The giant freshwater prawn, Macrobrachium rosenbergii
, is an economically important crustacean and is farmed in many countries. According to the United Nations Food and Agriculture Organization (FAO) (2009), the total production of cultured M. rosenbergii
reached 207,749 tons in 2008, of which Asia produced 127,627 tons. China is the largest farming country of M. rosenbergii
. To date, several viral diseases have been documented in M. rosenbergii
. One is due to a parvo-like virus affecting the digestive tract [1
]. The second viral disease is white tail disease (WTD) caused by M. rosenbergii
nodavirus and is associated with postlarvae mortality in M. rosenbergii
]. In addition, infectious hypodermal and hematopoietic necrosis virus (IHHNV), macrobrachium muscle virus (MMV), and hepatopancreatic parvovirus (HPV) have also been described in cultures of M. rosenbergii
]. Covert mortality nodavirus (CMNV), associated with the covert mortality disease of shrimp were also detected in the cultured M. rosenbergii
] and personal communication).
In 2009, a larval mortality syndrome of M. rosenbergii broke out in a M. rosenbergii hatchery located in Huzhou, Zhejiang Province, China. Afterwards, similar diseases were found in other main breeding areas of M. rosenbergii, including Zhejiang, Jiangsu, Guangxi, and Guangdong Provinces in China. This disease mainly threatened M. rosenbergii larvae, especially zoeal stage V. The clinical signs of the diseased larvae include moulting obstacles, red shed shell, decreased response to stimuli, sinking to the bottom, and eating difficulties. In general, the mortality rate of this disease ranges from 80% to 90%, and the peak mortality rate occurs in the seven-day-old larvae.
To investigate the causative agent of the larval mortality syndrome of M. rosenbergii, efforts were made to isolate the potential pathogenic bacteria from moribund larvae initially. Different bacteria were isolated from different sources of larvae, but similar clinical signs could not be replicated by experimental infection, which suggested that bacteria were not the main pathogen. Therefore, we analyzed viral pathogens using classical methods of virology. Hence, we reported a novel virus, which was isolated from the diseased larvae on the south bank of Taihu Lake and provided the molecular characterization of this novel virus, named Macrobrachium rosenbergii Taihu virus (MrTV).
Dicistroviruses are members of a rapidly growing family of picoranvirus-like RNA viruses, which are named as the Dicistroviridae
]. Dicistroviruses are pathogenic to beneficial arthropods, such as honey bees, shrimp, and insect pests of medical and agricultural importance [10
]. Currently, only two species of dicistroviruses have been reported in crustaceans. One was the TSV in Penaeus vannamei
shrimp, which was first reported in Ecuador in 1991 [14
]. The other was the mud crab dicistrovirus-1 (MCDV-1), which caused 100% mortality in crabs [15
]. This study reported a third member of dicistroviruses, MrTV, in crustaceans that was confirmed to be the causative agent of the larval mortality syndrome in M. rosenbergii
In dicistrovirus, the RNA genome is monopartite and dicistronic with two non-overlapping ORFs separated and flanked by UTRs. Genomic structural analysis revealed that the MrTV genome was arranged in this typical organization. In MrTV, two separated ORFs, flanked by UTRs, were identified. The 5’-proximal ORF encode a replicase protein and the 3’-proximal ORF encode a capsid polyprotein. Phylogenetic analysis using the full-length genome sequence, the putative amino acid sequences of the capsid protein and the RdRp (or polyprotein of ORF1) revealed that MrTV was more closely related to TSV than to any other viruses (Figure 4
and Figure S1
). According to these two features, MrTV could be a novel member of Dicistroviridae
Dicistroviridae contains two genera, Cripavirus
, which can be distinguished by the conserved IGR-IRES sequence. The Cripaviruses
have a conserved bulge sequence (UGAUCU and UGC) in the IGR IRES, while the Aparavirus
es have a different bulge sequence (UGGUUACCCAU and UAAGGCUU) [16
]. Additional stem loop structures and nucleotide sequences in the bulge regions are present in Aparavirus
es. Upon comparing the IGR IRES sequences of the MrTV with other dicistrovirues, the results indicated that MrTV contains the following bulge sequence: UGGAUACCCAU and UAAGGCUU. Considering the identity of the deduced structural protein less than 72% (Table 1
), we proposed that MrTV is a new species in the genus Aparavirus
Viral diseases are a threat to the aquaculture industry of M. rosenbergii. An emerging viral disease caused by MrTV was identified, which caused huge losses to the cultivation of M. rosenbergi. In accordance with clinic observations, experimental infections suggested that MrTV was lethal to larvae of M. rosenbergii and that its mortality ranged from 53% to 82% within 20 days. However, no mortality was observed when adult M. rosenbergii were injected with MrTV, although the viral RNA was detected in gill tissue at 10 days post-infection (data not shown). These results indicated that adult shrimp can carry MrTV and not be affected. Although the transmission of MrTV has not been confirmed, we suspected that vertical transmission of MrTV was conceivable. Additionally, a preliminary survey suggested that the wild M. rosenbergii collected in natural water bodies was negative for MrTV. These data suggest that breeding of specific pathogen-free M. rosenbergii stock is a feasible measure to prevent MrTV.
