Production of a Sindbis/Eastern Equine Encephalitis chimeric virus inactivated cell culture antigen.

Eastern Equine Encephalitis virus (EEEV) is a medically important pathogen that can cause severe encephalitis in humans, with mortality rates ranging from 30 to 80%. Unfortunately there are no antivirals or licensed vaccines available for human use, and laboratory diagnosis is essential to differentiate EEEV infection from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the EEEV immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). However, EEEV is classified as a HHS select agent and requires biosafety level (BSL) three containment, limiting EEEV antigen production in non-select agent and BSL-2 laboratories. A recombinant Sindbis virus (SINV)/EEEV has been constructed for use under BSL-2 conditions and is not regulated as a select agent. Cell culture production of inactivated EEEV antigen from SINV/EEEV for use in the EEEV MAC-ELISA is reported here. Cell culture conditions and inactivation procedures were analyzed for SINV/EEEV using a recently developed antigen production algorithm, with the MAC-ELISA as the performance indicator.

Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Use of sindbis/eastern equine encephalitis chimeric viruses in plaque reduction neutralization tests for arboviral disease diagnostics.

Eastern equine encephalitis virus (EEEV) is a highly virulent, mosquito-borne alphavirus that causes severe and often fatal neurological disease in humans and horses in eastern North American, the Caribbean, and Mexico and throughout Central and South America. EEEV infection is diagnosed serologically by anti-EEEV-specific IgM detection, with confirmation by the plaque reduction neutralization test (PRNT), which is highly specific for alphaviruses. Live virus is used in the PRNT procedure, which currently requires biosafety level 3 containment facilities and select agent security in the case of EEEV. These requirements restrict the ability of public health laboratories to conduct PRNTs. Sindbis virus (SINV)/EEEV recombinant constructs have been engineered to express the immunogenic structural proteins from 2 wild-type EEEV strains in an attenuated form. These SINV/EEEVs, which are not classified as select agents, were evaluated as alternative diagnostic reagents in a PRNT using human, equine, and murine sera. The results indicate that the chimeric viruses exhibit specificity comparable to that of wild-type EEEV, with only a slight reduction in sensitivity. Considering their benefits in increased safety and reduced regulatory requirements, these chimeric viruses should be highly useful in diagnostic laboratories throughout the Americas.

Laparoscopic-assisted placement of ventriculo-peritoneal shunt tips in children with multiple previous open abdominal ventriculo-peritoneal shunt surgeries.

BACKGROUND: Placing a ventriculo-peritoneal shunt in children with hydrocephalus is the standard of care. Many of these children will require revision of this portion of the shunt for a variety of reasons. Previously, it was thought that in a child with multiple previous ventriculo-peritoneal shunt (VPS) revisions, laparoscopy was contraindicated. This study aims to show that laparoscopy can be used safely and effectively in children with multiple previous ventriculo-peritoneal shunt surgeries. MATERIALS AND METHODS: Laparoscopically assisted placement of the peritoneal portion of the ventriculo-peritoneal shunt in children with multiple previous VPS revisions was performed in 8 consecutive children (4 female) with ages ranging from 7 months to 18 years between May 2003 and September 2007. All eight children had undergone more than two previous VPS operations. All shunts were placed in areas free of adhesions and flow was observed under direct visualization. RESULTS: All of the procedures were successful; none needed conversion to the standard mini-laparotomy approach. No obvious or occult injury to the abdominal components was noted during hospitalization or during follow-up. Six of 8 patients required lysis of adhesions at the time of the revision. Average length of hospital stay was 2.6 days and no revisions of the abdominal portion of the VPS have been required by any of the 8 patients after laparoscopic revision. Previously unknown complications of shunt surgery were corrected in 1 of 8 children. CONCLUSIONS: Laparoscopic placement of the peritoneal portion of a ventriculo-peritoneal shunt can be done safely and effectively in children with multiple previous VPS revisions due to improved visualization and placement of the shunt tip in a virgin area of the abdomen. Additionally, any known or unknown complications from previous VPS surgeries can be corrected with the laparoscopic approach. When combined with the reduction in pain, shorter hospital stay, and fewer immediate and future complications, this is the procedure of choice for patients requiring revision VPS surgeries in our hospital.

Culex flavivirus isolates from mosquitoes in Guatemala.

A new strain of Culex flavivirus (family Flaviviridae, genus Flavivirus, CxFV), an insect virus first described in Japan, was isolated from adult Culex quinquefasciatus Say (Diptera: Culicidae) collected in 2006 from Izabal Department on the Caribbean coast of Guatemala. Mosquito pools were assayed for flavivirus RNA by using flavivirus group-specific primers that amplified a 720-bp region of the nonstructural (NS) 5 gene by standard reverse transcriptase-polymerase chain reaction. From 210 pools (1,699 mosquitoes), eight tested positive, and six of these mosquito pools produced virus isolates in Aedes albopictus Skuse C6/36 cells. Nucleotide sequence comparison of the eight flavivirus RNA-positive pools showed that there was 100% identity among them, and phylogenetic analysis of the NS5 and envelope gene regions indicated that they represent a strain of the recently described CxFV from Japan. This is the first report of an insect flavivirus from Central America.

