aligned via the CLUSTAL W method in ClustalX2 2,1. The phylogenetic tree was constructed via the MEGA 7 software package. Numbers along the branches refer to bootstrap values for 1,000 replicates. The scale bar represents the number of substitutions per site. Chicken parvovirus (ChPV) was used as the outgroup. Sequences obtained in this study are in blue and marked with ●.

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  Figure 4. Phylogenetic relationships between the ANV sequences obtained here and other ANV sequences from China, South Korea, Brazil, Italy, the United States, Tanzania, India, Israel, the United Kingdom, Australia, Japan, and Poland based on a portion of the ORF 1b gene nucleotide sequence. Sequences were aligned via the CLUSTAL W method in ClustalX2 2,1. The phylogenetic tree was constructed via the MEGA 7 software package. Numbers along the branches refer to bootstrap values for 1,000 replicates. The scale bar represents the number of substitutions per site. Chicken parvovirus (ChPV) was used as the outgroup. Sequences obtained in this study are in red and marked with ■.

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  Figure 5. Phylogenetic relationships between the sequences of IBV obtained here and other sequences of IBV belonging to the GI groups of IBV classification based on part of the S1 gene nucleotide sequence. Sequences were aligned via the CLUSTAL W method in ClustalX2 2,1. The phylogenetic tree was constructed via the MEGA 7 software package. Numbers along the branches refer to bootstrap values for 1,000 replicates. The scale bar represents the number of substitutions per site. Sequences obtained in this study are pink and marked with ▲.

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  Figure 6. Phylogenetic relationships between the sequences of ARV obtained here and other sequences of ARV belonging to genotypes (G) of ARV based on part of the S1 nucleotide sequence coding to the σC protein. Sequences were aligned via the CLUSTAL W method in ClustalX2 2,1. The phylogenetic tree was constructed via the MEGA 7 software package. Numbers along the branches refer to bootstrap values for 1,000 replicates. The scale bar represents the number of substitutions per site. Sequences obtained in this study are purple and marked with ▼.

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  Figure 7. Phylogenetic relationships between the sequences of AvRV obtained here and other sequences of AvRV belonging to Groups A-H classification on the basis of a fragment of NSP4 gene nucleotide sequences. Sequences were aligned via the CLUSTAL OMEGA method in ClustalX2 2,1. The phylogenetic tree was constructed via the MEGA 11 software package. Numbers along the branches refer to bootstrap values for 1,000 replicates. The scale bar represents the number of substitutions per site. Sequences obtained in this study are in green and marked with ♦.