Generic placeholder image

Anti-Infective Agents

Editor-in-Chief

ISSN (Print): 2211-3525
ISSN (Online): 2211-3533

Research Article

In Vitro Validation of Camphene as a Potential Antiviral Agent Against Betanodavirus Causing Viral Nervous Necrosis in Barramundi

Author(s): Ruby Singh, Prachi Srivastava*, Anand Deepika and Pani K. Prasad*

Volume 20, Issue 2, 2022

Published on: 11 January, 2021

Article ID: e130621190183 Pages: 11

DOI: 10.2174/2211352519999210111223529

Price: $65

Abstract

Viral infections are a major threat to the aquaculture industry throughout the world. Betanodavirus is one of the most infectious viruses that cause the highest mortality in larval and juvenile stages of Latescalcarifer commonly known as Barramundi. It is a single-stranded positive- sense RNA virus and causes viral nervous necrosis(VNN).VNN is caused by a RNA virus that gets transmitted both horizontally and vertically so the most effective method against this virus is to vaccinate the fish, however, vaccination becomes difficult since the disease is associated with outbreaks in larval and juvenile stages which are not that much immunocompetent. In our previous in silico studies, we proved the stability of camphene as a better phytochemical agent. In continuation to prove the authenticity of camphene as a potential antiviral agent against betanodavirus, its in vitro validation was performed. Sea bass kidney cell line (SISK) was selected for carrying out the in vitro studies and cytotoxicity studies of camphene in the SISK were done by MTT assay. Based on the analysis of the MTT assay, different dosages of camphene were selected viz.,0.2, 0.5, 1, 1.5, 2, 2.5, 5, 10, 20, 30 μg/ml. The SISK cells were infected with a virus inoculum(200μl). Further, the antiviral activity of camphene on infected SISK cells by Betanodaviruswas elucidated with the help of quantitative Real-time PCR(qPCR) on the 3rd and 5th day of infection. Analysis of results depicted that the dose of camphene 2 to 10 μg/ml is the safest dose against Betanodavirus. Hence this is aptly revealed that camphene can be used as a potential antiviral agent against Betanodavirus.

Keywords: Viral nervous necrosis, Barramundi, phytochemical, molecular docking, molecular dynamic simulation, camphene.

