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2021, 42(5):77-85.
DOI: 10.19663/j.issn2095-9869.20200331001
Abstract:
For the establishment of the TaqMan RT-PCR detection method for the shrimp movement disorder nodavirus (MDNV), primers and probes were designed based on the RNA-dependent RNA polymerases (RdRp). The pMD18-MDNV plasmid and RNA standard containing the MDNV target gene were used as templates to optimize the reaction mixtures and program. The optimized reaction with 20.0 μL master mix included the following components: 11.0 μL one-step RT-PCR buffer, 0.8 μL enzyme mixture, 0.3 μmol/L forward primer, 0.3 μmol/L reverse primer, 0.4 μmol/L probe, 1.0 μL template, and 5.2 μL RNA-free H2O. The optimized reaction procedure was as following: incubation at 54.5℃ for 15 min, incubation at 95℃ for 1 min, then 45 thermal cycling amplifications (95℃ for 10 s, 60.3℃ for 30 s). The newly established method was specific for MDNV detection, showing a good linear relationship between the log value (Starting quality, Sq) and number of reaction cycles within the range of 1.4×1010~1.4×101 copies/μL pMD18-MDNV standard plasmid. The method could detect as low as 5.5×101 copies of the RNA standard or 1.4×101 copies of the pMD18-MDNV standard plasmid. Meanwhile, the newly developed assay showed that the coefficient of variation of the Ct value intra-assay and the Ct value inter-assay were less than 1.27% and 1.83%, respectively, indicating a good repeatability and stability. In the samples collected from shrimp farming provinces in China in 2019 using this new method, the positive detection rate of MDNV in Litopenaeus vannamei was 23.5% (16/68). The TaqMan RT-PCR method established in this study was specific, fast, and sensitive in the detection of MDNV. This method could provide technical support for the qualitative and quantitative detection and monitoring of MDNV in shrimp farming practices, as well as effective prevention and control of MDNV.
For the establishment of the TaqMan RT-PCR detection method for the shrimp movement disorder nodavirus (MDNV), primers and probes were designed based on the RNA-dependent RNA polymerases (RdRp). The pMD18-MDNV plasmid and RNA standard containing the MDNV target gene were used as templates to optimize the reaction mixtures and program. The optimized reaction with 20.0 μL master mix included the following components: 11.0 μL one-step RT-PCR buffer, 0.8 μL enzyme mixture, 0.3 μmol/L forward primer, 0.3 μmol/L reverse primer, 0.4 μmol/L probe, 1.0 μL template, and 5.2 μL RNA-free H2O. The optimized reaction procedure was as following: incubation at 54.5℃ for 15 min, incubation at 95℃ for 1 min, then 45 thermal cycling amplifications (95℃ for 10 s, 60.3℃ for 30 s). The newly established method was specific for MDNV detection, showing a good linear relationship between the log value (Starting quality, Sq) and number of reaction cycles within the range of 1.4×1010~1.4×101 copies/μL pMD18-MDNV standard plasmid. The method could detect as low as 5.5×101 copies of the RNA standard or 1.4×101 copies of the pMD18-MDNV standard plasmid. Meanwhile, the newly developed assay showed that the coefficient of variation of the Ct value intra-assay and the Ct value inter-assay were less than 1.27% and 1.83%, respectively, indicating a good repeatability and stability. In the samples collected from shrimp farming provinces in China in 2019 using this new method, the positive detection rate of MDNV in Litopenaeus vannamei was 23.5% (16/68). The TaqMan RT-PCR method established in this study was specific, fast, and sensitive in the detection of MDNV. This method could provide technical support for the qualitative and quantitative detection and monitoring of MDNV in shrimp farming practices, as well as effective prevention and control of MDNV.
2019, 40(2):25-32.
