Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavirus molecules

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.     

抗菌肽Alloferon-1串联式重组表达及其表达产物体外抗肿瘤活性的研究

目的:构建串联式表达Alloferon-1的原核重组表达系统,检测合成及重组表达的Alloferon-1体外抗肿瘤细胞活性。方法:采用连接引物PCR法,构建含6×His-EK-8×Alloferon-1-EK-6×His序列的人工融合基因;采用常规分子生物学方法克隆并构建该人工融合基因及其原核表达系统;采用SDS-PAGE和BioRad凝胶图象分析系统了解目的重组产物8×rAlloferon-1-EK表达情况和产量;采用Ni-NTA亲和层析及EK酶切和Sephadex G-50层析法提纯8×rAlloferon-1-EK及rAlloferon-1-EK;采用MTT法检测rAlloferon-1-EK体外抑制KB、SGC和HL-60肿瘤细胞增殖的作用,并与直接合成的Alloferon-1(sAlloferon-1)和Alloferon-1-EK(sAlloferon-1-EK)的抗肿瘤作用比较。结果:获得了序列正确的目的人工融合基因克隆及其原核表达系统pET42a-8×rAlloferon-1-EK-E.coliBLDE3。在IPTG诱导下,该原核重组表达系统可表达串联式目的重组蛋白8×rAlloferon-1-EK,其产量约为细菌总蛋白的30%。Ni-NTA和Sephadex G-50层析后,可分别获得8×rAlloferon-1-EK和rAlloferon-1-EK。在25~100μg/ml剂量范围内,sAlloferon-1、sAlloferon-1-EK和rAlloferon-1、rAlloferon-1-EK均有明显抑制KB、SGC和HL-60肿瘤细胞生长及增殖的效果(P<0.01),且四者抑制作用无明显差异(P>0.05)。结论:本研究成功构建了串联表达rAlloferon-1的原核表达系统,其表达产物具有与合成的Alloferon-1和Alloferon-1-EK相似的体外抗肿瘤细胞活性。     

Inversions and recombinations in mitochondrial DNA of the (SG-1) cytoplasmic mutant in two Neurospora species

Summary The mitochondrial DNAs of [SG-1] cytoplasmically-mutant and wild-type strains of Neurospora crassa and Neurospora sitophila were examined by comparative restriction endonuclease analyses. The mtDNA of N. sitophila wild type of Whitehouse differs from type II mtDNA of N. crassa by insertions of 3.3 kb in EcoRI-9, and 1.2 kb in EcoRI-3, and a deletion of 1.1 kb in EcoRI-5. These DNA heteromorphisms provided convenient markers for tracing N. crassa [SG-1] mtDNA during and after its transfer into N. sitophila. The [SG-1] cytoplasmic mutant in both N. crassa and N. sitophila has a distinctive inversion that connects the fragment EcoRI-4 with HindIII-10a. The [SG-1] mtDNA from N. crassa remained essentially intact after it was transferred by crosses into N. sitophila. In each species, a unique second inversion occured in the [SG-1] mtDNA after the transfer was made. In N. sitophila, polar recombination in heteroplasmons between [SG-1] and wild-type preferentially yields strains with mtDNAs that contain the maximum possible number of insertions in the cob and co-1 loci of the EcoRI-3 region of the mitochondrial chromosome.     

Comparison of clinical and environmental samples of Legionella pneumophila at the nucleotide sequence level

Legionella pneumophila serogroup 1 is the most common etiological agent of legionellosis. We have used clinical and environmental isolates from different sources to compare their genetic variability. We have obtained the nucleotide sequence for six protein-coding loci, included in the SBT scheme for L. pneumophila, and three intergenic regions from 127 samples, 47 of environmental origin and 80 from clinical samples. Levels of genetic variability were found to be higher in the environmental than in the clinical samples, but these did not represent a mere subset of the former. Not a single case of full identity between clinical and environmental isolates was found, which raises the possibility that only a specific subset of environmental isolates is actually capable of producing infection in humans. A phylogenetic analysis of the concatenate alignment of the nine loci sequences showed four main groups, each including clinical and environmental isolates, although their distribution was not uniform among them. The comparison of each individual gene tree with the others revealed several cases of incongruence involving samples from both origins, thus suggesting the presence of recombination in the two groups.     

