Infection of cultured human and monkey cell lines with extract of penaeid shrimp infected with Taura syndrome virus. (Dispatches).

Taura syndrome virus (TSV) affects shrimp cultured for human consumption. Although TSV is related to the Cricket Paralysis virus, it belongs to the "picornavirus superfamily," the most common cause of viral illnesses. Here we demonstrate that TSV also infects human cell lines, which may suggest that Penaeus is a potential reservoir of this virus.

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The Taura syndrome virus (TSV) causes a disease affecting penaeid shrimp, the most important commercial family of crustaceans (1). The causal agent is a single-stranded (+) RNA virus, recently reported to be genomically related to the Cricket Paralysis virus of the Cripavirus genus, family Dicistroviridae of the "picornavirus superfamily" (2-5). This superfamily includes several human pathogens, for example, the common cold viruses and several enteroviruses (e.g., polioviruses). Traditionally, research on the replication of shrimp viruses has been based on the use of cultured fish cellular lines (6). However, because TSV could potentially represent a public health threat, we explored whether this viral agent might be capable of infecting cultured mammalian cells.

The Study

Since Sabin strain LSc 2ab (Sabin 1), the poliovirus used for human vaccination, is usually replicated in monolayer culture cells of human rhabdomyosarcoma (RD), human larynx carcinoma (Hep-2C), or Buffalo green monkey kidney (BGM) (7), we injected these cell lines with a 0.22-[micro]m membrane-filtered whole extract of the hepatopancreas of shrimp (Penaeus stylirostris) affected with TSV. The animals were collected from farms located in northwestern Mexico. Control cell lines were injected with filtered hepatopancreas extracts from healthy shrimp. Cultures were incubated at 37[degrees]C and periodically observed under a microscope until any sign of cytopathic effect was detected (usually from 19-23 hours). Cells were then harvested and lysed. Fresh cell lines were inoculated with the lysate, incubated, and processed in a similar way. A third inoculation was again performed with the second lysate (8).

The cytopathic effect in RD cells began with a partial destruction of the cellular layer. Next, small cellular groups and some isolated round cells were observed. The cells showed an apparent increase in size, diffuse cell rounding, and a refringent aspect (Figure 1B). In Hep-2C cells, the cellular monolayer was partially destroyed. Most cells were individualized and clearly rounded; they also presented a refringent aspect. Hep-2C was the most affected of the three lines used (Figure 1D). The cytopathic effect in the BGM cell line began as a partial destruction of the cellular layer, which evolved to a syncytial-like formation of rounded, refringent cells. Some cells remained isolated but with altered morphology (Figure 1F). RD, Hep-2C, and BGM cells injected with an extract similarly processed but from healthy shrimp, showed no cytopathic effects, even after 7 days of culture (Figures 1A, 1C, and 1 E). As a positive control, RD cells were injected with Sabin viral extract and showed the characteristic cytopathic effect produced by an enterovirus infection.

[FIGURE 1 OMITTED]

To confirm the presence of TSV in the cell culture media, a bioassay was performed by using media from the third passage. For this assay, healthy P. stylirostris shrimp were injected with the infected medium in 10% volume of their corporal mass in the third abdominal segment. Twenty-four hours later, these animals were clearly infected, showing fragile antennas and soft cuticle as well as chromatophore expansion along the whole surface of the body, particularly at the tail fan (telson and uropods). These signs were clinically indistinguishable from those occurring in naturally infected animals and are considered as pathognomonic of the acute phase of infection by TSV (9). Presence of the viral genome in different subcuticular tissues (gills and pleopods) of these animals was confirmed by in situ hybridization by using TSV ShrimProbe (DiagXotics, Inc., Wilton, CT). RNA-DNA hybrids were clearly visible as black spots after the samples were stained with Bismarck brown (Figure 2). Shrimp injected with culture media from control cell lines showed no signs of infection after 7 days of observation.

[FIGURE 2 OMITTED]

Conclusions

If one takes into consideration the capacity of viruses to modify receptor recognition and host cell tropism and the fact that cell receptors for many of the picornavirus superfamily members seem to be ubiquitous membrane molecules (e.g., decay-accelerating factor, different type of integrins, low-density lipoprotein receptor, sialic acid [10-12]), the potential wide range of host cells for TSV should not come as a surprise. To our knowledge, these cultured human and monkey cell lines are the first reported to be infected with a viral agent isolated from shrimp. Because many members of the picornavirus superfamily are the most common causes of viral illnesses worldwide (including nonspecific febrile illnesses, myocarditis, aseptic meningitis, and sepsis-like disease), such illnesses lead to frequent unnecessary prescription of antibiotics (13). Penaeus could be considered as a reservoir of a virus that could become a potential pathogen to humans and other mammals (11,14).

Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.

Table 1. Origin of 354 Campylobacter jejuni isolates from travelers
returning to Finland and proportion of ciprofloxacin-resistant isolates
during two collection periods

                          1995-1997               1998-2000

                                    %                        %
                       All     Ciprofloxacin   All     Ciprofloxacin
Geographic area      isolates   resistant    isolates    resistant

Africa                24         17             8         38
America                4          0             3         67
Asia                  78         45 (a)        57         72 (a)
 Southeast Asia       28         61            39         77
 Other areas          50         36            18         61
Europe                93         44            73         51
 Mediterranean area   64         59            48         69
 Other areas          29         10            25         16
Total                205 (b)     40 (a)       149 (c)     60 (a)

(a) Difference is statistically significant (p<0.01).

(b) One isolate from Australia and five
isolates of unknown origin included.

