Ya-Li Hsu

Development of an in vitro subculture system for the oka organ (Lymphoid tissue) of Penaeus monodon

Abstract We tried to establish a subculture system for cells from the Oka organ (lymphoid tissue) of the grass prawn Penaeus monodon . The basic culture medium was tested for osmolality, serum concentration, serum sources and pH. It was found that Leibovitzs L-15 medium supplemented with 10% fetal bovine serum, 5 g l −1 NaCl, pH 7.63–8.1, with final osmolality at 470–500 mmol kg −1 allowed for enhanced cell attachment and growth; however, cells could not be maintained for more than 5 days. The supplementary nutrients were also tested for carbohydrates, amino acids, L-ascorbic acid, Buffalo rat liver(BRL) -condition medium and selenium. The basic culture medium + l g l −1 glucose were found to enhance cell attachment and growth. Our collected lymphoid cells required 3 days of incubation to obtain 80% confluency; however, cells did not grow in subcultures. Several growth factors were tested for developing a subculture system of shrimp cells. Epidermal growth factor (EGF) or transforming growth factor β (TGF β) did not foster cell growth. Cells treated with insulin or insulin-like growth factor I (IGF I) were capable of being subcultured, but resultant cells differed in terms of feeder layer, and were eventually discarded due to yeast contamination. Cells treated with basic fibroblast growth factor (bFGF) 20 ng ml −1 (F-20) could be subcultured for more than 90 passages without a feeder layer. Some F-20-treated cells were capable of extending their extracellular matrices for cell attachment and piled up; some of them became suspended and lost their anchorage-dependent and contact inhibition properties.

Dual challenges of infectious pancreatic necrosis virus and Vibrio carchariae in the grouper, Epinephelus sp.

The grouper industry in Taiwan faces serious threats from various disease problems. The present study investigated dual challenges with infectious pancreatic necrosis virus (IPNV) and Vibrio carchariae in the grouper (Epinephelus sp.). The fish were infected with IPNV for 2 weeks prior to a secondary infection with the bacteria, or vice versa, by either immersion (10(3)-10(4) TCID50 IPNV per ml, 10(6)-10(7) colony forming units (CFU) Vibrio per ml) or by intraperitoneal injection (10(3)-10(4) TCID50 IPNV per g fish or 10(7) CFU Vibrio/g fish) challenges. Mass mortalities occurred in fish infected with IPNV for 2 weeks prior to the infection with the bacteria, or vice versa, in either immersion or intraperitoneal injection challenges. The bacterium could only survive in seawater or brackish water similar to that of cultured groupers.

Molecular relationships in infectious pancreatic necrosis virus

Infectious pancreatic necrosis virus (IPNV), a Birnaviridae with a double-stranded RNA genome of two segments, is an important aquatic pathogen. Previous characterizations of the RNA and polypeptide patterns of different IPNV isolates have uncovered a wide variety of electropherotypes. We used RNA fingerprinting analysis to study the quasispecies, heterogeneity, and rapid mutation characteristics of IPN viruses. Via cluster analysis of the RNA fingerprints, IPNV standard serotypes as well as T42G-, T24K- and T34G-related isolates were classified into 3 clusters corresponding to the AB, SP, and VR-299 serological types. The IPNV SP cluster represents a different evolutionary route from those of the AB and VR-299 clusters. The VR-299 clusters were separated into two groups, the T34G group were divided into two subgroups. The molecular relationships between these isolates can be correlated with the biological characteristics of these IPN viruses, for example, selective growth in EPC cells and adaptive replication at high temperatures.

Involvement of serine proteinase in infectious pancreatic necrosis virus capsid protein maturation and NS proteinase cleavage in CHSE-214 cells

An investigation of virus-specific protein maturation in infectious pancreatic necrosis virus (IPNV) infected Chinook salmon embryo cells (CHSE-214) was undertaken. The precursor protein (pVP2-1) of the major mature capsid protein (VP2) was processed sequentially from pVP2-1 to pVP2-2 and VP2. Experiments using serine proteinase inhibitors showed that the maturation of the VP2 was blocked in the pVP2-1 post-translational cleavage steps. A protinin, a potent proteinase inhibitor, at 800 μg ml(-1) blocked pVP2-2 to VP2 and the cleavage of VP4 (28 kDa) to VP4-1 (25 kDa). Therefore, our data showed that the maturation of the capsid protein (VP2) and cleavage of VP4 (NS proteinase) can be blocked by serine proteinase inhibitors.

Comparison of RNAs and Polypeptides of Infectious Pancreatic Necrosis Virus Isolates from Eel and Rainbow Trout

Thirteen isolates of infectious pancreatic necrosis virus (IPNV) from eel and rainbow trout in Taiwan were compared with the selected serotypes AB, SP and VR-299 by PAGE analysis of their RNA genomes and early polypeptides. All the IPNV isolates from eels (1984 to 1986) from Lu Kang and Ping Tung, and from rainbow trout eggs and fry from Dan Sway (1985 to 1986) were most closely related to the AB serotype. However four rainbow trout isolates from Dan Sway (1984) had a similar RNA pattern to that of VR-299, whereas their early polypeptides were different, showing evidence of some mutations. Both RNA and polypeptide PAGE patterns were used to distinguish the isolates from known selected IPNV strains showing that this approach can be used for epizootiological studies.

Inhibition of Aquatic Birnavirus Replication Process in Fish Cells by Viral Antisense RNA

In order to study the inhibitory effect of viral antisense RNAs upon the replication of infectious pancreatic necrosis virus (IPNV) in fish cells. We used three fragments of IPNV-E1S virus cDNA, isolated from cloned pA8, pA28 and pB19 for this purpose. Fragment A28(nt 1346–1764 of segment A) and A8(nt 2040–2350 of segment A), encode the junction between the virus protein VP2 and VP4, and the junction between VP4 and VP3, respectively. Fragment B 19(nt 515–915 of segment B) encodes the partial VP1. The three fragments were constructed to the pRSVneo vector in antisense orientation. The resulting expression recombinants pA8(AS), pA28(AS) and pB19(AS) were lipofected into CHSE- 214 cells. After 24 hr transfection, the cells were tested for virus protecting effect. At M.O.I. 10, the titers of released virion from pA8(AS), pA28(AS) and pB19(AS) transfected cells decreased from 1010 TCID50 /ml to 4.65×107, 4.65×106, 2.15×106 TCID50/ml, respectively. The virus protein synthesis and the level of [α-32P]UTP incorporation rate in antisense cDNA transfected cells was also reduced to 18-24% of control cells. These results indicated that expression of the virus antisense RNA in cells can protect them from virus infection.