Association Between Symptom Cluster Endorsement at Initiation of a Graduated Return-to-Activity Protocol and Time to Return to Unrestricted Activity After Concussion in United States Service Academy Cadets.

BACKGROUND: The endorsement of symptoms upon initiation of a graduated return-to-activity (GRTA) protocol has been associated with prolonged protocols. It is unclear whether there are specific symptom clusters affecting protocol durations. PURPOSE: To describe the endorsement of specific concussion symptom clusters at GRTA protocol initiation and examine the association between symptom cluster endorsement and GRTA protocol duration. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: This study was conducted among cadets enrolled at 3 US service academies. Participants completed an evaluation upon GRTA protocol initiation. Participants endorsing symptoms were binarized based on 6 symptom clusters (cognitive, emotional, insomnia, physical, sensitivity, and ungrouped). The primary outcome of interest was GRTA protocol duration based on symptom cluster endorsement severity. Prevalence rates were calculated to describe symptom cluster endorsement. Kaplan-Meier survival estimates and univariate and multivariable Cox proportional hazards regression models were calculated for all 6 symptom clusters to estimate GRTA protocol duration while controlling for significant covariates. RESULTS: Data from 961 concussed participants were analyzed. Of these, 636 participants were asymptomatic upon GRTA protocol initiation. Among the 325 symptomatic participants, the physical symptom cluster (80%) was most endorsed, followed by the cognitive (29%), insomnia (23%), ungrouped (19%), sensitivity (15%), and emotional (9%) clusters. Univariate results revealed a significant association between endorsing cognitive (hazard ratio [HR], 0.79; p = .001), physical (HR, 0.84; p < .001), insomnia (HR, 0.83; p = .013), sensitivity (HR, 0.70; p < .001), and ungrouped (HR, 0.75; p = .005) symptom clusters and GRTA protocol duration. Endorsing physical (HR, 0.84; p < .001) and sensitivity (HR, 0.81; p = .036) clusters maintained a significant association with GRTA protocol duration in the multivariable models. CONCLUSION: Participants endorsing physical or sensitivity symptom clusters displayed GRTA protocols prolonged by 16% to 19% compared with participants not endorsing that respective cluster after controlling for significant covariates.

Factors Associated with Symptom Reporting in U.S. Service Academy Cadets and NCAA Student Athletes without Concussion: Findings from the CARE Consortium.

BACKGROUND: Symptom resolution is a key marker in determining fitness for return to activity following concussion, but in some cases, distinguishing persistent symptoms due to concussion versus symptoms related to other factors can be challenging. OBJECTIVE: To determine base rates of postconcussional syndrome (PCS) diagnostic categorization in healthy cadets and student athletes with no recent concussion. METHODS: 13,009 cadets and 21,006 student athletes completed baseline preseason testing. After inclusion/exclusion criteria were applied, the final sample included 12,039 cadets [9123 men (75.8%); 2916 women (24.2%)] and 18,548 student athletes [10,192 men (54.9%); 8356 women (45.1%)]. Participants completed the Sport Concussion Assessment Tool-3rd Edition (SCAT3) symptom evaluation as part of baseline preseason testing. The PCS diagnostic categorization was classified by the International Classification of Diseases, 10th Revision (ICD-10) symptom criteria for PCS. RESULTS: In the absence of recent concussion, subgroups of cadets (17.8% of men; 27.6% of women) and student athletes (11.4% of men; 20.0% of women) reported a cluster of symptoms that would meet the ICD-10 symptom criteria for PCS. Participants with insufficient sleep and/or preexisting conditions (e.g., mental health problems), freshmen cadets, and cadets at the U.S. Coast Guard Academy and at the U.S. Air Force Academy (freshmen were tested during basic cadet training) were more likely to report a cluster of symptoms that would meet the ICD-10 symptom criteria for PCS. CONCLUSION: The ICD-10 symptom criteria for PCS can be mimicked by preexisting conditions, insufficient sleep, and/or stress. Findings support person-specific assessment and management of symptoms following concussion.

