Comparison of PCR testing methods for white spot syndrome virus (WSSV) infections in penaeid shrimp

Sritunyalucksana, Kallaya; Srisala, Jiraporn; McColl, Kenneth A.; Nielsen, Linda Kærgaard; Flegel, Timothy W.

doi:10.1016/j.aquaculture.2005.12.002

Cited by 60 publications

(59 citation statements)

References 16 publications

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“…The TaqMan real-time PCR assay was carried out using TaqMan Universal PCR Master Mix (PE Applied Biosystems). A standard curve for WSSV was constructed as previously described (28). Quantitation of WSSV amplicons was accomplished by measuring the cycle threshold (C T ) value.…”

Section: Methodsmentioning

confidence: 99%

PmRab7 Is a VP28-Binding Protein Involved in White Spot Syndrome Virus Infection inShrimp

Sritunyalucksana

Wannapapho

et al. 2006

J Virol

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Our aim was to isolate and characterize white spot syndrome virus (WSSV)-binding proteins from shrimp. After a blot of shrimp hemocyte membrane proteins was overlaid with a recombinant WSSV envelope protein (rVP28), the reactive bands on the blot were detected using anti-VP28 antibody. Among three membraneassociated molecules identified by liquid chromatography-tandem mass spectrometry, there was a 25-kDa protein that bound to both rVP28 and WSSV. Since it had a primary structure with high homology to the small GTP-binding protein Rab7, we named it Penaeus monodon Rab7 (PmRab7). The full-length PmRab7 cDNA was obtained, and results from a glutathione S-transferase pull-down assay confirmed specific binding to rVP28. Reverse transcriptase PCR analysis revealed PmRab7 expression in many tissues, and real-time PCR analysis revealed that expression was constitutive. Binding of PmRab7 to rVP28 or WSSV occurred in a dose-dependent manner and was inhibited by anti-Rab7 antibody. In an in vivo neutralization assay, the number of dead shrimp after challenge with WSSV plus PmRab7 (15%) or WSSV plus anti-Rab7 antibody (5%) was significantly lower than after challenge with WSSV alone (95%). In contrast to the WSSV-injected group, shrimp injected with WSSV plus PmRab7 or WSSV plus anti-Rab7 showed no WSSV-type histopathology. We conclude that PmRab7 is involved in WSSV infection in shrimp. This is the first study to identify a shrimp protein that binds directly to a major viral envelope protein of WSSV.White spot syndrome virus (WSSV) is a viral pathogen that emerged in the early 1990s and has since spread throughout Asia and to the Americas. Diseased shrimp are lethargic and slow swimming and show reduced feed consumption. Histopathology has revealed that WSSV-infected shrimp tissues are of ectodermal and mesodermal origin (3,6,19,39). WSSV is an ellipsoid to bacilliform, enveloped particle of about 275 nm in length and 120 nm in width, with a tail-like appendage at one end. It is the type species of the genus Whispovirus in the family Nimaviridae (36). It is unique, with an infection strategy that does not match infection models of any other known virus, and must therefore be investigated ab initio.All three of the WSSV isolates that have been sequenced have a genome of about 300 kbp, and genetic comparisons have shown a high degree of genetic similarity (16). The availability of the complete WSSV sequence facilitates the global molecular characterization of the virus by genomic and proteomic approaches and has recently led to the discovery of many important WSSV genes, including latency-associated genes (10, 11), immediate-early genes (15), many other nonstructural genes (5,29,30,33), and more than 39 structural genes (6,13,19,31,32,35,43). To date, however, little is known of the interaction between shrimp and WSSV at the cellular and molecular levels.Neutralization experiments with a major WSSV envelope protein, VP28, have shown that it is involved in systemic infection of WSSV (34). It has further been shown that VP28 ...

