Abstract
Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.
Lymphocystis disease was discovered early in 1874 (34), but the viral agent was not detected until 1962 (36). The lymphocystis disease virus (LCDV) has been studied by a series of morphology and ultrastructure observations (2, 3, 15, 27, 36, 47), molecular characterization analysis (5, 7, 9, 12, 29, 30), and attempts at in vitro infection and propagation (25, 35, 38, 46). LCDV has been identified as an iridovirus (7, 39) and is distributed worldwide. The resulting lymphocystis disease has been reported to occur in over 100 different fish species in seawater and freshwater (34). In recent years, lymphocystis disease has been reported to occur frequently in cultured flounder (Paralichthys olivaceus) in China (31, 40), and the causative agent has also been identified as LCDV-C (LCDV isolated in China) (31, 40, 46).
Iridoviridae have been subdivided into four genera, including Iridovirus, Chloriridovirus, Ranavirus, and Lymphocystivirus (26). LCDV belongs to Lymphocystivirus and is the type species in the genus. LCDV-1, isolated in the United States, has been extensively studied, and its genome was characterized by molecular cloning and physical mapping about 20 years ago (5, 6). The genome structure, found to be common to other iridoviruses, is circularly permuted and terminally redundant (5, 6, 28, 37). In 1997, the LCDV-1 complete genomic DNA sequence was determined. The genome is 102,653 bp in length and contains 195 open reading frames (ORFs) (33). Recently, three other genomes of vertebrate iridoviruses, those of the mandarin fish infectious spleen and kidney necrosis virus (ISKNV) (13), the tiger frog virus (TFV) (14), and salamander Ambystoma tigrinum virus (ATV) (19), have been fully sequenced and characterized. Because lymphocystis diseases have been reported to occur in more than 100 different fish species in seawater and freshwater worldwide (34), some differences in genome structure, gene organization, and DNA sequence may exist in the virus isolates from different fish species or from different geographic regions. To reveal the genomic characterization of LCDV-C and to perform comparative-genomics studies on iridoviruses, we initiated a project to sequence the LCDV-C genome. Here we report the LCDV-C complete genome sequence and analyze the structural differences between LCDV-C and other iridoviruses.
MATERIALS AND METHODS
LCDV-C and its viral-DNA preparation.
LCDV-C used in this study was originally isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease from Shandong Province of China (31). The lymphocystis tissues were sampled from the tumor-like dermal lesions of diseased fish and homogenized in phosphate-buffered saline (PBS) containing antibiotics (penicillin [100 IU ml−1] and streptomycin [100 μg ml−1]). Extracts were stored overnight at −20°C, thawed, and clarified by low-speed centrifugation, and the supernatants containing LCDV-C were ultracentrifuged in a Beckman (rotor type, SW41) at 36,000 rpm (160,000 × g) for 40 min. The pellet was resuspended in 1 ml of PBS and further purified by using discontinuous sucrose (20, 30, 40, and 50%) gradient centrifugation at 36,000 rpm (160,000 × g) for 40 min. The virus particle band was collected, and sucrose was removed by further centrifugation. The purified virus particles were used to extract the LCDV-C genomic DNA by incubating virus with 0.2 mg of proteinase K/ml-1% sodium dodecyl sulfate at 37°C for 2 h. Then the DNA was subjected to phenol-chloroform extraction and ethanol precipitation as described previously (43, 44).
DNA sequencing.
The LCDV-C genome was sequenced by a shotgun approach (41). Briefly, the viral genome DNA was randomly sheared by sonication at 0°C, and blunt ends of the sonicated fragments were generated with T4 polymerase. The DNA fragments were size fractionated by gel electrophoresis, and different-size fragments, such as 1.6 to 2.0 kb, 2.0 to 2.5 kb, and 2.5 to 3.0 kb, were extracted from the gels by a QIAEXII gel extraction kit. Then the DNA fragments were cloned into the EcoRV site of the pUC18 vector with T4 DNA ligase. After transformation into Escherichia coli XL-100 competent cells, the recombinant plasmid DNAs were extracted, and the cloned viral DNA fragments were sequenced in both directions with M13 universal primers and other synthesized primers according to the sequences obtained with an ABI 3700 automated DNA sequencer. A total of 2,929 sequencing reactions were performed, and 2,517 high-quality sequence fragments were assembled with InnerPeace software. The average reading frame length was about 600 bp with eightfold coverage of the whole genome. During the final stages of assembly, gaps were filled by sequencing PCR products amplified directly from the whole virus DNA with 32 oligonucleotide primers.
Computer-assisted analysis.
Nucleotide and amino acid sequences, restriction enzyme patterns, and repeated sequences were compiled and analyzed with the programs of the DNASTAR software package (Lasergene). Putative ORFs were predicted one by one by finding the start codon AUG and the rest of the coding sequence with the DNASTAR software package; ORFs encoding more than 40 amino acids (120 bp) were considered putative ORFs. The putative viral genes were obtained from the putative ORFs of more than 40 codons by selecting nonoverlapping ORFs. When two ORFs overlapped, the larger ORF was generally chosen as the putative viral gene. DNA and protein comparisons with entries in the sequence databases were performed with BLAST programs (1, 24). Comparison of the homological sequence regions of LCDV-C, LCDV-1, and other iridoviruses was performed with BLAST programs. A phylogenetic tree was constructed by the MegAlign program of DNASTAR software on the basis of amino acid sequence alignment of the known major capsid protein (MCPs) of different iridoviruses, including LCDV-C, LCDV-1 (LCDV isolated from United States), CIV (Chilo iridescent virus), ISKNV, RSIV (Red Sea bream disease iridovirus), FV3 (frog virus 3), BIV (Bohie iridovirus), TFV, and EHNV (epizootic hematopoietic necrosis virus).
Nucleotide sequence accession number.
The complete nucleotide sequence of the LCDV-C genome was deposited in GenBank under accession no. AY380826.
RESULTS AND DISCUSSION
Determination of the nucleotide sequence of the LCDV-C genome.
