Surveillance of Influenza A and its sub-type inmigratory wild birds in Nepal

Wild migratory birds whose migratory path often crisscross south and east Asian countries, are known to carry the highly pathogenic avian influenza (HPAI). This study is designed to detect and characterize circulating Influenza A and its subtypes in migratory bird populations in Nepal. This kind of disease surveillance will help to prevent and/or minimize economic losses and public health risks caused by HPAI outbreaks.

Putative migratory bird environmental (faecal) samples were collected (N=1811) from seven potential migratory bird sanctuary sites (n=7) in Nepal from February through March of 2014 to screen Influenza A virus and subsequently characterize its subtypes. These samples were pooled (n=364) based on sites in the BSL2 laboratory of the Center for Molecular Dynamics (CMDN) in Kathmandu. They were then screened for Influenza A virus targeting well conserved Matrix (M) gene using a Real Time PCR assay.Pooled samples that were positive in Influenza A screening were further screened to identify the exactinfluenza A present individual sample. Subtypecharacterization for H and N were then carried outusing PCR- DNA sequencing.A separate round of InfluenzaA screening on domestic backyard poultry (n=100) and duck(n=300) were carried out fromsurrounding villages based onidentified positive Influenza Amigratory bird sample. Using aDNA barcoding technique, wealso identified the migratory bird species that had InfluenzaA virus in its faeces.

Only one pooled samples (Sunsari site) showedPCR amplification of M gene. Individual samples(n=5) within that pool was processed separately; and one sample showed positive PCR amplificationfor Influenza A. Geo-spatial map shows that thissample was collected near KoshiTapu in southeastern Nepal, which is less than 1 km away fromhuman settlement. The species of the putativemigratory bird was identified as Tadornaferruginea(Ruddy Shelduck) using mitochondrial cytochromeC oxidase subunit I (COI) DNA analysis. None ofthe poultry and ducks screened from KoshiTapucame out positive for Influenza A.

Our results indicate that there is potential forInfluenza A being carried by migratory birds andits spill over to domestic poultry and even humansin Nepal. Proper surveillance of Influenza A ingeneral and HPAI in particular in migratory wildbirds in Nepal is important, as Nepal is an importantmigratory path many wild birds; this kind ofsurveillance will also help devise early warningand preventive strategies for HPAI management.