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

项目概述：SARS-CoV-2是一种主要的人类呼吸道病原体，可导致COVID-19， 症状与甲型流感（Flu A）和B流感（Flu B）病毒引起的症状相似的疾病。几 已经确定了主要的SARS-CoV-2变异体（VOC）， 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、加州变体B.1.427/B.1.429/Epoxy和印度变体 B.1.6.7.1/B.1.6.7.2/Delta（CDC，2021 a）。SARS-CoV-2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。 已报告（Halfmann et al，2020; OIE，2021），以及动物至人类的有限报告 传输（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 甲型流感和B流感是主要的人类和动物呼吸道病原体，引起的症状有时很难诊断。 区别于COVID-19。该方案有三个主要目标：1）开发和验证多路实时 用于检测SARS-CoV-2、Flu A和Flu B病毒的PCR方法; 2）筛选动物呼吸道样本 从授权期的第7-30个月（2年）提交给KSVDL;以及3）对刺突蛋白进行测序- 所有SARS-CoV-2阳性样本的编码基因（S基因），以识别和表征潜在的变异体， 与人类菌株的关系 从2018年到2020年，堪萨斯州兽医诊断实验室（KSVDL）每年平均收到 11，065个动物呼吸道样本，包括平均69个猫科动物、491个犬科动物、123个马科动物、1，070个猪和 每年采集9，296份牛样本。所有猫和犬呼吸道样本以及来自 在资助期内第7-30个月接收的其他物种将通过多重SARS-CoV-2/Flu 本研究开发了甲型/流感B检测试剂盒。将对所有SARS-CoV-2阳性样本进行S基因测序， 尝试使用本研究中优化的程序，并将结果与主要VOC菌株进行比较。的全额或接近全额 本研究将通过对S基因序列的同源性分析，进一步研究其变异和遗传多样性。 从动物物种中鉴定出SARS-CoV-2菌株。分析还将揭示，如果在一个 动物物种之间的遗传关系比物种之间的品系更密切。的S基因序列 将比较动物和人源性菌株，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，持续两个月 年，从狗、猫、马、牛和猪收集的样本中; 2）识别潜在的SARS-CoV-2 VOC 3）对来自所有可测序阳性样品的S-基因进行系统发生学分析 (Ct≤ 32）确定遗传多样性; 4）鉴定和遗传学分析 动物和人源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.