Brenna Connolly
brc2020@wildcats.unh.edu

Introduction



(Source: ViralZone:www.expasy.org/viralzone, SIB Swiss Institute of Bioinformatics)
(Source: ViralZone:www.expasy.org/viralzone, SIB Swiss Institute of Bioinformatics)


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

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

Results


MEGA.PNG
Representative sample of alignment, produced using MEGA 7 (Kumar et al., 2016)


Nucleotide_Tree.png
Phylogenetic tree produced through UPGMA method, with bootstrap values, using MEGA 7 (Kumar et al., 2016)


Host_Tree.png
Phylogenetic tree of host species, color coded by viral clade. Produced using NCBI Taxonomy (Coordinators, 2016), phyloT (GmbH, 2016), and the Interactive Tree of Life (Letunic & Bork, 2016).


BasebyBase.png
Visual summary of deviations from the consensus, produced using Base-by-Base (Hillary et al., 2011).


percent_id.png
Percent identity table, computed using Base-by-Base (Hillary et al., 2011)

GraphDNA.png
Keto skew graph, created using GraphDNA (Thomas et al., 2007)

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



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