A WIDESPREAD PICORNAVIRUS AFFECTS THE HAEMOCYTES OF THE NOBLE PEN SHELL (PINNA NOBILIS) LEADING TO IMMUNOSUPPRESSION

Abstract

The widespread mass mortality of the noble pen shell (Pinna nobilis) has occurred in several Mediterranean countries in the past seven years. Single-stranded RNA virus affecting immune cells and leading to immune disfunction have been widely reported in human and animal species. Here we present data linking P. nobilis mass mortality events (MMEs) to haemocyte picornavirus (PV) infection. This study was performed on 30 specimens, from wild and captive populations. We sampled P. nobilis from two regions of Spain, Catalonia [24 animals] and Murcia [two animals]), and one region in Italy (Venice [four animals]). The low number of analyzed specimens was due to the scarcity of remaining individuals in the Mediterranean Sea. In 100% of our samples, ultrastructure revealed the presence of a virus (20nm diameter), capable of replicating within granulocytes and hyalinocytes, leading to the accumulation of complex vesicles of different dimensions within the cytoplasm. As the PV infection progressed, dead haemocytes, infectious exosomes, and budding of extracellular vesicles were visible, along with endocytic vesicles entering other cells. The THC (total haemocyte count) values observed in both captive (eight animals) (3.5 x 104 - 1.60 x105 ml-1 cells) and wild animals (14 samples) (1.90 - 2.42 x105 ml-1 cells) were lower than those reported before MMEs. Sequencing of P. nobilis (six animals) haemocyte cDNA libraries revealed the presence of two main sequences of Picornavirales, family Marnaviridae. The highest number of reads belonged to animals that exhibited active replication phases and abundant viral particles from Trasmission Electron Microscopy (TEM) observations. These sequences correspond to the genus Sogarnavirus - a picornavirus identified in the marine diatom Chaetoceros tenuissimus (named C. tenuissimus RNA virus type II). Real time PCR performed on the two most abundant RNA viruses previously identified by in silico analysis revealed positive results only for the sequences similar to C. tenuissimus RNA virus. These results may not be considered conclusive of picornavirus identification in noble pen shell haemocytes, and require further studies. Our findings suggest that picornavirus infection likely causes immunosuppression, making individuals prone to opportunistic infections which is a potential cause for the MMEs observed in the Mediterranean.