Megalocytiviruses

Brenna Connolly brc2020@wildcats.unh.edu toc

=Introduction=



//Megalocytivirus// is a recently discovered DNA-virus within the //Iridoviridae// family. It possesses isocahedral symmetry, and has a size range of 140-200nm diameter. Based on differences in the genome, //Megalocytivirus// can be divided into three major groups (sometimes called 'clades' or 'genotypes'); infectious spleen and kidney necrosis virus which reportedly causes disease in a large number of marine and freshwater fish species, red sea bream iridovirus which mainly infects red sea bream (//Pagrus major)//, and turbot reddish body iridovirus which is reported to infect Asian flounder species. //Megalocytivirus// induced disease has the potential to cause significant mortality and economic losses in the aquaculture industry , especially in East and Southeast Asian maricultured fish species. The first outbreak of megalocytivirus-induced disease was recorded in Japan in 1990, in cultured red sea bream (//Pagrus major//).This viral outbreak was designated red sea bream iridovirus disease (RSIVD). Since 1991, more than 30 species of cultured marine fish in the western part of Japan have experienced mass mortalities as a result of this disease. Susceptible hosts are mostly restricted to species within the order //Perciformes//, but some species in orders //Pleuronectiformes// and //Tetraodontiformes// can also be affected. Higher water temperature results in larger and more frequent RSVID outbreaks. Diseased fish are lethargic with severe difficulty swimming, and show petechiae of the gills (appearing as red or dark dots), severe anemia, and enlarged spleens. RSVID can approach 100% mortality rates in juvenile fish. (Kurita and Nakajima 2012)

Taxonomy
He et al (2001) worked with a virus isolate from diseased red drum (//Sciaenops ocellata//) and suggested a new genus within the //Iridoviridae// family to encompass this virus. He tentatively referred to it as cell hypertrophy iridovirus. Later, another new name was proposed for this genus -- 'tropical iridoviruses' or 'tropiviruses', due to the sequence homology between the viruses and their presence in the tropics (Sudthongkong et al 2002a,b; Go et al 2006). The genus //Megalocytivirus// includes the individual viruses infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), sea bass iridovirus (SBIV), African lampeye iridovirus (ALIV), grouper sleepy disease iridovirus (GSDIV), dwarf gourami iridovirus (DGIV), Taiwan grouper iridovirus (TGIV), and spotted knifejaw iridovirus (SKIV) (Faquet et al 2005; Dong et al 2008).

Phylogeny
Early phylogenetic analysis of the major capsid protein (MCP) gene suggests that there may be two clusters within the genus //Megalocytivirus//, with cluster I being more closely related to ISKNV and cluster II more closely related to orange-spotted grouper iridovirus (OSGIV) and rock bream iridovirus (RBIV) (Wang et al, 2007). Murray cod iridovirus (MCIV), DGIV, and ALIV are all variants of ISKNV (Go et al, 2006), but MCP gene analysis of seven other //Megalocytivirus// isolates by Wang et al (2009) from King grouper (//Epinephelus lancelolatus),// barramundi perch (//Lates calcarifer//), silver sea bream (//Rhabdosargus sarga//) and common ponyfish (//Leigonathus equulus//) showed higher homology with RSIV than with ISKNV, leading towards the belief that these isolates are variants of RSIV.

Song et al (2008) divided //Megalocytivirus// isolates into three genotypes based on the MCP gene sequences. Genotype I encompasses viruses that are closely related to OSGIV and RSIV, genotype II is those that are related to ISKNV, and genotype III consists almost entirely of flounder iridovirus (FLIV) and turbot iridovirus (TBIV) which are isolated from flafish species in Korea and China.