Graphical Abstract

[1]
Shetty, M.; Maiti, B.; Shivakumar Santhosh, K.; Venugopal, M.N.; Karunasagar, I. Betanodavirus of marine and freshwater fish: distribution, genomic organization, diagnosis and control measures. Indian J. Virol., 2012, 23(2), 114-123.
[http://dx.doi.org/10.1007/s13337-012-0088-x] [PMID: 23997435]
[2]
Nakai, T. Diagnostic and preventive practices for viral nervous necrosis (VNN). Disease Control in Fish and Shrimp Aquaculture in Southeast Asia - Diagnosis and Husbandry Techniques: Proceedings of the SEAFDEC-OIE Seminar-Workshop on Disease Control in Fish and Shrimp Aquaculture in Southeast Asia - Diagnosis and Husbandry Techniques Iloilo City, 2002, 80-89.
[3]
Glazebrook, J.S.; Heasman, M.P.; de Beer, S.W. Pcorna-like viral particles associated with mass mortalities in larval barramundi, Lates Calcarifer Bloch. J. Fish Dis., 1990, 13, 245-249.
[http://dx.doi.org/10.1111/j.1365-2761.1990.tb00780.x]
[4]
Kuo, H.C.; Wang, T.Y.; Hsu, H.H.; Chen, P.P.; Lee, S.H.; Chen, Y.M.; Tsai, T.J.; Wang, C.K.; Ku, H.T.; Lee, G.B.; Chen, T.Y. Nervous necrosis virus replicates following the embryo development and dual infection with iridovirus at juvenile stage in grouper. PLoS One, 2012, 7(4)
[http://dx.doi.org/10.1371/journal.pone.0036183] [PMID: 22563447]
[5]
Souto, S.; Olveira, J.G.; Vázquez-Salgado, L.; Dopazo, C.P.; Bandín, I. Betanodavirus infection in primary neuron cultures from sole. Vet. Res. (Faisalabad), 2018, 49(1), 86.
[http://dx.doi.org/10.1186/s13567-018-0580-4] [PMID: 30185222]
[6]
Nishi, Shinnosuke Cell Culture Isolation of Piscine Nodavirus (Betanodavirus) in Fish-Rearing Seawater. Applied and environmental biology., 2016, 82(8), 2537-2544.
[7]
Yoshikoshi, K.; Inoue, K. Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Oplegnathusfasciatus (Temminck & Schlegel). J. Fish Dis., 1990, 13, 69-77.
[http://dx.doi.org/10.1111/j.1365-2761.1990.tb00758.x]
[8]
Munday, B.L.; Nakai, T. Nodaviruses as pathogens in larval and juvenile marine finfish. World J. Microbiol. Biotechnol., 1997, 13(4), 375-481.
[http://dx.doi.org/10.1023/A:1018516014782]
[9]
Praveenraj, J.; Praveena Ezhil, P.; Bhuvaneswari, T. Navaneeth Krishnan, Jithendran KP. Experimental infection of Betanodavirus in freshwater fish Gambusia affinis(Baird and Girard, 1853)- a potential infection model for viral encephalopathy and retinopathy. Aquacult. Int., 2018, 26, 617-627.
[http://dx.doi.org/10.1007/s10499-018-0241-7]
[10]
Banerjee, D; Hamod, MA; Suresh, T; Karunasagar, I Isolation and characterization of a nodavirus associated with mass mortality in Asian seabass (Lates calcarifer) from the west coast of India. Virus Dis., 2014, 25(4), 425-429.
[11]
Azad, I.S.; Shekhar, M.S.; Thirunavukkarasu, A.R.; Poornima, M.; Kailasam, M.; Rajan, J.J.S.; Ali, S.A.; Abraham, M.; Ravichandran, P. Nodavirus infection causes mortalities in hatchery produced larvae of Lates calcarifer: first report from India. Dis. Aquat. Organ., 2005, 63(2-3), 113-118.
[http://dx.doi.org/10.3354/dao063113] [PMID: 15819426]
[12]
Chi, S.C.; Wu, Y.C.; Cheng, T.M. Persistent infection of betanodavirus in a novel cell line derived from the brain tissue of barramundi Lates calcarifer. Dis. Aquat. Organ., 2005, 65(2), 91-98.
[http://dx.doi.org/10.3354/dao065091] [PMID: 16060261]
[13]
Chia, TJ; Wu, YC; Chen, JY; Chi, SC Antimicrobial peptides(AMP) with antiviral activity against fish Nodavirus. Fish & Shellfish immunology., 2009, 28(3), 434-9.
[14]