The complete nucleotide sequence of the LCDV-C genome was determined by applying the whole-genome shotgun sequencing strategy. The LCDV-C genome consists of 186,250 bp (Table 1). Among the sequenced vertebrate iridoviruses, LCDV-1 is 102,653 bp (33), TFV is 105,057 bp (14), ISKNV is 111,362 bp (13), and ATV is 106,332 bp (19). LCDV-C has of the largest genome among them. However, another invertebrate iridovirus, CIV, which was analyzed by Jakob et al. (16), has a genome larger (212,482 bp) than that of the LCDV-C. The base composition of the LCDV-C genome was found to be 27.25% G+C. The low G+C ratio is similar to those of LCDV-1 (29.07%) (33) and CIV (28.63%) (16) but is significantly different from those of ISKNV (54.78%) (13), TFV (55.01%) (14), and ATV (54%) (19). Therefore, the markedly low G+C content is a characteristic of the genus Lymphocystivirus.
TABLE 1.
Comparison of genome size and genome characterization for the sequenced iridoviruses
| Virus | Genus | Genome size (bp) | GC content (%) | No. of
|
Coding density (%) | Avg length of ORF (bp) | No. of encoded amino acids | Yr determined | GenBank accession no. | Reference or source | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Potential ORFsa | Putative genes | ||||||||||
| LCDV-C | Lymphocystivirus | 186,247 | 27.25 | 240 | 176 | 67 | 702 | 40-1,193 | 2004 | AY380826 | This paper |
| LCDV-1 | Lymphocystivirus | 102,653 | 29.07 | 195 | 110 | 82 | 822 | 40-1,199 | 1997 | L63545 | 32 |
| CIV | Iridovirus | 212,482 | 28.63 | 468 | 234 | 85 | 843 | 40-2,432 | 2001 | AF303741 | 17 |
| ISKNV | Undetermined | 111,362 | 54.78 | 124 | 105 | 93 | 834 | 40-1,208 | 2001 | AF371960 | 13 |
| TFV | Ranavirus | 105,057 | 55.01 | 105 | 105 | 94 | 873 | 40-1,294 | 2002 | AF389451 | 14 |
| ATV | Ranavirus | 106,332 | 54 | 96 | 96 | 79 | 933 | 32-1,294 | 2003 | AY150217 | 19 |
Determination of the numbers of ORFs involved different criteria. See references.
In addition, about 0.4% nucleotide replacement heterogeneity has been observed from the repeatedly sequenced LCDV-C genome sequences, and the majority of the replacements are in the noncoding regions. The polymorphism might be related to the virus materials used for sequencing, because the virus materials could be potentially heterogenous, containing sequences from a number of different variant viruses.
Organization and coding capacity of the LCDV-C genome.
Computer-assisted analysis of the LCDV-C genomic DNA sequence revealed the presence of 240 potential ORFs. As shown in Table 2, these ORFs encode polypeptides ranging from 40 to 1,193 amino acids. The analysis of the coding strategy of the 240 potential ORFs revealed 176 largely nonoverlapping ORFs that are likely to represent putative viral genes. As shown in Table 1, the numbers of total potential ORFs and putative genes are related to the sizes of the genomes of these characterized iridoviruses. The percent coding densities and the average lengths of ORFs for the five sequenced iridoviruses were analyzed and compared. As shown in Table 1, the percent coding density of LCDV-C is 67% and is the lowest among the iridoviruses. Moreover, the average length of each ORF in the LCDV-C genome is 702 bp, also the smallest among the iridoviruses. The unusual low coding density may be related to the presence of large noncoding regions within the gene organization and structure of LCDV-C. In the sequenced iridoviruses, the coding densities of lymphocystiviruses LCDV-1 and LCDV-C are all low and LCDV-C contains a large number of repeated sequences, which are predominantly concentrated in the gaps between two neighbor ORFs. For example, the longest gap is up to 1,895 bp and is located between ORF086L and ORF087L (Fig. 1). Thus, the LCDV-C low coding density is consistent with the high degree of large noncoding regions.
TABLE 2.
Potential ORFs of the LCDV-C genome and comparison to those of LCDV-1 and other iridoviruses
| ORFb | Nucleotide position | No. of amino acids | Conserved domain or signatures | CD accession no. | Homologues to LCDV-1
|
Homologues to other iridoviruses in Iridoviridae
|
||||
|---|---|---|---|---|---|---|---|---|---|---|
| % Identity of amino acidsc | Accession no. | Predicted function and/or similaritya | % Identity of amino acids | Accession no. | Species | |||||
| 001L | 649-524 | 42 | ||||||||
| 002L | 1661-1362 | 100 | Caspase recruitment domain; motif contained in proteins involved in apoptotic signaling; predicted to possess a DEATH (pfam00531) domain-like fold | pfam00619.8 | ||||||
