Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.

A novel tumor necrosis factor ligand superfamily member (CsTL) from Ciona savignyi: Molecular identification and expression analysis

A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi. The CsTL cDNA consisted of 995 nucleotides and encoded 281 amino acids. A conserved TNF family signature and several motifs of TNF ligand superfamily were identified in deduced amino acid sequence of CsTL. Phylogenetic analysis grouped CsTL, CiTNF (predicted TNF ligand superfamily homolog in Ciona intestinalis) and urchin TL1A with their own cluster apart from mammalian TNF alpha, LTA, TNFSF15 and fish TNFa proteins. Expression studies demonstrated that CsTL mRNA is present in all tested tissues from unchallenged ascidians and its expression was significantly upregulated in hemocytes following LIPS injection. The recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells. Present results indicated that TNF ligand superfamity molecules are present in marine invertebrates. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

The Jeryl Lynn (JL) vaccine against mumps virus (MuV) contains two components, MuV(JL5) and MuV(JL2), which differ by over 400 nt. Due to the occurrence of bias in the direction of mutation, these differences and those found in nucleotide sequences of different isolates of the minor component in the vaccine (MuV(JL2)) might be due to the effect of ADAR-like deaminases on MuV grown in tissue-cultured cells. A molecular clone Of MuV(JL2) (pMuV(JL2)) and MuV(JL2) -specific helper plasmids were constructed in order to investigate molecular interactions between MuV(JL5) and MuV(JL2), to augment the existing molecular clone Of MuV(JL)5 (pMuV(JL5)) and MuV(JL5) -specific helper plasmids. Genome and mRNA termini Of MuV(JL2) were characterized, and an unusual oligo-G insertion transcriptional editing event was detected near the F mRNA polyadenylation site of MuV(JL2), but not Of MuV(JL5). Genes encoding glycoproteins of rMuV(JL2) and rMuV(JL5) have been exchanged to characterize the oligo-G insertion, which associated with the specific sequence of the IF gene of MuV(JL2) and not with any other genes or the RNA-dependent RNA polymerase of strain MuV(JL2). The results indicate that a single G-to-A sequence change obliterates the co-transcriptional editing of the F mRNA and that this oligo-G insertion does not affect the growth of the virus.

Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee

We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6–28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.

萤火虫的荧光素酶基因克隆表达及发光行为的初步研究

本研究围绕萤火虫的荧光素酶克隆表达和发光行为两方面来进行。首先对萤火虫的发光体系和发光行为进行了概述，然后对萤火虫荧光素酶cDNA进行了克隆表达分析，探讨了日行性和夜行性萤火虫荧光素酶的差异。最后通过萤火虫的饲养和发光行为观察研究，对卵发光的意义进行了探讨。本研究克隆了三种云南萤火虫的荧光素酶基因，并成功表达出两种有活性的荧光素酶。其中日行性萤火虫云南窗萤(Pyrocoelia pygidialis)的荧光素酶基因完成了首次克隆，并在大肠杆菌中成功表达出有功能的荧光素酶。测序结果表明：P. pygidialis荧光素酶的cDNA序列有1647个碱基，编码548个氨基酸残基。系统发育分析表明云南窗萤与同属三种发光很亮的萤火虫不形成单系，在一定程度上印证了P. pygidialis以及同属微弱发光的日行性种类与持续发出很亮荧光的夜行性种类构成窗萤属两个分支的观点；并且从发光器官的形态差异初步推断这两个分支之间是独立进化的。荧光素酶的结构模拟分析表明，云南窗萤荧光素酶的N端和C端结构域之间的裂沟处存在着五个多肽环，这是已知荧光素酶推测的活性位点。日行性的云南窗萤和三种夜行性萤火虫的荧光素酶有13个不同氨基酸，位于模拟的分子结构表面。这些氨基酸的差异，有利于进一步分析日行性和夜行性萤火虫荧光素酶结构的不同。对栉角雪萤(Diaphanes pectinealis)的荧光素酶cDNA也完成了克隆表达。结果表明其cDNA序列有1647个碱基，编码548个氨基酸残基，比从基因组中获得基因序列多3个碱基。体外表达产物在底物存在时发出黄绿色光。对雌光萤科Rhagophthalmus sp.的荧光素酶基因进行了克隆和氨基酸序列比对。得出其荧光素酶基因与来自同一科的荧光素酶基因有最高的一致性为90.7％，和鞘翅目另外三个科的物种的一致性在65％以下，显示出雌光萤科独特的分类地位。通过对采自昆明郊区的云南窗萤和一种扁萤属萤火虫的饲养和发光行为的观察，得出萤火虫的卵和蛹阶段持续发微弱的光的结论，且首次发现了卵发光在发育过程中的变化。本文对卵发光行为进行了描述，并在此基础上对萤火虫卵和蛹两阶段发光的原因和意义进行了探讨。

Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Because it plays a key role in the in vitro dimerization of short HIV-1 RNA transcripts (M. Laughrea and L. Jette, Biochemistry 35:1589-1598, 1996, and references therein; M. Laughrea and L. Jette, Biochemistry 35:9366-9374, 1996, and references therein) and because dimeric RNAs may be preferably encapsidated into the HIV-1 virus, alterations of the kissing-loop hairpin might affect the in vivo dimerization and encapsidation processes. Accordingly, substitution and deletion mutations were introduced into the kissing-loop hairpin of an infectious HIV-1 molecular clone in order to produce viruses by transfection methods. The infectivity of the resulting viruses was decreased by at least 99%, the amount of genomic RNA packaged per virus was decreased by 50 to 75%, and the proportion of dimeric genomic RNA was reduced from >80 to 40 to 50%, but the dissociation temperature of the genomic RNA was unchanged. There is evidence suggesting that the deletion mutations moderately inhibited CAp24 production but had no significant effect on RNA splicing. These results are consistent with the kissing-loop model of HIV-1 RNA dimerization. In fact, because intracellular viral RNAs are probably more concentrated in transfected cells than in cells infected by one virus and because the dimerization and encapsidation processes are concentration dependent, it is likely that much larger dimerization and encapsidation defects would have been manifested within cells infected by no more than one virus.

New approach for inhibiting Rev function and HIV-1 production using the influenza virus NS1 protein.

The Rev protein of HIV-1, which facilitates the nuclear export of HIV-1 pre-mRNAs, has been a target for antiviral therapy. Here we describe a new strategy for inhibiting Rev function and HIV-1 replication. In contrast to previous approaches, we use a wild-type rather than a mutant Rev protein and covalently link this Rev sequence to the NS1 protein of influenza A virus, a protein that inhibits the nuclear export of mRNAs. The NS1 protein contains an RNA-binding domain mutation (RM), so that the only functional RNA-binding domain in the chimeric protein (NS1RM-Rev) is in the Rev protein sequence. In the presence of the NS1RM-Rev chimeric protein, HIV-1 pre-mRNAs were retained in, rather than exported from, the nucleus. In addition, this chimeric protein effectively inhibited Rev function in trans in transfection experiments and effectively inhibited the production of HIV-1 in tissue culture cells transfected with an infectious molecular clone of HIV-1 DNA. The inhibitory activities of the NS1RM-Rev chimera were at least equivalent to those of the Rev M10 mutant protein, which has been considered to be the prototype trans inhibitor of Rev function and is currently in phase I clinical trials for the treatment of AIDS patients. We discuss (i) the potential for increasing the inhibitory activity of NS1-Rev chimeras against HIV-1 and (ii) the need for additional studies to evaluate these chimeras for the treatment of AIDS.

Comparative interleukin (IL-2)/interferon IFN-gamma and IL-4/IL-10 responses during acute infection of macaques inoculated with attenuated nef-truncated or pathogenic SICmac251 virus.

