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Download Download บทความวิจัย สัตวแพทย์มหานครสาร JOURNAL OF MAHANAKORN VETERINARY MEDICINE Available online: www.tci-thaijo.org/index.php/jmvm/ ความชุกและความหลากหลายทางพันธุกรรมของเชื้อ Torque Teno Sus Viruses 1 และ 2 จากสุกรในเขตจังหวัดเชียงใหม่และลาพูน ประเทศไทย ประยุทธ แซ่โค้ว1,# และวีณา จูเปีย1 1หน่วยพาราคลินิก ภาควิชาชีวศาสตร์ทางสัตวแพทย์และสัตวแพทย์สาธารณสุข คณะสัตวแพทยศาสตร์ มหาวิทยาลัยเชียงใหม่ ต าบลแม่เหียะ อ าเภอเมือง จังหวัดเชียงใหม่ บทคัดย่อ: เชื้อ Torque teno sus viruses 1 และ 2 (TTSuV1 และ 2) เป็นเชื้อไวรัสในแฟมิลี่ Anelloviridae พบ รายงานการติดเชื้อในสุกรแต่ยังไม่ทราบพยาธิก าเนิดของโรคอย่างแน่ชัด อย่างไรก็ตามมีรายงานการศึกษาถึงความชุก และความหลากหลายทางพันธุกรรมของเชื้อไวรัสดังกล่าวได้ทั่วโลก ซึ่งเชื้อไวรัส TTSuV1 และ 2 ยังไม่เคยมีรายงาน จากสุกรในเขตจังหวัดเชียงใหม่และล าพูน ดังนั้นวัตถุประสงค์ของการศึกษาในครั้งนี้เป็นการตรวจหาความชุกและ ความหลากหลายทางพันธุกรรมของเชื้อ TTSuV1 และ 2 จากทอนซินของสุกรที่มีสุขภาพปกติจ านวน 100 ตัวอย่าง ในจังหวัดเชียงใหม่และล าพูนด้วยวิธีปฏิกิริยาลูกโซ่โพลีเมอเรส ผลการตรวจพบว่าความชุกของ TTSuV1, TTSuV2, และการติดเชื้อ TTSuV1 และ 2 สองชนิดร่วมกันเป็นดังนี้ 62% (62/100), 68% (68/100), และ 50% (50/100) ตามล าดับ การตรวจวิเคราะห์ความหลากหลายทางพันธุกรรมด้วยการใช้ความสัมพันธ์เชิงวิวัฒนาการบริเวณ 5' untranslated regions จาก 19 ล าดับนิวคลีโอไทด์ของ TTSuV1 และ 2 ผลการวิเคราะห์พบว่าเชื้อไวรัสทั้งสองชนิด สามารถตรวจพบได้เป็น 2 กลุ่มกล่าวคือ กลุ่มแรกพบว่าไม่มีความสัมพันธ์กับบริเวณที่ศึกษาเนื่องจากมีความแตกต่าง ของล าดับนิวคลีโอไทด์ของเชื้อไวรัสเป็นจ านวนมาก (ความเหมือนของล าดับนิวคลีโอไทด์จาก TTSuV1 เป็นร้อยละ 85 และความเหมือนของล าดับนิวคลีโอไทด์จาก TTSuV2 เป็นร้อยละ 87) กลุ่มที่สองพบว่าไม่พบความแตกต่างของ ล าดับนิวคลีโอไทด์ของเชื้อไวรัสที่ได้ท าการศึกษาในครั้งนี้ จากผลการศึกษาในครั้งนั้นแสดงให้เห็นว่าเชื้อไวรัสทั้งสอง ชนิดอาจพบว่ามีการปรับตัวภายในเขตจังหวัดเชียงใหม่และล าพูน ค าส าคัญ: ความหลากหลายทางพันธุกรรม เชื้อ Torque teno sus viruses Anelloviridae สุกร #ผู้รับผิดชอบบทความ สัตวแพทย์มหานครสาร. 2562. 14(1): 33-46. E-mail address: [email protected] Submitted date 2019-03-15 Revised date 2019-06-05 Accepted date 2019-07-01 Saekhow, P. and V. Chupia / J. Mahanakorn Vet. Med. 2019. 14(1): 33-46. Prevalence and Genetic Diversity of Torque Teno Sus Viruses 1 and 2 in Pigs in Chiang Mai and Lamphun, Thailand Prayuth Saekhow1,# and Vena Chupia1 1Division of veterinary Paraclinic, Department of veterinary bioscience and veterinary public health, Faculty of Veterinary medicine, Tumbon Maehea, Amphoe Maung, Chiang Mai 50100 THAILAND Abstract: Torque teno sus viruses 1 and 2 (TTSuV1 and 2) belong to the family Anelloviridae, which is known to commonly infect pigs; however, their pathogenesis is unclear. Studies of TTSuV1 and TTSuV2 in pigs worldwide, especially with regard to their prevalence and genetic diversity, have been conducted; however, the prevalence and genetic diversity of both viruses have not been studied in Chiang Mai and Lamphun, Thailand. Therefore, this study aimed to investigate both viruses in terms of their prevalence and genetic diversity using polymerase chain reaction with specific primers in the tonsils of 100 apparently clinically healthy pigs that are being raised in this