Comparison of Taura syndrome virus (TSV) detection methods during chronic-phase infection in Penaeus vannamei

Abstract

Methods to detect Taura syndrome virus (TSV) were assessed for their ability to detect the virus during chronic phase infection in the Pacific white shrimp Penaeus vannamei. In situ hybridization (ISH), immunohistochemistry (IHC) using monoclonal antibody 1A1, conventional RT-PCR and real-time quantitative (q)RT-PCR were compared using shrimp sampled over 60 wk following experimental TSV infection. Between Weeks 7 and 60, hematoxylin-eosin histology confirmed the presence of lymphoid organ spheroids (LOS) and an absence of lesions in the cuticular epithelium. ISH detected TSV in LOS over the duration of the study. IHC was generally less sensitive than ISH, and after Week 24, was often unable to confirm TSV infection. Detection of TSV by RT-PCR was highly dependent on sample source after Week 43, where viral RNA was detected in 12 of 14 hemolymph samples but only 5 of 16 pleopod samples. qRT-PCR detected TSV over the 60 wk in both hemolymph and pleopods, although RNA copy numbers in pleopods were consistently lower throughout the study. This study demonstrates that ISH and qRT-PCR are the most reliable methods for detecting TSV during late chronic phase infection. RT-PCR was also reliable if hemolymph was used as the sample source. KEY WORDS: Taura syndrome · TSV · RT-PCR · qRT-PCR · In situ hybridization · Immunohistochemistry Resale or republication not permitted without written consent of the publisherDis Aquat Org 82: [179][180][181][182][183][184][185] 2008 lasting ~5 d and a chronic phase that lingers in survivors of ecdysis. Shrimp with a chronic phase infection present no melanized lesions or abnormal behavior and histologically present normal cuticular epithelium. However, they display marked hypertrophy of the lymphoid organ (LO) with the presence of numerous LO spheroids (LOS) (Hasson et al. 1999b, Srisuvan et al. 2005.Over 48 wk following TSV infection of juvenile Penaeus vannamei, in situ hybridization (ISH) was able to detect TSV within the LOS up to Week 32, even though LOS remained evident thereafter up to Week 48 post-infection (pi) (Hasson et al. 1999b). RT-PCR and real-time quantitative (q)RT-PCR have been developed to detect TSV (Nunan et al. 1998) and the latter test was able to detect the virus in LO, gill and pleopod tissues in the chronic phase of the disease up to 23 wk pi when the study was terminated . A monoclonal antibody (1A1) generated to a TSV isolate from Hawaii (circa 1994) used for immunohistochemistry can detect the virus in fixed tissue sections but the reactivity is limited to related isolates (Poulos et al. 1999, Erickson et al. 2002. However, the utility of IHC has not been investigated during chronic phase infection.Studies to date have shown that accurate diagnosis of TSV is dependent upon the stage of infection and the nature of the isolate. The selection of diagnostic specimens and TSV detection methods becomes more important to diagnose the infection once disease signs abate as infection passes through the transition stage and the virus becomes sequestered...

Protection from yellow head virus (YHV) infection in Penaeus vannamei pre-infected with Taura syndrome virus (TSV)

“…This TSV distribution has been reported previously (Tang et al 2004), and it is related to the typical TSV chronic phase when most of the TSV viral particles are harbored in this organ and circulating in the hemolymph (Poulos et al 2008). When comparing the TSV and YHV viral genetic loads of dead or surviving shrimp from the same group, a higher copy number of YHV was found in the dead shrimp (3.52 × 10 9 copies µg RNA −1 in the dead shrimp versus 8.91 × 10 8 copies µg RNA −1 in the surviving shrimp) (p < 0.05), suggesting that the mortalities observed were caused by YHV and not by TSV.…”

“…TSV in the acute phase does not infect the LO. A strong positive reaction is observable using ISH in the LO only during the transition and the chronic phases (Hasson et al 1999b, Poulos et al 2008. At that time, a higher viral genetic load is present in the LO than in pleopods (Tang et al 2004).…”

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of yellow head virus in shrimp

“…Real-time RT-PCR assays have been developed for laboratory diagnosis of shrimp viruses (Poulos et al, 2008;Xie et al, 2008;Dhar et al, 2009); however, these techniques have the intrinsic disadvantage of requiring either a high-precision instrument for the amplification or a complex method for the detection of amplified products. The sensitive real-time RT-LAMP assay has been successfully applied to detect many human pathogenic RNA viruses, resulting in rapid and simple diagnostic measures (Parida et al, 2004;Imai et al, 2006;Shirato et al, 2007).…”

Taura syndrome virus loads in Litopenaeus vannamei hemolymph following infection are related to differential mortality

“…We stopped on Day 61 because there were insufficient shrimp to continue the trial. Sampling of hemolymph has been shown to be more reliable than sampling of pleopod for monitoring TSV loads by real-time quantitative reverse transcription-PCR (qRT-PCR) (Poulos et al 2008). Shrimp that had been sampled were not returned to the tank.…”