Dong Van Quyen

Molecular detection and phylogenetic analysis of Anaplasma bovis from Haemaphysalis longicornis feeding on grazing cattle in Korea

Ticks are vectors of various pathogens that affect humans and animals throughout the world. Anaplasma bovis is one of the most important tick-borne pathogens that cause cattle diseases but there is still very little information available about this agent in Korea. In the present study, 535 Haemaphysalis longicornis tick pools were analyzed from grazing cattle in five Korean provinces. A. bovis was detected in 50 (9.3%) of 535 tick pools using 16S rRNA-based PCR. A. bovis infections were detected for the first time in ticks feeding on cattle in Chungbuk, Geongbuk, and Jeonbuk provinces in Korea. The 50 positive PCR products were sequenced successfully and compared with sequences in GenBank. Phylogenetic analysis of the Korean isolates classified them into four genotypes with nucleotide sequence identities of 99.4-100%. Two of the four genotypes had high similarity (99.8-100%) with known sequences. The other two genotypes have never been identified.

Phylogenetic analysis of black queen cell virus genotypes in South Korea.

The black queen cell virus (BQCV), a picorna-like honeybee virus, was first isolated from queen larvae and pupae of honeybees found dead in their cells. BQCV is the most common cause of death in queen larvae. Phylogenetic analysis of two Apis cerana and three Apis mellifera BQCV genotypes collected from honeybee colonies in different regions of South Korea, central European BQCV genotypes, and a South African BQCV reference genotype was performed on a partial helicase enzyme coding region (ORF1) and a partial structural polypeptide coding region (ORF2). The phylogeny based on the ORF2 region showed clustering of all the Korean genotypes corresponding to their geographic origin, with the exception of Korean Am str3 which showed more similarity to the central European and the South African reference genotype. However, the ORF1-based tree exhibited a different distribution of the Korean strains, in which A. cerana isolates formed one cluster and all A. mellifera isolates formed a separate cluster. The RT-PCR assay described in this study is a sensitive and reliable method for the detection and classification of BQCV strains from various regions of Korea. BQCV infection is present in both A. cerana and A. mellifera colonies. With this in mind, the present study examined the transmission of honeybee BQCV infections between A. cerana and A. mellifera.

Seroprevalence of Toxoplasma gondii and Trichinella spiralis infections in wild boars (Sus scrofa) in Korea.

Toxoplasma gondii and Trichinella spiralis are important zoonotic pathogens with worldwide distributions. In Korea, several outbreaks of human toxoplasmosis and trichinellosis due to the consumption of infected wild animals have been reported. The purpose of this study was to determine the seroprevalence of T. gondii and T. spiralis infections in wild boars killed in Korea from December 2009 to October 2011. A total of 521 wild boars hunted in eight provinces were examined for antibodies to T. gondii and T. spiralis by using commercial ELISA kits. Overall, 25.1% of serum samples from individual boars were seropositive for T. gondii and 1.7% were seropositive for T. spiralis. Seropositive for T. gondii was found in the boars in all the eight provinces investigated and for T. spiralis in four provinces. This is the first report on the seroprevalence of T. gondii and T. spiralis infections in wild boars in Korea. The consumption of undercooked wild boar meat may expose humans to a high risk of infection.

A rapid molecular strategy for early detection and characterization of Vietnamese foot-and-mouth disease virus serotypes O, A, and Asia 1

A one-step RT-PCR method using newly designed primers VN-VP1F/VN-VP1R targeting the full VP1 capsid protein-coding gene, combined with direct sequencing of its PCR product, has been developed successfully for universal detection and characterization of Vietnamese FMDV serotypes O, A, and Asia 1 directly from clinical samples. The one-step RT-PCR amplified 821-bp dsDNA products covering the entire VP1 gene of FMDV serotypes O, A, and Asia 1. The obtained dsDNA products were suitable for direct sequencing, cloning, and other molecular epidemiology studies of Vietnamese FMDV strains, which eliminated the need for cell culture and virus purification. This one-step RT-PCR system was applied to detect and characterize 55 field FMDV strains, including 34 serotype O, 17 serotype A, and 4 serotype Asia 1 isolates collected from endemic outbreaks in Vietnam from 2005 to 2010. Interestingly, the PCR products obtained from the present PCR method could be used as DNA templates for the second PCR typing method using serotypes O, A, and Asia 1-specific primers (Le et al., 2011). The use of the second PCR amplification increased markedly the sensitivity of the test for FMDV detection. The present RT-PCR method promises to be an effective tool for molecular epidemiological studies of FMD in Vietnam.