Adverse Events and Discontinuation Rates Associated with Sirolimus Treatment in Adult Renal Transplant Patients in Latin America vs Non-Latin American Countries.

BACKGROUND: Sirolimus is approved for prophylaxis of organ rejection following renal transplantation. Rates of treatment-emergent adverse events (TEAEs) leading to sirolimus discontinuation differ geographically. METHODS: Rates of TEAEs, serious AEs (SAEs), and discontinuations were evaluated in 3 clinical trials of conversion from calcineurin inhibitors to sirolimus. Posttransplantation, patients were treated over 4 years (study 1), over 1 year (study 2), and over 2 years (study 3). TEAEs, SAEs, and discontinuation rates were compared between Latin America (LATAM) vs North America (NA) and Europe/rest of world (EU/ROW). Data from studies 2 and 3, with similar times to conversion, were pooled. RESULTS: Study 1 comprised 551 patients (LATAM, n=189); studies 2/3 comprised 395 (LATAM, n=111). LATAM patients were significantly younger than NA or EU/ROW patients in study 1 and studies 2/3 (P < .0001), with a lower proportion of white patients and higher proportion of patients of other races in LATAM vs NA (P < .0001) and EU/ROW (P = .02) groups. Almost all patients reported TEAEs. Discontinuation because of medical events was significantly lower (P < .05) in LATAM vs NA or EU/ROW. Hypercholesterolemia and hypertriglyceridemia were more common, and anemia and peripheral edema less common in LATAM; diarrhea and proteinuria did not differ by region. Types of AEs leading to discontinuation did not differ by region. CONCLUSION: LATAM renal transplant recipients converted to sirolimus were more likely to remain on therapy than patients in other regions.

Influence of Multiple Conformations and Paths on Rate Constants and Product Branching Ratios. Thermal Decomposition of 1-Propanol Radicals.

The potential energy surface involved in the thermal decomposition of 1-propanol radicals was investigated in detail using automated codes (tsscds2018 and Q2DTor). From the predicted elementary reactions, a relevant reaction network was constructed to study the decomposition at temperatures in the range 1000-2000 K. Specifically, this relevant network comprises 18 conformational reaction channels (CRCs), which in general exhibit a large wealth of conformers of reactants and transition states. Rate constants for all the CRCs were calculated using two approaches within the formulation of variational transition-state theory (VTST), as incorporated in the TheRa program. The simplest, one-well (1W) approach considers only the most stable conformer of the reactant and that of the transition state. In the second, more accurate approach, contributions from all the reactant and transition-state conformers are taken into account using the multipath (MP) formulation of VTST. In addition, kinetic Monte Carlo (KMC) simulations were performed to compute product branching ratios. The results show significant differences between the values of the rate constants calculated with the two VTST approaches. In addition, the KMC simulations carried out with the two sets of rate constants indicate that, depending on the radical considered as reactant, the 1W and the MP approaches may display different qualitative pictures of the whole decomposition process.

Theoretical design of molecular grippers for anion recognition based on subporphyrazines and subphthalocyanines.

Subphthalocyanines (SubPcs) are 14 π-electron aromatic macrocycles containing three 1,3-diiminoisoindol units N-fused around a central boron atom. Their particular concave π-extended structure makes them potentially useful in fields related to optoelectronics and molecular electronics. Also, their structure makes them potential receptors of complementary convex-shaped molecules. Inspired by the properties of subphthalocyanines and by previous studies about the possibility of obtaining neutral receptors composed of aromatic rings that can bind anions, we performed a theoretical study to analyze the ability of subphthalocyanines and some SubPc analogues to capture anions by non-covalent interactions. This may be relevant, since they can produce a variety of chromogenic reagents for anion detection that are not destroyed in the detection process, since the non-covalent interactions are weak enough to reverse the procedure. We characterized complexes formed between several SubPc derivatives and different anions, and also studied the influence of this process on the optical properties of these molecules. The stabilization trend reflected by the energetic results and the changes shown in the absorption wavelengths of the host molecule under complexation confirm our hypothesis about the possibility of these kinds of molecules being used as chromogenic reagents for anion capture, even in solvents with a large dielectric constant like water.

