Journal of Invertebrate Pathology 127 (2015) 1–5

Contents lists available at ScienceDirect

Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip

Short Communication

Isolation and characterization of a novel invertebrate iridovirus from adult Anopheles minimus (AMIV) in China Yong Huang a,1, Shasha Li a,1, Qiumin Zhao a,1, Guangqian Pei a, Xiaoping An a, Xiaofang Guo b, Hongning Zhou b, Zhiyi Zhang a, Jiusong Zhang a,⇑, Yigang Tong a,⇑ a b

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dong-Da Street, Fengtai District, Beijing 100071, China Yunnan Institute of Parasitic Diseases, Pu’er, Yunnan, China

a r t i c l e

i n f o

Article history: Received 9 September 2014 Revised 20 January 2015 Accepted 21 January 2015 Available online 28 January 2015 Keywords: Invertebrate iridovirus Cytopathic effect Complete genome Electron microscope

a b s t r a c t An invertebrate iridovirus (designated AMIV) was isolated from adult wild-captured Anopheles minimus mosquitoes in China. AMIV was pathologically and morphologically characterized and sequenced using the Ion Torrent™ sequencing platform. Phylogenetic analysis based on both the major capsid protein and core genes revealed that AMIV differs from all the members of the family Iridoviridae. The AMIV negatively strained virion has a diameter of about 130 nm. AMIV contains a linear DNA molecule of 163,023 bp, with 39% G+C content and 148 coding sequences. The genome analysis revealed that AMIV genome encodes a high content of replication associated genes including BRO-like genes. This is the ninth complete genome of IIV reported. Ó 2015 Published by Elsevier Inc.

1. Introduction The family Iridoviridae consists of five genera: Chloriridovirus, Iridovirus, Lymphocystivirus, Megalocytivirus, and Ranavirus (King et al., 2012). Members of the last three genera infect only poikilothermic vertebrates, which are economically important, especially in aquaculture (Do et al., 2004; Zhang et al., 2004). In contrast, viruses of the genera Iridovirus and Chloriridovirus are known as invertebrate iridescent viruses (IIVs), infecting predominantly insects (Webby and Kalmakoff, 1998; Williams et al., 2005). The dsDNA genomes of IIVs are circularly permuted, with terminal redundancy, and range from 100 to 210 kb in length (Fauquet et al., 2005). IIVs are icosahedral particles with a diameter in the range of 110–300 nm (Fauquet et al., 2005). The important effects of IIVs on insects, including sublethal effects, debilitation, and the induction of apoptosis, have been examined (Marina et al., 2003a, 1999). Unfortunately, many functional activities associated with the viruses and the interactions between the viruses and host cells remain to be clarified. Furthermore, there is little complete sequence information on IIVs, leading to considerable confusion, ⇑ Corresponding authors. Tel./fax: +86 10 66948446 (Y. Tong). Tel./fax: +86 10 66948447 (J. Zhang). E-mail addresses: [email protected] (J. Zhang), [email protected] (Y. Tong). 1 Yong Huang, Shasha Li and Qiumin Zhao contributed equally to this work. http://dx.doi.org/10.1016/j.jip.2015.01.007 0022-2011/Ó 2015 Published by Elsevier Inc.