Development of one-step multiplex RT-PCR method for simultaneous detection and differentiation of foot-and-mouth disease virus serotypes O, A, and Asia 1 circulating in Vietnam

A one-step multiplex RT-PCR method using new primers was developed for the simultaneous detection and differentiation of Vietnamese FMDV serotypes O, A, and Asia 1 directly from clinical samples. The RT-PCR method used a cocktail of one universal minus-sense primer designed in the 2B gene and three serotype-specific plus-sense primers designed in the hypervariable regions of the capsid VP1 coding gene of FMDV. These serotype-specific primer pairs amplified 658, 535, and 427 bp PCR products corresponding to FMDV serotypes O, Asia 1, and A, respectively. In this study, six well-characterized FMDV strains belonging to serotypes O, A, and Asia 1 were used as reference strains for validation tests. Among these six FMDV strains were three vaccine strains for type O (O1/Manisa), A (A22/Iraq), and Asia 1 (As1/Shamir/89). The other reference strains included one pandemic strain of FMDV serotype Asia 1 (Asia1/MOG/05) and two pandemic strains of FMDV serotype O (O/UKG/34/2001 and O/SKR/2000). For field application, 37 positive-clinical samples and 18 cell culture-adapted viruses belonging to serotypes O, A, and Asia 1, as confirmed previously by antigen ELISA for FMDV detection, were used. The present method showed high sensitivity and specificity and can be adapted for detection and typing of FMDV serotypes O, A, and Asia 1 circulating in Vietnam.

Analysis of the nonstructural and structural polyprotein regions, and complete genome sequences of Israel acute paralysis viruses identified from honeybees (Apis mellifera) in Korea

Phylogenetic trees were constructed for 24 partial nucleotide sequences of the nonstructural polyprotein (ORF1) and structural polyprotein regions (ORF2) of Korean IAPV genotypes, as well as eight previously reported IAPV sequences from various countries. Most of the Korean genotypes formed a distinct cluster, separate from other country genotypes. To investigate this phenomenon in more detail, three complete IAPV genome sequences were identified from different regions in Korea, i.e., Korea1, Korea2, and Korea3. These sequences were aligned with eight previously reported complete genome sequences and various genome regions were compared. The Korean IAPVs were very similar to those from China and Israel, but highly diverged from USA and Australian genotypes. Interestingly, they showed greater variability than the USA and Australian genotypes in ORF1, but highly similar to the Australian genotype in the ORF2 region. Thus, genetic recombination may account for the spatial distance between the Korean IAPV genotypes and those from other countries.

Comparative Genomic Analysis for Genetic Variation in Sacbrood Virus of Apis cerana and Apis mellifera Honeybees From Different Regions of Vietnam

Abstract Sacbrood virus (SBV) is one of the most common viral infections of honeybees. The entire genome sequence for nine SBV infecting honeybees, Apis cerana and Apis mellifera, in Vietnam, namely AcSBV-Viet1, AcSBV-Viet2, AcSBV-Viet3, AmSBV-Viet4, AcSBV-Viet5, AmSBV-Viet6, AcSBV-Viet7, AcSBV-Viet8, and AcSBV-Viet9, was determined. These sequences were aligned with seven previously reported complete genome sequences of SBV from other countries, and various genomic regions were compared. The Vietnamese SBVs (VN-SBVs) shared 91–99% identity with each other, and shared 89–94% identity with strains from other countries. The open reading frames (ORFs) of the VN-SBV genomes differed greatly from those of SBVs from other countries, especially in their VP1 sequences. The AmSBV-Viet6 and AcSBV-Viet9 genome encodes 17 more amino acids within this region than the other VN-SBVs. In a phylogenetic analysis, the strains AmSBV-Viet4, AcSBV-Viet2, and AcSBV-Viet3 were clustered in group with AmSBV-UK, AmSBV-Kor21, and AmSBV-Kor19 strains. Whereas, the strains AmSBV-Viet6 and AcSBV-Viet7 clustered separately with the AcSBV strains from Korea and AcSBV-VietSBM2. And the strains AcSBV-Viet8, AcSBV-Viet1, AcSBV-Viet5, and AcSBV-Viet9 clustered with the AcSBV-India, AcSBV-Kor and AcSBV-VietSBM2. In a Simplot graph, the VN-SBVs diverged stronger in their ORF regions than in their 5′ or 3′ untranslated regions. The VN-SBVs possess genetic characteristics which are more similar to the Asian AcSBV strains than to AmSBV-UK strain. Taken together, our data indicate that host specificity, geographic distance, and viral cross-infections between different bee species may explain the genetic diversity among the VN-SBVs in A. cerana and A. mellifera and other SBV strains.