region. Overall, the prevalence rates of TTSuV1, TTSuV2, and both in combination, in the pigs were 62% (62/100), 68% (68/100), and 50% (50/100), respectively. Phylogenetic analysis of 5' untranslated regions of 19 nucleotide sequences of both viruses was conducted. Both viruses were separated into 2 groups. The first group displayed a lack of geographic clustering because the nucleotide sequences in these groups showed extreme differences (TTSuV1 minimum nucleotide sequence identity was recorded at 87% and TTSuV1 minimum nucleotide sequence identity was recorded at 85%). The second group revealed the identical nucleotide sequences as no differences were observed in terms of the nucleotide sequences among the specimens. Our data indicate that the nucleotide sequences of TTSuV1s and s that were detected in Chiang Mai and Lamphun, Thailand, likely were adapted from their genome. Keywords: Genetic diversity, Torque teno sus viruses, Anelloviridae, Pigs #Corresponding author J. Mahanakorn Vet. Med. 2019 14(1): 33-46. E-mail address: [email protected] 34 Saekhow, P. and V. Chupia / J. Mahanakorn Vet. Med. 2019. 14(1): 33-46. Introduction TTSuV2 (Kekarainen et al., 2006; Taira et al., Torque teno sus viruses include two 2009; Zhai et al., 2013). distinct species, namely TTSuV1 and TTSuV2. Although the pathogenic significance of TTSuV1 is a member of the genus these viruses is currently unclear, TTSuV1 and Alphatorquevirus, whereas TTSuV2 is a TTSuV2 genomes have been detected in pigs member of the genus Kappatorquevirus; that have been diagnosed with clinical both of these genera belong to the family diseases, namely, porcine circovirus Anelloviridae (Hino and Miyata, 2007; Taira et associated disease (Kekarainen et al., 2006; al., 2009). Torque teno virus (TTV) was first Taira et al., 2009) and respiratory disease discovered in a patient suffering from (Gimenez-Lirola et al., 2014; Lee et al., 2015). hepatitis. Since that time, it has been However, TTSuV1 and TTSuV2 genomes have reported in many species of mammals. It has also been detected in apparently clinically not only been identified in domestic animals, healthy pigs. Moreover, TTSuV1 and TTSuV2 such as pigs, dogs, cats, cattle, and sheep, but have been detected in many tissues and also in wildlife, including wild boars, tupaias, samples, including the semen, lungs, kidneys, sea lions, and primates (Meng, 2012; liver, intestine, bone marrow, lymph nodes, Okamoto, 2009). The genome of anellovirus and tonsils of aborted fetuses (Bigarre et al., is negative sense and single-stranded DNA 2005; Kekarainen et al., 2007; Lee et al., 2010; with a genome size ranging from 2 to 3.9 kb Martelli et al., 2006; Novosel et al., 2012; Taira (Spandole et al., 2015). This viral genome et al., 2009). The total prevalence rates of consists of at least three regions: the 5′ TTSuV1 and TTSuV2 in pigs have been untranslated region (5′UTR), open reading reported at multiple levels, including 52.2% frame 1 (ORF1), and open reading frame 2 in the USA (Xiao et al., 2012), 46.0% in Canada (ORF2) (Ninomiya et al., 2007). Although a (McKeown et al., 2004), 93.0% in France transcription profile of TTV showed that it has (Bigarre et al., 2005), 40.1% in Italy (Martelli et at least three mRNAs, the mechanism of viral al., 2006), 97.0% in Spain (Kekarainen et al., replication is unclear (Kamahora et al., 2000). 