Kathryn E. Wright

Evaluation of a Neonatal Rat Model for Prediction of Mumps Virus Neurovirulence in Humans

ABSTRACT Neurovirulence of several mumps virus strains was assessed in a prototype rat neurovirulence test and compared to results obtained in the monkey neurovirulence test. The relative human neurovirulence of these strains was proportional to the severity of hydrocephalus in rats but not to lesion scores in the monkeys.

Mutations in the hemagglutinin and matrix genes of a virulent influenza virus variant, A/FM/1/47-MA, control different stages in pathogenesis

The mouse adapted strain of influenza A/FM/1/47 virus, FM-MA, has increased virulence due to mutations in HA, M1 and at least one other, unmapped, genome segment. Genetic reassortants that differ due to the HA or M1 mutations were used to define the role of these mutations in pathogenesis. Pathological changes in lungs of infected mice were assessed by hematoxylin phloxine saffron (HPS) staining, and viral infection was measured by fluorescent antibody staining of thin sections and flow cytometry of lung parenchymal cells. HA played a role in bronchiolar pathology by increasing necrosis of bronchiolar epithelium, peribronchiolar lymphocytes, and airway obstruction. The HA mutation was shown to be responsible for a 0.2 unit decreased in the pH optimum of fusion and controlled resistance to alpha and beta inhibitors of hemagglutination. Both these changes in biology may confer a replicative advantage in bronchioles seen in the first day of infection. Thus the HA mutation may have conferred a survival advantage in the extracellular lung environment. The M1 mutation resulted in improved growth in the lung and cultured cells and was associated with increases in recruitment of macrophages, spread of infection into the alveoli of the lung and interstitial pneumonia. Sequence analysis indicated that the unmapped mutation in the control of FM-MA virulence is either the K482-->R substitution in the PB2 protein or the D538-->G substitution in the PB1 protein. One or other of these mutations results in a growth advantage in infected lung but not in cultured cells as well as a further increased recruitment and infection of macrophages in the lung. Infection with virulent strains of influenza that induced increases in macrophage recruitment caused hypothermia in the mouse.

The Mumps Virus Neurovirulence Safety Test in Rhesus Monkeys: A Comparison of Mumps Virus Strains

Wild type mumps viruses are highly neurotropic and a frequent cause of aseptic meningitis in unvaccinated humans. To test whether attenuated mumps viruses used in the manufacture of mumps vaccines have neurovirulent properties, a monkey neurovirulence safety test (MNVT) is performed. However, results with several mumps virus MNVTs have raised questions as to whether the test can reliably discriminate neurovirulent from nonneurovirulent mumps virus strains. Here, various mumps virus strains representing a wide range of neuropathogenicity were tested in a standardized MNVT. A trend of higher neurovirulence scores was observed in monkeys inoculated with wild type mumps virus versus vaccine strains, although differences were not statistically significant. Results indicated the need for further examination and refinement of the MNVT or for development of alternative MNVTs.

Maturation of hantaan virus glycoproteins G1 and G2

Hantaan virus-infected Vero E6 cell lysates were used for immunoprecipitation with monoclonal antibodies against glycoprotein G1 (MAbG1) or G2 (MAbG2). When cell lysates were prepared with buffer containing nonionic detergent, both G1 and G2 glycoproteins were precipitated with either MAbG1 or MAbG2. In contrast, when cell lysates were prepared with a buffer containing ionic detergents MAbG1 precipitated only glycoprotein G1 and MAbG2 precipitated only glycoprotein G2. Heterodimers and possibly higher oligomeric forms of the glycoproteins were detected on nonreducing SDS-polyacrylamide gels only after chemical cross-linking and immunoprecipitation with either MAbG1 or MAbG2. In order to determine the sites of Hantaan virus glycoproteins maturation and the G1-G2 complex formation, infected cells were treated with inhibitors that prevent specific steps of oligosaccharide processing. Furthermore, glycoproteins G1 and G2 immunoprecipitated from infected cell lysates or from isolated virus particles were tested for sensitivity to endoglycosidase H, endoglycosidase F, and endoglycosidase D. The results of these experiments show that maturation of both G1 and G2 takes place in the endoplasmic reticulum (ER). Furthermore, G1-G2 complex formation occurs in the ER as well, since the two glycoproteins co-precipitated with either MAbG1 or MAbG2 from infected cell lysates treated with brefeldin A and prepared with buffer containing nonionic detergent.