Wu, Y.C.; Lu, Y.F.; Chi, S.C. Anti-viral mechanism of barramundi Mx against betanodavirus involves the inhibition of viral RNA synthesis through the interference of RdRp. Fish Shellfish Immunol., 2010, 28(3), 467-475.
[http://dx.doi.org/10.1016/j.fsi.2009.12.008] [PMID: 20034570]
[15]
Costa, J.Z.; Thompson, K.D. Understanding the interaction between Betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy. Fish Shellfish Immunol., 2016, 53, 35-49.
[http://dx.doi.org/10.1016/j.fsi.2016.03.033] [PMID: 26997200]
[16]
Ayyanar, M.; Ignacimuthu, S.; Ignacimuthu, S. Ethnobotanical survey of medicinal plants commonly used by Kani tribals in Tirunelveli hills of Western Ghats, India. J. Ethnopharmacol., 2011, 134(3), 851-864.
[http://dx.doi.org/10.1016/j.jep.2011.01.029] [PMID: 21291981]
[17]
Jassim, SAA; Naji, MA Novel antiviral agents:a medicinal plant perspective. Journal of applied microbiology., 2003, 95.95.doi.org/10.1046
[18]
fratini, Filippo; felicioli, Antonio Bees wax: a minireview of its antimicrobial activity and its application in medicine. Asian Pac. J. Trop. Med., 2016, 9(9), 839-843.
[http://dx.doi.org/10.1016/j.apjtm.2016.07.003] [PMID: 27633295]
[19]
li, Bo; Xinjunwei, Feilu; Zhao, Ruixiang Fucoidan:structure and bioactivity. Molecules, 2008, 13, 1671-1695.
[http://dx.doi.org/10.3390/molecules13081671]
[20]
Nidhi, P.; Swati, P.; Krishnamurthy, R. Indian Tinospora species: Review article on naturalimmunomodulators and therapeutic agents. International Journal of Pharmaceutical Biological and Chemical Sciences., 2013, 2(2), 1-9.
[21]
Direkbusarakom, S.; Herunsalee, A.; Yoshimizu, M.; Ezura, Y. Antiviral Activity of Several Thai Traditional Herb Extracts against Fish Pathogenic Viruses. Fish Pathol., 1996, 31(4), 209-213.
[http://dx.doi.org/10.3147/jsfp.31.209]
[22]
Sivasankar, P.; Anix, Vivek; Santhiya, A.; Kanaga, V. A review on plants and herbal extracts against viral diseases in aquaculture. Journal of Medicinal Plants Studies., 2015, 3(2), 75-79.
[23]
Verma, Sonia; Sharma, Hitender; Garg, Munish PhyllanthusAmarus: A Review.Journal of Pharmacognosy and Phytochemistry. 2014, 3(2), 18-22.
[24]
Bártová, V.; Bárta, J.; Jarošová, M. Antifungal and antimicrobial proteins and peptides of potato (Solanum tuberosum L.) tubers and their applications. Appl. Microbiol. Biotechnol., 2019, 103(14), 5533-5547.
[http://dx.doi.org/10.1007/s00253-019-09887-9] [PMID: 31144014]
[25]
Gupta, Madhu; Sharma, Sushil; Gautam, Ajay K.; Bhadauria, Rekha Review article on Momordica Charantialinn (karela): Nature’s silent healer. International Journal of Pharmaceutical Sciences Review and Research Article-007., 2011, 1(11), 31-37.
[26]
Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev., 2001, 46(1-3), 3-26.
[http://dx.doi.org/10.1016/S0169-409X(00)00129-0] [PMID: 11259830]
[27]
Kakarala, K.K.; Jamil, K. Protease activated receptor-2 (PAR2): possible target of phytochemicals. J. Biomol. Struct. Dyn., 2015, 33(9), 2003-2022.
[http://dx.doi.org/10.1080/07391102.2014.986197] [PMID: 25386994]
[28]
Saladino, R.; Gualandi, G.; Farina, A.; Crestini, C.; Nencioni, L.; Palamara, A.T. Advances and challenges in the synthesis of highly oxidised natural phenols with antiviral, antioxidant and cytotoxic activities. Curr. Med. Chem., 2008, 15(15), 1500-1519.
[http://dx.doi.org/10.2174/092986708784638889] [PMID: 18537626]
[29]
Balasubramanian, G.; Sarathi, M.; Venkatesan, C.; Thomas, J.; Sahul Hameed, A.S. Oral administration of antiviral plant extract of Cynodondactylonon a large scale production against White spot syndrome virus (WSSV) in Penaeusmonodon. Aquaculture, 2008, 279, 2-5.
