돌돔(Oplgnathus fasciatus)에서의 Megalocytivirus 감염에 대한 효과적 vaccine 개발

Abstract

어류에서 질병을 유발하는 megalocytivirus는 우리나라를 비롯한 세계 각지의 해산어 및 담수 양식 산업에서 매우 큰 경제적 손실을 일으키며, 특히 우리나라에서는 주로 돌돔 양식 산업에서 큰 피해를 일으키고 있다. 하지만 현재 돌돔에 대한 megalocytivirus에 대한 연구는 부족한 실정이며, 예방책으로 생각되는 백신개발 역시 아직 이루어지지 않은 실정이다. 따라서 본 연구는 돌돔에서의 megalocytivirus에 효과적인 백신 개발에 중점을 두고 연구를 수행하였다. 우선 기존의 megalocytivirus vaccine의 경우 돌돔에서 효과적인 vaccine이 전무한 실정이므로 본 연구에서는 megalocytivirus에 감수성이 높은 돌돔에 효과를 높이기 위해 기존의 vaccine보다 더 높은 항원성 부여 및 돌돔에 감염되는 subgroup 2 의 megalocytivirus를 이용해서 백신을 제작하였다. 이러한 고농도의 백신의 경우 본 연구실에서는 qPCR에 의하여 결정 된 항원량을 기준으로 조제한 vaccine이 나타내는 효과를 PMF cell에 대한 감염력을 표시하는 TCID50 titer와 직접적인 비교가 이루어 질 수 있게 하였으며 이를 바탕으로 IVS-1의 persistent한 감염을 통한 PI-PMF cell 의 상등액 (1010.1 copies/ml)을 이용하여 고농도의 백신을 보다 용이하게 제작하였다. 하지만 기존의 vaccine들에 비해 본 실험의 vaccine의 경우 고농도로 제작되어지는 만큼 고농도의 virus의 수급과 그에 따른 경제성의 문제등을 해결할 필요가 있었으며 그에 따른 해결방안으로 adjuvant 및 FKC를 활용한 Dual vaccine을 이용해 기존 고농도 vaccine의 효과향상을 확인하였다. 또한 megalocytivirus 감염 폐사어의 조직을 이용하여 백신을 제작하여 효과를 확인하였으며, 백신 접종 후 공격실험에서의 생존어를 대상으로 1x106 copies/fish 로 고농도 challenge한 결과 100% 생존을 확인하였다. 또한 기존의 생존어 group 과 함께 종류별 백신투여 2주 후 group들의 혈청을 이용하여 중화항체가를 확인한 결과 백신의 농도에 따른 항체가의 경우 뚜렷한 차이를 확인할 수 없었다. 다음으로는 이러한 백신을 온도에 따른 백신의 효과 및 virus의 활성에 따른 폐사율의 차이를 확인하기 위해 기존 실험 온도였던 25℃ vaccination 이 아닌 21℃로 3주간 vaccine을 투여한 후 공격실험을 하루에 1℃씩 25℃로 맞춰준 후 폐사를 확인한 결과 기존의 실험과 동일한 결과를 확인하였다. 다음으로는 25℃로 2주간 vaccine 투여 후 공격실험을 하루에 1℃씩 21℃로 맞춰준 후 36일 간 폐사를 확인한 결과의 경우 25℃ maintain 결과에 비해 최대 25% 의 폐사율 감소를 확인하였다. 이후 생존어를 대상으로 (21℃→25℃) shifting-up 실험과 21℃ maintain 실험을 실시하여 40일간 누적폐사율을 관찰한 결과 shifting-up의 경우 25℃ maintain 에 비해 약 10% 정도의 페사 증가를 확인하였으며, 21℃ maintatin 의 경우에는 추가 폐사없는 결과를 확인하였으며. 21℃ 로 공격실험 후 40일 경의 생존어와 80일경의 생존어, 두 생존어를 각 group 별로 3 마리 씩 무작위로 선별하여 1st PCR을 통해 amplicon band를 확인한 결과 40일 경에서는 양성, 80일경에서는 음성으로 확인되었다. 다음으로는 기존의 challenge 농도인 1x104 copies/fish 보다 낮은 농도의 challenge (1x101, 102 copies/fish)를 복강주사를 통해 농도에 따른 백신의 효과 차이를 확인하였으며 이는 기존의 폐사율과 비교했을 때 최대 70% 이상의 결과 값을 확인하였다. 마찬가지로 기존의 cohabitation 방법 보다 donor 노출기간을 줄인 short expose cohabitation을 이용한 폐사를 확인 한 결과 폐사의 경우 거의 0%에 달했으며 두 결과 모두 기존의 방법과 비교해 보았을 때 훨씬 백신효과가 뚜렷하게 나타나는 것을 확인해 보았다.