A computational study of the protonation of simple amines in water clusters.

The microsolvation study of a group of amines with a variable number of water molecules was performed by conducting a theoretical analysis of the properties of the clusters formed by the amines with up to seven molecules of water. We describe the microsolvation of several amines focusing on the dissociation of a water molecule that transfers a proton to the amine and forms a hydroxide ion. Ab initio calculations were performed on these clusters employing the DFT/B3LYP and MP2 methods with the 6-311++G(2d,p) basis set. Several stationary points for each cluster were thus located and characterized as minima from frequency calculations. Intermolecular BSSE corrected interaction energies were obtained. The protonation mechanism of the amines was examined in terms of some parameters that include the lengths of the bonds involved in the process of proton transfer and the frequencies associated with certain O-H and N-H stretching modes. On the basis of the calculations, all studied amines present similar behavior but trimethylamine, whose limitations to be integrated in the water hydrogen bond network cause the instability of some of their complexes. The cyclic configurations are the most stable structures up to five water molecules due to the presence of cooperative effects associated with the hydrogen bonds of water molecules. However, when the number of water molecules increases the spatial forms become the most stable configurations. The dissociated forms were not found to have the most stable configuration in any of the studied systems but energetic differences between the dissociated and non-dissociated forms decrease with the number of water molecules.

Newcastle disease virus fusion and haemagglutinin-neuraminidase proteins contribute to its macrophage host range.

The fusion (F) and haemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are multifunctional proteins that play critical roles during infection. Here, we assessed the ability of NDV to replicate in macrophages and investigated the contribution of the F and HN proteins to NDV infection/replication in these cells. Results of our study revealed that, while presenting similar replication kinetics in a fibroblast cell line (DF1) or in primary non-adherent splenocytes, the NDV strain CA02 replicates better in macrophages (HD11 and primary adherent splenocytes) than the NDV strain Anhinga/93. Notably, exchange of the HN or both F and HN genes of NDV Anhinga/93 by the corresponding genes from NDV CA02 markedly improved the ability of the chimeric viruses to replicate in macrophages. These results indicate that the F and HN proteins are determinants of NDV macrophage host range. This represents the first description of productive NDV infection in macrophages.

Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination.

Birds transfer maternal antibodies (MAb) to their offspring through the egg yolk where the antibody is absorbed and enters the circulatory system. Maternal antibodies provide early protection from disease, but may interfere with the vaccination efficacy in the chick. MAb are thought to interfere with vaccine antigen processing that reduces the subsequent immune response. Once MAb titers are depleted, the chick will respond to vaccination, but they are also susceptible to viral infection. This study examines the effect of MAb on seroconversion to different viral-vectored avian influenza virus (AIV) vaccines. Chicks were given passively transferred antibodies (PTA) using AIV hyperimmunized serum, and subsequently vaccinated with a fowlpox-AIV recombinant vaccine (FPr) or a Newcastle disease virus-AIV recombinant vaccine (NDVr). Our results indicate that passively transferred antibodies led to significant reduction of seroconversion and clinical protection from virulent challenge in recombinant virus vaccinated chicks thus demonstrating maternal antibody interference to vaccination. The passive antibody transfer model system provides an important tool to evaluate maternal antibody interference to vaccination.

A duplex real-time polymerase chain reaction assay for the simultaneous detection of long terminal repeat regions and envelope protein gene sequences of Reticuloendotheliosis virus in avian blood samples.