ISKNV showed high pathogenicity in largemouth bass (//Micropterus salmoides)// and mandarin fish (//Siniperca chuatsi//) (He et al, 2002). Red sea bream was highly susceptible to RSIV (Jung & Oh 2000; Kim et al 2002), and TBIV was much more pathogenetic in turbot compared to rock bream (//Oplegnathus fasciatus//) and Japanese flounder (//Paralichthys olivaceus//) (Oh et al 2006). This, paired with PCR assays showing the distinction between TBIV, RSIV, and ISKNV (Shi et al, 2004) suggests that these three viruses are three different species of //Megalocytivirus.//

Relationships within //Iridoviridae//
//Megalocytivirus// belongs to the family //Iridoviridae//, which also includes //Ranavirus, Lymphocystivirus, Chloriridovirus,// and //Iridovirus// (Faquet et al, 2005). A feature that is used to distinguish //Megalocytivirus// from //Iridovirus// and //Chloriridovirus// is the ability of //Megalocytivirus// to infect vertebrates, as well as the presence of methyltransferase (He et al 2001; Williams et al 2005). In addition, sequence analysis reveals a characteristic difference in the genetic makeup of megalocytiviruses and other members of the family //Iridoviridae//, in reference to the large and small subunits of ribonucleotide reductase. //Megalocytiviruses// contain the RR-2 gene but not the RR-1 gene (which is considered to be of eukaryotic origin), while in contrast the other genera encode both genes (which are thought to originate from proteobacteria)(Kurita and Nakajima 2012). By using the cytopathological signs of //Megalocytivirus//, we can also identify infection. Inclusion body-bearing cells (IBC) as well as the difference shown by sequence analysis of ATPase and MCP genes against other genera from the //Iridoviridae// family are often used to identify //Megalocytivirus// infection. (Faquet et al, 2005).

=Primary Research=

Spread of infection
Some additional attention has been paid to the way that this disease spreads. International trade in live ornamental fish has been determined to be a major route of entry for megalocytiviruses in new geographic areas.(Whittington & Chong, 2007) In Australia, more stringent import measures have been implemented, with some backlash, in order to protect their local wildlife from megalocytivirus-induced diseases.(Cockburn, 2016)

Research was undertaken in 2008 in an attempt to determine the overall spread of viruses within each clade (or genotype), and it was determined that only one clade (referred to as genotype I in this paper) had seen any significant distribution outside of its original population. The author determined that genotypes II and III were likely to be more constrained by their host populations than genotype I, leading to the difference in dispersion.(Song et al., 2008)

Vaccine
A formalin-inactivated vaccine was developed to control RSVID in Japan.(Nakajima et al., 1999) It was developed for red sea bream, as well as for fish belonging to the genus //Seriola//, striped Jack (//Pseudocaranx dentex//), Malabar grouper (//Epinephelus malabaricus//) and orange-spotted grouper (//Epinephelus coioides//). This vaccine is commercially available in Japan, however it may not be enough. Fishes in the genus //Oplegnathus// (such as the rock bream, or barred knifejaw) can’t be protected by vaccination alone, as they are too susceptible to the disease.(Do et al., 2005) However, the vaccine is notable in being the first viral vaccine for marine fish worldwide. There has also been a vaccine developed in Taiwan for Taiwan grouper iridoviral disease, which is a similar megalocytiviral disease. (Kurita and Nakajima 2012)

=Materials and Methods=

To investigate further the genetic similarities and differences within the genera Megalocytivirus, a single gene that all isolates and variants of this gene will be guaranteed to have in their genome needs to be selected to work with. For Megalocytivirus, the gene that most researchers tend to use is the major capsid protein coding region (the MCP gene). The major capsid protein is the most abundant structural protein and can account for up to 45% of virion protein in iridoviruses.(Webby & Kalmakoff, 1999)

There is no shortage of useful tools available for bioinformatic work on the internet, many of them open source and free to use. I took advantage of a number of these tools throughout the course of my investigation, and found success with them. Three to four accession numbers from each clade listed by Rimmer et al.(2015) were selected, and then downloaded as a group into a FASTA file. I then used MEGA 7 (Kumar, Stecher, & Tamura, 2016) in order to align the nucleotides, and produce a phylogenetic tree with bootstrap values.

I also utilized phyloT (GmbH, 2016), and the Interactive Tree of Life (Letunic & Bork, 2016) to assemble a phylogenetic tree of host organisms represented by the viral nucleotides I was analyzing, for reference purposes. phyloT creates a Newick formatted phylogenetic tree from information parsed out of the NCBI Taxonomy database (Coordinators, 2016), and by importing it into the Interactive Tree of Life, I was able to visualize the tree itself and highlight which host organism was susceptible to viruses from which clade.

