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。     

SARS-CoV S蛋白抗原表位富集区的表达与初步应用

扩增并克隆编码SARS-CoV S蛋白中包含多个抗原表位的第539～630氨基酸残基区域的基因片段。基因片段经酶切处理后插入表达载体pGEX-6p-1的BamHⅠ和XhoⅠ位点之间,构建成融合表达质粒,转化宿主菌BL21经IPTG诱导后融合基因得到了表达,表达产物可用谷胱甘肽sepharose 4B RediPack亲和层析柱纯化。用5份SARS患者康复期血清对表达融合蛋白进行ELISA和免疫印迹试验,分析融合蛋白的免疫反应性,结果表明重组融合抗原在ELISA和免疫印迹试验中与5份SARS患者康复期血清均具有良好的免疫反应性。试验结果提示该SARS-CoV S蛋白第539～630氨基酸残基区域可作为SARS的诊断抗原。     

Characterization of bat coronaviruses: a latent global threat

It has been speculated that bats serve as reservoirs of a huge variety of emerging coronaviruses (CoVs) that have been responsible for severe havoc in human health systems as well as negatively affecting human economic and social systems. A prime example is the currently active severe acute respiratory syndrome (SARS)-CoV2, which presumably originated from bats, demonstrating that the risk of a new outbreak of bat coronavirus is always latent. Therefore, an in-depth investigation to better comprehend bat CoVs has become an important issue within the international community, a group that aims to attenuate the consequences of future outbreaks. In this review, we present a concise introduction to CoVs found in bats and discuss their distribution in Southeast Asia. We also discuss the unique adaptation features in bats that confer the ability to be a potential coronavirus reservoir. In addition, we review the bat coronavirus-linked diseases that have emerged in the last two decades. Finally, we propose key factors helpful in the prediction of a novel coronavirus outbreak and present the most recent methods used to forecast an evolving outbreak.     

SARS-CoV2 spike protein gene variants with N501T and G142D mutation–dominated infections in mink in the United States

Large numbers of mink have been infected with SARS-CoV2 containing the spike protein Y453F mutation in Europe, causing zoonosis concerns. To evaluate the genetic characteristics of the U.S. and Canadian mink–derived SARS-CoV2 sequences, we analyzed all animal-derived (977) and all Canadian (19,529) and U.S. (173,277) SARS-CoV2 sequences deposited in GISAID from December 2019 to March 12, 2021, and identified 2 dominant novel variants, the N501T-G142D variant and N501T-G142D-F486L variant, in the U.S. mink–derived SARS-CoV2 sequences. These variants were not found in mink from Canada or other countries. The Y453F mutation was not identified in the mink-derived sequences in the United States and Canada. The N501T mutation occurred 2 mo earlier in humans than in mink in the United States, and the novel N501T-G142D and N501T-G142D-F486L variants were found in humans prior to mink. Our results suggest that the novel SARS-CoV2 variants may have evolved during human infection and were then transmitted to mink populations in the United States.     

SARS冠状病毒与动物冠状病毒的分子生物学研究进展

冠状病毒是一类有囊膜的单股正链RNA病毒,经常引起动物和人群的感染。2003年,全世界多个国家和地区暴发了重症急性呼吸系统综合征,这也使冠状病毒的研究成为病毒学领域中最为关注的问题。文章主要叙述了动物冠状病毒和SARS冠状病毒的分子生物学特性及其最新研究进展。同时,也对SARS冠状病毒和其他冠状病毒的相关性进行了比较分析。     

Expression of feline angiotensin converting enzyme 2 and its interaction with SARS-CoV S1 protein

Feline angiotensin converting enzyme 2 (fACE2) gene was amplified from domestic cat lung with RT-PCR, cloned and sequenced. The complete coding region is 2418 bp in length and is the closest to human ACE2 among known ACE2 homologs of non-primate animals. The N terminal fragment 19– 367 aa was expressed in Escherishia coli. Both Western blotting and ELISA demonstrated that fACE2 could react with SARS-CoV S1 protein as efficiently as ACE2 of Vero E6 cells did.     

