Preliminary Study on Natural Infection of Shellfish with Movement Disorder Nodavirus (MDNV)

Abstract

China's marine aquaculture is rich and diverse. As China's marine aquaculture industry continues to expand, the impact of disease problems on this sector has become increasingly prominent, leading to substantial economic losses for the country's aquaculture enterprises. Shellfish is an important source of high-quality protein for human beings and simultaneously plays an important ecological service function in the marine ecosystem. Shellfish aquaculture in China mainly focuses on marine shellfish, accounting for the highest proportion of the total marine aquaculture output in China. Recently, pathogenic infections, including bacteria, fungi, and parasites, have caused notable disease outbreaks in China's mariculture shellfish. For example, oyster herpesvirus and its variant viruses can cause disease infections in many shellfish organisms and are important pathogens that lead to shellfish mortality. Additionally, shellfish farming in China's northern Yellow Sea area is often infected by Vibrio canis and Bacillus luminescens. Regarding the developmental changes of diseases, shellfish diseases in mariculture mainly include pathogenic and non-pathogenic diseases. Pathogenic diseases have harmful manifestations, such as bacteria, parasites, fungi, and pathogenic worms, whereas non-pathogenic diseases are mainly caused by mechanical injuries, drug abuse, and physical or chemical stimuli. Shellfish diseases in mariculture often involve multiple infections and cross-contamination, and non-pathogenic diseases can sometimes develop into pathogenic hazards. Around 2003, "viral covert mortality disease (VCMD)" appeared in the Penaeus vannamei culture area in southern China. VCMD is an emerging infectious disease caused by covert mortality nodavirus (CMNV). The cross-species transmission capacity of CMNV has caused huge economic losses to the aquaculture industry. Movement disorder nodavirus (MDNV) is similar to CMNV, both of which are members of the newly discovered α-nodavirus genus. P. vannamei, infected with MDNV, showed sinking characteristics and decreased swimming performance. To understand whether MDNV has the same cross-species transmission ability as CMNV, we collected wild shellfish samples in the offshore area of the Yellow Sea and the East China Sea and systematically analyzed the possibility of the shellfish carrying and infecting by MDNV. TaqMan real-time fluorescence quantitative PCR (TaqMan RT-qPCR), histopathology, and in situ hybridization were used in this investigation. The results of the TaqMan RT-qPCR assay showed that MDNV was detected in Crossostrea gigas and Azumapecten farreri collected in the offshore area of the Yellow Sea and East China Sea. The positive detection rates of MDNV in C. gigas and A. farreri was 16.67% (1/6) and 33.3% (2/6), respectively. MDNV was not detected in Scapharca broughtonii and Ruditapes philippinarum. Histopathology and in situ hybridization analysis showed many eosinophilic inclusions in the mantle tissue of C. gigas, and nuclear pyknosis occurred. In the gill tissue of C. gigas, epithelial cells were severely damaged, and the appearance of vacuolation and necrosis of karyopyknotic nuclei was observed in the gill. The gill filaments were filled with many eosinophilic inclusions. The epithelial cells of A. farreri chlamys were filled with eosinophilic substances, and the karyopyknotic nuclei of some epithelial cells were also observed. The positive hybridization signals of the purple MDNV RNA probe were observed in the lesion sites of C. gigas and A. farreri. In addition, the MDNV RNA-dependent RNA polymerase nucleic acid sequences were cloned and then submitted to NCBI web multi-sequence alignments using the online program. The multiple sequence alignment results showed that the similarity of MDNV sequence fragments of C. gigas and A. farreri was 99.3%, and the similarity between them and the original CMNV isolates was 83.3%. This study shows that MDNV can cross the species barrier under natural conditions, infect wild shellfish in the offshore area of the Yellow Sea, and cause evident pathological damage to target tissues. It also suggests that attention should focus on avoiding the use of MDNV-infected shellfish seed in aquaculture operations to prevent large-scale transmission or epidemic of MDNV in cultured shellfish.

Keywords

• movement disorder nodavirus (mdnv)
• natural infection
• host jumps
• crassostrea gigas
• azumapecten farreri