In the course of my investigating and researching, I located a website run by the Viral Bioinformatics Institute, which contained a variety of useful tools. Two of these tools I found useful enough to use in my research. The first tool, Base-by-Base (Hillary, Lin, & Upton, 2011), I used to compare the alignments of nucleotides I had produced earlier to an overall consensus and create a visual summary of discrepancies. I also used the tool to create a percent identity chart for the nucleotides I was working with. For the consensus comparison, I trimmed the alignment on either side so that there were no gaps in any of the columns at either end, in order to ensure a neater final result. The second tool from the Viral Bioinformatics Institute I used was the GraphDNA tool (Thomas, Horspool, Brown, Tcherepanov, & Upton, 2007). I used this tool to graph a keto skew of all of the nucleotides I was investigating. Graphing a DNA skew basically gives you a visual representation of the molecular makeup of a nucleotide from one end to the other, and more in-depth analysis can help a researcher to discover transcription sites and other information. For my purposes, I used DNA skew graphing to visually show the clustering of the three attested clades.

Nucleotide sequences
= = =Results=
 * Organism || Accession || Coding Sequence ||
 * Megalocytivirus paradise fish || GU168574.1 || MCP gene, partial ||
 * Turbot reddish body iridovirus || AY590687.2 || MCP gene, complete ||
 * Rock bream iridovirus || AY533035.1 || MCP gene, complete ||
 * Rock bream iridovirus || AY532611.1 || MCP gene, complete ||
 * Dwarf gourami iridovirus || AY989901.1 || MCP gene, complete ||
 * Murray cod iridovirus || AY936203.1 || MCP gene, complete ||
 * Red sea bream iridovirus || AY310918.1 || MCP gene, complete ||
 * Grouper sleepy disease iridovirus || AY285746.1 || MCP gene, complete ||
 * African lampeye iridovirus || AY285745.1 || MCP, complete ||
 * Sea bass iridovirus || AB109372.1 || MCP gene, complete ||
 * Turbot iridovirus || AB166788.1 || MCP, partial ||



= = =Discussion=

In the analyses I performed, the existence of the three attested clades is increasingly obvious. The distinct patterning shown in the keto skew graph, clustered by clade, is indicative of highly similar nucleotide sequences in each of these groups. Referring to the percent identity table leads one to the same conclusion. The issue still persists of how to best address this virus, and how it will influence aquaculture in the future. Because clade I is so dispersed across hosts and geographic regions, a more thorough investigation into it may be required in the future to ensure that food sources in Asia aren’t compromised by viral infections, not to mention the threat of imported ornamental fish carrying these diseases. = = =References=

