Covert Mortality Nodavirus Research Articles

Development of reliable in vitro long-term culture systems for hematopoietic cells of crayfish Cherax quadricarinatus and virus susceptibility assay

Long-term in vitro cultured crayfish cells can serve as a valuable tool for investigating various aspects of crayfish biology, encompassing cell physiology and viral mechanisms. Regrettably, they are difficult to maintain a highly active state and survive during long-term and continuous culture in vitro. In order to generate highly viable crayfish cell cultures, it is crucial to optimize dependable culture conditions and utilize a suitable cell medium. In this study, we have successfully developed reliable in vitro long-term culture systems for hematopoietic (HPT) cells of crayfish Cherax quadricarinatus by formulating an optimal crayfish cell growth medium (OCCM), including under two-dimensional (2D) and three-dimensional (3D) conditions. The novel medium was optimized by a mix of exogenous nutrients, crayfish plasma (CP) and shrimp serum-derived amino acids and saccharides within the Leibovitz's L-15 (L-15) medium. Our newly developed crayfish cell growth medium (CCM-28) enabled the culturing of HPT cells for over 142 days under 2D and over 60 days under 3D conditions, with consistent active cell proliferation observed over a 30-day incubation period. This proliferation was comparable to in vivo conditions, with a cell division rate exceeding 20%, suggesting that a high mitotic rate has been successfully achieved during long-term in vitro culture. Flow cytometry analysis further elucidated the mitosis-arrest of HPT cells cultured in vitro was predominantly observed in the G0/G1 phase. Furthermore, the cell subculture techniques have been established using mechanical dissociation method (in 2D) and substrate exchange method (in 3D). Additionally, the susceptibility of these cell cultures to covert mortality nodavirus (CMNV) and white spot syndrome virus (WSSV) was confirmed by capturing cytopathic effects (CPE) and analyzing viral copy numbers. Species identification using the cytochrome oxidase subunit I (COI) gene established the origin of the cell cultures from C. quadricarinatus. In conclusion, the successful development of highly viable crayfish cells in long-term in vitro culture systems, as demonstrated in this study, will significantly advance the research on immortalization in crayfish cells and the study of mechanisms related to viruses.

The development of a novel quantitative assay for the detection of convert mortality nodavirus (CMNV) in Litopenaeus vannamei

Due to the viral covert mortality disease (VCMD), a new disease brought on by covert mortality nodavirus (CMNV), the Litopenaeus vannamei aquaculture industry in China and Southeast Asian countries have experienced major economic losses over the past ten years. A rapid and accurate on-site pre-detection plays as the first step and a critical part in the efficient control of VCMD spreads and outbreaks. For the rapid and accurate on-site detection of CMNV, a novel real-time quantitative assay based on quantitative recombinase polymerase amplification (qRPA) was developed in this study. By optimizing the optimum primer pairs based on the conserved RNA-dependent RNA polymerase (RdRp) genes, appropriate reaction temperatures, and probe dosages. It was possible to perform the CMNV-qRPA test at 42 °C in 20 min without exhibiting any cross-reactivity with the four major aquatic infections. Then its ability was compared to real-time quantitative PCR (qPCR) as well as nested PCR assays for detection of CMNV. As results of the sensitivity tests, the limit of detection (LOD) for the CMNV-qRPA, CMNV nested PCR, and CMNV qPCR assays were 5.5 × 102 copies μL−1, 5.5 copies μL−1 and 5.5 × 102 copies μL−1, respectively. Based on these results, it is demonstrated that the CMNV-qRPA assay developed in this study is both sensitive and specific for the rapid on-site pre-diagnosis of CMNV. Those findings were also demonstrated the application potential of CMNV-qRPA assay for efficiently preventing and controlling the spread and outbreak of VCMD in the aquaculture industry and application prospect of this diagnosis method in other related fields.

