Effect of Myxovirus Infection on Synthesis of Cellular Ribonucleic Acid

Ribonucleic acid (RNA) synthesis of chick embryo fibroblasts was inhibited by two members of the myxovirus group, Newcastle disease virus (NDV) and fowl plague virus. It was also found that cellular deoxyribonucleic acid-dependent RNA polymerase was inhibited by a cytoplasmic factor induced by NDV infection.     

Myxovirus Infections in Acute Laryngotracheobronchitis,Toronto, 1961-62

Virological investigations were undertaken on nasopharyngeal secretions from 186 children who were admitted to the Hospital for Sick Children, Toronto, with acute laryngotracheobronchitis between December 1961 and March 1962. Patients who required tracheotomy for relief of respiratory obstruction were excluded from this series. Myxoviruses which were isolated from 47 patients included the following antigenic types: 29 Parainfluenza-1, four Parainfluenza-2, five Parainfluenza-3 and nine Influenza B. The average duration of hospitalization, 3.0 days, was identical for the 47 virus-positive patients and the entire group of 186 patients. Ages of 135 patients were less than three years.Between November 1960 and March 1962, myxoviruses were isolated from 150 of 445 laryngotracheobronchitis patients. The dominant strain, Parainfluenza-1, was isolated from 119 patients who became ill between November 1960 and April 1961, and between October 1961 and March 1962. Parainfluenza-3 virus was isolated irregularly from patients during both cool and warm months. Influenza B and Parainfluenza-2 isolations were made only during 1961-62 winter. Rising antibody levels in paired sera from 20 of 23 Parainfluenza-1 virus excretors and in four patients who yielded Parainfluenza-3 virus confirmed infection with these agents.     

Pathogen-driven Adaptive Evolution of Myxovirus Resistance (Mx) Genes in Fishes

Myxovirus resistance (Mx) proteins, which belong to the dynamin super-family, are known to inhibit RNA viral replication in a wide range of taxonomic groups, including fishes. Given their crucial role in host immune defense, the key amino acid residues in the GTP effector domain (GED) near the C-terminus are expected to evolve adaptively in order to protect the host against invading viral pathogens. The present study reveals the role of recombination and positive selection in the evolution of Mx proteins in fishes. While the GTP-binding domain in the N-terminal domain has experienced purifying selection, several amino acid residues in GED have evolved under positive selection, thus indicating adaptive evolution. Given the antiviral activity of GED, the adaptive evolutionary changes that were observed in this region are therefore predicted to be pathogen-driven.     

Dual Myxovirus Screen Identifies a Small-Molecule Agonist of the Host Antiviral Response

As we are confronted with an increasing number of emerging and reemerging viral pathogens, the identification of novel pathogen-specific and broad-spectrum antivirals has become a major developmental objective. Targeting of host factors required for virus replication presents a tangible approach toward obtaining novel hits with a broadened indication range. However, the identification of developable host-directed antiviral candidates remains challenging. We describe a novel screening protocol that interrogates the myxovirus host-pathogen interactome for broad-spectrum drug candidates and simultaneously probes for conventional, pathogen-directed hits. With resource efficiency and pan-myxovirus activity as the central developmental parameters, we explored coscreening against two distinct, independently traceable myxoviruses in a single-well setting. Having identified a pair of unrelated pathogenic myxoviruses (influenza A virus and measles virus) with comparable replication kinetics, we observed unimpaired coreplication of both viruses, generated suitable firefly and Renilla luciferase reporter constructs, respectively, and validated the protocol for up to a 384-well plate format. Combined with an independent counterscreen using a recombinant respiratory syncytial virus luciferase reporter, implementation of the protocol identified candidates with a broadened antimyxovirus profile, in addition to pathogen-specific hits. Mechanistic characterization revealed a newly discovered broad-spectrum lead that does not block viral entry but stimulates effector pathways of the innate cellular antiviral response. In summary, we provide proof of concept for the efficient discovery of broad-spectrum myxovirus inhibitors in parallel to para- and orthomyxovirus-specific hit candidates in a single screening campaign. The newly identified compound provides a basis for the development of a novel broad-spectrum small-molecule antiviral class.     