Comparison of immune responses to infection by a pathogenic or a nonpathogenic immunodeficiency virus in macaques may provide insights into pathogenetic events leading to simian AIDS. This work is aimed at exploring cytokine expression during infection by simian immunodeficiency virus (SIV). We used semiquantitative reverse transcription-PCR to monitor interleukin (IL)-2/interferon (IFN)-gamma (Th1-like), and IL-4/IL-10 (Th2-like) expression in unmanipulated peripheral blood mononuclear cells (PBMCs), during the acute phase of infection of eight cynomolgus macaques (Macaca fascicularis) with a pathogenic primary isolate of SIVmac251 (full-length nef), and of four other cynomolgus macaques by an attenuated molecular clone of SIVmac251 (nef-truncated). All the monkeys became infected, as clearly shown by the presence of infected PBMCs and by seroconversion. Nevertheless, PBMC-associated virus loads and p27 antigenemia in monkeys infected by the attenuated virus clone remained lower than those observed in animals infected with the pathogenic SIVmac251 isolate. A rise of IL-10 mRNA expression occurred in both groups of monkeys coincident with the peak of viral replication. In monkeys infected with the pathogenic SIVmac251, IL-2, IL-4, and IFN-gamma mRNAs were either weakly detectable or undetectable. On the contrary, animals infected by the attenuated virus exhibited an overexpression of these cytokine mRNAs during the first weeks after inoculation. The lack of expression of these cytokines in monkeys infected with the pathogenic primary isolate may reflect early immunodeficiency.

Persistent infection of rhesus macaques with T-cell-line-tropic and macrophage-tropic clones of simian/human immunodeficiency viruses (SHIV).

To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman primate model, we have constructed infectious recombinant viruses (chimeras) between the pathogenic molecular clone of simian immunodeficiency virus (SIV) SIVmac239 and molecular clones of HIV-1 that differ in phenotypic properties controlled by the env gene. HIV-1SF33 is a T-cell-line-tropic virus which induces syncytia, and HIV-1SF162 is a macrophage-tropic virus that does not induce syncytia. A DNA fragment encoding tat, rev, and env (gp160) of SIVmac239 has been replaced with the counterpart genetic region of HIV-1SF33 and HIV-1SF162 to derive chimeric recombinant simian/human immunodeficiency virus (SHIV) strains SHIVSF33 and SHIVSF162, respectively. In the acute infection stage, macaques inoculated with SHIVSF33 had levels of viremia similar to macaques infected with SIVmac239, whereas virus loads were 1/10th to 1/100th those in macaques infected with SHIVSF162. Of note is the relatively small amount of virus detected in lymph nodes of SHIVSF162-infected macaques. In the chronic infection stage, macaques infected with SHIVSF33 also showed higher virus loads than macaques infected with SHIVSF162. Virus persists for over 1 year, as demonstrated by PCR for amplification of viral DNA in all animals and by virus isolation in some animals. Antiviral antibodies, including antibodies to the HIV-1 env glycoprotein (gp160), were detected; titers of antiviral antibodies were higher in macaques infected with SHIVSF33 than in macaques infected with SHIVSF162. Although virus has persisted for over 1 year after inoculation, these animals have remained healthy with no signs of immunodeficiency. These findings demonstrate the utility of the SHIV/macaque model for analyzing HIV-1 env gene functions and for evaluating vaccines based on HIV-1 env antigens.

Interclonal Variations in the Molecular Karyotype of Trypanosoma cruzi: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure. © 2013 Lima et al.

A multi-genome analysis approach enables tracking of the invasion of a single Russian wheat aphid (Diuraphis noxia) clone throughout the New World

This study investigated the population genetics, demographic history and pathway of invasion of the Russian wheat aphid (RWA) from its native range in Central Asia, the Middle East and Europe to South Africa and the Americas. We screened microsatellite markers, mitochondrial DNA and endosymbiont genes in 504 RWA clones from nineteen populations worldwide. Following pathway analyses of microsatellite and endosymbiont data, we postulate that Turkey and Syria were the most likely sources of invasion to Kenya and South Africa, respectively. Furthermore, we found that one clone transferred between South Africa and the Americas was most likely responsible for the New World invasion. Finally, endosymbiont DNA was found to be a high resolution population genetic marker, extremely useful for studies of invasion over a relatively short evolutionary history time frame. This study has provided valuable insights into the factors that may have facilitated the recent global invasion by this damaging pest.

Global dissemination of a multidrug resistant Escherichia coli clone

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000–2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL–resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.

The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli Clone

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.

Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the β-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29 kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.