Single von Willebrand factor C-domain protein confers host defense against white spot syndrome virus by functioning as a pattern recognition receptor in Macrobrachium nipponense

The single von Willebrand factor C-domain proteins (SVWCs), also known as Vago, are primarily found in arthropods. Their expression was induced by nutritional status, bacterial and viral infections. Despite the prominence of SVWCs in antiviral immunity, the detailed molecular mechanisms remain poorly explained. SVWC has been proposed to elicit antiviral activities through its function as an interferon analog. In contrast, herein, we illustrate that an SVWC homolog from Macrobrachium nipponense (MnSVWC) confers host defense against white spot syndrome virus (WSSV) and covert mortality nodavirus (CMNV) as a pattern recognition receptor (PRR). qRT-PCR analyses demonstrated that the expression of MnSVWC was enhanced upon WSSV infection in all detected tissues, including gills, nerve cords, and hemocytes. Coating WSSV with recombinant MnSVWC (rMnSVWC) promoted the phagocytic activity of hemocytes and subsequent clearance of invasive WSSV from the prawn. On the other hand, the knockdown of MnSVWC with RNAi improved the proliferation ability of WSSV and CMNV in the prawn. Analysis of ELISA and Co-immunoprecipitation (Co-IP) showed that rMnSVWC could bind WSSV by interacting with the vesicle proteins VP26 and VP28. Co-IP analysis verified the interaction between MnSVWC and calmodulin, which implies a vesicle protein-SVWC-calmodulin-clathrin-dependent mechanism underlying the hemocyte-mediated phagocytosis against WSSV. Subsequently, MnSVWC was recognized to activate the expression of transcription factor STAT and an interferon-stimulating gene Viperin, illustrating its involvement in modulating humoral immunity via activation of the JAK/STAT pathway after WSSV infection. These findings indicate that MnSVWC could bind to WSSV as a PRR and participate in the promotion of hemocyte-mediated phagocytosis and the activation of the JAK/STAT pathway in prawns.

Disinfection of Perinereis aibuhitensis eggs with peroxymonosulfate to eliminate covert mortality nodavirus (CMNV)

Perinereis aibuhitensis is an essential bait for prompting ovarian development and sexual maturation in aquaculture shrimp broodstock. It is common for shrimp to become infected with pathogens by ingesting feed containing potential viruses. To prevent P. aibuhitensis being live feeds for shrimp broodstock from transmitting pathogens, we evaluated the disinfection effect of peroxymonosulfate (PMS) against covert mortality nodavirus (CMNV) by carrying out Oryzias melastigma (marine medaka) challenge assay and testing CMNV-exposed P. aibuhitensis eggs. In the marine medaka injected with PMS (15 and 20 ppm (ppm))-inactivated CMNV, no CMNV was detected at 6 and 12 days post-infection (d.p.i.) by using quantitative reverse transcription PCR (RT-qPCR). As well, no CMNV probe positive signal and pathological signs caused by CMNV in these marine medaka were present in situ hybridization (ISH) and histopathology assays. CMNV was not detected in the nectochaeta larvae originated from CMNV-exposed P. aibuhitensis eggs treated with PMS (5, 10, 15 and 20 ppm) for 1 min. In addition, nereids treated with 15 ppm PMS for 1 min had higher hatchability. Even at low concentrations of PMS-treated eggs, the hatchability was higher than that of the no-PMS-treated group, and no adverse effects on larval development after hatching were observed. Our study showed that disinfection of CMNV-exposed eggs by PMS is feasible. Disinfection of P. aibuhitensis eggs with PMS could reduce the risk of transmission of CMNV. Within acceptable limits for hatchery spawn losses, the recommended disinfection dose of PMS is 15 ppm for 1 min. This study laid the foundation for the culture of specific pathogen-free P. aibuhitensis and to provide biological baits with no pathogenic risk to farmed shrimp.

Routine development of long-term primary cell culture and finite cell line from the hemolymph of greasyback shrimp (Metapenaeus ensis) and virus susceptibility

