Macrobrachium rosenbergii nodavirus (MrNV) in juvenile crustacea
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In the giant freshwater prawn (Macrobrachium rosenbergii), white tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) has been found in many countries including, French West Indies, China, Taiwan, India and Thailand. In mid 2007, the index case of WTD in Australia presented in adult broodstock M. rosenbergii from Flinders River in western Queensland. In order to understand the phylogenetic relationship of the Australian isolate of MrNV with other MrNV isolates, the complete sequences of MrNV (RNA1 and RNA2) including protein B2 were determined in this study. Nucleotide sequence analysis showed that the identities of MrNV (RNA1) Australian strain were 94%, 95%, 95% and 97% similar to Malay, the French West Indies, Chinese and Thai strains, respectively. Also, MrNV (RNA2) Australian sequence showed 92% similar nucleotide sequence compared to the French West Indies, Chinese and Thai strains. The phylogenetic analysis demonstrated the Australian isolate of MrNV (RNA1) is closely related to the Thai and the French West Indies isolates, while MrNV (RNA2) is most distant to the other isolates. The phylogenetic tree of different nucleic acids (317 bp) of protein B2 also highlighted the differences between various nodavirus. Protein B2 of the Australian MrNV is different from fish, Penaeus vannamei and insect nodaviruses but it is closely related to black beetle nodavirus. The Australian protein B2 of MrNV is still in the MrNV cluster. WTD causes significant production losses to the Macrobrachium spp. industry worldwide. Therefore, cell culture and an animal model could be used to study MrNV infection. In this study, C6/36 cell line was used with different staining methods to observe cytopathic effect (CPE), count disrupted cells, and measure mitochondrial activity. Also, TaqMan real-time polymerase chain reaction (PCR) was used to detect the number of viral copies of MrNV in the cells. The typical CPE such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with Mayer’s haematoxylin and eosin stain (H&E) and Giemsa staining. Acridine orange was easier to detect single stranded nucleic acid, presumptive MrNV ribonucleic acid in the infected cells. The numbers of cells with disrupted cell membrane in infected treatment stained with trypan blue were higher than control treatment and rose to the maximum of 4 x 10⁵ cells at day 8. The absorbance reading of neutral red staining from infected samples peaked at day 4 (O.D. = 0.6) compared to control at day 12 (O.D. = over 3). However, TaqMan real-time PCR did not confirm the replication of MrNV in the cells over 14 days in the infected samples. The mean viral copies and mean cycle times of positive samples were stable at 2.07 x10⁴ and 24.12, respectively. TaqMan real-time PCR results from different passage times also showed the decreasing number of viral copy from 500 copies in passage 1 to less than 50 copies in passage 4. Different cell line and experimental techniques may need to be developed for MrNV in order to determine the replication of the Australian MrNV. As the replication of the Australian isolate of MrNV in the C6/36 cell line was not confirmed by TaqMan real-time PCR, an experimental animal model for the Australian MrNV was developed for this study. Macrobrachium can be hard to source due to their requirement for a saltwater environment for breeding and there is no operating farm in Australia. Instead, the Australian redclaw crayfish (Cherax quadricarinatus) were tested as a potential experimental animal model. In this experiment, the highest mortality (35%) was in the groups injected with MrNV and the lowest mortality (0%) was in the control groups. The inoculated crayfish had the smallest size (9.96 ± 0.99 cm) and lowest weight (22.34 ± 5.76 g) when compared to the feeding and control groups. The mean length of the control and feeding groups were 10.96 ± 0.68 cm and 10.33 ± 0.98 cm, respectively. The mean weight of the control treatment was 27.17 ±5.60 g, while feeding groups had a mean weight of 25.88 ± 7.79 g. The statistical analysis of length showed a significant difference (P < 0.05) between different treatments, while the weight did not indicate a significant difference (P > 0.05). Necrotic muscle and muscle degeneration with haemocytic infiltration were observed in infected crayfish. For the first time, a quantitative real-time polymerase chain reaction (qPCR) on clinical material was developed and it confirmed MrNV infection in infected animals. The mean viral titres (2.73 x 10² copies) and cycle times (Ct = 31.33) lead us to hypothesize that MrNV only poorly replicates in juvenile C. quadricarinatus when compared to the number of viral copies in the inoculums (10⁴). However, C. quadricarinatus may be a less than perfect but nevertheless a useable experimental animal model for MrNV infection in the future because of clinical signs, gross lesions, histopathological changes and qPCR titres that where present in experimentally infected C. quadricarinatus. RNA interference (RNAi) is an innate immune response which is triggered by the recognition of intracellular dsRNA that subsequently leads to inhibition of sequence-specific viral RNA in the cells. Also, RNAi has been shown to work against viral infection in prawns such as yellow head virus (YHV) and white spot syndrome virus (WSSV). However, no study of RNAi has been reported against MrNV infection. In this study, RNAi was designed against protein B2 of the Australian MrNV and tested in C. quadricarinatus. Mortality results at 10% and 60% showed in Stealth RNAi with MrNV and Stealth control RNAi with MrNV treatment, respectively. Percentage mortality (60%) of Stealth control RNAi with MrNV treatment was significantly different to other treatments (P < 0.05). Redclaw crayfish in control Stealth RNAi + MrNV treatment had the smallest size (8.7 ± 0.99 cm) and lowest weight (17.69 ± 3.55 g) when compared to other treatments which averaged 10.3 cm in length and 22.90 g in weight. Moreover, length and weight analyses of Stealth control RNAi with MrNV showed significant differences (P < 0.05) between different treatments. Clinical signs of MrNV and histopathological lesions such as myolysis with haemocytic infiltration in the muscle were found in infected redclaw crayfish. In two out of ten redclaw crayfish inoculated with Stealth RNAi with MrNV treatment and eight out of ten in the Stealth control RNAi with MrNV treatment MrNV was detected using qPCR. The mean number of viral copies from Stealth RNAi + MrNV treatment was 1.29 x 10¹ and cycle time was 34.88. The mean of viral copies from control Stealth RNAi + MrNV treatment was 3.54 x 10¹ and cycle time was 34.83. The mean viral copies and mean cycle times of positive samples were 3.45 x 10¹ and 34.84, respectively. No significant differences in viral copies or cycle time from qPCR between Stealth RNAi + MrNV treatment and control Stealth RNAi + MrNV treatment were found (P > 0.05). However, RNAi could be an effective tool to prevent MrNV infection and decrease mortality in infected redclaw crayfish. Thus, it may be possible to use RNAi to control viral infection on crustacean farms. After testing RNAi against protein B2 of the Australian MrMV with C. quadricarinatus, it can be concluded that RNAi can prevent MrNV infection and decrease mortality. Thus, RNAi may be used to control other viral infection in crustacean farming but each has to be redesigned to be specific to the viral strains. Moreover, the complete sequences of the Australian MrNV (RNA1 and RNA2) including protein B2 were different compared to fish and insect nodaviruses. To control nodavirus infection in different species, specific-sequence RNAi should be designed related to the particular viral strains. This study is the first development of experimental animal model for MrNV and the first use of RNAi against protein B2 of the Australian MrNV using C. quadricarinatus to control the disease. The information of RNAi from this study will be able to be applied for other nodavirus including fish and insect nodavirus to control the disease outbreak in the future.
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210
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DOI
10.25903/sea4-8e48