Toll-like receptors and interferon associated immune factors in viral haemorrhagic septicaemia virus-infected olive flounder (Paralichthys olivaceus)

Avunje, S.,Kim, W.S.,Park, C.S.,Oh, M.J.,Jung, S.J. Academic Press 2011 Fish & shellfish immunology Vol.31 No.3

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play pivotal role in antiviral innate immunity of a host. The present in-vivo experiment was conducted to investigate the role of these innate immune factors in early phase as well as during recovery of viral haemorrhagic septicaemia virus (VHSV) infection by quantitative real-time reverse transcriptase polymerase chain reaction. A less lethal VHSV infection was generated in olive flounder (Paralichthys olivaceus) and was sampled at 3, 6, and 12h post infection (hpi), and 1, 2, 4, and 7 days post infection (dpi). At 3 hpi, the VHSV N gene was detected in three out of five fish and all five fish showed a relative fold increase of TLR 2, TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7, and ISG 15. Viral copies rapidly increased at 12 hpi then remained high until 2 dpi. When viral copy numbers were high, a higher expression of immune genes IL 1β, IRF 3, IRF 7, Type I IFN, ISG 15 and Mx was observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immune response was considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. A fish with ascites sampled at 7 dpi displayed high viral copy but under-expressed immune genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against VHSV.

Temperature-dependent viral replication and antiviral apoptotic response in viral haemorrhagic septicaemia virus (VHSV)-infected olive flounder (Paralichthys olivaceus)

Avunje, S.,Kim, W.S.,Oh, M.J.,Choi, I.,Jung, S.J. Academic Press 2012 FISH AND SHELLFISH IMMUNOLOGY Vol.32 No.6

The olive flounder (Paralichthys olivaceus) shows a high rate of mortality to viral haemorrhagic septicaemia virus (VHSV) in the winter and spring but has zero mortality over 20 <SUP>o</SUP>C. In this experiment, we studied the effect of rearing temperature on viral replication, viral transcription and antiviral apoptotic immune response in VHSV-infected olive flounder by real-time polymerase chain reaction. Olive flounder were given intra-peritoneal injections of VHSV (10<SUP>7.8</SUP> TCID<SUB>50</SUB>/ml) and were reared at 15 <SUP>o</SUP>C or 20 <SUP>o</SUP>C. Five fish were randomly sampled for head kidney at 3, 6 and 12 h post-infection (hpi) and 1, 2, 4 and 7 days post-infection (dpi). Total RNA extracted from the tissue was reverse transcribed and used as template for real-time PCR. In the 15 <SUP>o</SUP>C group, the number of viral gRNA copies peaked after 2 dpi and remained high through 7 dpi, while in the 20 <SUP>o</SUP>C group, the copy number was at the highest at 1 dpi but drastically declined at later stages. Viral mRNA levels in the 15 <SUP>o</SUP>C group gradually increased starting at 3 hpi to reach their maximum value at 12 hpi and remained high until 2 dpi, whereas the other group showed much lower copy numbers that were undetectably low at 4 and 7 dpi. Type II IFN expression increased as the viral copies increased and the 20 <SUP>o</SUP>C group showed quicker and stronger expression than the 15 <SUP>o</SUP>C group. The MHC class I and CD8 expression was high in both the groups at early stage of infection (3-6 hpi) but at later stages (2-7 dpi) in 15 <SUP>o</SUP>C group expression reduced below control levels, while they expressed higher to control in 20 <SUP>o</SUP>C group. The expression of granzyme in 15 <SUP>o</SUP>C fish showed a single peak at 2 dpi, but was consistently expressing in 20 <SUP>o</SUP>C fish. Individuals expressed very high levels of perforin expressed very high levels of caspase 3. In 15 <SUP>o</SUP>C fish, TNFα, FasL and p53 expressed significantly higher than 20 <SUP>o</SUP>C only at initial stages of infection (3-6 hpi). Caspase 3 expression found to be low in 15 <SUP>o</SUP>C fish whereas it was significantly elevated in 20 <SUP>o</SUP>C group. Interestingly individual fish with high caspase 3 expression contained very low viral RNA. Thus, from our experiment, we can conclude that an effective apoptotic immune response in VHSV-infected olive flounder plays a crucial role in the survival of the host at higher temperatures.