Long-term and continuous shrimp cell cultures can offer an indispensable tool for studies on the isolation, multiplication and pathogenic mechanism of shrimp causative viruses at cellular and molecular levels. However, they are hard to be developed routinely and continuous shrimp cell line is still unavailable. This can be attributed to a great degree to the unsuitability of medium used. In this study, we have successfully developed a long-term culture condition for the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. Using the newly developed gelatin-containing optimal basic medium (SBM-12), MeH cell monolayer could be routinely developed and maintained healthily beyond 275 days. In the novel optimal growth medium (SGM-10), the proliferation potentials of MeH cells have been markedly improved, evidenced by the presence of dividing cells in the primarily cultured 30-day hemocytes, suggesting that the mitosis-arrest of MeH cells has been tentatively broken. It was also found that among the 4 types of hemocytes (granulocyte, semi-granulocyte, hyalinocyte and prohaemocyte), the hyalinocytes could not survive after a long-term in vitro culture, but the prohaemocytes could survive well and thus could serve as a good cell source. The addition of gelatin could significantly alter the attachment and cellular morphology of MeH cells and result in a loose attachment and round shape, which made it easy for the cells to be passaged by physical flushing for up to 32 times and a finite shrimp cell line can be developed routinely. Virus susceptibility test showed that the MeH cells cultured in the optimal growth medium were sensitive to shrimp covert mortality nodavirus (CMNV), and obvious cytopathic effects and viral multiplication were detected in the infected cells. In a word, the success in the routine development of long-term primary cell culture and finite cell line from shrimps in this study will greatly facilitate the establishment of shrimp continuous cell lines and studies on shrimp viruses.

Development of a Novel RT-qPCR Detecting Method of Covert Mortality Nodavirus (CMNV) for the National Proficiency Test in Molecular Detection.

Covert mortality nodavirus (CMNV), the pathogen of viral covert mortality disease (VCMD), has caused serious economic losses of shrimp aquaculture in Southeast Asian countries and China in the past decade. In view of that the rapid and accurate laboratory detection of CMNV plays a major role in the effective control of the spread of VCMD. The national proficiency test (NPT) for the detection of covert mortality nodavirus (CMNV) started in China from 2021. In this study, a novel TaqMan real-time reverse transcription quantitative PCR (RT-qPCR) detection method for CMNV with higher sensitivity than previous reports was established based on specific primers and probe designing from the conserved regions of the CMNV coat protein gene for using molecular detection of CMNV in NPT. The optimized RT-qPCR reaction program was determined as reverse transcription at 54.9 °C for 15 min and denaturation at 95 °C for 1 min, followed by 40 cycles including denaturation at 95 °C for 10 s, and annealing and extension at 54.9 °C for 25 s. The detection limit of the newly developed RT-qPCR method was determined to be as low as 2.15 copies of CMNV plasmids template per reaction, with the correlation coefficient (R2) at above 0.99. The new method showed no cross reaction with the six common aquatic animal pathogens and could be finished in one hour, which represents a rapid detection method that can save 50% detection time versus the previously reported assay. The CMNV TaqMan probe based RT-qPCR method developed in present study supplies a novel sensitive and specific tool for both the rapid diagnosing and quantitating of CMNV in NPT activities and in the farmed crustaceans, and will help practitioners in the aquaculture industry to prevent and control VCMD effectively.

Investigation of Pathogenic Mechanism of Covert Mortality Nodavirus Infection in Penaeus vannamei.

Viral covert mortality disease (VCMD), also known as running mortality syndrome (RMS), is caused by covert mortality nodavirus (CMNV) and has impacted the shrimp farming industry in Asia and Latin America in recent years. The pathogenic mechanism of CMNV infecting Penaeus vannamei was investigated in this study. In the naturally infected shrimp, histopathological and in situ hybridization (ISH) analysis verified that CMNV infection and severe cellar structural damage occurred in almost all cells of the ommatidium. Under transmission electron microscopic (TEM), vacuolation and necrosis, together with numerous CMNV-like particles, could be observed in the cytoplasm of most cell types of the ommatidium. The challenge test showed that a low CMNV infectious dose caused cumulative mortality of 66.7 ± 6.7% and 33.3 ± 3.6% of shrimp in the 31-day outdoor and indoor farming trials, respectively. The shrimp in the infection group grew slower than those in the control group; the percentage of soft-shell individuals in the infection group (42.9%) was much higher than that of the control group (17.1%). The histopathological and ISH examinations of individuals artificially infected with CMNV revealed that severe cellar damage, including vacuolation, karyopyknosis, and structural failure, occurred not only in the cells of the refraction part of the ommatidium, but also in the cells of the nerve enrichment and hormone secretion zones. And the pathological damages were severe in the nerve cells of both the ventral nerve cord and segmental nerve of the pleopods. TEM examination revealed the ultrastructural pathological changes and vast amounts of CMNV-like particles in the above-mentioned tissues. The differential transcriptome analysis showed that the CMNV infection resulted in the significant down-regulated expression of genes of photo-transduction, digestion, absorption, and growth hormones, which might be the reason for the slow growth of shrimp infected by CMNV. This study uncovered unique characteristics of neurotropism of CMNV for the first time and explored the pathogenesis of slow growth and shell softening of P. vannamei caused by CMNV infection.

