Molecular mechanisms of measles virus persistence.

As measles virus causes subacute sclerosing panencephalitis and measles inclusion body encephalitis due to its ability to establish human persistent infection, without symptoms for the time between the acute infection and the onset of clinical symptoms, it has been the paradigm for a long term persistent as opposed to chronic infection by an RNA virus. We have reviewed the mechanisms of persistence of the virus and discuss specific mutations associated with CNS infection affecting the matrix and fusion protein genes. These are placed in the context of our current understanding of the viral replication cycle. We also consider the proposed mechanisms of persistence of the virus in replicating cell cultures and conclude that no general mechanistic model can be derived from our current state of knowledge. Finally, we indicate how reverse genetics approaches and the use of mouse models with specific knock-out and knock-in modifications can further our understanding of measles virus persistence.

In vivo tropism of attenuated and pathogenic measles virus expressing green fluorescent protein in macaques

The global increase in measles vaccination has resulted in a significant reduction of measles mortality. The standard route of administration for the live-attenuated measles virus (MV) vaccine is subcutaneous injection, although alternative needle-free routes, including aerosol delivery, are under investigation. In vitro, attenuated MV has a much wider tropism than clinical isolates, as it can use both CD46 and CD150 as cellular receptors. To compare the in vivo tropism of attenuated and pathogenic MV, we infected cynomolgus macaques with pathogenic or attenuated recombinant MV expressing enhanced green fluorescent protein (GFP) (strains IC323 and Edmonston, respectively) via the intratracheal or aerosol route. Surprisingly, viral loads and cellular tropism in the lungs were similar for the two viruses regardless of the route of administration, and CD11c-positive cells were identified as the major target population. However, only the pathogenic MV caused significant viremia, which resulted in massive virus replication in B and T lymphocytes in lymphoid tissues and viral dissemination to the skin and the submucosa of respiratory epithelia. Attenuated MV was rarely detected in lymphoid tissues, and when it was, only in isolated infected cells. Following aerosol inhalation, attenuated MV was detected at early time points in the upper respiratory tract, suggesting local virus replication. This contrasts with pathogenic MV, which invaded the upper respiratory tract only after the onset of viremia. This study shows that despite in vitro differences, attenuated and pathogenic MV show highly similar in vivo tropism in the lungs. However, systemic spread of attenuated MV is restricted.

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.

Recent mumps outbreaks in vaccinated populations: no evidence of immune escape.

Recently, numerous large-scale mumps outbreaks have occurred in vaccinated populations. Clinical isolates sequenced from these outbreaks have invariably been of genotypes distinct from those of vaccine viruses, raising concern that certain mumps virus strains may escape vaccine-induced immunity. To investigate this concern, sera obtained from children 6 weeks after receipt of measles, mumps, and rubella (MMR) vaccine were tested for the ability to neutralize a carefully selected group of genetically diverse mumps virus strains. Although the geometric mean neutralizing antibody titer of the sera was lower against some virus strains than others, all viruses were readily neutralized, arguing against immune escape.

Anticipatory Planning Reveals Segmentation of Cortical Motor Output During Action Observation

It has been argued that the variation in brain activity that occurs when observing another person reflects a representation of actions that is indivisible, and which plays out in full once the intent of the actor can be discerned. We used transcranial magnetic stimulation to probe the excitability of corticospinal projections to 2 intrinsic hand muscles while motions to reach and grasp an object were observed. A symbolic cue either faithfully indicated the required final orientation of the object and thus the nature of the grasp that was required, or was in conflict with the movement subsequently displayed. When the cue was veridical, modulation of excitability was in accordance with the functional role of the muscles in the action observed. If however the cue had indicated that the alternative grasp would be required, modulation of output to first dorsal interosseus was consistent with the action specified, rather than the action observed-until the terminal phase of the motion sequence during which the object was seen lifted. Modulation of corticospinal output during observation is thus segmented-it progresses initially in accordance with the action anticipated, and if discrepancies are revealed by visual input, coincides thereafter with that of the action seen.

Determination of Spontaneous Mutation Frequencies in Measles Virus under Nonselective Conditions

There is a paradox between the remarkable genetic stability of measles virus (MV) in the field and the high mutation rates implied by the frequency of the appearance of monoclonal antibody escape mutants generated when the virus is pressured to revert in vitro (S. J. Schrag, P. A. Rota, and W. J. Bellini, J. Virol. 73: 51-54, 1999). We established a highly sensitive assay to determine frequencies of various categories of mutations in large populations of wild-type and laboratory-adapted MVs using recombinant viruses containing an additional transcription unit (ATU) encoding enhanced green fluorescent protein (EGFP). Single and double mutations were made in the fluorophore of EGFP to ablate fluorescence. The frequencies of reversion mutants in the population were determined by measuring the appearance of fluorescence indicating a revertant virus. This allows mutation rates to be measured under nonselective conditions, as phenotypic reversion to fluorescence requires only either a single-or a double-nucleotide change and amino acid substitution, which does not affect the length of the nonessential reporter protein expressed from the ATU. Mutation rates in MV are the same for wild-type and laboratory-adapted viruses, and they are an order of magnitude lower than the previous measurement assessed under selective conditions. The actual mutation rate for MV is approximately 1.8 x 10(-6) per base per replication event. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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.

Physical activity assessment in cystic fibrosis: A position statement

Background

The aim of this position statement was to inform the choice of physical activity tools for use within CF research and clinical settings.

Methods

A systematic review of physical activity tools to explore evidence for reliability, validity, and responsiveness. Narrative answers to “four key questions” on motion sensors, questionnaires and diaries were drafted by the core writing team and then discussed at the Exercise Working Group in ECFS Lisbon 2013.

Results and summary

Our current position is that activity monitors such as SenseWear or ActiGraph offer informed choices to facilitate a comprehensive assessment of physical activity, and should as a minimum report on dimensions of physical activity including energy expenditure, step count and time spent in different intensities and sedentary time. The DigiWalker pedometer offers an informed choice of a comparatively inexpensive method of obtaining some measurement of physical activity. The HAES represents an informed choice of questionnaire to assess physical activity. There is insufficient data to recommend the use of one diary over another. Future research should focus on providing additional evidence of clinimetric properties of these and new physical activity assessment tools, as well as further exploring the added value of physical activity assessment in CF.