Isolation and phylogenetic analysis of swinepox virus from pigs in China

Confirm whether the collected scab samples are caused by SWPV infection. According to the manufacturer's plan, we initially used the e.zn.a.® Viral DNA Kit (Omega, USA) to isolate DNA from clinical samples. Subsequently, the specific SWPV gene fragment was amplified by polymerase chain reaction (PCR) for identification. Primer A/1999/Nebraska (GenBank addition number: AF410153), the upstream and downstream primers for PCR amplification of the SWPV gene, was designed based on the SWPV/pig/U.S. reference isolation sequence. According to the identification method described by Thachamvally Riyesh et al., a 555 bp gene fragment was amplified, which spans the nucleotide region 12588-13142 of the SWPV genome. It is worth noting that this specific nucleotide region exists only in SWPV and has not been observed in other reported poxviruses. PCR was carried out using Phanta-Max ultra-high-fidelity DNA polymerase (Vazyme, China) in accordance with the manufacturer's protocol. The total PCR reaction volume was 50 μL, including 25 μL of 2× Phanta Max Buffer, 1 μL of 1 U/μL Phanta Max Super-Fidelity DNA polymerase, and 1 μL of 10 mM dNTP Mix. 2 μL of 20 μM upstream and downstream primers, 17 μL of nuclease-free water. The amplification parameters are set as follows: initial denaturation at 95°C for 5 minutes, initial denaturation at 95°C for 30 seconds, initial denaturation at 55°C for 40 seconds, initial denaturation at 72°C for 1 minute, and finally amplification at 72°C for 10 minutes. The PCR products were analyzed on 1% agarose gel. To ensure the accuracy of the identification results, we downloaded the published SWPV sequences (MW036632.1, MZ682626.1, MZ773480.1, MZ773481.1, NC003389.1 and OL456209.1) from NCBI for comparison and analysis. And primers were designed in its conserved region to amplify the gene fragment of 961 bp (87605-88565) for verification. The PCR reaction volume and parameters were the same as previously described: initial denaturation at 95°C for 5 minutes, followed by 30 seconds at 95°C, 1 minute and 30 seconds at 55°C, 1 minute at 72°C, and finally an extension of 10 minutes at 72°C for 34 cycles. The PCR products were analyzed again on 1% agarose gel.Based on the reference sequence SWPV/pig/U.S.A / 1999 / Nebraska (GenBank join no. : AF410153) We designed host genes such as KELch-like proteins (encoding SWPV-ORF006 and SWPV-ORF136), extracellular envelope virus proteins (encoding SWPV-ORF119 and SWPV-ORF120), anchor protein repeat sequence proteins (encoding SWPV-ORF143 and SWPV-ORF144), and diseases Primers of toxic late-stage transcription factor-3 (VLTF-3). We performed PCR amplification on the DNA extracted from the supernatant of the infected cell culture. Subsequently, the PCR products were cloned into the PMD18t vector (Takara, Bio USA) for sequencing. To conduct sequence comparison and analysis, we retrieved all published SWPV reference sequences from NCBI, performed molecular evolutionary genetic analysis using MEGA X software, and adopted the cluster W algorithm for multiple sequence alignment. A phylogenetic tree was constructed using the Neighbor-Joining method, which contained 1000 bootstrap repetitions to clarify the evolutionary relationship. Finally, the homology analysis of amino acid sequences was conducted using DNAStar MegAlign software.基于参考序列SWPV/pig/U.S.A/1999/Nebraska （GenBank加入号：AF410153），我们设计了宿主基因kelch样蛋白（编码SWPV-ORF006和SWPV-ORF136）、细胞外包膜病毒蛋白（编码SWPV-ORF119和SWPV-ORF120）、锚蛋白重复序列蛋白（编码SWPV-ORF143和SWPV-ORF144）和病毒晚期转录因子-3 （VLTF-3）的引物（表2）。我们对感染细胞培养的上清提取的DNA进行PCR扩增。随后，将PCR产物克隆到PMD18t载体（Takara， Bio USA）中进行测序。为了进行序列比较和分析，我们从NCBI中检索了所有已发表的SWPV参考序列，并使用MEGA X软件进行分子进化遗传分析，采用cluster W算法进行多序列比对。使用Neighbor-Joining方法构建了一个系统发育树，其中包含1000个bootstrap重复，以阐明进化关系（20,21）。最后，使用DNAStar MegAlign软件对氨基酸序列进行同源性分析（11）。