Cockburn, P. (2016, January 3). Lethal testing for viruses in ornamental fish postponed after complaints by pet industry. Retrieved November 26, 2016, from [] Coordinators, N. R. (2016). Database resources of the National Center for Biotechnology Information. Nucleic Acids Research, 44(D1), D7–19. Do, J. W., Cha, S. J., Kim, J. S., An, E. J., Park, M. S., Kim, J. W., … Park, J. W. (2005). Sequence variation in the gene encoding the major capsid protein of Korean fish iridoviruses. Archives of Virology, 150(2), 351–359. Dong CF, Weng SP, Shi XJ, Xu XP, Shi N, He JG (2008) Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV). Virus Research 135: 273–281. Eaton, H. E., Ring, B. A., & Brunetti, C. R. (2010). The genomic diversity and phylogenetic relationship in the family iridoviridae. Viruses, 2(7), 1458–1475. Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (2005) Virus Taxonomy Classification and Nomenclature of Viruses: Eighth Report of the International Committee on the Taxonomy of Viruses. Academic Press, Elsevier, San Diego. GmbH, B. S. (2016). phyloT. Retrieved November 26, 2016, from [] Go J, Lancaster M, Deece K, Dhungyel O, Whittington R (2006) The molecular epidemiology of iridovirus in Murray cod (Maccullochella peelii peelii) and dwarf gourami (Colisa lalia) from distant biogeographical regions suggests a link between trade in ornamental fish and emerging iridoviral diseases. Molecular and Cellular Probes 20: 212–222. He JG, Lu L, Deng M, He HH, Weng SP, Wang XH et al. (2001) Complete genome analysis of the mandarin fish infectious spleen and kidney necrosis iridovirus. Virology 291: 126–139. Hillary, W., Lin, S. H., & Upton, C. (2011). Base-By-Base version 2: single nucleotide-level analysis of whole viral genome alignments. Microbial Informatics and Experimentation, 1(1), 2. Hulo, C., de Castro, E., Masson, P., Bougueleret, L., Bairoch, A., Xenarios, I., & Le Mercier, P. (2011). ViralZone: a knowledge resource to understand virus diversity. Nucleic Acids Research, 39(Database issue), D576–82. Jung SJ, Oh MJ (2000) Iridovirus-like infection associated with high mortalities of striped beakperch, Oplegnathus fasciatus (Temminck et Schlegel), in southern coastal areas of the Korean peninsula. Journal of Fish Diseases 23: 223–226 Kim YJ, Jung SJ, Choi TJ, Kim HR, Rajendran KV, Oh MJ (2002) PCR amplification and sequence analysis of irido-like virus infecting fish in Korea. Journal of Fish Diseases 25:121–124. Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution, 33(7), 1870–1874. Kurita, J., & Nakajima, K. (2012). Megalocytiviruses. Viruses, 4(4), 521–538. Letunic, I., & Bork, P. (2016). Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Research, 44(W1), W242–5. Nakajima, K., Maeno, Y., Honda, A., Yokoyama, K., Tooriyama, T., & Manabe, S. (1999). Effectiveness of a vaccine against red sea bream iridoviral disease in a field trial test. Diseases of Aquatic Organisms, 36(1), 73–75. Oh MJ, Kitamura SI, Kim WS, Park MK, Jung SJ, Miyadai T et al. (2006) Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.). Journal of Fish Diseases 29: 415–421. Rimmer, A. E., Becker, J. A., Tweedie, A., Lintermans, M., Landos, M., Stephens, F., & Whittington, R. J. (2015). Detection of dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus) in populations of ornamental fish prior to and after importation into Australia, with the first evidence of infection in domestically farmed Platy (Xiphophorus maculatus). Preventive Veterinary Medicine, 122(1-2), 181–194. Shi CY, Wang YG, Yang SL, Huang J, Wang QY (2004) The first report of an iridovirus-like agent infection in farmed turbot, Scophthalmus maximus, in China. Aquaculture 236: 11–25 Song, J. Y., Kitamura, S., Jung, S. J., Miyadai, T., Tanaka, S., Fukuda, Y., … Oh, M. J. (2008). Genetic variation and geographic distribution of megalocytiviruses. Journal of Microbiology, 46(1), 29–33. Sudthongkong C, Miyata M, Miyazaki T (2002a) Iridovirus disease in two ornamental tropical freshwater fishes: African lampeye and dwarf gourami. Disease of Aquatic Organisms 48: 163–173. Sudthongkong C, Miyata M, Miyazaki T (2002b) Viral DNA sequences of genes encoding the ATPase and the major capsid protein of tropical iridovirus isolates which are pathogenic to fishes in Japan, South China Sea and Southeast Asian countries. Archives of Virology 147: 2089–2109. Thomas, J. M., Horspool, D., Brown, G., Tcherepanov, V., & Upton, C. (2007). GraphDNA: a Java program for graphical display of DNA composition analyses. BMC Bioinformatics, 8, 21. Wang YQ, Lu L, Weng SP, Huang JN, Chan SM, He JG (2007) Molecular epidemiology and phylogenetic analysis of a marine fish infectious spleen and kidney necrosis virus-like (ISKNV-like) virus. Archives of Virology 152: 763–773. Webby, R. J., & Kalmakoff, J. (1999). Comparison of the major capsid protein genes, terminal redundancies, and DNA-DNA homologies of two New Zealand iridoviruses. Virus Research, 59(2), 179–189. Whittington, R. J., & Chong, R. (2007). Global trade in ornamental fish from an Australian perspective: the case for revised import risk analysis and management strategies. Preventive Veterinary Medicine, 81(1-3), 92–116. Williams T, Solomieu VB, Chinchar VG (2005) A decade of advances in iridovirus research. Advances in Virus Research 65: 173–248.