VER-155008 induced Hsp70 proteins expression in fish cell cultures while impeding replication of two RNA viruses.

The heat-shock protein 70 (Hsp70) inhibitor, VER-155008 (VER), was explored as a potential antiviral agent for two RNA viruses important to fish aquaculture, viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Studies were done at a temperature of 14 °C, and with cell lines commonly used to propagate these viruses. These were respectively EPC from fathead minnow for VHSV and CHSE-214 from Chinook salmon embryo for IPNV. Additionally, both viruses were studied with the Atlantic salmon heart endothelial cell line ASHe. For both VHSV and IPNV, 25 µM VER impeded replication. This was evidenced by delays in the development of cytopathic effect (CPE) and the expression of viral proteins, N for VHSV and VP2 for IPNV, and by less production of viral RNA and of viral titre. As VER inhibits the activity of Hsp70 family members, these results suggest that VHSV and IPNV utilize one or more Hsp70s in their life cycles. Yet neither virus induced Hsp70. Surprisingly VER alone induced Hsp70, but whether this induction modulated VER's antiviral effects is unknown. Exploring this apparent paradox in the future should improve the usefulness of VER as an antiviral agent.

Fish viruses stored in RNAlater can remain infectious and even be temporarily protected from inactivation by heat or by tissue homogenates.

RNAlater is a commonly used transport and storage solution for samples collected for fish health investigations, particularly those potentially involving viruses. However, the infectivity of fish viruses after storage in RNAlater have not been determined. Nevertheless, knowledge of pathogen infectivity of preserved samples is crucial for ensuring safe transport and storage protocols. Therefore, the infectivity of three fish RNA viruses in RNAlater was examined at four temperatures: -80 °C, 4 °C, room temperature (RT, approximately 22 °C) and 37 °C. The viruses were viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV) and chum salmon reovirus (CSV). Overall, three consistent outcomes were observed. First, all three viruses remained infectious in RNAlater at RT or lower. High log titres of these viruses remained over 30 d of storage in either RNAlater or PBS. Second, RNAlater delayed the thermal inactivation of these viruses when compared to PBS at 37 °C. For VHSV, the titre remained high in RNAlater after one day of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For IPNV, the titre remained high in RNAlater after 30 d of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For CSV, the titre was slightly higher in RNAlater than PBS at 37 °C over 7 d, and by day 30, only samples stored in RNAlater proved infectious at titres above the detection threshold. Third, RNAlater delayed the inactivation of these viruses when they were stored together with head kidney homogenates. For VHSV, infectious virus was recovered from samples stored at 4 °C in RNAlater by day 7 of incubation, whereas it was inactivated to below threshold in PBS over the same period. For both IPNV and CSV, infectious virus was recovered from samples stored at 37 °C in RNAlater for 7 d, but not so in PBS. In summary, fish viruses can remain infectious and are even temporarily protected from inactivation while in RNAlater. This makes RNAlater a potentially useful solution for the transport of fish viruses. At the same time, precautionary measures must be taken when transporting potentially infectious samples in RNAlater.

Influence of chronic low-dose/dose-rate high-LET irradiation from radium-226 in a human colorectal carcinoma cell line.

PURPOSE: To evaluate potential damages of chronic environmentally relevant low-dose/dose-rate high-LET irradiation from a naturally occurring alpha-emitting radionuclide (radium-226, 226Ra) on a human colorectal carcinoma HCT116 p53+/+ cell line. METHODS: Clonogenic survival assays and mitochondrial membrane potential (MMP) measurement with a sensitive fluorescent MMP probe JC-1 were performed in HCT116 p53+/+ cells chronically exposure to low doses/dose rates of 226Ra with high-LET. Comparisons were made with the human non-transformed keratinocyte HaCaT cell line and acute low-dose direct low-LET gamma radiation. RESULTS AND CONCLUSION: The chronic low-dose/dose-rate alpha radiation (CLD/DRAR) did not reduce the clonogenic survival of HCT116 p53+/+ cells over the period of 70 days of exposure. Only one significant reduction in the HCT116 p53+/+ cells' clonogenic survival was when cells were grown with 10,000mBq/mL 226Ra for 40 days and progeny cells were clonogenically assessed in the presence of 10,000mBq/mL 226Ra. The cumulative doses that cells received during this period ranged from 0.05 to 46.2mGy. The mitochondrial membrane potential (MMP) dropped initially in both HCT116 p53+/+ and HaCaT cells in response to CLD/DRAR. The MMP in HCT116 p53+/+ cells recovered more quickly at all dose points than and that in HaCaT cells until the end of the exposure period. The highest dose rate of 0.66mGy/day depolarized the HaCaT's mitochondria more consistently during the exposure period. The faster recovery status of the MMP in HCT116 p53+/+ cells than that in HaCaT cells was also observed after exposure to acute low-dose gamma rays. Overall, it was found that CLD/DRAR had little impact on the MMP of human colorectal cancer and keratinocyte cell lines.

Characterizing responses to gamma radiation by a highly clonogenic fish brain endothelial cell line.

PURPOSE: The clonogenic property and radiobiological responses of a fish brain endothelial cell line, eelB, derived from the American eel were studied. METHODS: Clonogenic assays were performed to determine the plating efficiency of the eelB cells and to evaluate the clonogenic survival fractions after direct irradiation to low-dose low-LET gamma radiation or receiving irradiated cell conditioned medium in the bystander effect experiments. RESULT: eelB had the second highest plating efficiency ever reported to date for fish cell lines. Large eelB macroscopic colonies could be formed in a short period of time and were easy to identify and count. Unlike with other fish clonogenic cell lines, which had a relatively slow proliferation profile, clonogenic assays with the eelB cells could be completed as early as 12 days in culture. After direct irradiation with gamma rays at low doses ranging from 0.1Gy to 5Gy, the dose-clonogenic survival curve of the eelB cell line showed a linear trend and did not develop a shoulder region. A classical radio-adaptive response was not induced with the clonogenic survival endpoint when the priming dose (0.1 or 0.5Gy) was delivered 6h before the challenge dose (3 or 5Gy). However, a radio-adaptive response was observed in progeny cells that survived 5Gy and developed lethal mutations. eelB appeared to lack the ability to produce damaging radiation-induced bystander signals on both eelB and HaCaT recipient cells. CONCLUSION: eelB cell line could be a very useful cell model in the study of radiation impacts on the aquatic health.