| 003R | 1911-2930 | 340 | 3-β-Hydroxysteroid dehydrogenase/isomerase family | pfam01073 | 60 (163/263) | NP_078739.1 | Hydroxysteroid dehydrogenase; steroid D5-D4 isomerase; ORF31 | |||
| 004L* | 2062-1940 | 41 | ||||||||
| 005R | 2992-3243 | 84 | ||||||||
| 006R | 3686-4183 | 166 | 29 (26/89) | NP_078657.1 | ORF67 | |||||
| 007L | 5259-4765 | 165 | 61 (101/165) | NP_078618.1 | ORF70 | 37 (39/103) | NP_612278.1 | ISKNV | ||
| 008R* | 5126-5284 | 53 | 33 (35/106) | AAB94419.1 | CIV | |||||
| 009L | 6022-5741 | 94 | 68 (65/95) | NP_078638.1 | VLTF2-like late transcription factor, ORF102 | 31 (30/94) | AF397203 | Regina ranavirus | ||
| 010L | 6508-6371 | 46 | 31 (21/66) | AF303741 | CIV | |||||
| 011L | 7532-6675 | 286 | Thymidylate synthase. | pfam00303.8 | ||||||
| 012R | 7733-8116 | 128 | 74 (95/128) | NP_078640.1 | ORF88 | 36 (45/125) | AF368231 | Regina ranavirus | ||
| 013L | 10058-8502 | 519 | Serine/threonine protein kinases, catalytic domain; phosphotransferases; serine- or threonine-specific kinase subfamily. | smart00220.7 | 67 (342/509) | NP_078619.1 | ORF14 | 28 (110/380) | AF303741 | CIV |
| 54 (20/37) | AF303741 | CIV | ||||||||
| 014L | 10927-10643 | 95 | 60 (57/94) | NP_078639.1 | ORF97 | |||||
| 015L | 11410-11264 | 49 | ||||||||
| 016L | 12480-11647 | 278 | Tumor necrosis factor (TNF) receptor domain; superfamily of TNF-like receptor domains. | cd00185.2 | ||||||
| 017R* | 12300-12473 | 58 | ||||||||
| 018L | 13143-12955 | 63 | 36 (22/61) | NP_078710.1 | ORF130 | |||||
| 019R | 13401-14729 | 443 | 48 (212/417) | NP_078753.1 | ORF21 | |||||
| 020L* | 13912-13793 | 40 | ||||||||
| 021L* | 15085-14945 | 47 | ||||||||
| 022R | 15055-15702 | 216 | 50 (108/215) | NP_078727.1 | ORF52 | |||||
| 023R | 16260-16424 | 55 | 56 (30/53) | NP_078706.1 | ORF139 | |||||
| 024R | 16464-16793 | 110 | 51 (51/100) | NP_078705.1 | ORF95 | |||||
| 025R | 17018-20068 | 1,017 | RNA polymerase Rpb2, domain 6 | pfam00562.8 | 75 (770/1024) | NP_078633.1 | DNA-directed RNA polymerase subunit 2; ORF3 | 45 (519/1129) | NP_572001.1 | Rana tigrina ranavirus; Regina ranavirus |
| RNA polymerase beta subunit | pfam04563.2 | 46 (445/957) | AF397202 | ISKNV | ||||||
| 026L* | 19483-19343 | 47 | 43 (464/1079) | NP_612256.1 | ||||||
| 027R | 20577-21164 | 196 | Deoxynucleoside kinases (nucleotide transport and metabolism) | COG1428.1 | 71 (137/192) | NP_078725.1 | Deoxynucleoside kinase; ORF60 | 28 (50/175) | NP_149606.1 | Invertebrate iridescent virus 6 |
| 028L* | 20902-20765 | 46 | 26 (32/119) | NP_612254.1 | ISKNV | |||||
| 029R | 21472-23208 | 579 | 62 (33/580) | NP_078643.1 | ORF11 | |||||
| 030L* | 21935-21786 | 50 | ||||||||
| 031L* | 22136-22014 | 41 | ||||||||
| 032L* | 22635-22426 | 70 | ||||||||
| 033R | 23615-24976 | 454 | Membrane-bound metallopeptidase (cell division and chromosome partitioning) | COG4942.1 | 55 (253/454) | NP_078746.1 | ORF22 | |||
| 034L | 26151-25423 | 243 | 63 (149/233) | NP_078713.1 | Early iridovirus protein; ORF49 | 32 (79/241) | CAA07475.1 | EHNV | ||
| 32 (78/241) | NP_571993.1 | Rana tigrina ranavirus | ||||||||
| 32 (79/241) | CAA37177.1 | FV3 | ||||||||
| 25 (38/147) | NP_612340.1 | NP_612340.1 | ||||||||
| 035R* | 25820-25951 | 44 | ||||||||
| 036R* | 26074-26265 | 64 | ||||||||
| 077R | 58523-58762 | 80 | ||||||||
| 078R* | 59279-59452 | 58 | ||||||||
| 079R* | 59543-59668 | 42 | ||||||||
| 080L | 61359-58765 | 865 | Predicted ATPase (general function prediction only) | COG3378.1 | 71 (616/865) | NP_078717.1 | D5 family NTPase involved in DNA replication; ORF6 | 36 (319/885) | NP_612331.1 | ISKNV |
| 27 (259/936) | AAB94479.1 | CIV | ||||||||
| 081R* | 61166-61306 | 47 | ||||||||
| 082R | 61859-62260 | 134 | 27 (39/142) | NP_078759.1 | ORF18 | |||||
| 083R | 62853-63008 | 52 | ||||||||
| 084R | 63034-63153 | 40 | ||||||||
| 085L | 63384-63229 | 52 | ||||||||
| 086L | 64011-63688 | 108 | 76 (83/108) | NP_078617.1 | DNA methyltransferase; ORF51 | 52 (50/95) | NP_572009.1 | Rana tigrina ranavirus | ||
| 46 (45/97) | NP_612268.1 | ISKNV | ||||||||
| 087L | 66826-65909 | 306 | 57 (174/302) | NP_078740.1 | ORF38 | |||||
| 088R* | 66072-66224 | 51 | ||||||||
| 089R | 67518-67647 | 43 | ||||||||
| 090L | 68950-68408 | 181 | ||||||||
| 091L | 69880-69479 | 134 | 44 (53/120) | NP_078761.1 | ORF76 | |||||
| 092R* | 69604-69744 | 47 | ||||||||
| 093L | 70333-69947 | 129 | 50 (33/65) | NP_078760.1 | ORF93 | |||||
