[논문]구제역바이러스의 숙주 특이성 결정에 연관되어있는 구제역바이러스 cis-acting replication element 변이 분석 연구

강효린; 성미소; 구복경; 정재훈

doi:10.5352/jls.2020.30.11.947

구제역바이러스의 숙주 특이성 결정에 연관되어있는 구제역바이러스 cis-acting replication element 변이 분석 연구
Cis-acting Replication Element Variation of the Foot-and-mouth Disease Virus is Associated with the Determination of Host Susceptibility 원문보기

생명과학회지 = Journal of life science, v.30 no.11, 2020년, pp.947 - 955

강효린 (부산대학교 자연과학대학 분자생물학과) , 성미소 (부산대학교 자연과학대학 분자생물학과) , 구복경 (농림축산검역본부) , 정재훈 (부산대학교 자연과학대학 분자생물학과)

초록
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구제역바이러스(FMDV)는 피코나바이러스 과에 속하는 바이러스로서 야생과 가축화된 소와 돼지에 감염된다. FMDV는 제어되기 어려워서 가축의 생산과 국제통상에 큰 장애가 되고 있다. FMDV RNA 게놈의 복제 과정에서 3D 중합효소가 특이적인 복제 기능을 담당하는데 게놈에 결합하는 부위가 매우 중요하다. 이 사실은 FMDV 게놈의 비코딩영역 내에서 3D 중합효소에 의해 인지되는 특이 RNA 구조가 관여함을 제시한다. 이 과정에서 cis-acting replication element (CRE)는 RNA 복제를 위한 개시에 필요하다. FMDV CRE는 15-17 뉴클레오티드의 고리와 이를 지지하는 이중가닥으로 형성된 줄기-고리 모양을 가지며 이들을 구성하는 RNA 뉴클레오티드 서열의 차이가 다른 RNA 이차구조를 생성한다. CRE 이외에 FMDV 복제를 위해서 많은 바이러스와 세포 인자들이 단백질-단백질 결합과 단백질-RNA 결합을 통해 협조적인 네트워크를 만들어낸다. 이 연구에서 국내에서 2010년부터 2017년 까지 구제역이 발생한 소와 돼지에서 FMDV를 분리하여 CRE 서열을 분석하였으며 이들 FMDV들은 A형과 O형의 유전자형을 가졌다. 흥미롭게 국내 FMDV들의 CRE RNA 이차구조의 변이들은 바이러스 간의 계통유전학적 상관관련성과 일치하며 특정 숙주 동물종의 FMDV 감염의 특이성을 밝혀주었다. 이를 토대로 국내 FMDV의 각 유전군의 분류는 독특한 기능적 CRE에 의해 특징지을 수 있으며 새로운 유전적 계통의 진화학적 연속성은 특징있는 CRE 모티프의 구분과 연관지을 수 있다. 그러므로 CRE의 특이적 RNA 구조는 숙주 동물종 의존적인 FMDV 부류의 부가적인 단서로 활용할 수 있음을 제안한다. 이들 연구 결과들은 향후 FMDV 대량감염이 발생할 때 숙주동물종의 특이성을 CRE 서열로 조기에 정확히 분석하는데 도움이 될 것이다.

Abstract ▼ AI-Helper

The foot-and-mouth disease virus (FMDV), a member of the Aphthovirus genus in the Picornaviridae family, affects wild and domesticated ruminants and pigs. During replication of the FMDV RNA (ribonucleic acid) genome, FMDV-encoding RNA polymerase 3D acts in a highly location-specific manner. This suggests that specific RNA structures recognized by 3D polymerase within non-coding regions of the FMDV genome assist with binding during replication. One such region is the cis-acting replication element (CRE), which functions as a template for RNA replication. The FMDV CRE adopts a stem-loop conformation with an extended duplex stem, supporting a novel 15-17 nucleotide loop that derives stability from base-stacking interactions, with the exact RNA nucleotide sequence of the CRE producing different RNA secondary structures. Here, we show that CRE sequences of FMDVs isolated in Korea from 2010 to 2017 exhibit A and O genotypes. Interestingly, variations in the RNA secondary structure of the Korean FMDVs are consistent with the phylogenetic relationships between these viruses and reveal the specificity of FMDV infections for particular host species. Therefore, we conclude that each genetic clade of Korean FMDV is characterized by a unique functional CRE and that the evolutionary success of new genetic lineages may be associated with the invention of a novel CRE motif. Therefore, we propose that the specific RNA structure of a CRE is an additional criterion for FMDV classification dependent on the host species. These findings will help correctly analyze CRE sequences and indicate the specificity of host species for future FMDV epidemics.