DOI: 10.19663/j.issn2095-9869.20180420006
Abstract:
Viral covert mortality disease (VCMD) is an emerging disease caused by covert mortality nodavirus (CMNV). VCMD recently resulted in severe economic losses in China due to shrimp aquaculture infections. To identify the natural hosts of CMNV in the pond culture system, samples of Carassius auratus were collected for testing purposes from the drainage channel of a shrimp farm affected by VCMD. The results of CMNV reverse transcription nested PCR showed that the RNA of C. auratus sample could be amplified and produced the expected target gene fragment. Histopathological analysis showed that CMNV infection in C. auratus caused brain nervous tissue vacuolation. Obvious karyopyknosis occurred in the corpora bigemina granular cells and cortical pyramidal cells. In situ hybridization analysis showed CMNV positive signals in regions of the brain and cardiac muscle with pathological damage. Transmission electron microscopy analysis verified the brain nervous tissue vacuolation and the presence of CMNV particles in the cardiac muscle. The results of the present study indicate that CMNV can cross the species barrier to infect fish and cause pathological damage in the target tissues. Our findings emphasize the need for paying close attention to the high risk of CMNV infections in other fish species, especially freshwater fishes.
Viral covert mortality disease (VCMD) is an emerging disease caused by covert mortality nodavirus (CMNV). VCMD recently resulted in severe economic losses in China due to shrimp aquaculture infections. To identify the natural hosts of CMNV in the pond culture system, samples of Carassius auratus were collected for testing purposes from the drainage channel of a shrimp farm affected by VCMD. The results of CMNV reverse transcription nested PCR showed that the RNA of C. auratus sample could be amplified and produced the expected target gene fragment. Histopathological analysis showed that CMNV infection in C. auratus caused brain nervous tissue vacuolation. Obvious karyopyknosis occurred in the corpora bigemina granular cells and cortical pyramidal cells. In situ hybridization analysis showed CMNV positive signals in regions of the brain and cardiac muscle with pathological damage. Transmission electron microscopy analysis verified the brain nervous tissue vacuolation and the presence of CMNV particles in the cardiac muscle. The results of the present study indicate that CMNV can cross the species barrier to infect fish and cause pathological damage in the target tissues. Our findings emphasize the need for paying close attention to the high risk of CMNV infections in other fish species, especially freshwater fishes.
LI Xiaoping
,
WAN Xiaoyuan
,
ZHANG Qingli
,
HUANG Jie
,
DONG Xuan
,
WANG Xiuhua
,
QIU Liang
,
SONG Zenglei
,
CHENG Dongyuan
2019, 40(2):65-73.
DOI: 10.19663/j.issn2095-9869.20180420003
Abstract:
Covert mortality nodavirus (CMNV), a recently emerged RNA virus, is the pathogen responsible for viral covert mortality disease, which has caused serious losses of shrimp aquaculture in China in recent years. In order to determine the transmission, prevalence, and evolution of CMNV in the coastal provinces and cities in China, national wide survey of the molecular epidemiology of the CMNV and it’s variants was conducted, based on analysis of shrimp samples collected from 2016 and 2017 by using the methods of RT-nPCR, RT-LAMP, and TaqMan RT-qPCR in present study. The analyses found the presence of CMNV-positive specimens in the major species of cultured crustaceans, including Litopenaeus vannamei, Marsupenaeus japonicus, Fenneropenaeus chinensis, Penaeus monodon, and Macrobrachium rosenbergii. CMNV-positive specimens appeared in almost all the coastal provinces, such as Hebei, Shandong, Zhejiang, Fujian, Guangdong, and Hainan. The results based on RT-nPCR assays showed that the prevalence rates of CMNV among the collected samples were 11.8% (30/254) and 7.8% (30/387) in 2016 and 2017, respectively. The results based on RT-LAMP assays showed that the prevalence rates of CMNV were 6.7% (17/254) and 3.9% (15/387) in 2016 and 2017, respectively. The results based on TaqMan RT-qPCR assays showed that the prevalence rates of CMNV were 17.7% (45/254) and 12.4% (48/387) in 2016 and 2017, respectively. The total prevalence rates of CMNV were 26.8% (68/254) and 16.3% (63/387) in 2016 and 2017, respectively, based on the above mentioned three methods. The results based on RT-LAMP assays showed that the prevalence rate of MDNV was 9.4% (24/254) in 2016. The high prevalence of CMNV in the major shrimp species and in the main farming areas revealed that this virus still threatened shrimp aquaculture in China during 2016~2017. Moreover, mutation of the RNA-dependent RNA polymerase (RdRp) gene of CMNV will raise the risk of appearing of false-negative result in CMNV molecular tests, which is of great concern. Meanwhile, the high prevalence rates of the emerging CMNV variant, MDNV, is a reminder that close attention needs to be paid to the high risk of MDNV transmission widely in farmed crustaceans.
