PCR METHOD AND SANGER SEQUENCING FOR PB2 FRAGMENT DETECTION IN INFLUENZA VIRUS STRAINS FROM ROMANIA

Avian influenza (AI) is a highly contagious viral disease that affects several species of domestic birds raised for human consumption (chickens, turkeys, quails, pheasants, etc.), recreational birds, and wild birds. Occasionally, under well-defined circumstances, mammals, including humans, can contract the influenza virus from birds. Influenza A virus belongs to the genus Influenzavirus A of the family Orthomyxoviridae. These viruses have a genome of eight linear, negative-sense ssRNA segments. Influenza A virus isolates are designated by serotype/host species/place of origin/strain name/year of origin and (subtype HA [H] and NA [N]). The internal viral proteins needed for viral assembly and replication are encoded by the remaining six viral genes, PB2, PB1, PA, NS, M, and NP. The purpose of this work is to optimise the PCR detection method and Sanger sequencing of a PB2 fragment of the avian influenza virus genome in order to monitor the genetic evolution of the strains circulating/isolated in our country. The samples used in this study were nucleic acid extracts from different types of organs from birds detected with avian influenza virus in Romania during 2017-2024, from Braila, Galati, Ialomita, and Constanta counties. The RNA extraction was made by the Institute for Diagnosis and Animal Health according to the manufacturing kit they used. The amplification kit used was OneStep RT-PCR (kit OneStep RTPCR, Qiagen). The sequencing was made by outsourcing to a specialised company that performs Sanger sequencing (Antisel, CEMIA). The primers used for Sanger sequencing were the same as the classical PCR. Alignment of the sequence and bioinformatic processing was performed with BioEdit Software. All 9 samples were tested by classic PCR on both fragments of PB2: fragment 1 with the set of primers Bm-PB2-1 for forward and PB2-1250R for reverse and fragment 2 with the set of primers PB2-1105F for forward and Bm-PB2-2341R for reverse. Both of the fragments are migrated at 1200 bp. The purification of samples was appropriate and all sequences were free of errors and impurities. The Sanger sequencing method with the primers used in classical PCR facilitated the analysis of the PB2 fragment of the AIV virus and the subsequent phylogenetic analysis of the Influenza A virus isolates from Romania.