[http://dx.doi.org/10.1016/j.aquaculture.2008.03.052]
[30]
Kang, S.Y.; Kang, J.Y.; Oh, M.J. Antiviral activities of flavonoids isolated from the bark of Rhus verniciflua stokes against fish pathogenic viruses In Vitro. J. Microbiol., 2012, 50(2), 293-300.
[http://dx.doi.org/10.1007/s12275-012-2068-7] [PMID: 22538659]
[31]
Sharma, V.; Sarkar, I.N. Bioinformatics opportunities for identification and study of medicinal plants. Brief. Bioinform., 2013, 14(2), 238-250.
[http://dx.doi.org/10.1093/bib/bbs021] [PMID: 22589384]
[32]
Ichinose, T.; Musyoka, T.M.; Watanabe, K.; Kobayashi, N. Evaluation of antiviral activity of Oligonol, an extract of Litchi chinensis, against betanodavirus. Drug Discov. Ther., 2013, 7(6), 254-260.
[http://dx.doi.org/10.5582/ddt.2013.v7.6.254] [PMID: 24423657]
[33]
Gopiesh Khanna, V.; Kannabiran, K.; Sarath Babu, V.; Sahul Hameed, A.S. Inhibition of Fish Nodavirus by Gymnemagenol extracted from Gymnema sylvestre. J. Ocean Univ. China, 2011, 10(4), 402-408.
[http://dx.doi.org/10.1007/s11802-011-1841-2]
[34]
Krishnan, Kannabiran; Khanna, Venkatesan Gopiesh; Hameed, Sahul Antiviral activity of Dasyscyphin C extracted from Eclipta Prostrata against Fish Nodavirus. Journals of antivirals and antiretrovirals., 2016, 2(1), 029-032.
[35]
Singh, Ruby; Pani, Prasad K; Tiwari, Anshul; Ajey, Pathak; Srivastava, P. Molecular docking and simulation study to identify antiviral agent by targeting MX protein against Betanodavirus causing viral nervous necrosis in Barramundi. Research journal of pharmacy and technology., 2020, 14(3)
[36]
Esmael, A.; Mervat, G.Hassan; Amer, Mahmoud M; Abdelrahman, Soheir; Hamed, Ahmed M; Abd-raboh, Hagar A; Foda, Mohamed F Antimicrobial activity of certain natural based plant oil against the antibiotic-resistant acne bacteria. Saudi J. Biol. Sci., 2020, 27, 448-455.
[http://dx.doi.org/10.1016/j.sjbs.2019.11.006] [PMID: 31889869]
[37]
Gavanji, S.; Mohammadi, E.; Larki, B.; Bakhtari, A. Antimicrobial and cytotoxic evaluation of some herbal essential oils in comparison with common antibiotics in bioassay condition. Integr. Med. Res., 2014, 3(3), 142-152.
[http://dx.doi.org/10.1016/j.imr.2014.07.001] [PMID: 28664090]
[38]
Lewis, R.J., Sr Hawley’s Condensed Chemical Dictionary, 15th ed; John Wiley & Sons, Inc.: New York, NY, 2007, p. 225.
[http://dx.doi.org/10.1002/9780470114735]
[39]
Eggersdorfer, M Terpenes. Ullmann's Encyclopedia of Industrial Chemistry, (7th ed.), June 2000.
[40]
Sell, CS Terpenoids. Kirk-Othmer Encyclopedia of Chemical Technology, John Wiley and Sons, New York. 1991, 7, 127.
[41]
Girola, N.; Figueiredo, C.R.; Farias, C.F.; Azevedo, R.A.; Ferreira, A.K.; Teixeira, S.F.; Capello, T.M.; Martins, E.G.; Matsuo, A.L.; Travassos, L.R.; Lago, J.H. Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo. Biochem. Biophys. Res. Commun., 2015, 467(4), 928-934.
[http://dx.doi.org/10.1016/j.bbrc.2015.10.041] [PMID: 26471302]
[42]
Vallianou, I.; Peroulis, N.; Pantazis, P.; Hadzopoulou-Cladaras, M. Camphene, a plant-derived monoterpene, reduces plasma cholesterol and triglycerides in hyperlipidemic rats independently of HMG-CoA reductase activity. PLoS One, 2011, 6(11)
[http://dx.doi.org/10.1371/journal.pone.0020516] [PMID: 22073134]
[43]
Birmpa, A; Constantinou, P; Dedes, C; Bellou, M; Sazakli, E; Leotsinidis, M; Vantarakis, A Antibacterial and antiviral effect of essential oils combined with non thermal disinfection technologies for ready to eat Romaine Lettuce. Journal of food research and technology., 2018, 1(1), 24-32.