The Reticuloendotheliosis virus (REV) group of retroviruses infects a wide range of avian species, including chickens, turkeys, ducks, geese, quail, and prairie chickens. The objective of the present study was to develop a highly sensitive and specific diagnostic test for the detection of REV in whole blood samples. In order to increase the diagnostic sensitivity, a duplex real-time polymerase chain reaction (PCR) that detects both the envelope protein gene (env) and the long terminal repeat (LTR) region of REV was designed. This assay demonstrated greater analytical and diagnostic sensitivity than the gel-based PCR assay when using DNA extracted from whole blood by both phenol-chloroform and magnetic bead methods. In general, threshold cycle values in the duplex real-time PCR assay were lower from DNA extracted using the magnetic bead system compared to DNA extracted by the phenol-chloroform method. Data presented herein show the successful development of a rapid and accurate test procedure, with high-throughput capability, for the diagnosis of REV infection using avian blood samples.

Generation and evaluation of a recombinant Newcastle disease virus expressing the glycoprotein (G) of avian metapneumovirus subgroup C as a bivalent vaccine in turkeys.

Virulent strains of Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) can cause serious respiratory diseases in poultry. Vaccination combined with strict biosecurity practices has been the recommendation for controlling both NDV and aMPV diseases in the field. In the present study, an NDV based, LaSota strain recombinant vaccine virus expressing the glycoprotein (G) of aMPV subgroup C (aMPV-C) was generated as a bivalent vaccine using a reverse genetics approach. The recombinant virus, rLS/aMPV-C G was slightly attenuated in vivo, yet maintained similar growth dynamics, cytopathic effects, and virus titers in vitro when compared to the parental LaSota virus. Expression of the aMPV G protein in rLS/aMPV-C G-infected cells was detected by immunofluorescence assay. Vaccination of turkeys with one dose of rLS/aMPV-C G induced moderate aMPV-C-specific immune responses and comparable NDV-specific serum antibody responses to a LaSota vaccination control. Partial protection against pathogenic aMPV-C challenge and complete protection against velogenic NDV challenge was conferred. These results suggest that the LaSota recombinant virus is a safe and effective vaccine vector and that expression of the aMPV-C G protein alone is not sufficient to provide full protection against an aMPV-C infection. Expression of other immunogenic protein(s) of the aMPV-C virus alone or in conjunction with the G protein may be needed to induce a stronger protective immunity against the aMPV-C disease.

Ultrafast ring-opening/closing and deactivation channels for a model spiropyran-merocyanine system.

The photochemistry of a model merocyanine-spiropyran system was analyzed theoretically at the MS-CASPT2//SA-CASSCF(14,12) level. Several excited singlet states were studied in both the closed spiropyran and open merocyanine forms, and the paths to the different S(1)/S(0) conical intersections found were analyzed. After absorption of UV light from the spiropyran form, there are two possible ultrafast routes to efficient conversion to the ground state; one involves the rupture of the C(spiro)-O bond leading to the open form and the other involves the lengthening of the C(spiro)-N bond with no photoreaction. From the merocyanine side the excited state can reach a very broad S(1)/S(0) conical intersection region that leads the system to the closed form after rotation of the central methine bond. Alternatively, rotation of the other methine bonds connects the system through different S(1)/S(0) conical intersections to several merocyanine isomers. The present work provides a theoretical framework for the recent experimental results (Buback , J. J. Am. Chem. Soc. 2010, 132, 1610-1619) and sheds light on the complex photochemistry of these kinds of compounds.

Deletion of the M2-2 gene from avian metapneumovirus subgroup C impairs virus replication and immunogenicity in Turkeys.