2006), 85.0% in South Korea (Lee et al., 2010), The prominent 5′UTR is composed of a 78.9% in China (Zhai et al., 2013), and 40.0% conserved region and a region exhibiting in Thailand (McKeown et al., 2004). The variations that allowing for identification of genetic diversity, especially of the 5′UTR of the viral nucleotide sequences of TTSuV1 and both viruses, revealed heterogeneity and a lack of geographical clustering (McKeown et 35 Saekhow, P. and V. Chupia / J. Mahanakorn Vet. Med. 2019. 14(1): 33-46. al., 2004). Two previous studies on Thai 1,500 rpm for 5 min. These supernatant TTSuV1s and TTSuV2s (Cortey et al., 2012; homogenized tonsils were used for DNA McKeown et al., 2004) reported a lack of extraction by NucleoSpin® Tissue (Macherey- geographic clustering. However, information Nagel, GmbH, Duren, Germany), according to on both viruses has been insufficient in the manufacturer’s instructions. allowing us to understand the genetic Detection of TTSuV1 and TTSuV2 genomes diversity of the TTSuV1 and TTSuV2 viruses by PCR that are circulating in Chiang Mai and To detect the prevalence of TTSuV1 Lamphun, Thailand. Therefore, this study was and TTSuV2, PCR was carried out in separate established to investigate the prevalence of reactions with each pair of primers. The target the viruses, and to characterize the 5′ of the primers in this study were 5' UTR of untranslated region of TTSuV1 and TTSuV2 in both viruses (Kekarainen et al., 2006). The pair pigs located in Chiang Mai and Lamphun of primers in the PCR for TTSuV1 was TTSuV1- Provinces. F 5′ TACACTTCCGGGTTCAGGAGGCT 3′ and TTSuV1-R 5′ ACTCAGCCATTCGGAACCTCAC 3′, Materials and Methods whereas the pair of primers in the PCR for Sample collection TTsuV2 was TTSuV2-F 5′ AGTTACACATAACCA Tonsil samples were obtained from 100 CCAAACC 3′ and TTSuV2-R 5′ ATTACCGCCTGC apparently cadaveric pigs at approximately 6 CCGATAGGC 3′. Quick Taq HS DyeMix months of age from animals raised in Chiang (Toyobo, Osaka, Japan) was performed for Mai and Lamphun provinces. The status of all PCR amplification with specific primers. The pigs in this study were apparently clinically PCR was performed with an initial step of 94 healthy pigs. Sample collection was °C for 4 min; followed by 35 cycles of 94 °C performed in December, 2016. All tonsil for 1 min, 58 °C for 30 s, and 72 °C for 30 s; samples were kept at −20 °C until being used. and a final step of 72 °C for 7 min. Detection of TTSuV1 and TTSuV2 genomes Nucleotide sequencing and phylogenetic by polymerase chain reaction (PCR) analysis Viral DNA extraction The phylogenetic analyses of TTSuV1 Tonsil samples were homogenized in and TTSuV2 were carried out by directly 0.9% normal saline using zirconia beads and sequencing the PCR products of the viral 5' a Mini-Beadbeater1 (Bipspec, Bartlesville, OK, UTR regions. Sequencing primers, for TTSuV1, USA). The samples were then centrifuged at the 310 bp DNA of the 5' UTR used with the 36 Saekhow, P. and V. Chupia / J. Mahanakorn Vet. Med. 2019. 14(1):
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