Extracting turkey coronaviruses from the intestinal lumen of infected turkey embryos yields full genome data with good coverage by NGS

ABSTRACT

Currently, turkey coronaviruses (TCoV) are isolated from homogenized intestines of experimentally infected embryos to ensure a maximum recovery of viral particles from all components of the intestines. However, the process of homogenization also ensures release of a significant amount of cellular RNAs into the sample that hinders downstream viral genome sequencing. This is especially the case for next generation sequencing (NGS) which sequences molecules at random. This characteristic means that the heavily abundant cellular RNA in the sample drowns out the minority viral RNA during the sequencing process and, consequently, very little to no viral genome data are obtained. To address this problem, a method was developed, in which 10 descendent isolates of the European strain of TCoV were recovered uniquely from the intestinal lumen without homogenization of the tissue. For nine out of 10 samples, NGS produced viral RNA reads with good coverage depth over the entire TCoV genomes. This is a much-needed new, simple and cost effective method of isolating TCoV that facilitates downstream NGS of viral RNA and should be considered as an alternative method for isolating other avian enteric coronaviruses in the interest of obtaining full-length genome sequences.

KEYWORDS:

• Turkey
• coronavirus
• enrichment
• in ovo
• NGS
• sequencing

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was partly funded by Agence National de Sécurité Sanitaire de l’Alimentation, l’Environnement et du Travail (ANSES) [grant number 180019], the “Région Bretagne”, the “Université Bretagne Loire” and the “Université de Rennes 1” (UNIR) [code of convention: 180019] and partly funded by the European Joint Programme One Health METASTAVA [no 773830].