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.

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.

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.

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

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.

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.

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.

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.

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.