| 094R* | 70107-70229 | 41 | ||||||||
| 095R | 70395-71261 | 289 | ||||||||
| 096R | 71776-71934 | 53 | ||||||||
| 097L | 72846-72037 | 270 | ||||||||
| 098R* | 72277-72510 | 78 | ||||||||
| 099L | 73633-73307 | 109 | 46 (51/109) | NP_078751.1 | ORF94 | |||||
| 100L | 74643-73771 | 291 | 57 (168/293) | NP_078701.1 | ORF39 | 31 (58/184) | AF303741 | CIV | ||
| 101L | 75803-74982 | 274 | 22 (128/558) | NP_078764.1 | Putative filamentous protein; ORF10 | 30 (30/97) | NP_612318.1 | ISKNV | ||
| 102R* | 75004-75309 | 46 | ||||||||
| 103R* | 75641-75781 | 47 | ||||||||
| 104L | 77978-76956 | 341 | 54 (189/346) | NP_078702.1 | Hypothetical LCDV-1 paralog family 2; ORF30 | |||||
| 105R | 78572-78709 | 46 | ||||||||
| 106L | 79313-78897 | 139 | ||||||||
| 107L | 80761-79595 | 389 | 22 (84/372) | NP_078759.1 | ORF18 | |||||
| 108L | 80916-80767 | 50 | ||||||||
| 109L | 81476-81324 | 51 | ||||||||
| 110R | 81572-81934 | 121 | ||||||||
| 111L | 83147-82530 | 206 | 58 (116/200) | NP_078668.1 | Uncharacterized LCDV-1 paralog family 1; ORF56 | |||||
| 112R | 83202-83735 | 178 | 46 (79/169) | NP_078666.1 | Uncharacterized LCDV-1 paralog family 1; ORF61 | |||||
| 113L* | 83548-83429 | 40 | ||||||||
| 114L | 84942-84211 | 244 | 81 (199/244) | NP_078656.1 | Virion assembly protein; NTPase; ORF46 | 55 (136/244) | NP_571992.1 | Rana tigrina ranavirus | ||
| 55 (133/239) | NP_612345.1 | ISKNV | ||||||||
| 54 (131/242) | BAA28670.1 | RSIV | ||||||||
| 50 (125/248) | AAA43823 | FV3 | ||||||||
| 41 (100/240) | AAB94422 | CIV | ||||||||
| 115R | 85683-85919 | 79 | DNA-directed RNA polymerase, subunit M/transcription elongation factor TFIIS (transcription) | COG1594.1 | 59 (45/76) | NP_078754.1 | Transcription factor SII homolog; ORF42 | 34 (25/72) | NP_572006.1 | Rana tigrina ranavirus |
| 116R | 86212-87081 | 290 | 30 (102/330) | NP_612294.1 | ISKNV | |||||
| 117L | 88281-87349 | 311 | 48 (157/323) | NP_078623.1 | ORF35 | |||||
| 118R | 88741-88860 | 40 | ||||||||
| 119L | 90542-89130 | 471 | 31 (157/496) | NP_078676.1 | ORF23 | |||||
| 120R* | 89332-89475 | 48 | ||||||||
| 121R | 90758-91093 | 112 | ||||||||
| 122R | 91498-91881 | 128 | 34 (44/126) | NP_078670.1 | ORF85 | |||||
| 123L | 92090-91935 | 52 | ||||||||
| 124R | 92394-93368 | 325 | ||||||||
| 125R | 93740-93874 | 45 | ||||||||
| 126R | 93954-94124 | 57 | ||||||||
| 127L | 94383-94246 | 46 | ||||||||
| 128L | 94858-94586 | 91 | 38 (35/92) | NP_078642.1 | ORF104 | |||||
| 129R | 94922-95392 | 157 | ||||||||
| 130L* | 95223-95092 | 44 | ||||||||
| 131R | 96217-97197 | 327 | 21 (66/305) | NP_078702.1 | Hypothetical LCDV1 paralog family 2; ORF30 | |||||
| 132L* | 96401-96228 | 58 | ||||||||
| 133R | 97712-98722 | 337 | 48 (53/110) | NP_078708.1 | ORF69 | |||||
| 134L* | 99272-99039 | 78 | ||||||||
| 135R | 99061-99660 | 200 | ||||||||
| 136L | 100198-99839 | 120 | 40 (47/117) | NP_078707.1 | ORF86 | |||||
| 137L | 101109-100627 | 161 | ||||||||
| 138R* | 100988-101140 | 51 | ||||||||
| 139L | 101415-101275 | 47 | ||||||||
| 140L | 102507-101458 | 350 | 64 (233/361) | NP_078696.1 | Tristetraprolin-like zinc finger protein C3H; ORF28 | |||||
| 141R* | 102098-102247 | 50 | ||||||||
| 142L | 102988-102554 | 145 | Ervl/Alr family; biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. | pfam04777.2 | 51 (74/145) | NP_078699.1 | Thiol oxidoreductase; ORF79 | 38 (45/116) | NP_612265.1 | ISKNV |
| 27 (29/106) | AF303741 | CIV | ||||||||
| 143R | 103037-103993 | 319 | 57 (180/314) | NP_078700.1 | Ariadne-2 homologue; ORF36 | 29 (71/237) | NP_572004.1 | Rana tigrina ranavirus | ||
| 144L* | 103613-103419 | 65 | ||||||||
| 145R | 104543-104932 | 130 | 62 (79/127) | NP_078728.1 | Uncharacterized LCDV1 paralog family 1; ORF87 | |||||
| 146L | 105550-105146 | 135 | 60 (81/134) | NP_078686.1 | ORF83 | 27 (16/58) | NP_612232.1 | ISKNV | ||
| 147L | 105998-105555 | 148 | 75 (110/145) | NP_078685.1 | ORF75 | |||||
| 148L | 106813-106268 | 182 | Catalytic domain of ctd-like phosphatases | smart00577.6 | 58 (103/177) | NP_078678.1 | Putative NIF/NLI-interacting factor; ORF64 | 34 (57/165) | NP_612227.1 | ISKNV |
| 32 (58/177) | AF303741 | CIV | ||||||||
| 149R | 106822-107196 | 125 | 57 (70/121) | NP_078679.1 | ORF80 | |||||
| 150R | 107401-108923 | 506 | 49 (254/514) | NP_078684.1 | ORF15 | 28 (54/192) | AF303741 | CIV | ||
| 151R | 109535-110014 | 160 | 40 (52/130) | NP_078641.1 | ORF73 | |||||
| 152L* | 109787-109662 | 42 | ||||||||