Swine coronaviruses (SCoVs) and their emerging threats to swine population,inter-species transmission,exploring the susceptibility of pigs for SARS-CoV-2 and zoonotic concerns

Swine coronaviruses (SCoVs) are one of the most devastating pathogens affecting the livelihoods of farmers and swine industry across the world. These include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine delta coronavirus (PDCoV). Coronaviruses infect a wide variety of animal species and humans because these are having single stranded-RNA that accounts for high mutation rates and thus could break the species barrier. The gastrointestinal, cardiovascular, and nervous systems are the primary organ systems affected by SCoVs. Infection is very common in piglets compared to adult swine causing high mortality in the former. Bat is implicated to be the origin of all CoVs affecting animals and humans. Since pig is the only domestic animal in which CoVs cause a wide range of diseases; new coronaviruses with high zoonotic potential could likely emerge in the future as observed in the past. The recently emerged severe acute respiratory syndrome coronavirus virus-2 (SARS-CoV-2), causing COVID-19 pandemic in humans, has been implicated to have animal origin, also reported from few animal species, though its zoonotic concerns are still under investigation. This review discusses SCoVs and their epidemiology, virology, evolution, pathology, wildlife reservoirs, interspecies transmission, spill-over events and highlighting their emerging threats to swine population. The role of pigs amid ongoing SARS-CoV-2 pandemic will also be discussed. A thorough investigation should be conducted to rule out zoonotic potential of SCoVs and to design appropriate strategies for their prevention and control.     

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,...     

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%.     

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.     

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。     

大熊猫犬冠状病毒部分纤突蛋白基因的扩增与序列分析

大熊猫是我国特有的濒危物种，近年来人们在研究其生态环境、生理特点、繁殖性能、人工繁育等方面取得了显著成绩。然而，大熊猫疾病，特别是传染性疾病对大熊猫的生存构成了严重的威胁。     

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 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.     

Full-length ORF2 sequence-based genetic and phylogenetic characterization of Korean feline caliciviruses

Feline calicivirus (FCV) is a highly infectious pathogen in cats and widely distributed worldwide with high genetic variation. Full-length open reading frame 2 of 5 from recently isolated Korean FCV isolates were sequenced and compared with those of global isolates. The results of phylogenetic analysis supported dividing global FCV isolates into two genogroups (type I and II) and demonstrated the presence of genogroup II in Korea, indicating their geographic spread in East Asia. High sequence variations in region E of the FCV isolates emphasizes that a novel vaccine needs to be developed to induce protective immunity against various FCV strains.     

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.     

犬2型腺病毒毒株纤突蛋白基因的比较分析

根据GenBank中犬腺病毒（canine adenovirus,CAV)纤突基因序列，设计合成2对引物。用PCR方法，对犬2型腺病毒沈阳分离株（CAV-2SY株）第5代强毒（SY-V5）、经蚀斑克隆驯化的第60代毒弱毒（SY-V60）、SY-V60感犬体上传5代的回收毒（SY-CP5）、美国疫苗毒（US-V20）等4个毒株的纤突基因进行了扩增，PCR产物经纯化后进行基因序列测定，测序结果经拼接后得到1个由1629个核苷酸组成的纤突蛋白全基因序列，编码543个氨基酸。犬2型腺病毒与GenBank中的标准的CAV-2强毒株（Toronto A26/61)纤突蛋白基因序列的比较结果表明：CAV-2SY株强毒株与Tornto A26/61株相同；SY-V60株与SY-CP5相同，与驯化前的SY-V5相比，在1134位发生碱基颠换，编码的氨基酸由原来的天冬酰氨（N）变为赖氨酸（K），并导致N-X-S/T潜在糖基化位点发生改变，US-V20该部位与发生同样的突变，本试测定和比较的CAV-2强毒株有5个潜在的N-联糖基化位点，弱毒株仅有4个位点；US-V20与TorontoA26/61差异较大，有11个碱基发生变化，导致8个氨基酸的变异。     

犬冠状病毒的分离鉴定及其纤突蛋白基因的克隆与序列比较

以犬胎原代细胞，采用同步接种、带毒传代和添加新细胞的方法，从１例腹泻犬脾脏和１例临床健康犬肠内容物中分离出２株犬冠状病毒（ＣＣＶＹＳ１，ＣＣＶ ＣＩ１）。该２株病毒可使ＣＲＦＫ细胞出现典型的细胞融合病变，与从美国引进的ＣＣＶＮＬ－１８参考株形成的细胞病变相似；电镜负染和超薄切片观察，分离毒株细胞培养物中均含有典型的冠状病毒粒子，并形成胞浆内包涵体；经理化学、生物学鉴定，濉有ＣＣＶ的特性；间接免疫荧