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Letter
Taura Syndrome Virus and Mammalian
Cell Lines
Peng Luo,*
Chao-Qun Hu,* Chun-Hua Ren,* and Zhao-Feng Sun*
*The Chinese Academy of Sciences, Guangzhou, People's Republic of China
Suggested
citation for this article
To the Editor: Audelo-del-Valle et al. concluded that human and
monkey cell lines (rhabdomyosarcoma [RD], human larynx carcinoma [Hep-2C],
and Buffalo green monkey kidney [BGM]) could be infected by a penaeid
shrimp virus, Taura syndrome virus (TSV) (1). They also
concluded that Penaeus spp. could likely be a reservoir of a virus
that might become pathogenic to humans and other mammals (1).
Though researchers have tried to develop continuous marine crustacean
cell lines for >30 years, their efforts have not been successful. The
lack of continuous marine crustacean cell lines has become an obstacle
to conducting research on viral disease in shrimp (2,3).
During the last 20 years, many researchers searched for substitute cell
lines on which to study shrimp viruses (4,5). Audelo-del-Valle
et al. likely chose RD, Hep-2C, and BGM cell lines because TSV was "recently
reported to be genomically related to the cricket paralysis virus of the
Cripavirus genus, family Dicistrovirdae of the 'picornavirus
superfamily'" (1), and these cell lines were susceptible
to some picornaviruses. If their findings are correct, they may have found
substitute cell lines for isolating and studying TSV. To confirm their
findings, we selected two mammalian cell lines, Hep-2 and Vero, which
are highly sensitive to some picornaviruses (6,7), and
tested them to determine their susceptibility to TSV.
The TSV extract was prepared from frozen cephalothoraxes of shrimp, Litopenaeus
vananmei, that were infected with TSV (confirmed by standard reverse
transcriptase–polymerase chain reaction [RT-PCR]) (8).
To verify the TSV extract's validity, 50 μL of diluted TSV extract
(approximately 0.8% volume of shrimp body weight) was injected into each
of eight healthy shrimp, L. vannamei. Another eight healthy shrimp
(control group) were injected with a diluted extract prepared from frozen
cephalothoraxes of healthy shrimp. All of the TSV-injected shrimp died
within 6 days and were TSV-positive; control shrimp did not die and were
TSV-negative, which showed that our TSV extract was active and viable.
The TSV extract was transferred into cell culture flasks according to
a method previously reported (9). The cell monolayers
were exposed to 100 μL of diluted and filtered TSV extracts for 1 hour;
the extracts were then removed from the flasks, 2 mL of maintenance medium
was added to each flask, and the flasks were incubated in three separate
rooms at 37°C, 35°C, and 33°C, respectively. If a cytopathic
effect (CPE) was not evident within 7 days, cell monolayers were washed
with Hank's balanced salt solution (HBSS) six times to eliminate viral
particles from the primary extract or from infected cells. Then cells
were lysed in 2 mL HBSS, the lysate was clarified, and a portion of it
was used for the first passage. This procedure was repeated three times.
The control cell lines were injected with diluted extract from healthy
shrimp, and passage was conducted as described earlier. RNA samples were
extracted and purified from 150-μL lysates of primary cells and four
passage cells and used as templates for RT-PCR analysis to determine the
presence of TSV.
No CPE was observed in either the Hep-2 or Vero cell line that had been
injected with TSV after 7 days of culture at any of the three temperatures
tested, and CPE was not found after the fourth passage. The RT-PCR analysis
resulted in weak amplification (positive) from the first lysate, but no
amplification was found in lysates of four passage cells. Had TSV replicated
(productive infection) in either of the two cell lines, RT-PCR would have
shown a strong amplification from each lysate. Such a weak amplification
may have been the result of residual extracellular viruses that remained
in the cell culture flask after washing. However, after first passage
and repeated washing with HBSS, any remnants of the original medium were
not likely to have been present. Therefore, our result showed that TSV
was incapable of infecting Hep-2 and Vero cell lines.
Generally, aquatic viruses replicate in cells of aquatic animals at 20°C–35°C,
their natural environmental temperature. We incubated cultures as noted
earlier, as we did not know which temperature was most conducive for viral
replication; all attempts were unsuccessful. Hep-2 and Hep-2C derive from
the same tissue (human Caucasian larynx carcinoma), while Vero and BGM
derive from another organ (Africa green monkey kidney). Thus, Hep-2 and
Vero cell lines that we used are likely susceptible to TSV if the virus
can infect Hep-2C and BGM as reported by Audelo-del-Valle et al.