Investigation on Natural Infection of Covert Mortality Nodavirus in Farmed Giant Freshwater Prawn (Macrobrachium rosenbergii).

Simple SummaryCovert mortality nodavirus (CMNV) is a newly discovered aquatic animal virus in recent years. Here, we detected CMNV positive in farmed giant freshwater prawn (Macrobrachium rosenbergii) from Jiangsu, China by TaqMan RT-qPCR. Meanwhile, in situ hybridization and histological analysis indicated that the intestine, gill, hepatopancreas and ovary of giant freshwater prawn were the target organs of CMNV. In addition, a large number of CMNV-like particles were observed in the hepatopancreas and gill tissues under transmission electron microscopy. Overall, our study confirms that giant freshwater prawn is a susceptible host of CMNV, further expands the known host range of CMNV, and provided a new direction for further investigation and exploration of multiple pathogenic factors of giant freshwater prawn disease. Covert mortality nodavirus (CMNV), from the Nodaviridae family, is characterized by its unique cross-species transmission and wide epidemic distribution features. In this study, Macrobrachium rosenbergii was proved to be infected naturally by CMNV, which further expand the known host range of CMNV. Here, 61.9% (70/113) of the M. rosenbergii samples collected from Jiangsu Province were CMNV positive in the TaqMan RT-qPCR assay, which indicated the high prevalence of CMNV in M. rosenbergii. Meanwhile, the sequences of CMNV RdRp gene cloned from M. rosenbergii were highly identical to that of the original CMNV isolate from Penaeus vannamei. In situ hybridization (ISH) and histology analysis indicated that the intestine, gill, hepatopancreas and ovary were the targeted organs of CMNV infection in M. rosenbergii, and obvious histopathological damage including vacuolation and karyopyknosis were occurred in the above organs. Notably, the presence of CMNV in gonad alerted its potential risk of vertical transmission in M. rosenbergii. Additionally, numerous CMNV-like particles could be observed in tissues of hepatopancreas and gill under transmission electron microscopy. Collectively, our results call for concern of the potential negative impact of the spread and prevalence of CMNV in M. rosenbergii on its aquaculture, as well as providing a renewed orientation for further investigation and exploration of the diverse pathogenic factors causing M. rosenbergii diseases.

Investigation on Natural Infection of Covert Mortality Nodavirus in Large Yellow Croaker, Larimichthys crocea

Covert mortality nodavirus (CMNV) recently became more prevalent, severely affecting farmed shrimps, and also other invertebrates and teleost fish, in the coastal areas of China. For better understanding of its prevalence and susceptibility of large yellow croaker Larimichthys crocea to CMNV, an epidemiological survey was carried out in the main producing areas (Ningbo, Zhejiang, China, and Ningde, Fujian, China) and its offshore feeding grounds in the Southern Yellow Sea. The collected cultured L. crocea individuals showed clinical symptoms such as anorexia and abnormal swimming and diagnosed for CMNV infection by using RT-nPCR assay. The positive rates for CMNV in the collected L. crocea samples from Ningbo, Ningde, and the Yellow Sea were 14.29% (4/28), 20.00% (7/35), and 16.67% (1/6), respectively. Furthermore, CMNV diagnosis using TaqMan probe-based reverse transcription quantitative PCR (TaqMan RT-qPCR) yielded positive rates of 25.00% (7/28), 22.86% (8/35), and 16.67% (1/6) respectively in the above-mentioned samples, and with a range of 12.73–3,108.33 of CMNV copy numbers/μg total RNA. Phylogenetic tree analysis based on the RNA-dependent RNA polymerase (RdRp) and capsid protein genes showed that CMNV isolates from L. crocea samples were clustered tightly with the original isolate of CMNV from Penaeus vannamei. The histopathological studies indicated that severe cellular necrosis occurred in the heart, liver, spleen, eye, and gill of naturally infected fish. Stronger positive signals for CMNV-specific probes appeared in the necrotic cells and tissues in the in situ hybridization (ISH) analysis. Transmission electron microscopy (TEM) revealed the presence of numerous 30 nm diameter CMNV-like viral particles. The purified CMNV suspension could cause a significant cytopathic effect (CPE) in epithelioma papilloma cyprini (EPC) cells and resulted in the formation of typical inclusion bodies, and also visible CMNV particles around the lysed EPC cells under the TEM. Taken together, all results of this study demonstrated that L. crocea is susceptible to CMNV. The prevalence of CMNV and its potential impacts on the wild population of L. crocea see the need for further investigations.