especially regarding their taxonomic definition (Williams, 1994; Williams and Cory, 1994), and few proteomic analyses (Eaton et al., 2007). Eight complete IIV genomes have been reported to date: for IIV-3 (mosquito iridovirus) (Delhon et al., 2006), IIV-6 (Jakob et al., 2001), IIV-9 (Wiseana iridovirus; (Wong et al., 2011), IIV-22 (Piegu et al., 2013) and the recently reported four genomes (IIV-25 (Piegu et al., 2014b), IIV-22A (Piegu et al., 2014d), IIV-30 (Piegu et al., 2014a) and IIV-31 (Piegu et al., 2014c)). Of those viruses, Only IIV-3 was originally isolated from mosquitoes. Several IIVs have been reported that infect mosquitoes, the most important viral vector for human diseases (Fukuda, 1971a; Marina et al., 2003b). However, most of the previously reported mosquito isolates were found in Aedes, Ochlerotatus, and Psorophora, with no record of them in Anopheles or Uranotaenia (Williams, 2008). The literature suggests that IIVs have strong replicative capacities in a wide range of insect cell lines (Constantino et al., 2001; Williams et al., 2005) and broad host range, displayed in laboratory tests (Henderson et al., 2001; Jakob et al., 2002). Furthermore, almost all IIVs have been isolated in the larval but not adult stages. Here we describe a novel IIV isolated from wild captured adult Anopheles minimus in China, designated AMIV. The viral isolate was observed under electron microscopy and sequenced on the Ion Torrent™ sequencing platform. The complete genome of AMIV was characterized and submitted to GenBank under accession number KF938901.

2

Y. Huang et al. / Journal of Invertebrate Pathology 127 (2015) 1–5

2. Materials and methods 2.1. Biological material Mosquitoes were sampled during the active mosquito season (from June to September) in Yunnan Province, southwest China from 2007 to 2010 using light traps baited with CO2. The collected mosquitoes were frozen at 20 °C for 30 min and the blood-fed mosquitoes were removed. They were then grouped on the basis of collection site and mosquito species. A group of approximately 50– 100 mosquitoes was considered as one mosquito pool. A total of 20,256 mosquitoes were trapped and sorted into 247 pools according to species, location, and date of collection for viral isolation. Each mosquito pool was homogenized with RPMI-1640 medium containing 2% fetal calf serum and then centrifuged at 4000 rpm for 5 min. The supernatants were filtered through a 0.22 lm Millipore filter and were cultured with Aedes albopictus C6/36 cells. The cells were examined daily for evidence of cytopathic effect (CPE). A specimen was considered a positive isolate if it caused CPE in three successive passages. Two cell lines BHK21 and Vero-E6 were also used to explore the host range of AMIV: 500 ll virus supernatant from infected C6/36 cells was added to the new cultured cell lines (C6/ 36, BHK21 and Vero-E6) on 5 ML cell culture plate. As negative control, 500 ll cell culture was also added to the 3 cell lines. The virus particles were concentrated from positive isolate supernatant samples, and subsequently placed onto a formvar-coated copper grid, negatively stained with 1% uranyl acetate and viewed with a transmission electron microscope (TecnaiG2F20 S-TWIN) to examine the morphological characteristics of AMIV particles. 2.2. Genome sequencing and assembly The viral particles were obtained from the positive isolate supernatant. The DNA of the virus was extracted with the Qiagen Viral RNA Mini Kit (Qiagen, Valencia, CA), and used to construct a short-insert sequencing library (with 400 bp inserts) and a mate-paired sequencing library (with 4 kbp inserts), sequenced with the Ion Torrent™ sequencing platform. The raw data were assembled into two large scaffolds using Newbler (version 2.8). The interscaffold and intrascaffold gaps were closed with a combined strategy involving GapFiller (version 1.1) (Boetzer and Pirovano, 2012) and Sanger sequencing. 2.3. Genome analysis A phylogenetic tree based on the amino acid sequence of the major capsid protein (MCP) and one based on core genes of the family Iridoviridae were generated to explore the evolutionary relationships between members of the family Iridoviridae (Fig. 2). Sixteen reported viruses belonging to the genera Chloriridovirus and Iridovirus, together with four representative species from three other genera, were selected. The phylogenetic tree was inferred with the maximum likelihood method, with a 1000-replicate bootstrap test, using MEGA version 5 (Tamura et al., 2011) (Fig. 2A). Similarly, eleven complete genomes were selected to generate another phylogenetic tree based on core genes, using Pan-based software PGAP (Zhao et al., 2012) (Fig. 2B). The intermediate stage results show that six genes of AMIV (AMIV_039, AMIV_011, AMIV_067, AMIV_094, AMIV_069 and AMIV_096) have homology with that of other IIVs. 3. Results Three cell lines (BHK21, Vero-E6, and C6/36) were used to culture AMIV to explore its host cell range and pathogenic effects. The