The second matrix (M2) gene of avian metapneumovirus subgroup C (aMPV-C) contains two overlapping open reading frames (ORFs), encoding two putative proteins, M2-1 and M2-2. Both proteins are believed to be involved in viral RNA transcription or replication. To further characterize the function of the M2-2 protein in virus replication, the non-overlapping region of the M2-2 ORF was deleted from an infectious cDNA clone of the aMPV-C strain, and a viable virus was rescued by using reverse genetics technology. The recombinant virus, raMPV-C ΔM2-2, was characterized in vitro and in vivo. In Vero cells, raMPV-C ΔM2-2 replicated slightly less efficiently than the parental virus, 10-fold reduction at 48-h post-infection. The raMPV-C ΔM2-2 virus induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental virus infection. In specific-pathogen-free (SPF) turkeys, raMPV-C ΔM2-2 was attenuated and caused no clinical signs of disease. Less than 20% of the inoculated birds shed detectable virus in tracheal tissue during the first 5 days post-infection, and no virus shedding was detected afterward. Forty percent of infected birds produced a weak antibody response at 14 days post-infection. Upon challenge with a virulent aMPV-C strain, more than 80% of the raMPV-C ΔM2-2-inoculated birds showed typical disease signs and virus shedding in tracheal tissue. These results suggest that the M2-2 protein of aMPV-C virus is not essential for virus replication in vitro, but is required for sufficient virus replication to maintain pathogenicity and immunogenicity in the natural host.

Anion-π aromatic neutral tweezers complexes: are they stable in polar solvents?

The impact of the solvent environment on the stabilization of the complexes formed by fluorine (T-F) and cyanide (T-CN) substituted tweezers with halide anions has been investigated theoretically. The study was carried out using computational methodologies based on density functional theory (DFT) and symmetry adapted perturbation theory (SAPT). Interaction energies were obtained at the M05-2X/6-31+G* level. The obtained results show a large stability of the complexes in solvents with large dielectric constant and prove the suitability of these molecular tweezers as potential hosts for anion recognition in solution. A detailed analysis of the effects of the solvent on the electron withdrawing ability of the substituents and its influence on the complex stability has been performed. In particular, the interaction energy in solution was split up into intermonomer and solvent-complex terms. In turn, the intermonomer interaction energy was partitioned into electrostatic, exchange, and polarization terms. Polar resonance structures in T-CN complexes are favored by polar solvents, giving rise to a stabilization of the intermonomer interaction, the opposite is found for T-F complexes. The solvent-complex energy increases with the polarity of the solvent in T-CN complexes, nonetheless the energy reaches a maximum and then decreases slowly in T-F complexes. An electron density analysis was also performed before and after complexation, providing an explanation to the trends followed by the interaction energies and their different components in solution.

A single amino acid substitution in the haemagglutinin-neuraminidase protein of Newcastle disease virus results in increased fusion promotion and decreased neuraminidase activities without changes in virus pathotype.

Attachment of Newcastle disease virus (NDV) to the host cell is mediated by the haemagglutinin-neuraminidase (HN), a multifunctional protein that has receptor recognition, neuraminidase (NA) and fusion promotion activities. The process that connects receptor binding and fusion triggering is poorly understood and amino acid residues important for the functions of the protein remain to be fully determined. During the process of generating an infectious clone of the Anhinga strain of NDV, we were able to rescue a NDV with highly increased fusogenic activity in vitro and decreased haemagglutinating activity, as compared with the wild-type parental strain. Sequencing of this recombinant virus showed a single mutation at amino acid position 192 of the HN protein (Ile→Met). In the present study, we characterized that single amino acid substitution (I192M) in three strains of NDV by assessing the NA activity and fusogenic potential of the mutated versus wild-type proteins in cell cultures. The original recombinant NDV harbouring the mutation in the HN gene was also used to characterize the phenotype of the virus in cell cultures, embryonated chicken eggs and day-old chickens. Mutation I192M results in low NA activity and highly increased cell fusion in vitro, without changes in the viral pathotype of recombinant viruses harbouring the mutation in vivo. The results obtained suggest that multiple regions of the HN-protein globular head are important for fusion promotion, and that wild-type levels of NA activity are not absolutely required for viral infection.

Pathogenicity evaluation of different Newcastle disease virus chimeras in 4-week-old chickens.