| 153L | 110936-110439 | 166 | 63 (104/164) | NP_078627.1 | ORF71 | 46 (66/141) | AF303741 | CIV | ||
| 56 (58/114) | NP_612308.1 | ISKNV | ||||||||
| 43 (64/147) | AF367980 | Regina ranavirus | ||||||||
| 154R | 112593-113570 | 326 | ||||||||
| 155L* | 112795-112677 | 40 | ||||||||
| 156L* | 113550-113431 | 40 | ||||||||
| 157R | 114005-114463 | 153 | 54 (83/151) | NP_078659.1 | ORF74 | |||||
| 158R | 115020-116390 | 457 | 46 (214/461) | NP_078665.1 | Myristylated membrane protein A; ORF20 | 28 (110/385) | AF368229 | Regina ranavirus | ||
| 23 (113/477) | AAB94444.1 | CIV | ||||||||
| 23 (107/450) | NP_612229.1 | ISKNV | ||||||||
| 24 (96/385) | BAC66967.1 | RSIV | ||||||||
| 25 (51/197) | AF303741 | CIV | ||||||||
| 159R | 116374-116736 | 121 | 56 (68/121) | NP_078664.1 | ORF89 | |||||
| 160R | 117323-118495 | 391 | 32 (133/413) | NP_078759.1 | ORF18 | |||||
| 161L | 120785-118915 | 624 | Acetyl-coenyme A hydrolase (energy production and conversion) | COG0427.1 | 63 (395/622) | NP_078649.1 | ORF9 | |||
| 162R | 120803-121969 | 389 | 61 (228/371) | NP_078648.1 | Hypothetical immediate-early protein; ORF27 | 23 (85/360) | NP_572011.1 | Rana tigrina ranavirus | ||
| 23 (85/360) | AF367980 | Regina ranavirus | ||||||||
| 25 (80/309) | AF303741 | CIV | ||||||||
| 25 (68/269) | AF371960 | ISKNV | ||||||||
| 163L* | 121300-121067 | 78 | ||||||||
| 164L | 122674-122075 | 200 | ||||||||
| 165L | 123025-122816 | 70 | ||||||||
| 166L | 125548-123146 | 801 | Herpesvirus major outer envelope glycoprotein (BLLF1); serine/threonine protein kinases, catalytic domain; phosphotransferases; serine- or threonine-specific kinase subfamily | pfam05109.2; smart00220.7 | 44 (258/577) | NP_078689.1 | Uncharacterized conserved domain linked to protein kinase domain; ORF7 | |||
| 167R* | 124870-125001 | 44 | ||||||||
| 168L | 125867-125739 | 43 | ||||||||
| 169R | 125950-126960 | 337 | Xeroderma pigmentosum G N and I regions (XPGN, XPGI); contains the HhH2 motif; domain in nucleases | cd00128.2 | 54 (182/331) | NP_078767.1 | Putative XPG/RAD2-type nuclease; ORF34 | 36 (104/283) | NP_572012.1 | Rana tigrina ranavirus |
| 33 (87/259) | AF367980 | Regina ranavirus | ||||||||
| 30 (91/295) | NP_612249.1 | ISKNV | ||||||||
| 30 (89/295) | BAA82754.1 | RSIV | ||||||||
| 27 (91/331) | AF303741 | CIV | ||||||||
| 170R | 127874-128008 | 45 | ||||||||
| 171L | 128623-128483 | 47 | ||||||||
| 172L | 130914-129274 | 547 | Ribonucleotide reductase, alpha subunit (nucleotide transport and metabolism) | COG0209.1 | 74 (405/547) | Np_078756.1 | Ribonucleotide reductase large subunit; ORF12 | |||
| 173R | 131370-134168 | 933 | Putative lipopolysaccharide-modifying enzyme | smart00672.6 | 64 (468/728) | NP_078770.1 | ORF8 | 38 (200/515) | AAB94478.1 | CIV |
| 31 (204/651) | NP_571995.1 | Rana tigrina ranavirus | ||||||||
| 174L* | 133972-133838 | 45 | ||||||||
| 175R | 134663-136102 | 480 | Serine/threonine protein kinases, catalytic domain; phosphotransferases of the serine- or threonine-specific kinase subfamily | cd00180.2 | 54 (282/522) | NP_078677.1 | Phosphotransferase; ORF81 | 26 (109/413) | AF303741 | CIV |
| 176R | 136165-136347 | 61 | ||||||||
| 177R | 137000-137404 | 135 | ||||||||
| 178L | 139562-138150 | 471 | Mn2+-dependent serine/threonine protein kinase (signal transduction mechanisms) | COG3642.1 | 53 (249/468) | NP_078729.1 | Phosphotransferase; ORF17 | 28 (33/117) | NP_612235.1 | ISKNV |
| 179L | 140985-140065 | 307 | 37 (35/93) | NP_078708.1 | ORF69 | |||||
| 180R | 141161-142072 | 304 | ||||||||
| 181R | 142378-143133 | 252 | 67 (169/250) | NP_078747.1 | Putative replication factor and/or DNA binding/packing protein; ORF43 | 33 (58/172) | AF303741 | CIV | ||
| 23 (36/151) | NP_612283.1 | |||||||||
| ISKNV | ||||||||||
| 182R | 143285-143524 | 80 | 67 (53/79) | NP_078695.1 | ||||||
| 183L* | 143517-143373 | 48 | ||||||||
| 184L | 144548-144423 | 42 | ||||||||
| 185R | 144571-146019 | 483 | 55 (272/486) | NP_078744.1 | ORF16 | 24 (87/359) | AF303741 | CIV | ||
| 186R | 146201-146539 | 113 | ||||||||
| 187R | 146942-147694 | 251 | 76 (193/251) | NP_078726.1 | RNase III; ORF44 | 45 (111/246) | NP_572005.1 | Rana tigrina ranavirus; CIV | ||
| 30 (74/241) | AAB94459.1 | ISKNV | ||||||||
| 30 (66/216) | NP_612309.1 | |||||||||
| 188L* | 147656-147462 | 67 | ||||||||
| 189R | 148578-149099 | 174 | ||||||||
| 190L | 149785-149483 | 101 | ||||||||