[44]
Adachi, K.; Sumiyoshi, K.; Ariyasu, R.; Yamashita, K.; Zenke, K.; Okinaka, Y. Susceptibilities of medaka (Oryzias latipes) cell lines to a betanodavirus. Virol. J., 2010, 7, 150.
[http://dx.doi.org/10.1186/1743-422X-7-150] [PMID: 20624282]
[45]
Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immnunological methods, 1983, 65(1-2), 55-63.
[46]
Borenfreund, E.; Babich, H.; Martin-Alguacil, N. Comparisons of two in vitro cytotoxicity assays-The neutral red (NR) and tetrazolium MTT tests. Toxicol. In Vitro, 1988, 2(1), 1-6.
[http://dx.doi.org/10.1016/0887-2333(88)90030-6] [PMID: 20702351]
[47]
Sushila, N.; Hameed, A.S.S.; Prasad, K.P.; Majeed, S.A.; Tripathi, G. In vitro screening of selected antiviral drugs against betanodavirus. J. Virol. Methods, 2018, 259, 66-73.
[http://dx.doi.org/10.1016/j.jviromet.2018.06.005] [PMID: 29890241]
[48]
Nishizawa, T.; Mori, K.; Nakai, T.; Furusawa, I.; Muroga, k. Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis virus (SJNNV). Dis. Aquat. Organ., 1994, 18, 103-107.
[http://dx.doi.org/10.3354/dao018103]
[49]
Deepika, A.; Makesh, M.; Rajendran, K.V. Development of primary cell cultures from mud crab, Scylla serrata, and their potential as an in vitro model for the replication of white spot syndrome virus. In Vitro Cell. Dev. Biol. Anim., 2014, 50(5), 406-416.
[http://dx.doi.org/10.1007/s11626-013-9718-x] [PMID: 24357036]
[50]
Munday, B.L.; Langdon, J.S.; Hyatt, A.D.; Humphrey, J.D. Mass mortalities associated with a viral induced vacuolating encephalopathy and retinopathy of larval and juvenile Barramundi, Lates Calcarifer Bloch. Aquaculture, 1992, 103, 197-211.
[http://dx.doi.org/10.1016/0044-8486(92)90166-I]
[51]
Prem, K.; Sanjay, K.; Sudhakar, D.; kumar S, h; Himabindu, An overview of fisheries and aquaculture in India. Agroeconomist., 2015, 2, 16.
[52]
Keawcharoen, J.; Techangamsuwan, S.; Ponpornpisit, A.; Lombardini, E.D.; Patchimasiri, T.; Pirarat, N. Genetic characterization of a betanodavirus isolated from a clinical disease outbreak in farm-raised tilapia Oreochromis niloticus (L.) in Thailand. J. Fish Dis., 2015, 38(1), 49-54.
[http://dx.doi.org/10.1111/jfd.12200] [PMID: 24164433]
[53]
Ikenaga, T.; Tatecho, Y.; Nakai, T.; Uematsu, K. Betanodavirus as a novel transneuronal tracer for fish. Neurosci. Lett., 2002, 331(1), 55-59.
[http://dx.doi.org/10.1016/S0304-3940(02)00831-5] [PMID: 12359322]
[54]
Zorriehzahra, M.J.; Adel, M.; Dadar, M.; Ullah, S.; Ghasemi, M. Viral nervous necrosis (VNN) an emerging disease caused by Nodaviridae in aquatic hosts: Diagnosis, control and prevention: A review. Iranian Journal of Fisheries Sciences, 2019, 18(1), 30-47.
[55]
Thiéry, R.; Cozien, J.; Cabon, J.; Lamour, F.; Baud, M.; Schneemann, A. Induction of a protective immune response against viral nervous necrosis in the European sea bass Dicentrarchus labrax by using betanodavirus virus-like particles. J. Virol., 2006, 80(20), 10201-10207.
[http://dx.doi.org/10.1128/JVI.01098-06] [PMID: 17005697]
[56]
Silva, Joyce Kelly R da; Figueiredo, Pablo Luis Baia; Byler, Kendall G; N.Setzer., William Essential Oils as Antiviral Agents, Potential of Essential Oils to Treat SARS-CoV-2 Infection: An In-Silico Investigation. Int. J. Mol. Sci., 2020, 21, 3426.
[http://dx.doi.org/10.3390/ijms21103426]
[57]
Kumar, S.; Kumari, R.; Mishra, S. Pharmacological properties and their medicinal uses of Cinnamomum: a review. J. Pharm. Pharmacol., 2019, 71(12), 1735-1761.