The aim of this study was to evaluate the disease-inducing ability of four chimeric Newcastle disease viruses (NDV) by clinicopathological assessment. The infectious clones were previously generated by insertion of hemagglutinin-neuraminidase (HN) and/or fusion (F) genes from virulent strains (Turkey North Dakota and California 02) into a mesogenic strain (Anhinga) backbone. Groups of 4-week-old chickens were inoculated via eye drop instillation, clinical signs were monitored daily, and necropsies with collection of tissues were performed at 2, 5, 10, and 14 days post infection. Tissue sections were evaluated for histopathology and immunohistochemistry for NDV nucleoprotein. All viruses replicated successfully in the natural host, although viral recovery, seroconversion, and extent of immunohistochemical staining were greatest from birds infected with those viruses containing both F and HN genes from the same virulent virus. There was minimal to no increase in clinicopathologic disease due to infection with the chimeras compared to the recombinant backbone. However, all birds developed histological evidence of encephalitis. The results suggest that the inherent virulence of Turkey North Dakota and California 2002 strains is due to more than the simple presence of their F and HN genes.

Enhancing the interactions between neutral molecular tweezers and anions.

Several structural modifications to the original molecular tweezers of the Klärner's group were made with the aim of improving its binding capacity towards anions. The proposed modifications raise the molecular electrostatic potential inside the cavity and provide more conformational flexibility. The complexes of these new molecules with the halide anions Cl(-), Br(-), I(-) were optimized at the MPW1B95/6-31+G* level of theory. The molecular interactions were analyzed by single point density fitted local second-order Møller-Plesset perturbation theory (DF-LMP2) and DF-LMP2 spin-component-scaled MP2 (SCS-MP2), calculations were performed with the cc-pVTZ basis set. In view of the large magnitude of the interaction energies computed and the stability of the complexes in different solvents, this kind of molecule is a good candidate as molecular host for anion recognition.

Generation and biological assessment of recombinant avian metapneumovirus subgroup C (aMPV-C) viruses containing different length of the G gene.

Genetic variation in length of the G gene among different avian metapneumovirus subgroup C (aMPV-C) isolates has been reported. However, its biological significance in virus replication, pathogenicity and immunity is unknown. In this study, we developed a reverse genetics system for aMPV-C and generated two Colorado (CO) strain-based recombinant viruses containing either the full-length G gene derived from a Canadian goose isolate or a C-terminally truncated G gene of the CO strain. The truncated short G (sG) gene encoded 252 amino acids (aa), which is 333 aa shorter than the full-length G (585 aa). The biological properties of these two recombinant G variants were assessed in Vero cells and in specific-pathogen-free (SPF) turkeys. In Vero cells, the short G variant displayed a similar level of growth dynamics and virus titers as the parental aMPV-CO strain, whereas the full-length G variant replicated less efficiently than the sG variant during the first 72 h post-infection. Both of the G variants induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental aMPV-CO infection. In SPF turkeys, both of the G variants were attenuated and caused little or no disease signs, but the full-length G variant appeared to grow more readily in tracheal tissue than the sG variant during the first 5 days post-infection. Both G variants were immunogenic and induced a slightly different level of antibody response. These results demonstrated that the large portion (333 aa) of the extracellular domain of the viral attachment protein is not essential for virus viability in vitro and in vivo, but may play a role in enhancing virus attachment specificity and immunity in a natural host.

Comparison of viral shedding following vaccination with inactivated and live Newcastle disease vaccines formulated with wild-type and recombinant viruses.