Covert mortality nodavirus (CMNV), a recently emerged RNA virus, is the pathogen responsible for viral covert mortality disease, which has caused serious losses of shrimp aquaculture in China in recent years. In order to determine the transmission, prevalence, and evolution of CMNV in the coastal provinces and cities in China, national wide survey of the molecular epidemiology of the CMNV and it’s variants was conducted, based on analysis of shrimp samples collected from 2016 and 2017 by using the methods of RT-nPCR, RT-LAMP, and TaqMan RT-qPCR in present study. The analyses found the presence of CMNV-positive specimens in the major species of cultured crustaceans, including Litopenaeus vannamei, Marsupenaeus japonicus, Fenneropenaeus chinensis, Penaeus monodon, and Macrobrachium rosenbergii. CMNV-positive specimens appeared in almost all the coastal provinces, such as Hebei, Shandong, Zhejiang, Fujian, Guangdong, and Hainan. The results based on RT-nPCR assays showed that the prevalence rates of CMNV among the collected samples were 11.8% (30/254) and 7.8% (30/387) in 2016 and 2017, respectively. The results based on RT-LAMP assays showed that the prevalence rates of CMNV were 6.7% (17/254) and 3.9% (15/387) in 2016 and 2017, respectively. The results based on TaqMan RT-qPCR assays showed that the prevalence rates of CMNV were 17.7% (45/254) and 12.4% (48/387) in 2016 and 2017, respectively. The total prevalence rates of CMNV were 26.8% (68/254) and 16.3% (63/387) in 2016 and 2017, respectively, based on the above mentioned three methods. The results based on RT-LAMP assays showed that the prevalence rate of MDNV was 9.4% (24/254) in 2016. The high prevalence of CMNV in the major shrimp species and in the main farming areas revealed that this virus still threatened shrimp aquaculture in China during 2016~2017. Moreover, mutation of the RNA-dependent RNA polymerase (RdRp) gene of CMNV will raise the risk of appearing of false-negative result in CMNV molecular tests, which is of great concern. Meanwhile, the high prevalence rates of the emerging CMNV variant, MDNV, is a reminder that close attention needs to be paid to the high risk of MDNV transmission widely in farmed crustaceans.
2018, 39(3):167-172.
Abstract:
Globally, infectious hypodermal and haematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimp, causing huge economic loss to the shrimp farming industry. Further, IHHNV has been listed as a reportable crustacean disease pathogen by the World Organization for Animal Health (OIE). It is widely distributed in China and has become an important virus affecting shrimp culture. In the present paper, IHHNV pathogenicity-related progress geographically, host range, pathogenicity type, pathogenicity in host at different growth stages, interference with white spot syndrome virus (WSSV), and pathogensis mechanism of IHHNV were reviewed. The paper aimed to provide systematic references for the studies on control of IHHNV.
Globally, infectious hypodermal and haematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimp, causing huge economic loss to the shrimp farming industry. Further, IHHNV has been listed as a reportable crustacean disease pathogen by the World Organization for Animal Health (OIE). It is widely distributed in China and has become an important virus affecting shrimp culture. In the present paper, IHHNV pathogenicity-related progress geographically, host range, pathogenicity type, pathogenicity in host at different growth stages, interference with white spot syndrome virus (WSSV), and pathogensis mechanism of IHHNV were reviewed. The paper aimed to provide systematic references for the studies on control of IHHNV.