[http://dx.doi.org/10.1111/jphp.13173] [PMID: 31646653]
[58]
Senthilraja, P; Kathiresan, K. In vitro cytotoxicity MTT assay in Vero, HepG2 and MCF -7 cell lines study of Marine Yeast. Journal of applied pharmaceutical sciences, 2015, 5(3), 080-084.
[59]
Sarath Babu, V.; Abdul Majeed, S.; Nambi, K.S.N.; Taju, G.; Madan, N.; Sundar Raj, N.; Sahul Hameed, A.S. Comparison of betanodavirus replication efficiency in ten Indian fish cell lines. Arch. Virol., 2013, 158(6), 1367-1375.
[http://dx.doi.org/10.1007/s00705-013-1617-7] [PMID: 23392632]
[60]
Chi, S.; Hu, W.; Lo, B. Establishment and characterization of a continuous cell line (GF-1) derived from grouper, Epinephelus coioides (Hamilton): a cell line susceptible to grouper nervous necrosis virus (GNNV). J. Fish Dis., 1999, 22, 173-182.
[http://dx.doi.org/10.1046/j.1365-2761.1999.00152.x]
[61]
Iwamoto, T.; Okinaka, Y.; Mise, K.; Mori, K.; Arimoto, M.; Okuno, T.; Nakai, T. Identification of host-specificity determinants in betanodaviruses by using reassortants between striped jack nervous necrosis virus and sevenband grouper nervous necrosis virus. J. Virol., 2004, 78(3), 1256-1262.
[http://dx.doi.org/10.1128/JVI.78.3.1256-1262.2004] [PMID: 14722280]
[62]
Iwamoto, T.; Okinaka, T.; Iwamoto, T.; Mori, K.; Arimoto, M.; Nakai, T. A combined cell-culture and RT–PCR method for rapid detection of piscine nodaviruses. J. Fish Dis., 2001, 24, 231-236.
[http://dx.doi.org/10.1046/j.1365-2761.2001.00291.x]
[63]
Iwamoto, T.; Nakai, T.; Mori, K.; Arimoto, M.; Furusawa, I. Cloning of the fish cell line SSN-1 for piscine nodaviruses. Dis. Aquat. Organ., 2000, 43(2), 81-89.
[http://dx.doi.org/10.3354/dao043081] [PMID: 11145456]
[64]
Morit, K.; Mangyoku, T.; Iwamoto, T.; Arimoto, M.; Tanaka, S.; Nakai, T. Serological relationships among genotypic variants of betanodavirus. Dis. Aquat. Organ., 2003, 57(1-2), 19-26.
[http://dx.doi.org/10.3354/dao057019] [PMID: 14735917]
[65]
Mori, K.; Sugaya, T.; Nishioca, T.; Gomez, D.K.; Fujinamy, Y.; Oka, M.; Arimoto, M.; Okinaka, Y.; Nakai, T. Detection of Betanodaviruses from feed fish used in marine aquaculture. 12th International Conference Diseases of Fish and Shellfish, 2005,
[66]
Chi, S.C.; Shieh, J.R.; Lin, S.J. Genetic and antigenic analysis of betanodaviruses isolated from aquatic organisms in Taiwan. Dis. Aquat. Organ., 2003, 55(3), 221-228.
[http://dx.doi.org/10.3354/dao055221] [PMID: 13677508]
[67]
Dalla Valle, L.; Zanella, L.; Patarnello, P.; Paolucci, L.; Belvedere, P.; Colombo, L. Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT‐PCR plus nested PCR. J. Fish Dis., 2000, 23, 321-327.
[http://dx.doi.org/10.1046/j.1365-2761.2000.00255.x]
[68]
Dalla Valle, L.; Negrisolo, E.; Patarnello, P.; Zanella, L.; Maltese, C.; Bovo, G.; Colombo, L. Sequence comparison and phylogenetic analysis of fish nodaviruses based on the coat protein gene. Arch. Virol., 2001, 146(6), 1125-1137.
[http://dx.doi.org/10.1007/s007050170110] [PMID: 11504420]
[69]
Hodneland, K.; García, R.; Balbuena, J.A.; Zarza, C.; Fouz, B. Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain. J. Fish Dis., 2011, 34(3), 189-202.
[http://dx.doi.org/10.1111/j.1365-2761.2010.01227.x] [PMID: 21306586]
[70]
Dalla Valle, L.; Toffolo, V.; Lamprecht, M.; Maltese, C.; Bovo, G.; Belvedere, P.; Colombo, L. Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR. Vet. Microbiol., 2005, 110(3-4), 167-179.
[http://dx.doi.org/10.1016/j.vetmic.2005.07.014] [PMID: 16146676]

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy