Comparative genetic analyses of Korean bat coronaviruses with SARS-CoV and the newly emerged SARS-CoV-2

BackgroundBats have been considered natural reservoirs for several pathogenic human coronaviruses (CoVs) in the last two decades. Recently, a bat CoV was detected in the Republic of Korea; its entire genome was sequenced and reported to be genetically similar to that of the severe acute respiratory syndrome CoV (SARS-CoV).ObjectivesThe objective of this study was to compare the genetic sequences of SARS-CoV, SARS-CoV-2, and the two Korean bat CoV strains 16BO133 and B15-21, to estimate the likelihood of an interaction between the Korean bat CoVs and the human angiotensin-converting enzyme 2 (ACE2) receptor.MethodsThe phylogenetic analysis was conducted with the maximum-likelihood (ML) method using MEGA 7 software. The Korean bat CoVs receptor binding domain (RBD) of the spike protein was analyzed by comparative homology modeling using the SWISS-MODEL server. The binding energies of the complexes were calculated using PRODIGY and MM/GBGA.ResultsPhylogenetic analyses of the entire RNA-dependent RNA polymerase, spike regions, and the complete genome revealed that the Korean CoVs, along with SARS-CoV and SARS-CoV-2, belong to the subgenus Sarbecovirus, within BetaCoVs. However, the two Korean CoVs were distinct from SARS-CoV-2. Specifically, the spike gene of the Korean CoVs, which is involved in host infection, differed from that of SARS-CoV-2, showing only 66.8%–67.0% nucleotide homology and presented deletions within the RBD, particularly within regions critical for cross-species transmission and that mediate interaction with ACE2. Binding free energy calculation revealed that the binding affinity of Korean bat CoV RBD to hACE2 was drastically lower than that of SARS-CoV and SARS-CoV-2.ConclusionsThese results suggest that Korean bat CoVs are unlikely to bind to the human ACE2 receptor.     

SARS-CoV-2 natural infection in animals: a systematic review of studies and case reports and series

COVID-19 pandemic is essentially a zoonotic disease. In this context, early in 2020, transmission from humans to certain animals began reporting; the number of studies has grown since. To estimate the pooled prevalence of SARS-CoV-2 natural infection in animals and to determine differences in prevalence between countries, years, animal types and diagnostic methods (RT-PCR or serological tests). A systematic literature review with meta-analysis using eight databases. Observational studies were included but analyzed separately. We performed a random-effects model meta-analysis to calculate the pooled prevalence and 95% confidence interval (95% CI) for prevalence studies and case series. After the screening, 65 reports were selected for full-text assessment and included for qualitative and quantitative analyses. A total of 24 reports assessed SARS-CoV-2 infection by RT-PCR, combining a total of 321,785 animals, yielding a pooled prevalence of 12.3% (95% CI 11.6%–13.0%). Also, a total of 17 studies additionally assessed serological response against SARS-CoV-2, including nine by ELISA, four by PRTN, one by MIA, one by immunochromatography (rest, two studies, the method was not specified), combining a total of 5319 animals, yielding a pooled prevalence of 29.4% (95% CI 22.9%–35.9%). A considerable proportion of animals resulted infected by SARS-CoV-2, ranking minks among the highest value, followed by dogs and cats. Further studies in other animals are required to define the extent and importance of natural infection due to SARS-CoV-2. These findings have multiple implications for public human and animal health. One Health approach in this context is critical for prevention and control.     

SARS-CoV-2 in animals: potential for unknown reservoir hosts and public health implications

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously 2019-nCoV) is suspected of having originated in 2019 in China from a coronavirus infected bat of the genus Rhinolophus. Following the initial emergence, possibly facilitated by a mammalian bridge host, SARS-CoV-2 is currently transmitted across the globe via efficient human-to-human transmission. Results obtained from experimental studies indicate that animal species such as cats, ferrets, raccoon dogs, cynomolgus macaques, rhesus macaques, white-tailed deer, rabbits, Egyptian fruit bats, and Syrian hamsters are susceptible to SARS-CoV-2 infection, and that cat-to-cat and ferret-to-ferret transmission can take place via contact and air. However, natural infections of SARS-CoV-2 have been reported only in pet dogs and cats, tigers, lions, snow leopards, pumas, and gorillas at zoos, and farmed mink and ferrets. Even though human-to-animal spillover has been reported at several instances, SARS-CoV-2 transmission from animals-to-humans has only been reported from mink-to-humans in mink farms. Following the rapid transmission of SARS-CoV-2 within the mink population, a new mink-associated SARS-CoV-2 variant emerged that was identified in both humans and mink. The increasing reports of SARS-CoV-2 in carnivores indicate the higher susceptibility of animal species belonging to this order. The sporadic reports of SARS-CoV-2 infection in domestic and wild animal species require further investigation to determine if SARS-CoV-2 or related Betacoronaviruses can get established in kept, feral or wild animal populations, which may eventually act as viral reservoirs. This review analyzes the current evidence of SARS-CoV-2 natural infection in domestic and wild animal species and their possible implications on public health.     

SARS-CoV-2 infection in domestic and feral cats: current evidence and implications

Current evidence indicates that cats play a limited role in COVID-19 epidemiology, and pets are probably dead-end hosts of SARS-CoV-2 and pose negligible risks of transmission to humans. Still, one health concept is to be adopted widely as a component of mitigation strategies to tackle the ongoing pandemic. Therefore, in terms of the magnitude of infection and potential to transmit SARS-CoV-2 to humans, our surveillance efforts should mainly focus on mustelids (especially minks, ferrets, and others) for early detection and control of infection. This will ensure that SARS-CoV-2 will not get established in the wild animal population of these susceptible species. We agree with Dr. Passarella Teixeira on the possibility of domestic and feral cats acting as an urban reservoir, subsequently transmitting the virus to human beings. However, it is less likely that such a phenomenon will be reported even if it has occurred due to the efficient and extensive human-to-human transmission of SARS-CoV-2.     

新型冠状病毒及其与猪冠状病毒的相关性和启示

2019年底新型冠状病毒(SARS-Co V-2)造成了对人类健康构成了巨大威胁的疫情,但其与猪冠状病毒基因组序列相似性低,在决定病毒宿主嗜性和毒力的纤突(spike,S)蛋白结构上具有较大差异,据此推测不能构成跨种传播。对蝙蝠携带冠状病毒多样性和分布区域的研究可有助于分析其对人类和养殖业的威胁,在公共卫生中具有积极意义。针对突发疫病,快速响应获得原始材料数据有助于疫病诊断、治疗和预防研究的开展。多种人冠状病毒S蛋白被证明是最适合开发的疫苗及治疗性抗体靶标,可在动物疫苗等产品的设计中借鉴。     

动物新型冠状病毒流行现状

新型冠状病毒(SARS-CoV-2)感染导致的新冠肺炎(COVID-19)疫情自2019年12月以来,已在全球221个国家和地区传播,导致1亿多人感染,230多万人死亡.SARS-CoV-2除了对公共卫生产生极大影响之外,全球已有24个国家和地区报告470余起动物感染疫情,涉及犬、猫、水貂、狮子、老虎等多种动物.因此,...     

SARS-CoV-2 infection in farmed minks,associated zoonotic concerns,and importance of the One Health approach during the ongoing COVID-19 pandemic

The coronavirus disease 2019 (COVID-19) pandemic has now affected over 72.5 million people worldwide, with nearly 1.6 million deaths reported globally as of December 17, 2020. SARS-CoV-2 has been implicated to have originated from bats and pangolins, and its intermediate animal hosts are being investigated. Crossing of the species barrier and exhibition of zoonosis have been reported in SARS-CoV-2 in farm (minks), domesticated (cats and dogs), and wild animals (tigers, puma, and lions). Recently, the rapid spread of SARS-CoV-2 infection was reported in mink farms, which led to the death of a myriad minks. The clinical and pathological findings of SARS-CoV-2 infection and the rapid animal-to-animal transmission in minks are almost similar to the findings observed in patients with COVID-19. Additionally, the rapid virus transmission among minks and the associated mutations resulted in a new mink-associated variant that was identified in both minks and humans, thereby providing evidence of mink-to-human transmission of SARS-CoV-2. The new mink-associated SARS-CoV-2 variant with a possible reduced sensitivity to neutralizing antibodies poses serious risks and is expected to have a direct effect on the diagnostic techniques, therapeutics, and vaccines that are currently under development. This article highlights the current evidence of SARS-CoV-2 infection in farmed minks, and provides an understanding of the pathogenesis of COVID-19 in minks and the associated zoonotic concerns of SARS-CoV-2 transmission from minks to humans with an emphasis on appropriate mitigation measures and on the necessity of adopting the One Health approach during the COVID-19 pandemic.     

鸡IL-2 mRNA SYBR Green Ⅰ实时荧光定量RT-PCR检测方法的建立与应用

根据GenBank提供的ChIL-2参考序列设计了特异性引物,并以鸡3-磷酸甘油脱氢酶(GAPDH)基因为内参,以二者标准质粒为模板,应用SYBR Green Ⅰ实时荧光定量PCR技术,并采用双标准曲线相对定量方法建立了ChIL-2 mRNA荧光定量RT-PCR检测方法.结果显示,该方法该可以在3h左右对低拷贝量的模板(200 copy/μL)进行检测,同时该检测方法具有很好的特异性和重复性.雏鸡免疫试验IL-2 mRNA检测结果显示,该方法可有效应用于鸡体内IL-2在核酸水平的检测,并为今后IL-2相对定量的检测研究提供参考依据.     

