Detection of SARS-CoV-2 and characterization of SARS-CoV-2 S-gene in major companion and food animals

项目摘要：SARS-CoV-2是一种主要的人类呼吸道病原体，可导致新冠肺炎，a 症状与甲型流感(流感A)和乙型流感(流感B)病毒相似的疾病。几个 主要的SARS-CoV-2病毒变异(VOC)已经被确定，并与增加的 在人类中的传播性，包括英国变体B.1.1.7/Alpha，南非变体B.1.351/Beta， 日本/巴西变体P.1/Gamma、加利福尼亚州变体B.1.427/B.1.429/Epsilon和印度变体 B.1.6.7.1/B.1.6.7.2/Delta(CDC，2021A)。SARS-CoV-2病毒也在不同的动物物种中发现， 包括狗、猫、老虎、狮子、水貂、雪貂等。 已被报告(Halfmann等人，2020；OIE，2021)，以及有限的动物对人类的报告 传播(Hedman等人，2021年)，但可能需要更广泛的研究才能得出结论。两种流感 甲型和乙型流感是人类和动物呼吸道的主要病原体，引起的症状有时很难 与新冠肺炎区分开来。该方案有三个主要目标：1)开发和验证多路实时 用聚合酶链式反应检测SARS-CoV-2、流感A和流感B病毒；2)筛选动物呼吸道样本 在授权期的7-30个月(2年)内提交给KSVDL；以及3)对尖峰蛋白进行测序- 对所有SARS-CoV-2阳性样本的编码基因(S基因)进行鉴定和特征分析 它们与人类菌株的关系。 从2018-2020年，堪萨斯州立兽医诊断实验室(KSVDL)每年平均收到 11,065个动物呼吸道样本，包括平均69只猫，491只狗，123只马，1,070只猪和 每年9296个牛样本。所有猫和狗的呼吸道样本和精选样本 在赠款期间7-30个月收到的其他物种将通过SARS-CoV-2/Flu复合病毒进行测试 A/Flu B试验是在研究中建立的。S将对所有SARS-CoV-2阳性样本进行基因测序 尝试了一种在本研究中优化的程序，并与主要VOC菌株进行了比较。满载或接近满载 将对S的基因序列进行系统发育分析，以进一步研究该物种的变异和遗传多样性。 从动物中鉴定出SARS-CoV-2毒株。分析还将揭示，如果在 动物物种之间的亲缘关系比菌株亲缘关系更密切。中国人S基因序列分析 将对动物和人类来源的菌株进行比较，以确定是否在 人类。主要目标包括：1)提供全面的SARS-CoV-2阳性率，持续两年 从从狗、猫、马、牛和猪身上收集的样本中收集数据；2)确定潜在的SARS-CoV-2挥发性有机化合物 3)对所有可测序的阳性标本进行S基因的系统发育分析 (CT≤32)测定遗传多样性；4)鉴定和系统发育分析 动物和人源性SARS-CoV-2毒株的S基因。

Project Summary: SARS-CoV-2 is a major human respiratory pathogen that can cause COVID-19, a disease with symptoms similar to those caused by Influenza A (Flu A) and Influenza B (Flu B) viruses. A few major SARS-CoV-2 variants of concern (VOC) have been identified and associated with increased transmissibility in humans, including the UK variant B.1.1.7/Alpha, South Africa variant B.1.351/Beta, Japan/Brazil variant P.1/Gamma, California variants B.1.427/B.1.429/Epsilon, and India variants B.1.6.7.1/B.1.6.7.2/Delta (CDC, 2021a). SARS-CoV-2 has also been found in different animal species, including dogs, cats, tigers, lions, minks, ferrets etc. Transmission among individuals within an animal species has been reported (Halfmann et al, 2020; OIE, 2021), as well as limited reports of animal to human transmission (Hedman et al., 2021), yet more extensive studies may be needed to draw a conclusion. Both Flu A and Flu B are major human and animal respiratory pathogens, and cause symptoms sometimes difficult to differentiate from COVID-19. This proposal has three main goals: 1) Develop and validate a multiplex real-time PCR assay for detection of SARS-CoV-2, Flu A and Flu B viruses; 2) Screening animal respiratory samples submitted to KSVDL from Months 7-30 (2 years) of the grant period; and 3) Sequencing the spike protein- encoding gene (S-gene) of all SARS-CoV-2 positive samples to identify and characterize potential variants and their relationship to human strains. From 2018-2020, Kansas State Veterinary Diagnostic Laboratory (KSVDL) received an annual average of 11,065 animal respiratory samples, including an average of 69 feline, 491 canine, 123 equine, 1,070 swine and 9,296 bovine samples for each year. All feline and canine respiratory samples and selected samples from other species received in Months 7-30 during the grant period will be tested by the multiplex SARS-CoV-2/Flu A/Flu B assay developed in the study. S-gene sequencing of all SARS-CoV-2 positive samples will be attempted with a procedure optimized in this study and results compared to major VOC strains. Full or near-full S-gene sequences will be phylogenetically analyzed to further study the variation and genetic diversity of the SARS-CoV-2 strains identified from animal species. Analysis will also reveal if strains identified within an animal species have a closer genetic relationship than strains between species. The S-gene sequences of animal- and human-derived strains will be compared to determine if animal strains have been observed in humans. Main goals include: 1) Provide comprehensive SARS-CoV-2 positive rates, for a duration of two years, from samples collected from dogs, cats, horses, cattle and pigs; 2) Identify potential SARS-CoV-2 VOC strains from positive samples; 3) Phylogenetically analyze the S-gene from all sequenceable positive samples (Ct ≤ 32) to determine genetic diversity; 4) Identify and phylogenetically analyze similarities and differences of the S-gene of animal- and human-derived SARS-CoV-2 strains.