Pathogenicity study of covert mortality nodavirus (CMNV) infection in zebrafish model

Covert mortality nodavirus (CMNV), a new emerging shrimp virus, was capable of crossing species barriers to infect fish. However, impending studies in elucidating pathogenicity of CMNV in fish have been limiting due to the lack of appropriate animal disease models. An attempt of establishing an in vivo model of CMNV infection in zebrafish (Danio rerio) were conducted in present study. Further, CMNV infection in zebrafish important tissues was also investigated by using the methods of fatality rate statistics, histopathology, in situ hybrization (ISH) and transmission electron microscopy (TEM). Behavioral observations of the infected zebrafish showed that the infected individuals exhibited an abnormal spiraling swimming activity, and some individuals had exceptionally protruding eyes. The eye and brain tissues of artificially infected zebrafish were proved to be CMNV-positive by ISH, and pathological changes like vacuolation of nervous tissue were observed in this two tissues. Analysis of TEM also confirmed masses of CMNV-like virus particles in the brain and eye tissues of the infected zebrafish. CMNV positive signals of ISH were also detected in muscle, intestine and gill tissues, and myolysis in the muscle, dropping of epithelial cells in gill were found in the same site with positive signals. In addition, CMNV could cause 53.33% mortality of zebrafish within 14 days of post challenge injection. The establishment of the zebrafish infection model of CMNV offered a valuable tool for further investigations of the host-virus interactions, especially the underlying molecular mechanisms of CMNV for crossing species barrier to infect fish.

Histopathological study of covert mortality nodavirus infection in sea cucumber (Apostichopus japonicus)

Covert mortality nodavirus (CMNV), a unique aquatic pathogen first isolated from shrimp, has a broad host range that includes crustaceans and teleosts. The sea cucumber Apostichopus japonicus, an Echinoderm, was recently shown to be a host for CMNV. To investigate the histopathology of CMNV infection in sea cucumber, four out of 61 A. japonicus individuals that tested positive in reverse transcription nested PCR (RT-nPCR) assays were furtherly analyzed through in situ hybridization (ISH), histology, and transmission electron microscopy (TEM). Extensive bluish-purple positive hybridization signals of CMNV probe were found in tissues of the respiratory trees, intestine, and gonad in conjunction with a variety of pathological lesions, including vacuolation, karyopyknosis, and muscle necrosis. Also, masses of CMNV-like particles were observed through TEM in these tissues, as well as in the body wall and longitudinal muscle. Sea cucumber is important in Chinese aquaculture, and CMNV infection of gonad tissue indicates a potential for vertical transmission that could negatively impact larval rearing.