cells were regularly observed under light microscopy at different times after AMIV inoculation. A CPE was only observed in C6/36 cells. The C6/36 cells showed a significant CPE, starting about 9 h after infection with AMIV. Within 36 h, almost all the cells in the plate were lysed, indicating the pathogenic infection of C6/36 cells by AMIV. The electron microscope results showed that the AMIV virion has a diameter of about 130 nm (Fig. 1A). The diameter of the particle was close to that of the members in the genus Iridovirus (Williams, 1998). The proposed defining differences between the Chloriridovirus and Iridovirus genera are particle sizes (King et al., 2012), AMIV should be classified into genus Iridovirus. The complete genome of AMIV is a linear DNA molecule of 163,023 bp, with a G+C content of 39%. The genome was annotated using the RAST server (Aziz et al., 2008) and verified with BLAST against the non-redundant database of NCBI. In total, 148 open reading frames (ORFs) were annotated, 94 of which encode hypothetical proteins, and 54 were functionally annotated. The coding ORFs cover 83.3% of the entire genome. The characteristics of AMIV and the other eight published complete IIV genomes are listed in Supplementary Table 2. Nine AMIV ORFs (0 0 3, 0 0 4, 0 3 2, 0 5 9, 0 7 3, 1 0 2, 1 3 5, 1 4 6, and 1 4 8) have no orthologs in the other IIV genomes. Repeated sequences were identified within the genome, known as BRO-like genes. In total, three BRO-like genes were found in AMIV with the same number in IIV-6. Previous researches have shown that BRO-like gene may enhance replication of invertebrate virus (Bideshi et al., 2003). Interestingly, replication associated genes (17 genes) were widely distributed on the complete genome (Fig. 1B). This high content of replication associated genes, together with the 3 BRO-like genes, may explain why the AMIV has a potent CPE on cultured cell lines. As shown in both trees of Fig. 2, AMIV clustered in the group of IIVs but formed a single clade that was distant from the other IIVs. The phylogenetic analysis also showed that AMIV is more closely related to members in the genus Iridovirus than to IIV-3 in the genus Chloriridovirus, suggesting that AMIV is quite different from the previously reported mosquito-derived IIV-3. Interestingly, the genome size, G+C content and ORF number of AMIV are intermediate between those of IIV-3 and members of the genus Iridovirus, which is consistent with the phylogenetic trees in Fig. 2. The discovery of AMIV provides one more record of a natural infection of mosquitoes by an IIV, revealing the genetic diversity of these viruses. AMIV can be defined as a novel species isolated from a natural infection of Anopheles minimus. The full sequence extends our knowledge of the Iridovirus genomes and may have great reference value for virologists and entomologists in defining interspecies variations in the IIVs.

4. Discussion High-throughput sequencing (HTS), a frequently used tool, makes the identification of unknown isolates easy. With the paucity of complete genomic data available and the strong demand for genomic analyses, HTS is undoubtedly an efficient approach to the large-scale and economically feasible determination of the complete genomes of the iridescent viruses. Insect vector diversity in the Yunnan region may be conducive to the spread of arthropod-borne viruses. The origin of the AMIV host and information about its geographic distribution extends the known distribution of the IIVs. This is the first natural IIV infection recorded in Anopheles mosquitoes, except for the IIV-6 infections established in the laboratory (Fukuda, 1971b). Infection of cell lines with AMIV in this study revealed the very rapid onset of CPE. Although there is insufficient evidence to show

Y. Huang et al. / Journal of Invertebrate Pathology 127 (2015) 1–5

3

A

B

Fig. 1. Electron micrograph of negatively stained AMIV virion and the coding replication associated genes. (A): The size of AMIV virion is approximately 130 nm. (B): The coding ORFs of AMIV. The colored frames are replication associated. Blue: BRO-like genes. Green: genes associated with virus replication. (For interpretation of the references to colour in this Fig. 1 legend, the reader is referred to the web version of this article.)