Impaired TLR2 and TLR7 response in olive flounder infected with viral haemorrhagic septicaemia virus at host susceptible 15 ^oC but high at non-susceptible 20 ^oC

Avunje, S.,Oh, M.J.,Jung, S.J. Academic Press 2013 Fish & shellfish immunology Vol.34 No.5

The olive flounder (Paralichthys olivaceus) is susceptible to viral haemorrhagic septicaemia virus (VHSV) at 15 <SUP>o</SUP>C but no mortality is observed at 20 <SUP>o</SUP>C even though the virus can grow profusely in vitro. Thus, we designed an experiment to better understand the immune response of olive flounder to VHSV when the host reared at 15 <SUP>o</SUP>C or 20 <SUP>o</SUP>C and infected with the virus. Olive flounder (18-22 g) reared at 15 +/- 0.5 <SUP>o</SUP>C or 20 +/- 0.5 <SUP>o</SUP>C were intra-peritoneally injected with VHSV (10<SUP>7.8</SUP> TCID<SUB>50</SUB>/fish) and sampled (n = 5) for head kidney at 3, 6, 12 hpi, 1, 2, 4 and 7 dpi; similarly, mock injected control groups (n = 5). Real-time PCR-based absolute quantification method was followed to quantify copies of VHSV gRNA and mRNA, while the immune gene expression of the olive flounder was quantified relative to internal control, β-actin. Viral infection resulted in a cumulative mortality of 24% in olive flounder reared at 15 <SUP>o</SUP>C, but no mortality was recorded in the 20 <SUP>o</SUP>C group or control groups. TLR2 and TLR7 expression at 15 <SUP>o</SUP>C was enhanced during early-infection phase (3-6 hpi) and recovery phase (4-7 dpi) when viral transcription was low, but expression was significantly reduced (12 hpi-1 dpi) at peak-infection period. However, the 20 <SUP>o</SUP>C group showed low viral transcription and expressed high level of TLR7 and a moderately higher unchanged level of TLR2. In both the groups, TLR3 expression was unaffected. Nevertheless, expression of MDA5 and LGP2 increased significantly irrespective of rearing temperature at the time of peak infection, hence at 15 <SUP>o</SUP>C VHSV down-regulated expression of TLR2 and TLR7 but not MDA5 or LGP2. Comparatively, at 15 <SUP>o</SUP>C IRF3 expressed high but IRF7 remained very low. Interleukins (IL-1β, IL-6 and IL-8) were significantly elevated in both the groups, but quicker and for a shorter period at 20 <SUP>o</SUP>C. In the 15 <SUP>o</SUP>C group, an extended period of expression of ILs could create an unsafe prolonged inflammatory condition. The olive flounders expressed high ISGs at 15 <SUP>o</SUP>C but were lagging by 12 h than 20 <SUP>o</SUP>C group. Based on these findings, we concluded that viral-mediated disruption of TLR2 and TLR7 expression in the 15 <SUP>o</SUP>C group could have delayed the host interferon response and provided a window for high viral growth. However, an effective host immune response at 20 <SUP>o</SUP>C contained VHSV from reaching the critical limit.

Efficacy of Solanum nigrum L extract for Viral hemorrhagic septicemia virus (VHSV) replication in fathead minnow cell line and Paralichthysolivaceus

Han-Kook Yang,Satheesha Avunje,Young Bae Ryu,Sung-Ju Jung 한국수산과학회 양식분과 2014 한국수산과학회 양식분과 학술대회 Vol.2014 No.7

기후변화에 기인한 연안 수온상승에 따른 연안 어류의 바이러스성 질병 발생 예측

김위식 ( Wi Sik Kim ),김석렬 ( Seok Ryel Kim ),박명애 ( Myoung Ae Park ),이준수 ( Joon Soo Lee ),사티샤에분제 ( Satheesha Avunje ),김도형 ( Do Hyung Kim ),오명주 ( Myung Joo Oh ) 한국수산과학회 2013 한국수산과학회지 Vol.46 No.5

The impact of global warming on aquatic systems has been a priority research area in the past decade. However, the possibility that increased temperatures will cause shifts in viral disease outbreaks has not been well addressed. In the present study, with increasing water temperature (WT) in the coastal area of Korea, we estimated the possibility of changes in fish viral diseases. From the present time, WT may rise between 0.62 and 1.7 ℃ by 2050, and the effect on aquaculture could be more adverse than benefitial. Red seabream iridovirus disease (RSIVD) and viral nervous necrosis (VNN) cause high mortality above 22 and 24 ℃, respectively, and outbreaks could commence earlier and persist for prolonged periods. Nevertheless, the period of occurrence of viral hemorrhagic septicemia (VHS), which outbreaks at a lower WT (<18℃), could be shorter than the current infectious period. Thermal stress in fish causes reductions in growth and immunocompetence; thus, increases in summer WT can lead to the development of new viral diseases. WT has a strong influence on fish population dynamics; therefore, entry of new viruses and changes in the prevalence of infection can be expected if carrier fishes are introduced or migrate to Korean waters.