Virulent Newcastle disease virus isolates from the 1971 and 2002 U.S. outbreaks are of the same serotype but a different genotype than current vaccine strains. Prior experiments with inactivated vaccines in chickens show significantly less virus shed in birds vaccinated with a homologous vaccine (same genotype as challenge) compared to chickens vaccinated with genotypically heterologous vaccines. Subsequent experiments have compared the protection induced in chickens by live vaccines of B1 and LaSota (genotype II), Ulster (genotype I), and recombinant viruses that express the hemagglutinin neuraminidase gene (HN) or the HN and fusion gene (F) of CA 2002 (genotype V). Vaccinates were challenged with virulent viruses CA 2002 (genotype V) or Texas GB (TXGB, genotype II). After challenge with CA 2002 the birds vaccinated with a live recombinant genotype V virus containing the HN of CA 2002 shed significantly less virus in oropharyngeal swabs compared to B1 and had fewer birds shedding virus compared to B1, LaSota, and Ulster vaccinates. After challenge with CA 2002 birds vaccinated with the recombinant containing both the HN and F of CA 2002 (rA-CAFHN) shed less virus, and fewer birds shed virus compared to LaSota-vaccinated birds. TXGB-challenged LaSota-vaccinated birds shed less virus, and fewer birds shed virus compared to TXGB-challenged rA-CAFHN-vaccinated birds. Genotypic differences between vaccine and challenge did not diminish ability of vaccines to protect against disease, but genotypic similarity did reduce virus shed and may reduce transmission. The development and use of vaccines of the same genotype as the expected field challenge may provide an additional tool for control of this important poultry pathogen.

Differential host gene expression in cells infected with highly pathogenic H5N1 avian influenza viruses.

In order to understand the molecular mechanisms by which different strains of avian influenza viruses overcome host response in birds, we used a complete chicken genome microarray to compare early gene expression levels in chicken embryo fibroblasts (CEF) infected with two avian influenza viruses (AIV), A/CK/Hong Kong/220/97 and A/Egret/Hong Kong/757.2/02, with different replication characteristics. Gene ontology revealed that the genes with altered expression are involved in many vital functional classes including protein metabolism, translation, transcription, host defense/immune response, ubiquitination and the cell cycle. Among the immune-related genes, MEK2, MHC class I, PDCD10 and Bcl-3 were selected for further expression analysis at 24 hpi using semi-quantitive RT-PCR. Infection of CEF with A/Egret/Hong Kong/757.2/02 resulted in a marked repression of MEK2 and MHC class I gene expression levels. Infection of CEF with A/CK/Hong Kong/220/97 induced an increase of MEK2 and a decrease in PDCD10 and Bcl-3 expression levels. The expression levels of alpha interferon (IFN-alpha), myxovirus resistance 1 (Mx1) and interleukin-8 (IL-8) were also analyzed at 24 hpi, showing higher expression levels of all of these genes after infection with A/CK/Hong Kong/220/97 compared to A/Egret/Hong Kong/757.2/02. In addition, comparison of the NS1 sequences of the viruses revealed amino acid differences that may explain in part the differences in IFN-alpha expression observed. Microarray gene expression analysis has proven to be a useful tool on providing important insights into how different AIVs affect host gene expression and how AIVs may use different strategies to evade host response and replicate in host cells.

Glycoprotein gene truncation in avian metapneumovirus subtype C isolates from the United States.

The length of the published glycoprotein (G) gene sequences of avian metapneumovirus subtype-C (aMPV-C) isolated from domestic turkeys and wild birds in the United States (1996-2003) remains controversial. To explore the G gene size variation in aMPV-C by the year of isolation and cell culture passage levels, we examined 21 turkey isolates of aMPV-C at different cell culture passages. The early domestic turkey isolates of aMPV-C (aMPV/CO/1996, aMPV/MN/1a-b, and 2a-b/97) had a G gene of 1,798 nucleotides (nt) that coded for a predicted protein of 585 amino acids (aa) and showed >97% nt similarity with that of aMPV-C isolated from Canada geese. This large G gene got truncated upon serial passages in Vero cell cultures by deletion of 1,015 nt near the end of the open reading frame. The recent domestic turkey isolates of aMPV-C lacked the large G gene but instead had a small G gene of 783 nt, irrespective of cell culture passage levels. In some cultures, both large and small genes were detected, indicating the existence of a mixed population of the virus. Apparently, serial passage of aMPV-C in cell cultures and natural passage in turkeys in the field led to truncation of the G gene, which may be a mechanism of virus evolution for survival in a new host or environment.