Megalocytivirus belongs to the Iridoviridae family and is the etiological agent of red sea bream iridovirus Disease (RSIVD)occurring in red sea bream (Pagrus major)and other fish species. We isolated a Megalocytivirus sachun-1 (IVS-1) strain in 2006 from rock bream suffering from RSIVD with high mortality rate. To culture IVS-1, the PMF cell line developed in our laboratory was inoculated with megalocytivirus and subjected to multiple subcultures to generate a persistently infected cell line (PI-PMF) with Megalocytivirus, The propagation of megalocytivirus in PI-PMF cells was consistent and reached 1010.1 copies/ml, in contrast to the GF cell line, in which the virus titer decreased gradually to zero. titers determined by Q-PCR were~10-fold higher than those by CPE observation in the PMF cell line, and did not differ between the PMF and PI-PMF cell lines. Additinally, the pathogenicity against rock bream did not differ between megalocytivirus produced using PMF and PI-PMF cells. The higher concentration of megalocytivirus in the supernatant of PI-PMF cells compared to PMF cells facilitated development of a high-dose megalocytivirus vaccine that may protect rock bream against infection. Formalin-inactivated iridovirus vaccine was prepared using PI-PMF cell culture supernatant by treatment of 0.1% formalin for 7days at 4℃ without dilution (high-supernatant of cultured megalocytivirus, HSCMV). Rock bream vaccinated by intraperitoneal injection of 0.1 ml HSCMV showed <50% cumulative mortality, compared to 100% cumulative mortality in the control group. 10-fold and 100-fold dillution of HSCMV yielded SCMV ( supernatant of cultured megalocytivirus) and LSCMV (low-supernatant of cultured megalocytivirus) Vaccination with SCMV and LSCMV resulted in 60% and 70% cumulative mortality rates, respectively, suggestive of a dose-dependent effect. The effects on vaccine efficacy of several adjuvants were evaluated; squalene+aluminum hydroxide with HSCMV decreased the cumulative mortlity rate of vaccinated rock bream to 30%~20% higher than HSCMV vaccination. Water temperature influences the onset and severity of virus infections of fish by affecting viral replication and augmenting the efficacy. To assess the influence of shifts in rearing water temperature, fish vaccinated at 21℃ for 3 weeks were inoculated at 25℃ with IVS using the cohabitation method-rearing temperature was increased by ~1℃ per day. The vaccinated group showed a similar or slightly lower mortality rate (depending on the vaccine formulation) compared to the group vaccinated and inoculated at 25℃. moreover fish vaccinated at 25℃ for 2 weeks were challenged with IVS by cohabiattion at 21℃, and showed <25% cumulative mortality rates after 36 days of observation before water temperature was returned to 25℃, with the exception of one group vaccinated with SCMV (40%). Fish vaccinated at 25℃ and inoculated with IVS at 21℃ were then reared at 25℃. Immediately after temperature shifting-up, fish in the vaccinated group began to die at a similar or higher rate than that of fish maintatined at 25℃. The RSIV genome was detected by primary PCR due to the low level of replication of RSIV in fish collected immediately prior to increased the water temperature to 25℃ (38 days after decreasing the water temperature to 21℃). The stress induced by a decrease in water temperature would enhance virus replication in experimnetal fish compared to fish vaccinated and challenged at 25℃ without a change in water temperature. However, these RSIV genome levels in fish shifted to water at 21℃ . may not result in disease and the virus was enetually cleared, as evidenced by >90% survival and negative PCR results after 80 days of exposure to 21℃ water. Thus, rock bream vaccinated at 25℃, with HSCMV without adjuvant were protected against megalocytivirus infection at 21℃. Furthermore, a high proportion of vaccinated rock bream survived after RSIV infection irrespective of rearing temperature and were protected against re-challenge with homologous RSIV (similar to the results of red sea bream). Serum neutralizing antibody levels in vaccinated fish did not differ significantly among the various vaccine formulations , but exhibited marked increases in vaccinated fish that survived viral challenge. This suggested the importance of T cell activity for the protection of virus infection. In this study, megalocytivirus challenge of vaccinated fish was performed by I.p injection of 104 viral particles/fish; this dose is 100-fold higher than that required to induce 100% mortality in rock bream. Additionally, in a cohabitation challenge experiment, the megalocytivirus dose administered to donor rock bream was sufficient to induce 100% mortality in the control group within 7 days after exposure. Use of challenge doses higher than those occuring under natural condition may result in a lesser protective effect than would use of lower doses. As low dose of megalocytivirus, we used 102 viral particles/fish for I.p injection or decresed the exposure time from 7-9 days to 5 days for cohabitation inoculation. Use of these low dose and decreaseed exposer time decreased the cumulative mortality rates to almost zero. Therefore, field tests of the megalocytivirus vaccine developed in this study against rock bream should involve use of lower viral doses to better simulate natural conditions. Further study to develop a lower-cost megalocytivirus vaccine by enhancing the virus yield in cell culture and a mass production system is warranted.