Natural infection of covert mortality nodavirus affects Zebrafish (Danio rerio)

Covert mortality nodavirus (CMNV), a novel aquatic pathogen, causes viral covert mortality disease (VCMD) in shrimps and also known to infect farmed marine fish. To date, there has no report regarding the ability of this virus to infect freshwater fish. In this study, we screened and discovered CMNV-positive freshwater zebrafish individuals by reverse transcription-nested PCR (RT-nPCR). The sequence of CMNV amplicons from zebrafish was found to share 99% identity with RNA-dependent RNA polymerase (RdRp) gene of the original CMNV isolate. Histopathological examination of the CMNV-positive zebrafish samples revealed extensive vacuolation and karyopyknosis lesions in the retina of the eye and the midbrain mesencephalon. CMNV-like virus particles were visualized in these tissues under transmission electron microscope. Different degrees of pathological damages were also found in muscle, gills, thymus and ovarian tissues. Strong positive signals of CMNV probe were observed in these infected tissues by in situ hybridization. Overall, all results indicated that zebrafish, an acknowledged model organism, could be infected naturally by CMNV. Thus, it is needed to pay close attention to the possible interference of CMNV whether in assessment of toxic substances, or in studying the developmental characterization and the nerval function, when zebrafish was used as model animal.

Natural Infection of Covert Mortality Nodavirus in Small Yellow Croaker in Coastal Water

Covert mortality nodavirus (CMNV) is an alphanodavirus mainly infecting marine shrimp and co-inhabiting organisms in aquaculture ponds. To evaluate the possibility of CMNV prevalence in the wild fish species, epidemiological survey of CMNV infection in Larimichthys polyactis, the dominant species in the Yellow Sea and East China Sea, were conducted in the present study. We performed CMNV RT-LAMP assay in samples of L. polyactis collected in August 2018 and 2019 and found that CMNV prevalence was 18% and 7%, respectively. The nucleotide sequences of both RdRp and capsid protein genes of CMNV from L. polyactis were 99% similar to those of CMNV isolated from shrimp. CMNV-positive L. polyactis exhibited necrosis of cardiac muscle, oocytes loosely arranging, severe cytoplasmic vacuolation of hepatocytes, moderate pyknosis of brain pyramidal cells, degenerate renal tubular cells with ill-defined margins, and declined spleen cells in the histological examination. Moreover, CMNV-positive signals were further observed in pyramidal cells of the brain, the cortical area of the kidney, oocyte growth rings, and in necrotic tissues of cardiac muscle, liver, and spleen in the in situ hybridization assay. The results revealed that CMNV had colonized in the wild populations of L. polyactis and the ecological risk of CMNV spread and epidemic in wild fish in the coastal water was non-negligible.

First Report on Natural Infection of Nodavirus in an Echinodermata, Sea Cucumber (Apostichopus japonicas).

Cross-species transmission of emerging viruses happens occasionally due to epidemiological, biological, and ecological factors, and it has caused more concern recently. Covert mortality nodavirus (CMNV) was revealed to be a unique shrimp virus that could cross species barrier to infect vertebrate fish. In the present study, CMNV reverse transcription-nested PCR (RT-nPCR)-positive samples were identified from farmed sea cucumber (Apostichopus japonicas) in the CMNV host range investigation. The amplicons of RT-nPCR from sea cucumber were sequenced, and its sequences showed 100% identity with the RNA-dependent RNA polymerase gene of the original CMNV isolate. Histopathological analysis revealed pathologic changes, including karyopyknosis and vacuolation of the epithelial cells, in the sea cucumber intestinal tissue. The extensive positive hybridization signals with CMNV probe were shown in the damaged epithelial cells in the in situ hybridization assay. Meanwhile, transmission electron microscopy analysis revealed CMNV-like virus particles in the intestine epithelium. All the results indicated that the sea cucumber, an Echinodermata, is a new host of CMNV. This study supplied further evidence of the wide host range of CMNV and also reminded us to pay close attention to its potential risk to threaten different aquaculture animal species.