4

Y. Huang et al. / Journal of Invertebrate Pathology 127 (2015) 1–5

IIV_25YP_009010611

93

IIV_2AAC97168

60

IIV_9GQ918152

IIV_9AAB82568 62

IIV_25NC_023613

IIV_22ACCV01919 IIV_29AAC97172

78 94

IIV_30AAC97169

IIV_22HF920633

IIV_24AAC97173

IIV_22AHF920634

55

IIV_23AAC97175

52 99

IIV_30NC_023611

AgIVAAC97176

71

IIV_22AAA66585 AMIV

97

IIV_1AAA46245

74

IIV_3DQ643392

IIV_16AAB82569 65 AMIV

ISKNVAF371960 IIV_3ABF82044

95

IIV_6AAK82135

LCDV_CAY380826

IIV_31AAC97170

100

SGIVAY521625 100

PjIVAAC97171 ISKNVAAL98730

IIV_6AF303741

SGIVAAS47819 IIV_31NC_024451

LCDV_1AAC24486

91 99

LCDV_CAAS47819 50.000

B

A

0.05

invertebrateiridovirus

Ranavirus

Lymphocystivirus

Megalocytivirus

Fig. 2. Phylogenetic trees of AMIV and other tentative IIVs. (A): the major capsid protein based tree using maximum likelihood method. The protein ID of each virus is attached to its clade. The number at each node indicates the bootstrap % values for 1000 replications. ISKNV, infectious spleen and kidney necrosis virus in the genus Megalocytivirus; LCDV-1, lymphocystis disease virus 1 in the genus Lymphocystivirus; LCDV-C, LCDV-China in the genus Lymphocystivirus; SGIV, Singapore grouper iridovirus in the genus Ranavirus. AgIV, Anticarsia gemmatalis iridovirus; PjIV, Popillia japonica iridovirus. (B): the Pan-based distance tree using PGAP. The Genbank ID of each virus was attached to its clade.

that AMIV is a significant etiological agent of mosquito disease, this does not preclude its possible application to insect control. Acknowledgments This research was supported by a grant from the National Hi-Tech Research and Development (863) Program of China (No. 2012AA022003 and No.2014AA021402), China Mega-Project on Infectious Disease Prevention (No. 2013ZX10004605 and No. 2011ZX10004001), the National Natural Science Fund of China (No. 81273138), and the State Key Laboratory of the Pathogen and Biosecurity Program (No. SKLPBS1113). Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jip. 2015.01.007. Reference Aziz, R. et al., 2008. The RAST server: rapid annotations using subsystems technology. BMC Genomics. 9, 75. Bideshi, D.K. et al., 2003. Phylogenetic analysis and possible function of bro-like genes, a multigene family widespread among large double-stranded DNA viruses of invertebrates and bacteria. J. Gen. Virol. 84, 2531–2544.