Seroprevalence of antibodies against severe acute respiratory coronavirus 2 (SARS-CoV-2) in household dogs in Japan

We investigated the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among dogs in the Tokyo area via enzyme-linked immunosorbent assay (ELISA) using the spike protein as the target antigen. Plasma samples from 494 household dogs and blood-donor dogs were tested from July 2020 to January 2021. Of these samples, three showed optical densities that were higher than the mean plus two standard deviations of the mean of the negative-control optical densities (ODs). Of these three samples, only the sample with the highest OD by ELISA was confirmed positive by virus neutralization testing. The positive dog presented no SARS-CoV-2-related symptoms. The positivity rate of SARS-CoV-2 infections among dogs in the Tokyo area was approximately 0.2%.     

狂犬病病毒荧光定量RT-PCR快速检测试剂盒的评价及应用

基于目前在我国人群和动物中流行的狂犬病病毒（RV）均属于基因Ⅰ型,在本室所建立的基因Ⅰ型RV荧光定量RT-PCR方法的基础上,组装了可以便捷使用的RV快速检测试剂盒。对4 577份临床样品的检测结果表明,该试剂盒的检测灵敏度达4.68个TCID50,与《狂犬病防治技术规范》规定的套式RT-PCR灵敏度相当,而且稳定性良好,可以冷冻保存8个月以上。所研制的荧光定量RT-PCR快速检测试剂盒具有操作简便、快速、特异性强、灵敏度高、重复性好、稳定性强等优点,既适合单个临床样品的确诊,又适合RV流行病学监测的大规模筛查。     

实时荧光定量RT-PCR检测禽白血病病毒方法的建立与应用

建立了TaqMan实时荧光定量RT-PCR方法检测禽白血病病毒(ALV)。选取ALV病毒的LTR序列设计引物和探针,以梯度稀释的含有ALV目的扩增片段的质粒作为标准品,进行定量PCR反应以确定检测灵敏度。阳性标准品在3.0×102~3.0×107个拷贝共6个数量级的范围内,定量PCR反应有"S"型扩增曲线,检测灵敏度最低为30个拷贝。根据病毒拷贝数与定量反应Ct值的关系,绘制了标准曲线。该方法具有特异性,对新城疫病毒、禽流感病毒、传染性支气管炎病毒、传染性囊病病毒、鸡传染性贫血病毒和马立克病病毒核酸都没有扩增反应。实时定量PCR检测ALV的方法,灵敏度高,特异性好,可以进行定量分析,在禽病的快速检测上具有重要意义。     

Second round of an interlaboratory comparison of SARS-CoV2 molecular detection assays used by 45 veterinary diagnostic laboratories in the United States

The COVID-19 pandemic presents a continued public health challenge. Veterinary diagnostic laboratories in the United States use RT-rtPCR for animal testing, and many laboratories are certified for testing human samples; hence, ensuring that laboratories have sensitive and specific SARS-CoV2 testing methods is a critical component of the pandemic response. In 2020, the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) led an interlaboratory comparison (ILC1) to help laboratories evaluate their existing RT-rtPCR methods for detecting SARS-CoV2. All participating laboratories were able to detect the viral RNA spiked in buffer and PrimeStore molecular transport medium (MTM). With ILC2, Vet-LIRN extended ILC1 by evaluating analytical sensitivity and specificity of the methods used by participating laboratories to detect 3 SARS-CoV2 variants (B.1; B.1.1.7 [Alpha]; B.1.351 [Beta]) at various copy levels. We analyzed 57 sets of results from 45 laboratories qualitatively and quantitatively according to the principles of ISO 16140-2:2016. More than 95% of analysts detected the SARS-CoV2 RNA in MTM at ≥500 copies for all 3 variants. In addition, for nucleocapsid markers N1 and N2, 81% and 92% of the analysts detected ≤20 copies in the assays, respectively. The analytical specificity of the evaluated methods was >99%. Participating laboratories were able to assess their current method performance, identify possible limitations, and recognize method strengths as part of a continuous learning environment to support the critical need for the reliable diagnosis of COVID-19 in potentially infected animals and humans.     

鸡传染性法氏囊病病毒实时荧光定量RT—PCR检测方法的建立

本试验根据GenBank中鸡传染性法氏囊病病毒(IBDV)基因组设计一对针对病毒vp5基因的引物和一条特异性Taq Man探针,建立了一种快速检测IBDV核酸载量的Taq Man荧光定量RT-PCR方法.通过对反应条件和反应体系的优化,使得该方法在108拷贝/μL～101拷贝/μL范围内具有良好的线性关系.能够灵敏地检测初始模板中30个拷贝的病毒核酸,其灵敏度是常规RT-PCR检测方法的100倍.该方法不与其它的禽源病毒发生非特异性反应,并且具有良好的重复性,为IBDV的定性定量检测提供了有效的工具.     

