Nonsusceptibility of primate cells to Taura syndrome virus

Abstract

Taura syndrome virus (TSV), a pathogen of penaeid shrimp and member of the family Dicistroviridae, was recently reported to have the ability to infect primate cells. We independently retested this hypothesis. Three lines of primate cells FRhK-4, MA-104, and BGMK, which are highly susceptible to infection by human picornaviruses, were challenged with TSV. Viral replication was assayed by real-time reverse transcription-polymerase chain reaction using cell media samples collected on days 0, 4, and 7 postchallenge. By day 7, genome copy numbers had decreased 25%-99%. No cytopathic effect was observed after 7 days. An in situ hybridization assay, with gene probes specific for detection of TSV, was negative for TSV in challenged cells. The infectivity of residual virus in the cell culture media at day 7 was confirmed by bioassay using TSV-free indicator shrimp (Litopenaeus vannamei). TSV did not infect the primate cells tested, and no evidence of zoonotic potential was found.

Figure 1

Absence of cytopathic effect after a 7-day exposure of three lines of primate cells (FRhK-4, MA-104, and BGMK) to an inoculum prepared with hemolymph from Taura syndrome virus (TSV)-infected shrimp (Litopenaeus vannamei) during the acute phase of the disease compared to a control inoculum containing hemolymph from specific pathogen free (SPF) shrimp. A) FRhK-4 cells exposed to SPF hemolymph; B) FRhK-4 cells exposed to TSV hemolymph; C) MA-104 cells exposed to SPF hemolymph; D) MA-104 cells exposed to TSV hemolymph; E) BGMK cells exposed to SPF hemolymph; F) BGMK cells exposed to TSV hemolymph (no stain; 25x).

Figure 2

Example of the decrease on Taura syndrome virus (TSV) genome copy number within tissue cell culture flasks exposed to TSV. A) Real time reverse transcription–polymerase chain reaction plots and mean quantity of TSV copies/μL from tissue cell culture media collected at days 0, 4, and 7 postinfection from MA-104 cell flasks injected with TSV-infected shrimp hemolymph. Samples of tissue cell culture media collected from FrhK-4 and BGMK cell culture flasks inoculated with TSV-infected hemolymph or hepatopancreas also decreased by >1 log in concentration of viral copies as a function of time. The value of 33.79 obtained for one of the no template control (NTC) replicates is considered an artifact. B) Standard curve of TSV copy number versus threshold cycle (C_t) value. Purified TSV plasmid was serially diluted and used as templates in real-time polymerase chain reaction. The resulting C_t values are plotted against the logarithm of their respective copy numbers (C0). R_n, fluorescence signal; SPF 1 and 2, specific pathogen free.

Figure 3

Histologic section through the anterior gastric chamber of a moribund juvenile Litopenaeus vannamei that was injected with an inoculum prepared with tissue cell culture media from BGMK cells exposed to Taura syndrome virus (TSV) (day 7 postexposure). A) The arrows point to a portion of cuticular epithelium displaying diagnostic acute-phase TSV lesions (hematoxylin/eosin-phloxin stain; 50x). B) The dashed arrows point to a portion of the stomach epithelium from the same shrimp, where digoxigenin (DIG)-labeled TSV-specific gene probes were reacted by in situ hybridization (ISH), resulting in the deposition of a black precipitate on areas where the probe hybridized with target TSV (Bismarck Brown counterstain; 50x).

Figure 4

Covert Taura syndrome virus (TSV) infection (transition/chronic phase of TS disease) in indicator specific pathogen free–Litopenaeus vannamei shrimp was confirmed by in situ hybridization (ISH) with digoxigenin-labeled gene probes specific for detection of TSV. A) Histologic section through the dorsal cuticular epithelium showing a melanized resolving lesion (MZ) and hemolytic congestion (HCg), indicative of the transition phase of TSV infection (hematoxylin/eosin-phloxin stain; 50x) . B) TSV ISH on the consecutive section to that shown in 5A, where binding of the TSV probes is shown by the black precipitate (arrow) indicating the presence of TSV within the cytoplasm of cells of the cuticular epithelium (Bismarck Brown counterstain; 50x)

Figure 5

Absence of reaction by in situ hybridization (ISH) to the digoxigenin (DIG)-labeled Taura syndrome virus (TSV) probes within the BGMK cells harvested at day 7 postinjection with TSV. A) No cytopathic effect suggestive of TSV infection was evident by conventional hematoxylin/eosin-phloxin (H&E) histology (H&E stain; 100x). B) Consecutive histologic section to that shown in Figure 4A, but subjected to ISH with DIG-labeled TSV probes specific for TSV. No reaction to TSV is apparent in the challenged cells (Bismarck Brown counterstain; 100x).