Interestingly, two mortality peaks of larvae (7 and 15 days) were observed after immersion with MrTV (Figure 6
B), which may be associated with the special developmental stages of larvae. According to larval development, of M. rosenbergii
endure exposure to eclosin 11 times from larvae to juvenile shrimp (ZI–ZXI). During each time, they are fragile and sensitive to changes in the water environment or other stresses, especially in the larvae of seven days and 15 days. The former is during zoeal stage V and begins by changing the diet from chirocephalus
to egg custard, while the latter is the key time point for M. rosenbergii
to develop into the post-larval stage from zoeal stage XI and is accompanied with changes in swimming behavior and diet. The MrTV infection made the situation worse in these two stages, which was in accordance with the clinical observations. In fact, the larval mortality syndrome disease was called the “disease of seven days” in the breeding process of M. rosenbergii
In conclusion, MrTV, a lethal virus of M. rosenbergii, was isolated and characterized as a novel member of the Dicistroviridae according to its genomic features. This expands the family of Dicistroviridae and may shed light on controlling the larval mortality syndrome of M. rosenbergii.
4. Materials and Methods
4.1. Larvae of M. rosenbergii
The diseased larvae were collected from hatcheries located in the Huzhou district, Zhejiang Province, China. The larvae were washed in sterile saline, transported to the laboratory on dry ice, and stored at −80 °C for further study.
One-day-old healthy larvae of M. rosenbergii were purchased from a hatchery in Huzhou, Zhejiang Province, China. The larvae were maintained in 50 × 38 × 23 cm3 disinfected tanks and fed chirocephalus three times per day.
4.2. Virus Isolation, Purification, and Examination by Electron Microscopy
Approximately 20 g of moribund larvae samples were collected for virus isolation. After thawing, the larvae were weighed and ground by mortar and pestle with beads of alundum in TN buffer (50 mM Tris–HCl, 100 mM NaCl, pH 7.4) in a 1:10 proportion. The tissue homogenates were centrifuged at 10,000× g for 15 min. The supernatant was collected and filtered through a nylon net (400 mesh) for virus purification. Viral particles in the supernatant were concentrated by centrifugation through a 30% sucrose cushion at 184,000× g for 3 h at 4 °C using a Ty70 rotor (Beckman, Brea, CA, USA). Next, the pelleted viruses were dissolved in TN buffer and further purified by discontinuous sucrose gradient centrifugation (30%–60%) at 114,000× g for 3 h at 4 °C using a Ty90 rotor (Beckman). The band at approximately 40% sucrose was collected and centrifuged at 114,000× g for 3 h after being washed with TN buffer. The pelleted viruses were dissolved with TN buffer, and aliquots were stored at −70 °C.
Purified viruses were checked by electron microscopy using Formvar- and carbon-coated copper grids (200 mesh) (Zhongjingkeyi Inc., Beijing, China), negatively stained with 2% phosphotungstic acid (pH 7.0), and examined at 75 kV with a Hitachi H-7000FA transmission electron microscope (Hitachi, Tokyo, Japan).
4.3. Extraction of Viral Genome or Total RNA
The viral genome was extracted for characterization. Briefly, viral suspensions were digested with 200 μg/mL proteinase K in TE buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8.0) containing 0.5% sodium dodecyl sulphate (SDS) at 37 °C for 1 h. The viral genome was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and chloroform/isoamyl alcohol (24:1, v/v) and then precipitated with 2.5 volume of absolute ethanol after addition of 0.3 M sodium acetate (final concentration) at −20 °C for 2 h, followed by washing with 75% ethanol and dissolving as above. Then, the viral genome was divided into three parts, and two parts were digested with RNase A (10 ng/μL) or DNase I (0.2–0.3 U/μL) at 37 °C for 30 min. The remaining part was used as a control. The treated viral genome was stored at −70 °C.
Total RNA was extracted from whole larvae with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The final RNA was resuspended in 40 to 50 μL of diethy pyrocarbonate (DEPC) water and stored at −70 °C.