猪捷申病毒TaqMan实时定量RT-PCR方法的建立

为建立猪捷申病毒(PTV)的早期检测及定量分析方法,本研究基于PTV 11个血清型基因组5′端非编码区保守序列,设计引物和TaqMan探针,建立了检测PTV的TaqMan实时定量RT-PCR方法.应用该方法对PTV、猪细小病毒、猪繁殖与呼吸综合症病毒、猪圆环病毒2型、猪伪狂犬病毒以及猪瘟病毒进行特异性试验,结果除PTV为阳性外其它均为阴性;针对PTV最低可检测到10个拷贝;批内、批间重复试验的变异系数均小于3％.应用建立的方法与病毒分离方法分别对91份临床样品进行检测,检出率分别为79.12％和57.14％,两者的符合率是78.02％.经临床应用表明,该实时定量RT-PCR方法可为PTV的早期诊断及定量分析提供技术手段.     

荧光定量RT-PCR检测雏鸡感染NDV强毒后胸腺和法氏囊中TLR2与TLR4 mRNA的表达

Toll样受体2(TLR2)和Toll样受体4(TLR4)在识别病原微生物过程中发挥重要作用。为了定量检测TLR2、TLR4 mRNA表达水平,研究病原与机体的相互作用,本研究建立了检测鸡TLR2、TLR4 mRNA表达水平的SYBR GreenⅠ荧光定量RT-PCR(RRT-PCR)方法,检测了新城疫病毒强毒(vNDV)感染SPF雏鸡后36h、48h时胸腺、法氏囊中TLR2、TLR4 mRNA表达量变化。结果显示该方法特异性好,RRT-PCR产物分别在85.5℃、83.3℃出现单特异峰,对TLR2、TLR4的扩增效率分别为105.91%和95.30%,相关系数分别为0.9980、0.9996,最低检测限分别为108拷贝/反应和461拷贝/反应。感染后36h vNDV显著抑制TLR2、TLR4基因在法氏囊、胸腺中的表达;感染后48h时,法氏囊、胸腺中TLR2基因的表达水平显著升高,胸腺中TLR4基因的表达显著升高,而法氏囊中TLR4基因的表达仍处于抑制状态。本研究证明TLR2和TLR4参与了鸡体对NDV的感染应答。     

口蹄疫病毒TaqMan荧光定量PCR检测方法的建立

为建立检测口蹄疫病毒(FMDV)的方法,本研究根据GenBank中FMDV的2B基因序列,设计合成一对引物和一条TaqMan探针,将2B基因克隆到pBlueScriptSK(-)载体中,利用T7体外转录试剂盒制备标准品,通过优化反应条件,建立了TaqMan荧光定量PCR检测方法.结果表明,该检测方法的敏感性达到102拷贝/μL;与其它主要相关病毒均不发生交叉反应,批内和批间试验重复性的变异系数(CV)均小于3％.本研究建立的FMDV TaqMan荧光定量PCR方法对FMDV的快速检测具有重要意义.     

山东省部分地区水貂养殖场户貂源冠状病毒和新型冠状病毒认知调查及病原检测

国外水貂感染新型冠状病毒(SARS-CoV-2)遭扑杀事件引起了国内水貂养殖业关注。为了解SARS?CoV-2对山东省水貂养殖场户的影响,通过问卷调查方法了解水貂养殖场户对貂源冠状病毒和SARS-CoV-2的认知情况,同时采集样本应用双重荧光定量PCR方法进行核酸检测,确定水貂中两种病毒的感染状况。结果显示:47.83%的养殖场户对国外水貂感染SARS-CoV-2被扑杀事件有所了解,36.23%的养殖场户认为该事件会对水貂养殖业带来影响,其中10.14%的养殖场户对水貂进行过SARS-CoV-2核酸检测;84.06%的养殖场户认为貂源冠状病毒不会感染人,57.49%认为国外水貂感染SARS-CoV-2的主要途径是通过养殖场其他动物(如犬猫等)感染,仅0.97%认为貂源冠状病毒与SARS-CoV-2存在一定关系,更多的(52.17%)是不清楚;310份样品的双重荧光定量PCR检测结果均为阴性。结果表明,山东省水貂养殖场户普遍意识到貂源冠状病毒与SARS?CoV-2相关性很小,其对水貂养殖业影响不大。但部分养殖场户对这两种病毒及其对水貂养殖业的潜在威胁尚缺乏足够认知,因此需要加强对养殖场户的认知宣传和病毒监测。