| 191R | 150198-153776 | 1,193 | DNA-directed RNA polymerase beta′ subunit/160-kDa subunit (transcription); RNA polymerase Rpb1, domain 5 | COG0086.1; pfam04998.2 | 69 (820/1188) | NP_078624.1 | DNA-dependent RNA polymerase, largest subunit; ORF5 | 43 (558/1274) | NP_571990.1 | Rana tigrina ranavirus; ISKNV |
| 38 (467/1226) | NP_612250.1 | |||||||||
| 37 (463/1226) | BAA82753.1 | |||||||||
| 192L* | 150408-150283 | 42 | ||||||||
| 193L* | 151287-151156 | 44 | ||||||||
| 194L* | 152516-152328 | 63 | ||||||||
| 195L* | 153063-152944 | 40 | ||||||||
| 196L | 153897-153775 | 41 | ||||||||
| 197L | 154686-153940 | 249 | 58 (146/248) | NP_078615.1 | Proliferating cell nuclear antigen; ORF45 | 25 (64/247) | NP_612334.1 | ISKNV | ||
| 198R* | 154169-154297 | 43 | ||||||||
| 199L* | 155080-154934 | 49 | ||||||||
| 200R* | 154959-155129 | 57 | ||||||||
| 201L | 156417-155221 | 399 | 64 (259/400) | NP_078687.1 | ORF25 | 29 (55/185) | AF303741 | CIV | ||
| 202L | 157950-157021 | 310 | ||||||||
| 203L | 161193-158389 | 935 | DNA polymerase family B; DNA polymerase elongation subunit (family B) | pfam00136.8; COG0417.1 | 65 (613/930) | NP_078724.1 | DNA polymerase family B; ORF5 | 40 (411/1007) | NP_572000.1 | Rana tigrina ranavirus; RSIV |
| 37 (349/942) | BAA28669.1 | ISKNV | ||||||||
| 36 (353/959) | NP_612241.1 | CIV | ||||||||
| 32 (228/692) | AF083915 | Regina ranavirus | ||||||||
| 42 (176/414) | AF368230 | |||||||||
| 204R* | 158963-159220 | 85 | ||||||||
| 205R | 161257-161703 | 149 | 87 (36/41) | NP_078723.1 | ORF90 | |||||
| 206R* | 161818-161940 | 41 | ||||||||
| 207L* | 161986-161849 | 46 | ||||||||
| 208L | 163448-163005 | 148 | ||||||||
| 209R | 163864-165933 | 690 | Cell division protein 48 (CDC48), N-terminal domain | pfam02359.8 | ||||||
| ATPase family associated with various cellular activities (AAA) | pfam00004.8 | |||||||||
| 210L* | 164576-164388 | 63 | ||||||||
| 211R | 166014-166280 | 89 | ||||||||
| 212L | 166664-166446 | 73 | ||||||||
| 213L* | 166783-166694 | 41 | ||||||||
| 214R | 166685-167239 | 185 | 23 (46/188) | NP_078681.1 | ORF59 | |||||
| 215L | 167845-167714 | 44 | ||||||||
| 216L | 168452-167850 | 201 | Collagen triple helix repeat | pfam01391.8 | ||||||
| 217R | 168693-169028 | 112 | 51 (58/112) | NP_078751.1 | ORF94 | |||||
| 218R | 169041-169225 | 62 | 39 (23/58) | NP_078752.1 | ORF121 | |||||
| 219R | 169337-169600 | 88 | ||||||||
| 220R | 169862-169990 | 43 | ||||||||
| 221R | 170159-170605 | 149 | NUDIX domain | pfam00293.8 | 54 (80/146) | NP_078631.1 | Putative antimutator GTP pyrophosphohydrolase MutT; ORF78 | |||
| 222L | 171030-170830 | 67 | ||||||||
| 223R* | 170894-171013 | 40 | ||||||||
| 224L | 172370-171090 | 427 | Papain family cysteine protease | pfam00112.8 | 66 (269/407) | NP_078647.1 | Papain-like proteinase; ORF24 | 35 (121/338) | AF303741 | CIV |
| 225R* | 171510-171647 | 46 | ||||||||
| 226R | 172401-172751 | 117 | 46 (53/115) | NP_078646.1 | ORF92 | |||||
| 227L | 172966-172838 | 43 | ||||||||
| 228R | 173468-173887 | 140 | ||||||||
| 229R | 174258-174627 | 124 | ||||||||
| 230L | 174853-174713 | 47 | ||||||||
| 231L | 176937-176209 | 243 | 58 (123/209) | NP_078737.1 | ORF53 | |||||
| 232R* | 176774-176959 | 62 | ||||||||
| 233L* | 177093-176962 | 44 | ||||||||
| 234R | 176981-177439 | 153 | 34 (49/141) | NP_078738.1 | ORF72 | |||||
| 235R | 177645-180923 | 1,093 | 57 (627/1086) | NP_078748.1 | ORF2 | 26 (304/1127) | AAK37740.1 | Regina ranavirus | ||
| 23 (304/1127) | NP_612298.1 | ISKNV | ||||||||
| 236L* | 180717-80574 | 48 | 22 (90/409) | AF303741_ | CIV | |||||
| 237L | 184512-181744 | 923 | Chromosome segregation ATPases (cell division and chromosome partitioning) | COG1196.1 | 31 (134/432) | NP_078764.1 | Putative filamentous protein; ORF10 | |||
| 238R* | 182066-182242 | 59 | ||||||||
| 239R | 184871-186256 | 462 | 31 (67/212) | NP_078629.1 | ORF54 | |||||
| 240L* | 185613-185331 | 94 | ||||||||
The ORF numbers of LCDV-1 are from GenBank (accession no. NC_001824).
Asterisks indicate that the ORFs are not likely to represent viral genes because they overlap other large ORFs.
Percentage of residues identical to those of the homologous protein or domain in the protein deduced from the ORF.
FIG. 1.
Genomic organization of the LCDV-C. Arrows, locations of 176 potentially putative genes with respect of their sizes, positions, and orientations. The scale is in kilobase pairs. Black arrows, ORFs that are homologous to the potentially putative genes of LCDV-1; white arrows, potentially novel genes that were not found in LCDV-1 and other iridoviruses.