2017, 38(2):158-166.
DOI: 10.11758/yykxjz.20160123001
Abstract:
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the pathogens causing the significant diseases of crustaceans listed by the OIE (Office internaptional despizooties) Manual of Diagnostic Tests for Aquatic Animals. Infection with IHHNV does not produce any pathognomonic gross clinical signs, although runt-deformity syndrome (RDS) has been noted in infected juvenile Litopenaeus vannamei and Penaeus monodon. Enterocytozoon hepatopenaei (EHP) was first found and isolated in Thailand in 2009, which caused slow growth of P. monodon. IHHNV and EHP were found in the samples of L. vannamei postlarvae which were collected from hatchery farms in Tianjin and Hebei Province in 2013. The diseased shrimps showed some clinical signs with high mortality, slow growth and difference of individual growth rate. The IHHNV and EHP individual detection was conducted on these samples using the real-time PCR technique. In total, 108 out of 108 (100%) L. vannamei postlarvae samples were IHHNV positive, and 53 out of 108 (49.1%) L. vannamei postlarvae samples were EHP positive. Positive samples contained approximately 103–107 copies/(μg DNA) of IHHNV and big size of individual shrimp carried higher number of virus copies. Positive samples contained approximately 103–105 copies/(μg DNA) of EHP and mainly the small sized individual shrimp. The analysis showed that the IHHNV loads were positively correlated with the shrimp growth rate. IHHNV loads above 8.51×104 copies/μg DNA represented a high risk level. EHP loads were negatively correlated with the shrimp growth rate. EHP loads above 2.19×104 copies/μg DNA represented a high risk level. Our study showed that the diseased L. vannamei postlarvae samples occurred in the hatchery farms of Tianjin and Hebai Province infected with shrimp pathogens of IHHNV and EHP. Data provides scientific basis on the effects of pathogens infection on the growth of cultured penaeid shrimp.
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the pathogens causing the significant diseases of crustaceans listed by the OIE (Office internaptional despizooties) Manual of Diagnostic Tests for Aquatic Animals. Infection with IHHNV does not produce any pathognomonic gross clinical signs, although runt-deformity syndrome (RDS) has been noted in infected juvenile Litopenaeus vannamei and Penaeus monodon. Enterocytozoon hepatopenaei (EHP) was first found and isolated in Thailand in 2009, which caused slow growth of P. monodon. IHHNV and EHP were found in the samples of L. vannamei postlarvae which were collected from hatchery farms in Tianjin and Hebei Province in 2013. The diseased shrimps showed some clinical signs with high mortality, slow growth and difference of individual growth rate. The IHHNV and EHP individual detection was conducted on these samples using the real-time PCR technique. In total, 108 out of 108 (100%) L. vannamei postlarvae samples were IHHNV positive, and 53 out of 108 (49.1%) L. vannamei postlarvae samples were EHP positive. Positive samples contained approximately 103–107 copies/(μg DNA) of IHHNV and big size of individual shrimp carried higher number of virus copies. Positive samples contained approximately 103–105 copies/(μg DNA) of EHP and mainly the small sized individual shrimp. The analysis showed that the IHHNV loads were positively correlated with the shrimp growth rate. IHHNV loads above 8.51×104 copies/μg DNA represented a high risk level. EHP loads were negatively correlated with the shrimp growth rate. EHP loads above 2.19×104 copies/μg DNA represented a high risk level. Our study showed that the diseased L. vannamei postlarvae samples occurred in the hatchery farms of Tianjin and Hebai Province infected with shrimp pathogens of IHHNV and EHP. Data provides scientific basis on the effects of pathogens infection on the growth of cultured penaeid shrimp.
