Detection and molecular characterization of Avipoxvirus in Culex spp. (Culicidae) captured in domestic areas in Rio de Janeiro, Brazil.

Avian pox is a highly contagious poultry disease that causes significant economic losses. Mosquitoes belonging to the genus Culex (Diptera: Culicidae) have a fundamental role in disseminating Avipoxvirus (Poxviridae). This study proposes investigating the presence of Avipoxvirus (APV) DNA in Culex spp. from Rio de Janeiro to determine its frequency and perform a phylogenetic analysis based on the core like the 4b protein (p4b) gene. The detection of APVs was conducted individually on four hundred Culex spp. mosquitoes. A total of 12.23% (47/384) of the Culex spp. were positive in the PCR. Sequencing the p4b gene revealed that this study's sequences displayed 98.8-99% identity with Fowlpoxvirus (FWPW) sequences available in GenBank. In the phylogenetic analysis, these APVs were clustered in the A1 subclade together with FWPW sequences from several countries. The evolutionary distance of the p4b gene was 0.61 ± 0.21% in rural areas and 0.38 ± 0.16% in peri-urban areas. The current investigation is the first study to report the detection of APVs in field-caught mosquitoes. Moreover, a high frequency of APV DNA was observed in Culex spp. captured in domestic areas, where backyard poultry is present. This data demonstrates the importance of implementing control measures for Culex spp. to mitigate the transmission of APVs in backyard poultry in Rio de Janeiro.

Robust comparative performance of genomic DNA extraction methods from non-engorged phlebotomine sandflies.

The present study is a comparative analysis of DNeasy Blood & Tissue Qiagen® kit (Qiagen®, Hilden, Alemanha), salting out, HotShot and phenol-chloroform protocols to extract DNA from sandflies. In addition, a comparative test using sandflies with and without eyes evaluated the potential inhibitory effect in the cPCR. An inhibition test was performed using an exogenous DNA added to the qPCR. The genomic DNA quality of each sample was evaluated by cPCR based on the cytochrome c oxidase subunit I (cox1) gene. The DNA extraction protocols showed the following percentage of amplification: HotShot (91.6% [55/60]), salting out (71.6% [43/60]), phenol-chloroform (95% [57/60]) and kit DNeasy Blood & Tissue Qiagen® (73.3% [44/60]). The phenol-chloroform method achieved a significantly higher frequency of cox1 gene amplification. The pigment present in the phlebotomine's eyes seems to inhibit cPCR reactions since the frequency of amplification of the cox1 gene increased in the sandflies without eyes (p < 0.0001). The HotShot method showed the highest inhibitory potential. These manual extraction techniques can be an inexpensive and effective alternative to study vector-pathogen interactions.