Figure 1 shows a linear map of the 176 largely nonoverlapping ORFs and their sizes, positions, and orientations in the LCDV-C genome. In the 176 putative genes, 103 genes have significant homology to the corresponding ORFs of LCDV-1, but there are still 73 potential genes that were not found in LCDV-1 and other iridoviruses (Fig. 1; Table 2). Among the 73 genes, it was found that 8 genes, ORF002L, ORF011L, ORF016L, ORF047R, ORF051L, ORF058L, ORF209R, and ORF216L, contained coding sequences for conserved domains of other cellular proteins (Table 2). For example, ORF002L contains the coding sequence for the caspase recruitment domain involved in apoptotic signaling. ORF016L contains the coding sequence for tumor necrosis factor receptor domains. ORF209R and -216L contain the coding sequences for an N-terminal domain of cell division protein 48 (CDC48) and a collagen triple-helix repeat. ORF011L, ORF047R, and ORF058L may encode thymidylate synthase, a site-specific recombinase, and a transmembrane receptor, respectively (Table 2). Interestingly, the protein product deduced from ORF051L (Table 2) is highly related to reverse transcriptase (RVT), and the C-terminal region from amino acid 191 to 446 has 26.3% identity to the consensus 200-amino-acid sequence of RVT (CD accession no. pfam00078.11, RVT) (21). Furthermore, there are 65 novel genes that do not show any significant homology with the sequences in public databases (Table 2).
Repeated sequences.
Searching by the program GeneQuest of the DNASTAR software package revealed a large number of tandem and overlapping direct repeated and inverted repeated sequences in the LCDV-C genome. Although they are distributed randomly, two concentrated regions of direct repeated sequences were discovered. The first concentrated region is located from bp 1 to 530 of the genome. In the 530 bp of sequence, there are eight almost identical repeated sequences. Each repeat is composed 66 bp. As shown in Fig. 2, only six nucleotide changes occur in the first seven repeats. In the eighth repeat, the first 55-bp segment is also identical to those of the first seven repeats. And each repeat sequence includes three short AAAGAA repeated sequences (Fig. 2). The second concentrated region, located from 124334 to 124755, includes 10 repeated sequences, and each repeat consists of 36 bp. In addition, the 52-bp repeated sequence TATATATATATA…was observed at positions 25336 to 25387. Furthermore, other short repeated sequences were found dispersed all over the genome. For example, there are 73 copies of the 12-bp direct repeated sequence TTAACCCTTTAA in the genome, and 95% of them are located in the noncoding region.
FIG. 2.
Repeated sequences of bp 1 to 530 in the LCDV-C genome. There are eight direct repeated sequences with 66 bp, and each repeat sequence includes three short AAAGAA repeated sequences (boxes). The individual changed nucleotides and different nucleotides beyond the repeats are in boldface.
In previous studies, some repeated sequences have been found in certain regions of several iridoviruses, such as FV3, LCDV-1 (27), CIV (7, 8), RSIV (35), and ISKNV (13), but the extensive and concentrated repeat sequences were observed only in the LCDV-C genome. He et al. revealed a complex cluster of multiple tandem and overlapping direct repeated sequences of 496 bp at positions 23273 to 23768 in the complete genome of ISKNV (13), but the characterization and function were unknown.
Relatedness of LCDV-C gene products to other proteins in databases.
The comparison of amino acid sequences deduced from the LCDV-C ORFs with entries in protein databases led to the identification of several kinds of functionally characterized proteins in other species. These proteins included some enzymes involved in virus replication, transcription, and modification, such as DNA polymerase (ORF203L), RNA-dependent DNA polymerase (ORF051L), DNA-directed RNA polymerase (ORF115R and ORF191R), DNA methyltransferase (ORF086L), RNA polymerase (ORF025R), site-specific recombinase (ORF047R), ribonucleotide reductase (ORF041L and ORF172L), helicase (ORF75L), deoxynucleoside kinase (ORF027R), thymidylate synthase (ORF011L), protein kinase (ORF013L, ORF045R, ORF166L, ORF175R, and ORF178L), phosphatase (ORF148L), acetyl-coenzyme A hydrolase (ORF161L), and papain-like proteinase (ORF224L) (Table 2). Some of the viral proteins that might be involved in virus-host interaction were also identified from LCDV-C ORFs by significant amino acid sequence homology, such as tumor necrosis factor receptor (ORF016L), β-hydroxysteroid dehydrogenase (ORF003R), membrane-bound metallopeptidase (ORF033R), histone-like transcription factor (ORF054R), ATPase (ORF080L, ORF209R, and ORF237L), transmembrane receptor (ORF058L), and caspases (ORF002L) (Table 2). Just as for other sequenced iridoviruses (13, 14, 16, 17, 18, 33), the majority of these enzymes for LCDV-C represent homologues of cellular enzymes involved in virus replication and transcription and are shared by all iridoviruses (Table 3). Since iridoviruses form a viromatrix in cytoplasm and since their replication, transcription, and nucleotide metabolism mainly occur outside of the nucleus (42), they must establish their own replication and transcription machinery (18). Further studies on these shared genes, therefore, have significant implications for understanding the evolution and phylogeny of iridoviruses.
TABLE 3.
The common genes involved in virus replication and transcription in the LCDV-C, LCDV-1, TFV, and ISKNV genomes and their ORF numbers
| Function | Protein(s) | ORF for:
|
|||
|---|---|---|---|---|---|
| LCDV-C | LCDV-1a | TFVb | ISKNVb | ||
| DNA repication, modification and processing | DNA polymerase, DdDP | 203L | ORF5 | 63R | 19R |
| DNA methyltransferase, DMet | 86L | ORF51 | 89R | 46L | |
| Helicase | 75L | ORF4 | 9L, 56L | 63L | |
| XPG/RAD2-type nuclease | 169R | ORF34 | 101R | 27L | |
| Transcription of DNA | Subunit 1 of DdRP I | 191R | ORF1 | 8L | 28L |
| DdRP II | 25R | ORF3 | 65R | 34R | |
| RNase III | 187R | ORF44 | 85L | 87R | |
| RBRD | 41L | ORF26 | 71L | 24R | |
Comparison of LCDV-C to LCDV-1.