2015, 36(1):67-73.
DOI: 10.11758/yykxjz.20150110
Abstract:
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a kind of common virus in shrimp culture industry in China, influencing prawn breeding all over the world. IHHNV is harmful to Litopenaeus stylirostris and the mortality can be as high as 90%. For Litopenaeus vannamei, the mortality is not high, but can suffer from runt deformity syndrome (RDS). IHHNV infection in Penaeus monodon is usually subclinical, but RDS, reduced growth rates and reduced culture performance have been reported in IHHNV-infected stocks. OIE recommended 5 sets of primer pairs to detect different genotypes of IHHNV. In order to know the detecting types in China, we used four sets of primer pairs in the PCR protocols recommended by the OIE standards to detect penaeid shrimp samples collected in 2011 and 2012 from different provinces. These four sets of primer pairs are 389F/389R, 392F/392R, 77012F/77353R, 309F/309R, respectively. The detection results showed that IHHNV positives were detected in the samples of L. vannamei, P. monodon, Fenneropenaeus chinensis and Melicertus latisulcatus, but not in the sample of Palaemon carincauda. The positive percentage of L. vannamei was the highest, 70.8% (389F/R) in 2011 and 34.3% (389F/R) in 2012, and that of F. chinensis was the lowest, which is 8.3%. The more positive of penaeid shrimp samples in 2011 were detected than that in 2012. The positive percentage of IHHNV in penaeid shrimp samples from East China is higher than that from North China and South China. We got four detecting types from the PCR detection with 4 sets of primer pairs recommended by the OIE standards. For detected typeⅠ, the positive rate is 90% for all the four primer sets. For detected typeⅡ, the positive rate is 4% for primer set 389F/R. For detected type Ⅲ, the positive rate is 4% for primer sets of 389F/389R, 392F/392R and 309F/309R. For detected type Ⅳ, the positive rate is 2% for primer sets of 389F/389R and 309F/309R. The survey provided valuable information for the future study and surveillance on epidemiology of IHHNV.
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a kind of common virus in shrimp culture industry in China, influencing prawn breeding all over the world. IHHNV is harmful to Litopenaeus stylirostris and the mortality can be as high as 90%. For Litopenaeus vannamei, the mortality is not high, but can suffer from runt deformity syndrome (RDS). IHHNV infection in Penaeus monodon is usually subclinical, but RDS, reduced growth rates and reduced culture performance have been reported in IHHNV-infected stocks. OIE recommended 5 sets of primer pairs to detect different genotypes of IHHNV. In order to know the detecting types in China, we used four sets of primer pairs in the PCR protocols recommended by the OIE standards to detect penaeid shrimp samples collected in 2011 and 2012 from different provinces. These four sets of primer pairs are 389F/389R, 392F/392R, 77012F/77353R, 309F/309R, respectively. The detection results showed that IHHNV positives were detected in the samples of L. vannamei, P. monodon, Fenneropenaeus chinensis and Melicertus latisulcatus, but not in the sample of Palaemon carincauda. The positive percentage of L. vannamei was the highest, 70.8% (389F/R) in 2011 and 34.3% (389F/R) in 2012, and that of F. chinensis was the lowest, which is 8.3%. The more positive of penaeid shrimp samples in 2011 were detected than that in 2012. The positive percentage of IHHNV in penaeid shrimp samples from East China is higher than that from North China and South China. We got four detecting types from the PCR detection with 4 sets of primer pairs recommended by the OIE standards. For detected typeⅠ, the positive rate is 90% for all the four primer sets. For detected typeⅡ, the positive rate is 4% for primer set 389F/R. For detected type Ⅲ, the positive rate is 4% for primer sets of 389F/389R, 392F/392R and 309F/309R. For detected type Ⅳ, the positive rate is 2% for primer sets of 389F/389R and 309F/309R. The survey provided valuable information for the future study and surveillance on epidemiology of IHHNV.