4.4. Random PCR Procedure
A random PCR procedure covering both RNA and DNA was used to identify the unknown viral genome [18
]. Briefly, purified virus particles were treated with 10 ng/μL RNase A and 100 U DNase I (Promega, Madison, WI, USA) in a total volume of 140 μL at 37 °C for 30 min. A QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) was used to extract the viral nucleic acid (either DNA or RNA) according to the manufacturer’s protocol. cDNA synthesis was performed by incubating the extracted viral nucleic acid at 90 °C for 5 min, followed by quenching on ice for 2 min. A 20-μL reaction mixture containing the following was prepared: 10 pmol of universal primer-dN6 (5’-GCCGGAGCTCTGCAGAATTCNNNNNN-3’), 10 μL of denatured nucleic acid, 0.6 mM aliquots of each deoxynucleoside triphosphate (dNTP), 20 U RNase inhibitor, and 200 U M-MLV reverse transcriptase (Promega). The mixture was incubated at 25 °C for 10 min, followed by 37 °C for 1 h. To synthesize second-strand cDNA/DNA, the reaction mixture was boiled for 2 min and cooled rapidly on ice, followed by incubation at 37 °C for 30 min in the presence of 5 U Klenow fragment (New England Biolabs, Ipswich, MA, USA) and 10 pmol of universal primer-dN6. Polymerase chain reaction (PCR) was conducted using a universal primer (5’-GCCGGAGCTCTGCAGAATTC-3’). PCR products larger than 500 bp in length were purified using an E.Z.N.A Gel Extraction Kit (Omega Bio-Tek, Norcross, GA, USA) and cloned into the pGEM-T Easy Vector (Promega). Recombinant plasmids were sequenced on an ABI Prism 3730 DNA Analyzer (Applied Biosystems, Foster, CA, USA) using the primer pair of M13 forward and reverse primer.
4.5. Full Genome Sequencing
Based on the sequences obtained by random PCR, the full-length sequence of the viral genome was acquired by PCR using specific primers (Table S1 in supplementary files
). The 5’ and 3’ end sequences of the genome were obtained using a RACE kit (Takara, Dalian, China).
4.6. Phylogenetic Analysis
Routine sequence management and analyses were performed using DNAStar (DNAStar Inc., Madison, WI, USA). Using the Open Reading Frame Finder and GeneMark (version 2.8a), ORFs were predicted and identified by a translated BLAST search (BLASTx at http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/blast/Blast.cgi
). Sequence alignment was performed using ClustalW and corrected manually [20
]. The phylogenetic trees based on the full-length genomic sequences or deduced amino acid sequences of capsid proteins of all available dicistroviruses were constructed via the neighbor-joining (NJ) method with the MEGA program (version 6) with a bootstrap of 1000 replicates [21
]. Gaps were regarded as a complete deletion.
4.7. Experimental Infection
One gram of moribund larvae was homogenized in 10 mL of TN buffer. After centrifugation at 3000× g for 5 min, the supernatant was filtered (0.45 μm) and diluted serially with TN buffer (1:100, 1:200, and 1:400) for infection. Three groups of healthy larvae (200 for each group) were immersed in the corresponding viral suspensions for 10 min in a volume of 500 mL and subsequently transported to 50 × 38 × 23 cm3 disinfected tanks. Control groups were treated with PBS. All of the tested or control larvae were fed chirocephalus three times per day. All the tanks were aerated gently and its water temperature was maintained at 28 to 30 °C. Excreta and food remains were removed and one-third of the freshwater was exchanged each day. Clinical signs and mortality were monitored daily. The dead larvae were collected and stored at −70 °C for detection.
4.8. RT-PCR Assay
RT-PCR with a specific primer pair was performed to detect the novel virus. Reverse transcription was performed in a 20 μL volume consisting of 6 μL of RNA and 10 pmol of random primer (dN6). The mixture was firstly denatured at 70 °C for 10 min, immediately quenched on ice, and subsequently added to the RT mixture containing 0.6 mM aliquots of each dNTP, 16 U RNasin (BioStar) and 200 U M-MLV reverse transcriptase (Promega). The reverse transcription reaction was conducted at 37 °C for 60 min, followed by heating to 70 °C for 5 min and holding at 4 °C.
For the amplification, the 25-μL reaction mixture contained 2 μL of cDNA, 2.5 μL of PCR buffer, 20 pmol of primer pair (MrTV472F: 5′-TGCTTCTATTTCGGCTCG-3′ and MrTV472R 5′-CAACGAATTAGGGAGAGG-3′), 0.2 mM dNTP, and 0.5 U Taq DNA polymerase (Promega). After an initial incubation step at 95 °C for 2 min, 35 cycles of amplification were carried out, consisting of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 40 s and a final extension step at 72 °C for 10 min. The expected products were gel purified by an E.Z.N.A. gel extraction kit (Omega Bio-Tek, Norcross, GA, USA) and sequenced for confirmation.
4.9. Nucleotide Sequence Accession Numbers
The genome sequence for MrTV was submitted to GenBank under accession No. HQ113110 or NC 018570.