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Section: Methodsmentioning

confidence: 99%

PmRab7 Is a VP28-Binding Protein Involved in White Spot Syndrome Virus Infection inShrimp

Sritunyalucksana

Wannapapho

et al. 2006

J Virol

Self Cite

236

145

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show abstract

“…Here, we have attempted Further, Rab proteins were identified and revealed their role during viral infection in several organisms. Similarly, PmRab7 might be receptor for VP28, which is reducing mortality of shrimp after WSSV challenge [24]. In earlier studies, it has also been shown that WSSV-binding activity with the expressed rPmRab7 where Rab7 plays a vital role in the trafficking of endocytosis vesicles within the cell, subsequent fusion of early endosome to late endosome and further in lysosome.…”

Section: Discussionmentioning

confidence: 96%

Revealing localization and regulation of GTPase PmRab7 in lymphoid cells of Penaeus monodon after WSSV infection

2016

J Coast Life Med

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“…Pada pengujian ini hanya menggunakan 2 standar kopi, tetapi untuk hasil yang lebih akurat sedikitnya harus menggunakan 3 standar kopi sehingga didapat nilai kurva standar yang lebih baik. Sebaiknya standar dibuat selalu baru setiap saat melakukan pengujian, standar pada pengujian dengan q-PCR ini sangat penting karena dapat mempengaruhi tingkat sensitivitas diagnosa itu sendiri (Sritunyalucksana et al, 2006). Hasil analisa kuantitatif dari beberapa sampel uji ikan mas ini dapat dilihat pada Tabel 2.…”

Section: Analisa Real-time Quantitative Pcr -Sybr Greenunclassified

APLIKASI KUANTIFIKASI KOI HERPESVIRUS : REAL TIME – QUANTITATIVE POLYMERASE CHAIN REACTION (RT-Q PCR) MENGGUNAKAN SYBR GREEN PADA IKAN MAS (Cyprinus carpio)

Koesharyani¹,

Gardenia

Mufidah

et al. 2017

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ABSTRAKKoi Herpes Virus (KHV) di Indonesia sejak tahun 2002 merupakan penyakit mematikan yang menyerang ikan koi Cyprinus carpio koi dan ikan mas Cyprinus carpio carpio, dan sampai saat ini, infeksi KHV dilaporkan sudah menyebar hampir di seluruh dunia. Untuk mengetahui adanya infeksi KHV perlu cara diagnosa yang sangat akurat/sensitif, sehingga keberadaan KHV dapat diketahui secara pasti dengan tingkat sensitivitas yang lebih baik pada ikan budidaya. Tujuan dari penelitian ini adalah untuk mengaplikasikan teknik deteksi dengan real time quantitative polymerase chain reaction (RT-qPCR/qPCR) guna mengetahui adanya infeksi KHV secara kuantitatif pada ikan mas dengan mengetahui kandungan virus (viral load). Sebanyak masing-masing 3 ekor sampel diperoleh dari sentra budidaya ikan mas di Cirata-Jawa Barat, Maninjau-Sumatera Barat, dan Banjarmasin-Kalimantan Selatan. Sampel-sampel tersebut selanjutnya dianalisa keberadaan KHV-nya dengan RT-qPCR menggunakan SYBR Green. Hasil pengujian menunjukkan bahwa jumlah tertinggi (viral load) diperoleh dari ikan mas asal Cirata-3 dengan nilai Threshold Cycle (Ct.) 18,24 atau setara dengan 3,4

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Comparison of PCR testing methods for white spot syndrome virus (WSSV) infections in penaeid shrimp

Cited by 60 publications

References 16 publications

PmRab7 Is a VP28-Binding Protein Involved in White Spot Syndrome Virus Infection inShrimp

PmRab7 Is a VP28-Binding Protein Involved in White Spot Syndrome Virus Infection inShrimp

Revealing localization and regulation of GTPase PmRab7 in lymphoid cells of Penaeus monodon after WSSV infection

APLIKASI KUANTIFIKASI KOI HERPESVIRUS : REAL TIME – QUANTITATIVE POLYMERASE CHAIN REACTION (RT-Q PCR) MENGGUNAKAN SYBR GREEN PADA IKAN MAS (Cyprinus carpio)

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