Growth of Myxovirus parainfluenza type 3 in organ cultures of guinea pig tissue

Craighead, J. E. (Harvard Medical School, Boston, Mass.). Growth of Myxovirus parainfluenza type 3 in organ cultures of guinea pig tissue. J. Bacteriol. 92:751-761. 1966.-Organ cultures of adult guinea pig nasal mucosa, lung, and pleura were infected with Myxovirus parainfluenza type 3. Observations were made on the growth of virus at intervals after inoculation. An inoculum of 10(2.5) tissue culture infectious doses (tcid(50)) initiated infection in each of the tissues. Cultures of nasal mucosa yielded up to 10(6.0)tcid(50) per 6 hr for periods of as long as 2 weeks. Virus production was not affected by the "immune" status of the animal used as a source of tissue. Introduction of antiserum into the medium appeared to suppress virus release but failed to "cure" the infection. Interferon was not detected in fluids bathing the nasal mucosa. Cultured fragments of lung produced virus for 28 days after inoculation. As much as 10(5.0)tcid(50) per 6 hr was released by the tissue. Pleural mesothelial cells lining the diaphragm yielded up to 10(6.0)tcid(50) per 6 hr over a 14-day period. Histological sections showed that the tissues retained differentiated morphological features during maintenance in vitro. Cytological changes unequivocally associated with infection were not recognized. The techniques described give reproducible, quantitative results. Organ cultures are feasible for the study of virus growth and cytopathology in differentiated tissues.     

Systemic Dissemination by Intrarectal Infection with Listeria monocytogenes in Mice

Orally ingested Listeria monocytogenes is known to penetrate into Peyer's patches (PP) and translocate to the spleen and liver. Herein, extraintestinal dissemination of the bacterium independent of PP was investigated. Dissemination of Listeriae to the spleen and liver was observed in intrarectally infected mice as well as in intragastrically infected animals in spite that no Listeriae were detected in the small intestines of mice infected intrarectally. Decreased numbers of intestinal intraepithelial lymphocytes (iIEL) and increased numbers of lymphocytes in the contents of the small and large intestines were observed after intragastric infection and in the large intestine after intrarectal infection, giving the assumption that the leakage of iIEL caused by injury of epithelial layers in intestines might occur during infection. These results suggest that L. monocytogenes might be able to disseminate through small and large intestines in part by a PP-independent mechanism.     

Biofilm as an Environment for Dissemination of stx Genes by Transduction

Dissemination of Shiga toxin (Stx)-encoding bacteriophages is the most likely mechanism for the spread of Stx-encoding genes and the emergence of new Stx-producing Escherichia coli (STEC). Biofilm has been reported to be a place where horizontal gene transfer by plasmid conjugation and DNA transformation may occur, and in this study, horizontal gene transfer by transduction has been demonstrated. Transfer of Stx-encoding bacteriophages to potentially pathogenic E. coli in biofilm was observed at both 20°C and 37°C. The infection rates were higher at 37°C than at 20°C. To our knowledge, this study is the first to show lateral gene transfer in biofilm mediated by a temperate bacteriophage. The study shows that the biofilm environment can be suitable for transduction events and can thereby be an environment for the emergence of new pathogenic E. coli.     

Dissemination of Spotted Fever Rickettsia Agents in Europe by Migrating Birds

Migratory birds are known to play a role as long-distance vectors for many microorganisms. To investigate whether this is true of rickettsial agents as well, we characterized tick infestation and gathered ticks from 13,260 migratory passerine birds in Sweden. A total of 1127 Ixodes spp. ticks were removed from these birds and the extracted DNA from 957 of them was available for analyses. The DNA was assayed for detection of Rickettsia spp. using real-time PCR, followed by DNA sequencing for species identification. Rickettsia spp. organisms were detected in 108 (11.3%) of the ticks. Rickettsia helvetica, a spotted fever rickettsia associated with human infections, was predominant among the PCR-positive samples. In 9 (0.8%) of the ticks, the partial sequences of 17kDa and ompB genes showed the greatest similarity to Rickettsia monacensis, an etiologic agent of Mediterranean spotted fever-like illness, previously described in southern Europe as well as to the Rickettsia sp.IrITA3 strain. For 15 (1.4%) of the ticks, the 17kDa, ompB, gltA and ompA genes showed the greatest similarity to Rickettsia sp. strain Davousti, Rickettsia japonica and Rickettsia heilongjiangensis, all closely phylogenetically related, the former previously found in Amblyomma tholloni ticks in Africa and previously not detected in Ixodes spp. ticks. The infestation prevalence of ticks infected with rickettsial organisms was four times higher among ground foraging birds than among other bird species, but the two groups were equally competent in transmitting Rickettsia species. The birds did not seem to serve as reservoir hosts for Rickettsia spp., but in one case it seems likely that the bird was rickettsiemic and that the ticks had acquired the bacteria from the blood of the bird. In conclusion, migratory passerine birds host epidemiologically important vector ticks and Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents and their diseases.     

