Characterization of a recombinant canine coronavirus with a distinct receptor-binding (S1) domain

Canine alphacoronaviruses (CCoV) exist in two serotypes, type I and II, both of which can cause severe gastroenteritis. Here, we characterize a canine alphacoronavirus, designated CCoV-A76, first isolated in 1976. Serological studies show that CCoV-A76 is distinct from other CCoVs, such as the prototype CCoV-1-71. Efficient replication of CCoV-A76 is restricted to canine cell lines, in contrast to the prototypical type II strain CCoV-1-71 that more efficiently replicates in feline cells. CCoV-A76 can use canine aminopeptidase N (cAPN) receptor for infection of cells, but was unable to use feline APN (fAPN). In contrast, CCoV-1-71 can utilize both. Genomic analysis shows that CCoV-A76 possesses a distinct spike, which is the result of a recombination between type I and type II CCoV, that occurred between the N- and C-terminal domains (NTD and C-domain) of the S1 subunit. These data suggest that CCoV-A76 represents a recombinant coronavirus form, with distinct host cell tropism.     

ANG-1基因重组腺病毒载体的构建及其转染骨髓问充质干细胞的实验研究

目的构建携带大鼠血管生成素．1（angiopoietin-1,ANG．1）基因的重组腺病毒载体,并检测其转染对大鼠骨髓间充质干细胞（bone marrow mesenchymal stemcells,BMSCs）活力的影响。方法RT-PCR法获取大鼠ANG-1基因并亚克隆至穿梭质粒pAdTrack-CMV,经测序无误后与骨架质粒pAdEasy-1在BJ5183中同源重组。重组质粒pAdEasy-1-ANG-1经鉴定后转染293细胞进行病毒包装扩增。体外转染BMSCs,检测转染后BMSCs中ANG-1的表达。MTT法评估常氧及缺氧环境下ANG-1对BMSCs的影响。结果重组腺病毒载体pAdEasy-1-ANG-1经测序及酶切鉴定正确;BMSCs经转染ANG-1基因后表达ANG-1。MTT法检测提示常氧及缺氧条件下转染前后BMSCs活性的差异均无统计学意义（缺氧组与缺氧下转染组相比,P〉0-05;常氧组与常氧下转染组相比,P〉0-05）。结论成功构建大鼠ANG-1基因重组腺病毒载体,且其转染在体外不影响BMSCs的活性。     

Analysis of recombination and natural selection in human enterovirus 71

The development of effective vaccines and antiviral prophylaxis against human enterovirus 71 (EV71) has been hampered by the extensive antigenic diversity of the virus. To gain new insights into the evolutionary processes that create this genetic diversity, the TreeOrder Scan Method and RDP program were employed to detect recombination events in the genome, and then parsimony-based and maximum-likelihood-based methods were used to detect natural selection effects on viral proteins. Recombination analysis provided strong evidence for recombination events in the majority of the sequences analyzed. Recombination events were found to be distributed nonrandomly with the highest frequency at the 3D region. Furthermore, positive selection was only detected at site 145 of VP1 by the maximum likelihood-based method. These results reveal that EV71 proteins are extensively influenced by stabilizing selection. We conclude that recombination may play a more important role than positive selection in the formation of genetic diversity.     

Insertion of host DNA into PVL-encoding phages of the Staphylococcus aureus lineage ST80 by intra-chromosomal recombination

Temperate bacteriophages play a critical role in the pathogenicity of the human pathogen Staphylococcus aureus by mediating positive lysogenic conversion for different virulence factors such as Panton-Valentine leukocidin (PVL) or by interrupting chromosomal virulence genes. PVL-encoding phages are integrated in the S. aureus genome within a conserved ORF which is surrounded by a cluster of tandemly repeated genes. Here we demonstrate that in S. aureus clonal complex ST80 strains PVL-phage induction led to the acquisition of host DNA into the phage genome probably due to a homologous recombination event between direct repeats of the two paralogous genes adjacent to the phage integration site. Phage excision was accompanied by an additional chromosomal deletion in this region. This so far unrecognized mechanism of DNA uptake into the phage genome may play an important role in the co-evolution of phages and bacteria.     

High throughput sequence analysis reveals hitherto unreported recombination in the genus Norovirus

Viruses of the Norovirus genus (Caliciviridae family) are a major cause of human gastroenteritis. In some viruses, recombination is an important evolutionary process and therefore we should try to discover the quantity and characteristics of such events in Noroviruses. In order to identify recombination events, multiple sequence alignments were assembled from publicly available strains, and were tested using RAT, a recently developed software tool. Strains identified by RAT as putative recombinants were tested further, using a phylogenetic approach, the LARD software, and a Monte Carlo method, to gain additional support for their status. The identification of two previously described recombinants, WUG1 and Snow Mountain, was made. Furthermore, three instances of hitherto unreported recombination implicating Norovirus strains MD 145-12, Gifu'96 and Saitama U4 were found, with good statistical support for the latter two of these cases. Lordsdale-like viruses were highlighted as major contributors to recombination events during Norovirus evolution. Finally, the relevance of recombinants to the worldwide transmission of Norovirus is discussed.     

Cross-species recombination in the haemagglutinin gene of canine distemper virus

Canine distemper virus (CDV) has high prevalence in the world dog population and poses an important conservation threat to many carnivore species. In this study, extensive phylogenetic and recombination analyses were performed on all available complete haemagglutinin gene sequences and a strain (AF178038) isolated from giant panda was identified as putative recombinant. Interestingly, the mosaic was produced by recombination between genotypes European wildlife and Asia-1 and the recombination event involves viruses infecting different host species. This finding may have important implications for the evolution of CDV.     