A search of the GenBank database with the 176 individual ORFs revealed 103 homologues to those in the LCDV-1 genome (Fig. 1), accounting for 58.5% of the LCDV-C ORFs. However, comparison of the genome organizations, i.e., the putative gene orders, revealed less similarity between LCDV-C and LCDV-1. The most similar sequence between LCDV-C and LCDV-1 was located at positions 15055 to 25423 (∼11 kb). It includes eight ORFs and shows 68% identity of nucleotide sequences with those of LCDV-1. Although some similarity between putative gene products of LCDV-C and the corresponding viral proteins of LCDV-1 was revealed, no whole colinearity was detected when the ORF arrangements and the coding strategies of the LCDV-C and LCDV-1 genomes were compared. The significant differences between LCDV-C and LCDV-1 genomes in gene organization and gene order are similar to those between vertebrate fish LCDV-1 and invertebrate insect CIV (18). The data suggest that there have been a large number of genetic rearrangements between LCDV-C and LCDV-1 and that the rearrangements might be of high complexity.
During the last decades, lymphocystis diseases throughout the world have been extensively described (34) and have raised serious economic problems in modern aquaculture, fish farming, and wildlife fish. In recent years, many new iridovirus-like pathogens have been isolated from over 100 different species of fish and other cold-blooded vertebrates worldwide (4, 10, 45). Indeed, LCDV and iridovirus-like pathogens vary worldwide with respect to host range and virulence, but intraspecific variation between them has been less extensively characterized. The currently studied LCDV-C was isolated in China from cultured flounder (Paralichthys olivaceus) with lymphocystis disease (31, 46). LCDV-C and LCDV-1 have related hosts (LCDV-1 was isolated from the flounder Platichthys flesus), but their geographical and temporal distributions are very different. Obviously, the significant difference in genome organization between LCDV-C and LCDV-1 suggests that such genomic differences might exist in other isolates of fish. For this reason, more work on comparative genome analysis of LCDV and other unclassified iridovirus-like isolates from distinct sources remains to be done. Recently, Goldberg et al. (11) explored intraspecific strain variation within an emerging iridovirus of North American warm-water fishes, largemouth bass virus, by amplified fragment length polymorphism analysis and revealed that the most virulent viral strain replicated to the highest level in fish. As suggested by Jakob and Darai (18), a substantial revision of the taxonomy of LCDV isolates and other iridoviruses based on molecular anatomy and phylogeny is required.
Relationship of LCDV-C to other iridoviruses and its taxonomic position.
The highest homologies were detected between putative gene products of LCDV-C and the corresponding viral proteins of LCDV-1, but some important genes involved in virus replication, transcription, and modification in the LCDV-C genome have been identified previously in three other vertebrate iridovirus genomes that were completely sequenced, including the LCDV-1 (32), TFV (14), and ISKNV (13) genomes. As shown in Table 3, these genes included those encoding DNA polymerase, DNA methyltransferase, helicase, XPG/RAD2-type nuclease, subunit 1 of DNA-dependent RNA polymerase (DdRP), DdRP II, RNase III, and ribonucleotide reductase (RBRD).
The LCDV-C MCP is encoded by ORF043L and is composed of 459 amino acids (Table 2). It presents the highest identity to those of LCDV-1 and other iridoviruses among the putative gene products of LCDV-C. Homology analysis showed that the MCPs of LCDV-1 (33), CIV (16), TFV (14), FV3 (20), EHNV (22), BIV (3), RSIV (23), and ISKNV (13) had 87.6, 53.0, 51.1, 50.9, 50.7, 50.7, 49.0, and 49.2% identity to that of LCDV-C, respectively. Based on the multiple alignments of amino acid sequences of nine complete MCPs, a phylogenetic tree was constructed. As shown in Fig. 3, the nine iridoviruses are divided into four groups, the lymphocystiviruses LCDV-C and LCDV-1; the insect iridoviruses, including CIV; the ranaviruses, including FV3, BIV, TFV, and EHNV; and the unassigned viruses ISKNV and RSIV. Interestingly, LCDV-C and LCDV-1 are clustered together, but their amino acid identity is much less than that in the other three clusters. Recently, Jakob and Darai (18) drew the conclusion that a cricket iridovirus (CrIV) isolate and CIV are not different species because of the high identity (97.9%) of their MCP amino acid sequences and considered CrIV a variant or a strain of CIV. The MCPs of FV3, TFV, ENHV, and BIV have over 96.8% identity (Fig. 3), suggesting that these viruses might also be different variants of the same species. And the identities of MCPs of ISKNV and RSIV were also found to be up to 98.2%. However, LCDV-C was identified to be the Chinese LCDV variant on the basis of the infection symptoms (31, 40) and viral morphology (46), but the MCPs of LCDV-C and LCDV-1 have only 87.6% identity, and there are significant differences between their genome sizes (Table 2) and gene organizations (Fig. 1). The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a separate species, different from LCDV-1.
FIG. 3.
Phylogenetic tree based on the multiple alignments of the amino aced sequences of the MCPs of iridoviruses. The GenBank accession numbers for the virus nucleotide sequences are as follows: LCDV-1, AAC24486; CIV, AAK82135; ISKNV, AAL98730; RSIV, BAC66968; FV3, Q67473; TFV, AF389451; EHNV, AA032315; BIV, AY187046.
LCDV-C is the second LCDV isolate whose complete genomic sequence has been determined since the first complete genome of LCDV was sequenced from the LCDV-1 isolate in 1997 (33). Up to now, more than 100 new iridovirus-like isolates have been reported from over 100 different species of fish in seawater and freshwater worldwide (34). Of the numerous virus isolates, only two isolates have been completely sequenced, and a great number of divergences between them have been revealed. Obviously, a handicap for further analysis is the lack of genome sequence information for other iridovirus-like isolates (18). Therefore, the significant divergences between LCDV-C and LCDV-1 draw our attention to the different iridovirus-like isolates. The detailed molecular anatomy and functional analyses of these different iridovirus-like isolates will provide more novel and distinct knowledge about their relationship and taxonomic position among the iridoviruses.
Acknowledgments
We thank Sun Xiu-qin for her assistance on sampling the diseased fish, and we thank members of BGI Life Tech, Beijing, for their assistance on sequencing technologies.
This study was supported by National 863 Project of China (2001AA626030 and 2002AA626010), the National Natural Science Foundation of China (30170726), the Project of Chinese Academy of Sciences (STZ-00-13, KSCX2-SW-302), and the Frontier Science Project Program of the Institute of Hydrobiology, Chinese Academy of Sciences (220301).
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