IL-6 Production Is Positively Regulated by Two Distinct Src Homology Domain 2-Containing Tyrosine Phosphatase-1 (SHP-1)–Dependent CCAAT/Enhancer-Binding Protein β and NF-κB Pathways and an SHP-1–Independent NF-κB Pathway in Lipopolysaccharide-Stimulated Bone Marrow-Derived Macrophages

Comparison of the inflammatory cytokine profile in bone marrow-derived macrophages (BMDMs) from normal and Src homology domain 2-containing tyrosine phosphatase-1 (SHP-1)–deficient Motheaten (me/me) mice revealed a dramatic suppression of IL-6 transcript and protein in me/me BMDMs after LPS stimulation. Interfering with SHP-1 expression using antisense SHP-1 oligonucleotides led to a significant downregulation of IL-6 in normal BMDMs. Conversely, reconstitution of me/me BMDMs with the SHP-1 gene using adenoviral vectors restored IL-6 production. Expression of only SHP-1 Src homology region 2 domains in normal BMDMs inhibited IL-6 production, confirming that IL-6 regulation depends on SHP-1 phosphatase activity. We further demonstrated that loss of SHP-1 function affects proper phosphorylation of Erk1/2 MAPKs and, to a lesser degree, of NF-κB downstream of TLR4 in BMDMs. Inefficient phosphorylation of Erk1/2 MAPKs abrogated the activation of C/EBPβ transcription factor, which was reversed on restoration of SHP-1 function and led to a concomitant enhancement of IL-6 production. We demonstrate that IL-6 production is regulated by a complex network of signaling pathways that include SHP-1–dependent activation of Erk1/2–C/EBPβ and NF-κB, in addition to SHP-1–independent IκB pathway through the activation of protein tyrosine kinases downstream of TLR4. Taken together, these results revealed for the first time, to our knowledge, a positive and critical role of SHP-1 in IL-6 regulation and dependence of Erk1/2–C/EBPβ pathway in addition to that of IκB on SHP-1 activity required for IL-6 induction after LPS stimulation.

Breast Cancer Cells Proliferation Is Regulated by Tyrosine Phosphatase SHP1 through c-jun N-Terminal Kinase and Cooperative Induction of RFX-1 and AP-4 Transcription Factors

In this study, we show that proliferation of breast cancer cells is suppressed by IGF-1–activated JNK MAPK pathway. The molecular mechanism by which c-jun-NH,-kinase (JNK) activation induces antiproliferative signals in IGF-1–stimulated breast cancer cells remains unknown. Tyrosine phosphatase SHP1 is known to negatively regulate signal transduction pathways activated by cell surface receptors including IGF-1. Moreover, SHP1 transcript and protein levels are increased in epithelial tumors. Therefore, we hypothesized that IGF-activated JNK induces expression of SHP1 in breast cancer cells. To further clarify the role of SHP1 in tumor growth, we correlated the proliferation rates of breast adenocarcinoma cells with SHP1 expression and JNK activation. We show that proliferation of serum- or IGF-1–stimulated breast adenocarcinoma cells is negatively regulated by SHP1 and show for the first time that IGF-1–activated JNK induces SHP1 expression in MCF-7 cells used as experimental model. In an attempt to understand the mechanism by which serum- or IGF-1–activated JNK induces SHP1 expression resulting in suppression of cell proliferation, we reveal for the first time that in serum- or IGF-1–stimulated breast cancer MCF-7 cells, JNK induces SHP1 expression through the binding of AP-4 and RFX-1 transcription factors to the epithelial tissue–specific SHP1 promoter. Overall, we show for the first time that IGF-1–stimulated proliferation of breast adenocarcinoma cells is negatively regulated by SHP1 through activation of JNK. Mol Cancer Res; 9(8); 1112–25. ©2011 AACR.

Biological characteristics of genetic variants of Urabe AM9 mumps vaccine virus.

The Urabe AM9 mumps vaccine is composed of a mixture of variants distinguishable by a difference at nucleotide (nt) 1081 of the hemagglutinin-neuraminidase (HN) gene (Brown, E.G., Dimock, K., Wright, K.E., 1996. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid (aa) 335 of the hemagglutinin-neuraminidase gene with one form associated with disease. J. Infect. Dis. 174, 619-622.). Further genetic and biological variation was detected in plaque purified viruses from the Urabe AM9 vaccine by examining the HN gene sequence, plaque morphology, cytopathic effects and growth in Vero cells, and temperature sensitivity (ts). Infection of Vero cells with plaque purified viruses with a G at nt 1081 of the HN gene produced large, clear plaques, caused significant CPE early after infection but yielded lower titres of virus than other purified viruses. None of these viruses were ts. In contrast, half of the plaque purified viruses with an A at nt 1081 were sensitive to a temperature of 39.5 degrees C. These viruses produced small plaques, caused significant CPE and grew to low titres. Two ts viruses possessed a unique aa substitution at aa 468 of HN. The remaining A(1081) viruses were not ts, produced large plaques but little CPE, and grew to titres 10-fold higher than the G(1081) viruses. Isolates of Urabe AM9 associated with post-vaccination illness were similar to these non-ts A(1081) viruses, but could be further sub-divided into two groups on the basis of a difference at aa 464 of HN. The post-vaccination isolates may represent insufficiently attenuated components of the vaccine, while the G(1081) and ts subset of A(1081) viruses may be more fully attenuated.