(c) Eight isolates of unknown origin included.

Table 2. Rate ratios of travel-associated infections with
ciprofloxacin-resistant Campylobacter jejuni isolates (a)

                 Estimated no. of trips              Speculative
                  from Finland during    No. of all  infection
Country           the study months (b)    isolates    rate (c)

Spain (incl.           1,145,872             77        (0.1)
Canary Islands)
Thailand                 87,842              50        (0.6)
India                    27,591              23        (0.8)
China                    25,073              12        (0.5)
Portugal                148,647              11        (0.1)

                                                 No. of
                                             ciprofloxacin-
Country              Rate ratio (d)        resistant isolates

Spain (incl.      0.12 (0.08 to 0.17)              55
Canary Islands)
Thailand                   1                       39
India             1.46 (0.89 to 2.40)              11
China             0.84 (0.45 to 1.58)               8
Portugal          0.13 (0.07 to 0.25)               7

                 Speculative infection
                      rate (c) by           Rate ratio (d) by
                     ciprofloxacin-          ciprofloxacin-
Country            resistant isolates      resistant isolates

Spain (incl.             (0.05)          0.11 (0.07 to 0.16) (e)
Canary Islands)
Thailand                 (0.44)                  1 (e)
India                    (0.40)          0.90 (0.46 to 1.75)
China                    (0.32)          0.72 (0.34 to 1.54)
Portugal                 (0.05)          0.11 (0.05 to 0.24) (e)

(a) The five most frequent countries of origin of
ciprofloxacin-resistant C. jejuni isolates were
included in this analysis.

(b) Based on the numbers of Finnish travelers to
these countries; data collected from the reports
of Statistics Finland.

(c) Infections per 1,000 trips. Since the total
number of isolates from Finnish population was not
studied, the rate is speculative but could be used
to calculate comparable rate ratios.

(d) Thailand as the reference country; 95%
confidence intervals in parentheses.

(e) Differences between Thailand and Spain,
and Thailand and Portugal, are statistically
significant (p<0.01).

Acknowledgments

We gratefully acknowledge Dolores Correa and Mirza Romero for helpful and critical discussions.

References

(1.) Food and Agricultural Organization. Aquaculture production statistics 1987-1996. Rome: The Organization; 1998.

(2.) Bonami JR, Hasson KW, Mari J, Poulos BT, Lightner DV. Taura syndrome of marine penaeid shrimp: characterization of the viral agent. J Gen Virol 1997;78:313-9.

(3.) 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.

(4.) Robles-Sikisaka R, Garcia DK, Klimpel KR, Dhar AK. Nucleotide sequence of 3'-end of the genome of Taura syndrome virus of shrimp suggests that it is related to insect picornaviruses. Arch Virol 2001;146:941-52.

(5.) van Regenmortel MHV, Fauquet CM, Bishop DHL, Cartens EB, Estes MK, Lemon SM et al., editors. In: Virus taxonomy: seventh report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press, 2000.

(6.) Loh PC, Tapay LM, Lu Y, Nadala ECB Jr. Viral pathogens of the penaeid shrimp. Adv Virus Res 1997;48:263-312.

(7.) Ashkenazi A, Melnick JL. Enteroviruses: a review of their properties and associated diseases. Am J Clin Pathol 1962;38:209-29.

(8.) Mahy BWJ, Kangro HO. Virology methods manual. London: Academic Press, Ltd.; 1996.

(9.) Hasson KW, Lightner DV, Poulos BT, Redman RM, While BL, Brock JA, et al. Taura syndrome in Penaeus vannamei: demonstration of a viral etiology. Dis Aquat Organ 1995;23:115-26.

(10.) Evans DJ, Almond JW. Cell receptors for picornaviruses as determinants of cell tropism and pathogenesis. Trends Microbiol 1998;6:198-202.

(11.) Baranowski E, Ruiz-Jarabo CM, Domingo E. Evolution of cell recognition by viruses. Science 2001;292:1102-5.

(12.) Rossmann MG, He Y, Kuhn RJ. Picornavirus-receptor interactions. Trends Microbiol 2002;10:324-31.

(13.) Rotbart HA, Hayden FG. Picornavirus infections. A primer for the practitioner. Arch Faro Med 2000;9:913-20.

(14.) Weiss RA. The Leeuwenhoek Lecture 2001. Animal origins of human infectious disease. Phil Trans R Soc Lond B 2001;356:957-77.

Address for correspondence: Fernando Montiel-Aguirre, Universidad Nacional Autonoma de Mexico, Facultad de Quimica, Departamento de Biologia, Cd. Universitaria C.P. 04510, Mexico, D.F.; fax: [phone]; e-mail: [e-mail]

Josefina Audelo-del-Valle, * Oliva Clement-Mellado, ([dagger]) Anastasia Magana-Hernandez, 9([dagger]) Ana Flisser, ([double dagger]) Fernando Montiel-Aguirre, ([double dagger]) and Baltasar Briseno-Garcia ([dagger])

* Universidad de Occidente, Los Mochis, Sinaloa, Mexico; ([dagger]) Instituto de Diagnostico y Referencia Epidemiologicos, Mexico, D.F., Mexico; and ([double dagger]) Universidad Nacional Autonoma de Mexico, Mexico, D.F., Mexico

Ms. Audelo-del-Valle is a fisheries biologist and lecturer at the Universidad de Occidente, Campus Los Mochis, Sinaloa, Mexico. She founded and directs the Molecular Biology Aquaculture Laboratory in the Universidad de Occidente and is currently a Ph.D. candidate.