Boetzer, M., Pirovano, W., 2012. Toward almost closed genomes with gap filler. Genome Biol. 13, R56. Constantino, M. et al., 2001. A comparison of techniques for detecting invertebrate iridescent virus 6. J. Virol. Methods 98, 109–118. Delhon, G. et al., 2006. Genome of invertebrate iridescent virus type 3 (mosquito iridescent virus). J. Virol. 80, 8439–8449. Do, J.W. et al., 2004. Complete genomic DNA sequence of rock bream iridovirus. Virology 325, 351–363. Eaton, H.E. et al., 2007. Comparative genomic analysis of the family iridoviridae: reannotating and defining the core set of iridovirus genes. Virol J. 4, 11. Fauquet, C. M., et al., 2005. Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses. Access Online via Elsevier. Fukuda, T., 1971a. Per os transmission of Chilo iridescent virus to mosquitoes. J. Invertebr. Pathol. 18, 152–153. Fukuda, T., 1971b. Per os transmission of Chilo iridescent virus to mosquitoes. J. Invertebr. Pathol. 18, 152–153. Henderson, C. et al., 2001. Replication of Chilo iridescent virus in the cotton boll weevil, anthonomus grandis, and development of an infectivity assay. Arch. Virol. 146, 767–775. Jakob, N. et al., 2002. Comparative analysis of the genome and host range characteristics of two insect iridoviruses: Chilo iridescent virus and a cricket iridovirus isolate. J. Gen. Virol. 83, 463–470. Jakob, N.J. et al., 2001. Analysis of the first complete DNA sequence of an invertebrate iridovirus: coding strategy of the genome of Chilo iridescent virus. Virology 286, 182–196. King, A. M., et al., 2012. Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier. Marina, C. et al., 2003a. Sublethal iridovirus disease of the mosquito Aedes aegypti is due to viral replication not cytotoxicity. Med. Vet. Entomol. 17, 187–194. Marina, C.F. et al., 1999. Sublethal effects of iridovirus disease in a mosquito. Oecologia 119, 383–388.

Y. Huang et al. / Journal of Invertebrate Pathology 127 (2015) 1–5 Marina, C.F. et al., 2003b. Adverse ffects of covert iridovirus infection on life history and demographic parameters of Aedes aegypti. Entomol. Exp. Appl. 106, 53–61. Piegu, B. et al., 2013. Complete genome sequence of invertebrate iridescent virus 22 isolated from a blackfly larva. J. Gen. Virol. 94, 2112–2116. Piegu, B. et al., 2014a. Complete genome sequence of invertebrate iridovirus IIV30 isolated from the corn earworm, Helicoverpa zea. J. Invertebr. Pathol. 116, 43– 47. Piegu, B. et al., 2014b. Complete genome sequence of invertebrate iridovirus IIV-25 isolated from a blackfly larva. Arch. Virol. 159, 1181–1185. Piegu, B. et al., 2014c. Genome sequence of a crustacean iridovirus, IIV31, isolated from the pill bug, Armadillidium Vulgare. J. Gen. Virol. 95, 1585–1590. Piegu, B. et al., 2014d. Complete genome sequence of invertebrate iridovirus IIV22A, a variant of IIV22, isolated originally from a blackfly larva. Stand Genomic Sci. 9, 940–947. Tamura, K. et al., 2011. MEGA5: molecule evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739.

5

Webby, R., Kalmakoff, J., 1998. Sequence comparison of the major capsid protein gene from 18 diverse iridoviruses. Arch. Virol. 143, 1949–1966. Williams, T., 1994. Comparative studies of iridoviruses: further support for a new classification. Virus Res. 33, 99–121. Williams, T., 1998. Invertebrate Iridescent Viruses. The Insect Viruses. Springer, pp. 31–68. Williams, T., 2008. Natural invertebrate hosts of iridoviruses (Iridoviridae). Neotrop Entomol. 37, 615–632. Williams, T. et al., 2005. A decade of advances in iridovirus research. Adv. Virus Res. 65, 173–248. Williams, T., Cory, J.S., 1994. Proposals for a new classification of iridescent viruses. J. Gen. Virol. 75 (Pt 6), 1291–1301. Wong, C.K. et al., 2011. Genomic and proteomic analysis of invertebrate iridovirus type 9. J. Virol. 85, 7900–7911. Zhang, Q.Y. et al., 2004. Complete genome sequence of lymphocystis disease virus isolated from China. J. Virol. 78, 6982–6994. Zhao, Y. et al., 2012. PGAP: pan-genomes analysis pipeline. Bioinformatics 28, 416– 418.