Generic aspects of the airborne spread of human pathogens indoors and emerging air decontamination technologies

Indoor air can be an important vehicle for a variety of human pathogens. This review provides examples of airborne transmission of infectious agents from experimental and field studies and discusses how airborne pathogens can contaminate other parts of the environment to give rise to secondary vehicles leading air-surface-air nexus with possible transmission to susceptible hosts. The following groups of human pathogens are covered because of their known or potential airborne spread: vegetative bacteria (staphylococci and legionellae), fungi (Aspergillus, Penicillium, and Cladosporium spp and Stachybotrys chartarum), enteric viruses (noro- and rotaviruses), respiratory viruses (influenza and coronaviruses), mycobacteria (tuberculous and nontuberculous), and bacterial spore formers (Clostridium difficile and Bacillus anthracis). An overview of methods for experimentally generating and recovering airborne human pathogens is included, along with a discussion of factors that influence microbial survival in indoor air. Available guidelines from the U.S. Environmental Protection Agency and other global regulatory bodies for the study of airborne pathogens are critically reviewed with particular reference to microbial surrogates that are recommended. Recent developments in experimental facilities to contaminate indoor air with microbial aerosols are presented, along with emerging technologies to decontaminate indoor air under field-relevant conditions. Furthermore, the role that air decontamination may play in reducing the contamination of environmental surfaces and its combined impact on interrupting the risk of pathogen spread in both domestic and institutional settings is discussed.

Identification of Genetic Mutations Associated With Attenuation and Changes in Tropism of Urabe Mumps Virus

Although several effective mumps virus vaccines have been developed, almost nothing is known about the genetic changes responsible for loss of virulence. One vaccine, Urabe AM9, was withdrawn from the market because of insufficient attenuation. The vaccine was found to contain a mixture of viruses that could be distinguished based on the sequence of the hemagglutinin‐neuraminidase gene (HN). Viruses containing lysine at HN amino acid position 335 were isolated from cases of post‐vaccination parotitis or meningitis whereas viruses containing glutamic acid at this position were not associated with post‐vaccination disease. Using a rat based model of mumps neurovirulence, we demonstrate that this latter virus is significantly attenuated compared to a virus isolated from a patient with post‐vaccination meningitis. Complete sequence analysis of the genomes of the two viruses identified sixteen genetic differences, some or all of which must be responsible for differences in virulence. These same genetic differences also account for changes in tropism in cell culture. J. Med. Virol. 81:130–138, 2009.

Decontamination of indoor air to reduce the risk of airborne infections: Studies on survival and inactivation of airborne pathogens using an aerobiology chamber.

BACKGROUND Although indoor air can spread many pathogens, information on the airborne survival and inactivation of such pathogens remains sparse. METHODS Staphylococcus aureus and Klebsiella pneumoniae were nebulized separately into an aerobiology chamber (24.0 m3). The chambers relative humidity and air temperature were at 50% ± 5% and 20°C ± 2°C, respectively. The air was sampled with a slit-to-agar sampler. Between tests, filtered air purged the chamber of any residual airborne microbes. RESULTS The challenge in the air varied between 4.2 log10 colony forming units (CFU)/m3 and 5.0 log10 CFU/m3, sufficient to show a ≥3 log10 (≥99.9%) reduction in microbial viability in air over a given contact time by the technologies tested. The rates of biologic decay of S aureus and K pneumoniae were 0.0064 ± 0.00015 and 0.0244 ± 0.009 log10 CFU/m3/min, respectively. Three commercial devices, with ultraviolet light and HEPA (high-efficiency particulate air) filtration, met the product efficacy criterion in 45-210 minutes; these rates were statistically significant compared with the corresponding rates of biologic decay of the bacteria. One device was also tested with repeated challenges with aerosolized S aureus to simulate ongoing fluctuations in indoor air quality; it could reduce each such recontamination to an undetectable level in approximately 40 minutes. CONCLUSIONS The setup described is suitable for work with all major classes of pathogens and also complies with the U.S. Environmental Protection Agencys guidelines (2012) for testing air decontamination technologies.