Major viral diseases in culturable penaeid shrimps: a review

The practice and development of the culture of commercially important penaeid shrimp results in the occurrence of infectious and non-infectious diseases worldwide. Most of the diseases are caused by the opportunistic microorganisms which are part of the microflora and fauna of the penaeid shrimp. Once the pathogens get a favourable condition, it will cause diseases to the host organisms. Most of the organisms are ubiquitous and are found and reported all over the cultured area in the world. The pathogens are the bacteria, viruses, parasites and protozoans. Viruses are the most important disease-causing agents in penaeid shrimps. The penaeid viruses are widely spread in geographical areas of shrimp culture. The shipment of broodstock and post larvae from one geographical region to another region often results in spreading of viral diseases. The penaeid viruses are the Whispovirus, white spot syndrome virus (WSSV), Penaeus monodon baculovirus (monodon baculovirus), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV), Taura syndrome virus (TSV), yellow head virus (YHV), white tail disease (WTD), covert mortality nodavirus (CMNV), Laem–Singh virus (LSNV) and shrimp haemocyte iridescent virus (SHIV). The review examines and highlights the aquaculture penaeid shrimp viral diseases in detail and concludes the need and importance of severe disease surveillance of the global countries through networking and coordination to safeguard the aquaculture practice for our future generation.

Genomic characterization of covert mortality nodavirus from farming shrimp: Evidence for a new species within the family Nodaviridae

The prevalence of covert mortality nodavirus (CMNV) has become one of the major threats to the shrimp farming industry in Asia and South America recently. Here, the genomic RNA1 and RNA2 of CMNV were characterized by using transcriptome sequencing and RT-PCR. Our study revealed that RNA1 is 3228 bp in length, and contains two putative Open Reading Frames (ORFs), one encoding the RNA dependent RNA polymerase (RdRp) of length 1043 amino acids and another encoding the protein B2 with a length of 132 amino acids. RNA2 is 1448 bp in length and encodes a capsid protein of 437 amino acids. CMNV shared the highest similarity of 51.78 % for RdRp with the other known nodaviruses. Phylogenetic analyses on the basis of RdRp, B2 and capsid proteins indicated that CMNV might represent a novel viral species in the family Nodaviridae. This study reported the first genome sequence of CMNV and it would be helpful for further studies of CMNV in relation to its evolution, diagnostic technique and control strategy.

Infection of covert mortality nodavirus in Japanese flounder reveals host jump of the emerging alphanodavirus.

Interspecies transmission of viruses, where a pathogen crosses species barriers and jumps from its original host into a novel species, has been receiving increasing attention. Viral covert mortality disease, caused by covert mortality nodavirus (CMNV), is an emerging disease that has recently had a substantial impact on shrimp aquaculture in Southeast Asia and Latin America. While investigating the host range of CMNV, we found that this virus is also capable of infecting populations of the farmed Japanese flounder Paralichthys olivaceus, a vertebrate host. The infected fish were being raised in aquaculture facilities that were also producing marine shrimp. Through RT-nPCR, targeting the RNA-dependent RNA polymerase (RdRp) gene of CMNV, we found that 29 % of the fish sampled were positive. The amplicons were sequenced and aligned to the RdRp gene of shrimp CMNV and were found to have 98 % identity. Histopathological examination indicated that CMNV-positive fish showed vacuolation of nervous tissue in the eye and brain, as well as extensive necrosis of cardiac muscle. In situ hybridization showed positive reactions in tissues of the eye, brain, heart, liver, spleen and kidney of infected fish. Transmission electron microscopy showed the presence of CMNV-like particles in all of the above-mentioned tissues, except for brain. The novel finding of a shrimp alphanodavirus that can also infect farmed P. olivaceus indicates that this virus is capable of naturally crossing the species barrier and infecting another vertebrate. This finding will contribute to the development of efficient strategies for disease management in aquaculture.

Development and validation of a TaqMan RT-qPCR for the detection of convert mortality nodavirus (CMNV)

Covert mortality nodavirus (CMNV), an emerging RNA virus, is the pathogen of viral covert mortality disease (VCMD), which has emerged as a cause of serious losses in shrimp aquaculture in China. To improve VCMD diagnosis, a one-step, real-time TaqMan probe-based reverse transcription quantitative PCR (RT-qPCR) was developed in this study. The TaqMan RT-qPCR was optimized firstly, whereby the best results were obtained with 0.2 μM of each primer, 0.2 μM probe, and 0.5 μL Enzyme Mix II. The optimal reaction program was determined as 15 min at 51ºC for reverse transcription and 5 min at 95 ºC, followed by 40 cycles of denaturation at 94 ºC for 10 s, and annealing and extension at 52.7 ºC for 30 s. The optimized assay detected as little as 9.6 pg total RNA from CMNV-infected shrimp and 5.7 copies of the target plasmid. The RT-qPCR assay for CMNV with a high correlation coefficient (r2 = 0.996) was developed basing on the standard curve generated by plotting the threshold cycle values (y) against the common logarithmic copies (log10nc as x; nc is copy number) of pMD20-CMNV. The diagnostic sensitivity and specificity of this assay versus the previously reported RT-qPCR was 96.2% and 98.0%, respectively. This method is highly specific to CMNV, as it showed no cross-reactivity with other common shrimp viruses. It is anticipated that the newly developed and optimized RT-qPCR assay will be instrumental for the rapid diagnosis and quantitation of CMNV.