The difference between our methods and those used by Audelo-del-Valle
et al. may explain the discrepant result. If CPE occurred "usually
from 19–23 hours" and "cells were then harvested and lysed"
for next injection, TSV most likely persisted in the lysate after the
third passage because the cell monolayers had not been washed as they
were in our method. According to the time the CPE was observed and the
methods of Audelo-del-Valle et al., we assumed that, in their study, cells
might be passaged at least three times within 1 week, whereas TSV might
remain viable and infective for 1 week. Additionally, the Office International
des Epizooties recommended an injection volume of 1% of shrimp body weight
(8); Audelo-del-Valle et al. used 10%. With such a large
dose, shrimp could be infected easily with TSV from the initial medium
and die suddenly. Moreover, the evidence of successful infection from
photos of CPE only is not sufficient; Audelo-del-Valle et al. should offer
more convincing evidence from images of viral particles in cells by electron
microscope or in situ hybridization. Therefore, we think the CPE that
Audelo-del-Valle et al. reported was not caused by TSV but by a virus
contaminant or some harmful component from shrimp extract.
The structure of the TSV genome is similar to that of small insect–infecting
RNA viruses (10), which belong to a renamed virus genus,
Cripavirus (11). No published reports have shown
that other viruses in this genus are able to infect mammalian cells or
cell lines. Moreover, TSV is prevalent in shrimp farming areas in the
world, and L. vannamei (principal host for TSV) are eaten by people
worldwide (8). In China, some persons eat fresh shrimp
without disinfecting them; however, no evidence shows that TSV can infect
humans. The results of our study show that TSV cannot infect mammalian
cell lines or cells.
This work was supported
by innovation-projects funds provided by The Chinese Academy of Sciences
(Projects No. ZKCX2-211).
References
- Audelo-del-Valle J, Clement-Mellado O, Magaña-Hernández
A, Flisser A, Montiel-Aguirre F, Briseño-García B. Infection
of cultured human and monkey cell lines with extract of penaeid shrimp
infected with Taura syndrome virus. Emerg Infect Dis. 2003;9:265–6.
- Toullec JY. Crustacean
primary cell culture: a technical approach. Methods Cell Sci. 1999;21:193–8.
- Crane MS. Mutagenesis
and cell transformation in cell culture. Methods Cell Sci. 1999;21:245–53.
- Loh PC, Lu YA, Brock JA. Growth
of the penaeid shrimp virus infectious hypodermal and hematopoietic
necrosis virus in a fish cell line. J Virol Methods. 1990;28:273–80.
- Yue YL, Li CY, Yang SH, Lu Q, Tao ZS, Wang WD, et al. Cell isolation
and culture of penaied shrimp paramyxovirus-like virus. Chin J Vet Sci.
1997;17:306–7.
- Johnston SLG, Siegel CS. Presumptive
identification of enteroviruses with RD, Hep-2, and RMK cell lines.
J Clin Microbiol. 1990;28:1049–50.
- Kok T, Pryor T, Payne L. Comparison
of rhabdomyosarcoma, Buffalo green monkey kidney epithelial, A549 (human
lung epithelial) cells and human embryonic lung fibroblasts for isolation
of enteroviruses for clinical specimens. J Clin Virol. 1998;24:61–5.
- Office International des Epizooties. Diagnostic manual for aquatic
animal health diseases. 3rd ed. Paris: The Office; 2000.
- Yin Z. Animal virology. Beijing: Science Press; 1985.
- Mari J, Poulos BT, Lightner DV, Bonami JR. Shrimp
Taura syndrome virus: genomic characterization and similarity with members
of the genus Cricket paralysis-like viruses. J Gen Virol. 2002;83:915–26.
- Mayo MA. A
summary of taxonomic changes recently approved by ICTV. Arch Virol.
2002;147:1655–63.
Suggested citation
for this article:
Luo P, Hu CQ, Ren CH,
Sun ZF. Taura syndrome virus and mammalian cell lines [letter]. Emerg
Infect Dis [serial on the Internet]. 2004 Dec [date cited]. Available
from http://www.cdc.gov/ncidod/EID/vol10no12/04-0537.htm
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