주제어

표/그림 (5)

표 Table 1. Foot-and-Mouth Disease Viruses used in this study
그림 Fig. 1. Sequence alignment and nucleotide variation of FMDV CRE regions. (A) Nucleotide variations in the 55 nucleotides of the FMDV CRE. The FMDV strains were isolated in Korea from 2010 to 2017. (B) Summary of the variations at each 55 nucleotide position.
그림 Fig. 2. Sequence alignment and nucleotide variation of the FMDV CRE region classified by O/A serotypes and host species. (A) Nine virus strains had expected nucleotide sequences of the O serotype. (B) Two strains (A/PC/SKR/2010/C and A/YC/ SKR/2017/C) had expected nucleotide sequences of the A serotype. (C) Seven FMDV strains were isolated from cows. (D) Four FMDV strains from 2014 and 2016 were isolated from pigs.
그림 Fig. 3. Maximum Likelihood tree showing phylogenetic relationships of FMDV isolates based on the complete genomic sequence (A) and CRE region sequence (B). Three trees were rooted using the FMDV type O strain (O/AD/SKR/2010/C). Bootstrap support values above 70% out of 1,000 replicates are shown near the major nodes. Horizontal branch lengths are drawn to scale.
그림 Fig. 4. RNA secondary structures of individual FMDV strains depend on the host species infected. The secondary structure of the CRE RNA of FMDVs was determined using the Fold Web Server with the lowest free energy calculation. The blue-colored box indicates relatively variable regions of RNA secondary structures of FMDV CRE.

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가설 설정

Since 2010, FMDV epidemics in Korea seem to be sensitive and specific to one type of host. In this study, we determined that the secondary structure of the FMDV CRE plays a role in determining the host specificity of an infection. These results, thus, assist in shedding light on FMDV evolution and host adaptation.

제안 방법

Here, we provide novel results regarding the epidemiological trends of FMDV outbreaks in Korea over a recent 10-year period, and provide viral determinants for host susceptibility. The endemic viruses from clinically infected animals showed different genetic variations dependent on the number of endemic years and the host species.
The A²³A²⁴ A²⁵C²⁶A²⁷ motif in the CRE (Domain I) is essential for virus replication, and VPg uridylylation [17] was fully conserved amongst all examined strains. Since the CRE region is essential for FMDV replication, we focused on sequence variations within the CRE region and identified multiple nucleotide differences between FMDV isolates. We converted these RNA sequences into secondary RNA structures using the Fold Web Server (http://rna.
The entire genome was amplified using AccuPower® ProFi Taq PCR PreMix (BIONEER, Korea), according to the manufacturer’s instructions, with nine overlapping pairs of FMDV-specific primers.

대상 데이터

Clinical isolates were collected from suspected FMDV outbreaks in Korea from 2010 to 2017 (Table 1). These isolates were classified as either A or O.

이론/모형

Evolutionary distances were calculated using Kimura’s two parameter model.
The tree was rooted using the FMDV serotype O strains [O1/ Kaufbeuren (GenBank accession no: X00871), O1/Campos (AJ320488) and O2/Brescia (M55287)] as outgroups. Percentage similarities/differences were estimated using the Meg Align program from the Lasergene package (DNASTAR Inc., USA).

성능/효과

(A) Nine virus strains had expected nucleotide sequences of the O serotype. (B) Two strains (A/PC/SKR/2010/C and A/YC/ SKR/2017/C) had expected nucleotide sequences of the A serotype. (C) Seven FMDV strains were isolated from cows.
2A and 2B). Comparisons of the CRE variation between host species indicated that CRE sequences from pig isolates of FMDVs were extremely similar (Fig. 2D), while CREs from cow isolates of FMDVs showed large variations in divergence (Fig. 2C). Interestingly, comparisons of nucleotide sequences of FMDV isolates from cows, A/PC/SKR/2010/C, and two O serotypes from 2017 showed similar CRE variation patterns regardless of serotype.

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