The Biochemistry of Cancer Dissemination

Abstract

The progression of a tumor cell from one of benign delimited proliferation to invasive and metastatic growth is the major cause of poor clinical outcome of cancer patients. Recent research has revealed that this complex process requires many components for successful dissemination and growth of the tumor cell at secondary sites. These include angiogenesis, enhanced extracellular matrix degradation via tumor and host-secreted proteases, tumor cell migration, and modulation of tumor cell adhesion. Each individual component is multifaceted and is discussed within this review with respect to historical and recent findings. The identification of components and their interrelationship have yielded new therapeutic targets leading to the development of agents that may prove effective in the treatment of cancer and its metastatic progression.     

Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the IncX3-Type Plasmid

The emergence and spread of NDM-1-producing Enterobacteriaceae have resulted in a worldwide public health risk that has affected some provinces of China. China is an exceptionally large country, and there is a crucial need to investigate the epidemic of bla _NDM-1-positive Enterobacteriaceae in our province. A total of 186 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected in a grade-3 hospital in Zhejiang province. Carbapenem-resistant genes, including bla _KPC, bla _IMP, bla _VIM, bla _OXA-48 and bla _NDM-1 were screened and sequenced. Ninety isolates were identified as harboring the bla _KPC-2 genes, and five bla _NDM-1-positive isolates were uncovered. XbaI-PFGE revealed that three bla _NDM-1-positive K. pneumoniae isolates belonged to two different clones. S1-PFGE and southern blot suggested that the bla _NDM-1 genes were located on IncX3-type plasmids with two different sizes ranging from 33.3 to 54.7 kb (n=4) and 104.5 to 138.9 kb (n=1), respectively, all of which could easily transfer to Escherichia coli by conjugation and electrotransformation. The high-throughput sequencing of two plasmids was performed leading to the identification of a smaller 54-kb plasmid, which had high sequence similarity with a previously reported pCFNDM-CN, and a larger plasmid in which only a 7.8-kb sequence of a common gene environment around bla _NDM-1 (bla _NDM-1-trpF- dsbC-cutA1-groEL-ΔInsE,) was detected. PCR mapping and sequencing demonstrated that four smaller bla _NDM-1 plasmids contained a common gene environment around bla _NDM-1 (IS5-bla _NDM-1-trpF- dsbC-cutA1-groEL). We monitored the CRE epidemic in our hospital and determined that KPC-2 carbapenemase was a major risk to patient health and the IncX3-type plasmid played a vital role in the spread of the bla _NDM-1 gene among the CRE.     

Dissemination of the Transmissible Quinolone-Resistance Gene qnrS1 by IncX Plasmids in Nigeria

The plasmid-encoded quinolone resistance gene qnrS1 was recently found to be commonly associated with ciprofloxacin resistance in Nigeria. We mapped the qnrS1 gene from an Escherichia coli isolate obtained in Nigeria to a 43.5 Kb IncX2 plasmid. The plasmid, pEBG1, was sufficient to confer ciprofloxacin non-susceptibility, as well as tetracycline and trimethoprim resistance, on E. coli K-12. Deletion analysis confirmed that qnrS1 accounted for all the ciprofloxacin non-suceptibility conferred by pEBG1 and tetracycline and trimethoprim resistance could be attributed to tetAR and dfrA14 genes respectively. While it contained a complete IncX conjugation system, pEBG1 was not self-transmissible likely due to an IS3 element inserted between the pilX5 and pilX6 genes. The plasmid was however efficiently mobilizable. pEBG1 was most similar to another qnrS1-bearing IncX2 plasmid from Nigeria, but both plasmids acquired qnrS1 independently and differ in their content of other resistance genes. Screening qnrS1–positive isolates from other individuals in Nigeria revealed that they carried neither pEBG1 nor pNGX2-QnrS1 but that IncX plasmids were prevalent. This study demonstrates that the IncX backbone is a flexible platform that has contributed to qnrS1 dissemination in Nigeria.