1 Institute of Zoology, National Taiwan University, Taipei 106, Taiwan, Republic of China
2 Department of Entomology, National Taiwan University, Taipei 106, Taiwan, Republic of China
Correspondence
Chu-Fang Lo
gracelow{at}ntu.edu.tw
Guang-Hsiung Kou
ghkou{at}ccms.ntu.edu.tw
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ABSTRACT |
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These authors contributed equally to this work.
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY355309AY355311.
A table showing abbreviations and descriptions of TSV isolates used in this study is available as supplementary material in JGV Online.
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MAIN TEXT |
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In this study, TSV-positive samples from three sources were collected. The first sample, a P. monodon brooder (Tw2KPm), was selected by screening the gills and pleopods of a batch of 24 brooders that were captured from southern Taiwan coastal waters in 2000. These brooders were frozen, transported to our laboratory and stored at 70 °C. The sample brooder was used for TSV tissue-distribution analysis and to prepare a virus inoculum (see Supplementary Table, available in JGV Online, for the names and sources of the inocula used in this study). The second sample was from a batch of live, cultured M. ensis that were bought from a local fish market in 2000 and showed visible signs of Taura syndrome. The M. ensis specimen studied here (Tw2KMe) died 2 days after arrival in the laboratory, at which time its gills and two of its pleopods were excised and analysed for virus infection, while the rest of the body was stored at 70 °C until used as above. For the third sample, two pleopods were taken from an L. vannamei shrimp (Tw02Lv) collected in 2002 from a culture pond in southern Taiwan in which there had recently been an outbreak of TSV. This sample was used for sequence comparison only.
Total RNA of tested specimens was extracted by using TRIzol reagent (Invitrogen). First-strand cDNAs of these RNAs were synthesized by using an oligo-dT primer (Roche) and Superscript II reverse transcriptase (Invitrogen). A nested (two-step) PCR using three TSV-specific primers for amplifying cDNA in a region of the TSV orf2 gene was used to confirm TSV infection. The primers TSVF2 (5'-ACCCCAGAAATGTGAATAACC-3') and TSVR2 (5'-GGAAAAGCAATGTCAATACCC-3') served as the outer primer pair for the first PCR step and the primer TSVF3 (5'-ATACTTAGCACAGCGACCATA-3') combined with TSVR2 served as the inner primers for the nested amplification. Amplicons resulting from TSVF2/TSVR2 and TSVF3/TSVR2 were 910 and 360 bp in length, respectively. The cDNA from 0·1 µg total RNA was subjected to PCR in a 50 µl reaction. Plasmids containing TSV orf2 cDNA were diluted serially to estimate the PCR sensitivities of one-step and nested amplification. cDNAs from the testis of TSV-challenged P. monodon and from the pleopods of the other tested individuals were also analysed by real-time RT-PCR following the method of Dhar et al. (2002).
Two cultured, TSV-free P. monodon shrimps (body mass approx. 40 g) were challenged experimentally by intramuscular injection with Tw2KPmTSV or Tw2KMeTSV virus inoculum prepared from Tw2KPm and the Tw2KMe, respectively. Methods for inoculum preparation and injection challenge were as described by Lightner (1996), except that the Tw2KPmTSV inoculum was concentrated 10-fold. At the end of the experimental period (51 h post-infection), various tissues/organs from the two challenged individuals were screened for TSV.
To demonstrate more rigorously the different replication abilities of Tw2KMeTSV and Tw2KPmTSV, a batch of specific pathogen-free (SPF) Marsupenaeus japonicus juveniles (body mass approx. 12 g) was used (kindly provided by Dr C.-M. Kuo, Marine Research Station, Academia Sinica, Taiwan). These shrimp were specifically free of TSV, white spot syndrome virus (WSSV), yellow head virus (YHV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV). Before challenge, the inoculum prepared from either Tw2KMe or Tw2KPm was pre-treated with chloroform to exclude any possible low-level contamination of WSSV and then diluted to contain approximately 300 TSV genome copies in a volume of 30 µl (as determined by real-time RT-PCR). Challenge was by intramuscular injection. Four shrimps were sampled from each group at 72 h post-infection and screened for TSV, WSSV, IHHNV and YHV.
For TSV genomic sequence analysis, cDNA products from the pleopods of Tw2KPm, Tw2KMe and Tw02Lv were subjected to PCR using Pfu DNA polymerase (Promega). The entire orf2 gene of these three isolates was amplified and sequenced. Multiple nucleotide sequence alignments of the entire length of the TSV orf2 gene of these and three other TSV isolates (see Supplementary Table in JGV Online) were analysed by CLUSTAL X (Thompson et al., 1997) and GeneDoc (Nicholas et al., 1997
). Multiple alignments of the ORF2 amino acid sequences of all six isolates were used for phylogenetic analysis based on the neighbour-joining and maximum-parsimony (MP) methods of the PAUP 4.0b1 program (Swofford, 1998
). One thousand bootstrap replicates were generated to test the robustness of the trees.
Fig. 1(a) shows the calibration results for the nested TSV RT-PCR protocol that was used in this study. In this PCR test, a sample that is positive in the first amplification must have a viral load that is at least 104-fold greater than a sample that is positive only in the second (nested) step. The 24 P. monodon brooders that were screened had a low TSV prevalence rate, with only two specimens testing positive for TSV by nested PCR (Fig. 1b
). This was comparable to the positive result for one-step PCR for Tw2KMe (Fig. 1b
) and all of the other randomly tested M. ensis specimens (data not shown). Tissue-distribution results for the naturally infected Tw2KPm showed a low level of infection in all of the organs tested (Fig. 1c
), compared with positive results for one-step PCR for each organ in the Tw2KMe specimen (Fig. 1d
). An adult P. monodon shrimp that was challenged with concentrated Tw2KPmTSV was positive for nested PCR in only two tested organs (Fig. 1e
). Challenge with Tw2KMeTSV, however, produced positive results for nested PCR in all organs and positive results for one-step PCR in some (Fig. 1f
). These preliminary data suggested that in P. monodon (a species in which TSV is normally found only at very low levels), the Tw2KMeTSV isolate was able to replicate much more freely than the Tw2KPmTSV isolate. In an additional, more rigorous demonstration of the difference in replication ability between Tw2KPmTSV and Tw2KMeTSV, challenge tests were performed on SPF M. japonicus juveniles with Tw2KMeTSV (Fig. 1g
, lanes 14) virus loads (revealed by real-time RT-PCR) of 1·2x1053·7x106 copies in 0·1 µg total RNA. In the Tw2KPmTSV-challenged group (Fig. 1g
, lanes 58), virus loads were 33100 copies in 0·1 µg total RNA. WSSV, YHV and IHHNV were not detected in any of these challenged M. japonicus (data not shown). The results reconfirmed that these two isolates replicate differently in intramuscularly challenged shrimp.
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ACKNOWLEDGEMENTS |
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Received 25 March 2004;
accepted 2 July 2004.
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