表达新疆出血热病毒S基因片段的重组腺病毒特异性CTL功能测定

目的 通过表达新疆出血热病毒（XHFV）S基因片段的重组腺病毒特异性细胞毒性T淋巴细胞（CTL）功能测定，探求由XHFVS基闪片段编码的核衣壳蛋白作为抗原诱导的特异性CTL细胞对蓖组腺病毒致敏的靶细胞杀伤能力大小。方法 PCR扩增XHFVS基因的cDNA片段，将扩增后的cDNA片段克隆入腺病毒自主载体，使其成为重组腺病毒，用重组腺病毒感染Balb／C小鼠后，利用非放射性乳酸脱氰酶（LDH）释放法测定重组腺病毒的特异性CTL功能。结果 在一定比例范围内，效应细胞与靶细胞的比例越高，持异性CTL％越高。结论 由XHFVS基因片段编码的核衣壳蛋白抗原诱导的特异性CTL细胞，能特异性地杀伤重组腺病毒致敏的靶细胞；证实由腺病毒自主载体本身所表达的一些蛋白质也可诱导细胞免疫，由该载体所引发的细胞免疫对疫苗接种不利。     

核、膜蛋白的基因能抑制禽类流感病毒及其重组株的繁殖

对人流感病毒A/Udorn/72(H_3N_2)株与禽类流感病毒A/Mallard/NY/78/(H_2N_2)重组后的重组株分析表明,仅含禽类病毒的核蛋白(NP)或膜蛋白(M)的RNA片段的重组株,在松鼠猴的呼吸道繁殖是受限制的。另外。仅有禽类的RNAl和NS基因的重组株(Clone 12)在松鼠猴的气管内的繁殖也明显受限制,而只具有其中一个基因的Clone 9, Clone 2, 则限制就不明显。由此表明,禽类流感病毒的NP和M基因在宿主范围的繁殖限制中起主要作用,而RNAI和SN基因的结合,同样起着繁殖受限制作用。     

Molecular epidemiology, genome characterization, and recombination event of human parechovirus

Human Parechovirus (HPeV), a member of the Picornaviridae family, is an infectious agent mostly affecting children. There are 16 recognized genotypes which have globally spread. This study incorporated a total of 2957 nasopharyngeal (NP) swab and 759 fecal samples that were collected from different parts of Thailand. The NP of HPeV was detected in 0.4% of NP swab and 6.1% of fecal samples. The majority of HPeV infections occur in infants below the age of 2 years, while infections were detected in children above the age of 10 years as well. Various genotypes comprising 1A, 1B, 2, 3, 4, 5, 6, 10 and 14 have been characterized. This study revealed recombination events in 16 samples in which HPeV1B was shown as the highest frequency. In conclusion, HPeV can be detected in both the respiratory and GI tract. Moreover, HPeV which circulates in Thailand is highly diverse and subject to recombination.     

A tymovirus with an atypical 3′-UTR illuminates the possibilities for 3′-UTR evolution

We report the complete genome sequence of Dulcamara mottle virus (DuMV), confirming its membership within the Tymovirus genus, which was previously based on physical and pathology evidence. The 5′-untranslated region (UTR) and coding region of DuMV RNA have the typical characteristics of tymoviral RNAs. In contrast, the 3′-UTR is the longest and most unusual yet reported for a tymovirus, possessing an internal poly(A) tract, lacking a 3′-tRNA-like structure (TLS) and terminating at the 3′-end with –UUC instead of the typical –CC(A). An expressible cDNA clone was constructed and shown to be capable of producing infectious DuMV genomic RNAs with –UUC 3′-termini. A chimeric Turnip yellow mosaic virus (TYMV) genome bearing the DuMV 3′-UTR in place of the normal TLS was constructed in order to investigate the ability of the TYMV replication proteins to amplify RNAs with –UUC instead of –CC(A) 3′-termini. The chimeric genome was shown to be capable of replication and systemic spread in plants, although amplification was very limited. These experiments suggest the way in which DuMV may have evolved from a typical tymovirus, and illuminate the ways in which viral 3′-UTRs in general can evolve.     

Molecular variability and genetic structure of the population of Soybean mosaic virus based on the analysis of complete genome sequences

The complete genomes of 30 Soybean mosaic virus (SMV) isolates and strains were sequenced in this study. Together with fourteen previously reported sequences, we analyzed the genetic structure of the SMV population. Analyses of genetic diversity showed that different genomic regions of SMV are under different evolutionary constraints and that there was no significant genetic differentiation between East Asian and North American populations of SMV. Phylogenetic analyses revealed a significant correlation between phylogeny of the cylindrical inclusion (CI) gene of SMV and SMV resistance gene 3 (Rsv3)-relating pathogenicity of SMV, suggesting CI might be a pathogenic determinant in Rsv3-mediated disease response. Interestingly, recombination analyses identified 19 ‘clear’ recombination events in the SMV population. Furthermore, as several resistance-breaking strains were identified as recombinants, it appears that recombination might contribute to overcome host resistance in SMV-soybean pathosystem. Our finding suggests that recombination as well as mutation is an important evolutionary process in the genetic diversification of SMV population.