Optimizing environmental viral surveillance: bovine serum albumin increases RT-qPCR sensitivity for high pathogenicity avian influenza H5Nx virus detection from dust samples

ABSTRACT The latest outbreaks of high pathogenicity avian influenza viruses (HPAIVs) were the most widespread ever seen in Europe, entailing considerable economic costs and raising public health concerns. Virological surveillance protocols involve reverse transcription quantitative polymerase chain reactions (RT-qPCR) from tracheal and cloacal swabs, which are laborious to perform on a large scale and require special skills. Environmental sampling, and especially dust collection, may represent a rele vant alternative, as it is cheap, non-invasive for animals, simpler, and quicker to carry out. The main drawback of dust samples is that they may contain high amounts of organic substances that can inhibit RT-qPCR reactions. Bovine serum albumin (BSA) is a molecule known to facilitate DNA polymerization in the presence of numerous inhibitors, including those from feces, litter, or food. We tested its use on dust samples collected on 107 farms localized in areas affected by epizootics of clade 2.3.4.4b HPAIV H5N1. We used a latent class modeling approach to compare the performance of three detection protocols: (i) BSA addition to the RT-qPCR reaction mix, (ii) dilution of template RNA, and (iii) a control protocol. Our results indicate that BSA addition to the RT-qPCR reaction mix improved the sensitivity of the method, by neutralizing inhibitors' effect. Indeed, for hemagglutinin (HA) or matrix (M) RNA detection, the sensitivity of the BSA protocol was the highest, followed by that of the control protocol, and that of the dilution protocol. Our results suggest that the use of BSA could be routinely implemented in HPAIV dust monitoring RT-qPCR protocols. IMPORTANCE With the circulation of high pathogenicity avian influenza viruses having intensified considerably in recent years, the European Union is considering the vaccination of farmed birds. A prerequisite for this vaccination is the implementation of drastic surveillance protocols. Environmental sampling is a relevant alternative to animal sampling. However, environmental samples often contain inhibitory compounds in large enough quantities to inhibit RT-qPCR reactions. As bovine serum albumin is a molecule used in many fields to overcome this inhibitory effect, we tested its use on dust samples from poultry farms in areas heavily affected by HPAIV epizootics. Our results show that its use significantly increases the sensitivity of the method.