Evidence for Cross-Species Transmission of Covert Mortality Nodavirus to New Host of Mugilogobius abei.

Viral covert mortality disease (VCMD), caused by covert mortality nodavirus (CMNV), is a newly emerging disease affecting most cultured shrimp and other crustaceans, but not fish. However, we discovered for the first time that Mugilogobius abei, a common marine fish collecting from shrimp farming ponds and surrounding coastal waters in China, was tested to be CMNV positive based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Further investigation based on the quantitative RT-LAMP assay indicated that 39% individuals of sampled M. abei were CMNV positive. Sequencing and alignment of sequences revealed that the partial RNA-dependent RNA polymerase gene of CMNV isolated from M. abei shared 98% homology with that from the original CMNV isolates. Histopathological analysis showed that CMNV infection in M. abei could induce extensive skeletal muscle necrosis, nervous tissue vacuolation in retina of eye and cerebellum of brain. Positive signals were verified in skeletal muscle, eye, brain and intestine by in situ hybridization (ISH) with CMNV probes. Under transmission electron microscope (TEM), CMNV particles were further visualized in the cytoplasm of neurogliocytes, granulocytes and myocytes in the CMNV positive samples diagnosed by ISH. All findings suggested that CMNV, a typical alphanodavirus originated from shrimp, could switch their hosts to fish by cross-species transmission. Meanwhile, the results reminded us to pay close attention to the high risk of CMNV to use fish as intermediate or new host as well as potentially spread or cause epidemic among cultured marine fish.

Vectors and reservoir hosts of covert mortality nodavirus (CMNV) in shrimp ponds.

Covert mortality nodavirus (CMNV) is the pathogen that has been identified as the cause of viral covert mortality disease (VCMD) in marine and brackish water shrimp. Recent outbreaks of this disease have resulted, and continue to result, in substantial production and economic losses to shrimp aquaculture producers in China and elsewhere. To explore potential vectors and reservoir hosts of CMNV, we collected fifteen species of invertebrates from shrimp ponds affected by VCMD. Samples were tested through the use of: reverse transcription loop-mediated isothermal amplification (RT-LAMP), reverse transcription nested PCR (RT-nPCR) followed by gene sequencing, histopathology, and in situ RNA hybridization (ISH). The results of RT-LAMP and RT-nPCR assay indicated that CMNV positive samples were identified in eleven species including brine shrimp Artemia sinica, a barnacle Balanus sp., the rotifer Brachionus urceus, the amphipod Corophium sinense Zhang, the Pacific oyster Crassostrea gigas, a hermit crab Diogenes edwardsii, the common clam Meretrix lusoria, a ghost crab Ocypode cordimundus, the hyperiid amphipod Parathemisto gaudichaudi, a fiddler crab Tubuca arcuata, and an unidentified gammarid amphipod. The alignment of CMNV RNA-dependent RNA polymerase gene sequences from eight of the species demonstrated high identities (97-100% in nucleotide sequence) with that from the original CMNV isolates of Penaeus vannamei, which suggests that these species could either be infected with, or acting as mechanical vectors of, CMNV. The CMNV infection in C. sinense, D. edwardsii, O. cordimanus Zhang, P. gaudichalldi, and T. arcuata results, to varying degrees, in vacuolation and necrosis of targeted tissues, as was verified by ISH. The infection of CMNV in these five species suggests that they might act as reservoir hosts of CMNV. The results indicate that the common species of invertebrates inhabiting shrimp ponds may constitute biological risk factors for CMNV outbreaks.