小鼠NMNAT1基因的过表达和RNAi重组慢病毒的构建包装和检测

目的:针对小鼠烟酰胺单核苷酸腺苷酰转移酶1( NMNAT1)基因构建质粒并进行慢病毒包装,同时检测其表达水平和干扰效率,为进一步探讨该基因的功能提供研究工具和实验基础.方法:根据NMNAT1基因信息,用慢病毒载体pLenti6构建三种重组质粒,分别包含NMNAT1 cDNA全长、一个针对NMNAT1的小干扰序列和一个用于干扰对照的阴性序列.把这些质粒包装进慢病毒载体,并检测病毒滴度,再用慢病毒感染Hela细胞检测NMNAT1表达量和RNA干扰效率.结果:测序结果证明目的序列正确地插入到载体内.通过qPCR方法鉴定慢病毒包装成功,病毒滴度均为2×108 TU/ml以上.表达NMNAT1的重组慢病毒感染Hela细胞,该细胞能够高水平表达NMNAT1蛋白,而携带RNAi序列的慢病毒能够显著抑制其表达,干扰效率在70％以上.结论:针对NMNAT1的过表达和RNAi重组慢病毒制备成功,为进一步研究NMNAT1基因的功能和用慢病毒进行基因治疗提供了良好的研究工具.     

Molecular characterization of infectious bronchitis virus in Southwestern China for the protective efficacy evaluation of four live vaccine strains

The high mutation rate of infectious bronchitis virus (IBV) poses a significant threat to the protective efficacy of vaccines. This study aimed at analyzing the S1 genes of IBV field strains isolated in Southwestern China from 2018 to 2020, assessing the pathogenicity of four dominating strains, and evaluating the protective efficacy of four commercial vaccine strains against the endemic representative strains. Thirty-two field strains of IBV were isolated in Southwestern China from 2018 to 2020. Phylogenetic analysis of their S1 genes revealed the nucleotide homology ranged from 64.6% to 100%, and belonged to five genotypes [GI-19 (QX, 53.13%), GI-28 (LDT3-A,15.63%), GI-7 (TW, 12.50%), GI-1 (Mass, 6.23%), GVI-1 (TC07-2, 6.25%)], and two variant groups [variant-3 (3.13%) and variant-5 (3.13%)]. Recombination events between field and vaccine strains or between field strains were identified in the S1 genes of eight IBV field strains. The CK/CH/YNKM/191128 and CK/CH/CQBS/191203 strains of GI-19 showed morbidity rates of 66.7% and 73.7%, respectively, and mortality rates of 13.3% and 33.3%, respectively. Besides, the CK/CH/SCYC/191030 and CK/CH/GZGY/191021 strains of GI-28 caused morbidity rates of 60% and 86.7%, respectively, and mortality rates of 33.3%. The protective efficacy of the four commercial live vaccine strains (4/91, FNO-E55, LDT3-A, and QXL87) ranged from 70% ? 100% and reduced tissue lesions against CK/CH/GZGY/191021 and CK/CH/CQBS/191203 strains. LDT3-A strain was the most effective one but still could not completely prohibit IBV shedding. These findings provide a reference for IBV molecular evolution analysis and control of IB.     

Consensus summary report for CEPI/BC March 12–13, 2020 meeting: Assessment of risk of disease enhancement with COVID-19 vaccines

A novel coronavirus (CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 in Wuhan, China and has since spread as a global pandemic. Safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of Coronavirus Disease 2019 (COVID-19) disease and ease the major economic impact. There has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. However, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. A syndrome of “disease enhancement” has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. Animal models allowed scientists to determine the underlying mechanism for the former in the case of Respiratory syncytial virus (RSV) vaccine and have been utilized to design and screen new RSV vaccine candidates. Because some Middle East respiratory syndrome (MERS) and SARS-CoV-1 vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for SARS-CoV-2 vaccines. To address this challenge, the Coalition for Epidemic Preparedness Innovations (CEPI) and the Brighton Collaboration (BC) Safety Platform for Emergency vACcines (SPEAC) convened a scientific working meeting on March 12 and 13, 2020 of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. This report summarizes the evidence presented and provides considerations for safety assessment of COVID-19 vaccine candidates in accelerated vaccine development.     

Complementarity between RNA dimerization elements favors formation of functional heterozygous murine leukemia viruses

The cis-elements that direct packaging and dimerization of retroviral RNAs overlap, and it has been suggested that dimerization is required for RNA packaging. This also implies that heterodimerization would be necessary for co-packaging and recombination. Moreover, co-packaging of distinct RNAs may be reduced if incapable of heterodimerizing. In this study, we have designed a novel two-vector rescue system in which co-packaging and interstrand transfer are necessary for transduction. Thus, the rescue titer is a measure of the ability of a given vector combination to co-package and subsequently generate a provirus. In the current MLV-based set-up, we explored Akv- and MLV-like-endogenous virus (MLEV)-derived vectors with modulated dimerization signals. Results show that rescue is influenced by competition at the level of RNA packaging, as well as complementarity between dimerization elements. Altogether, the results support the hypothesis that complementarity between dimerization elements may favor co-packaging of distinct retroviral RNAs.