West Nile virus infection and serologic response among persons previously vaccinated against yellow fever and Japanese encephalitis viruses.

It is hypothesized that previous heterologous flaviviral exposure may modulate clinical illness among persons infected with West Nile virus (WNV). Little is known about the serological response in such persons. In summer 2003, a WNV outbreak occurred in Colorado, the location of the Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases (DVBID). DVBID employees, most previously vaccinated with yellow fever virus (YFV) or Japanese encephalitis virus (JEV) vaccines, were studied to determine whether previous vaccination affected symptom development among those subsequently infected with WNV during the outbreak, as well as their serological response. Serum samples collected in December 2003 and previously banked samples were tested using the plaque reduction neutralization test (PRNT) against WNV, Saint Louis encephalitis virus, dengue- 4 virus, JEV, and YFV. Specimens shown to have WNV antibody by PRNT were tested by IgM and IgG enzymelinked immunosorbent assays (ELISAs). Ten (9%) of 113 serosurvey participants had WNV neutralizing antibody titers in December 2003. PRNT titers from previous specimens showed that one of the ten had seroconverted to WNV before 2003. Of the remaining nine participants, seven reported illness in the summer of 2003, two of which were unvaccinated and five previously vaccinated. In the December 2003 specimens, five persons previously unvaccinated or vaccinated only against YFV had a fourfold or greater neutralizing titer with WNV than with other flaviviruses, whereas no persons previously vaccinated against JEV or JEV and YFV showed a similar difference in neutralizing titers. Eight of nine persons infected in 2003 had negative or indeterminate WNV MAC-ELISA results in the December 2003 sample; the ninth person was vaccinated against YFV one month previously, and was also YFV positive by MAC-ELISA. We conclude that previous flaviviral vaccination does not markedly affect the development of WNV fever and that the IgM antibody response in patients without neuroinvasive WNV disease is transient.

A single amino acid substitution in the envelope protein of chimeric yellow fever-dengue 1 vaccine virus reduces neurovirulence for suckling mice and viremia/viscerotropism for monkeys.

A chimeric yellow fever-dengue 1 (ChimeriVax-DEN1) virus was produced by the transfection of Vero cells with chimeric in vitro RNA transcripts. The cell culture supernatant was subjected to plaque purification for the identification of a vaccine candidate without mutations. Of 10 plaque-purified clones, 1 containing no mutation (clone J) was selected for production of the vaccine virus. During subsequent cell culture passaging of this clone for vaccine production, a single amino acid substitution (K to R) occurred in the envelope (E) protein at residue 204 (E204) (F. Guirakhoo, K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath, J. Virol. 78:4761-4775, 2004). The same mutation was observed in another clone (clone E). This mutation attenuated the virus in 4-day-old suckling mice inoculated by the intracerebral (i.c.) route and led to reduced viremia in monkeys inoculated by the subcutaneous or i.c. route. The histopathology scores of lesions in the brain tissue of monkeys inoculated with either the E204K or E204R virus were reduced compared to those for monkeys inoculated with the reference virus, a commercial yellow fever 17D vaccine (YF-VAX). Both viruses grew to significantly lower titers than YF-VAX in HepG2, a human hepatoma cell line. After intrathoracic inoculation into mosquitoes, both viruses grew to a similar level as YF-VAX, which was significantly lower than that of their wild-type DEN1 parent virus. A comparison of the E-protein structures of nonmutant and mutant viruses suggested the appearance of new intramolecular bonds between residues 204R, 261H, and 257E in the mutant virus. These changes may be responsible for virus attenuation through a change in the pH threshold for virus envelope fusion with the host cell membrane.

Changes in rolandic mu rhythm during observation of a precision grip.

We recorded 128-channel EEG from 16 participants while they observed, imitated, and self-initiated the precision grip of a manipulandum. Mu rhythm amplitudes were significantly lower during observation of a precision grip than during observation of a simple hand extension without object interaction. Scalp topographies for subtractions of observation, imitation, and execution conditions from the control condition showed a high degree of congruence, supporting the notion of a human observation-execution matching system. Surface Laplacian transformations suggest that the decrease in mu amplitude during precision grip observation reflects desynchronization of mu rhythm generators in the sensorimotor cortex. These results support the hypothesis that sensorimotor cortex is a neural substrate involved in the representation of both self- and other-generated actions and show the mu rhythm is sensitive to subtle changes in observed motor behavior.

Neuromagnetic responses associated with perceptual segregation of pitch.

In recent EEG investigations [Johnson, 2003] [Hautus, 2005], we described a novel late negative ERP component associated with binaural processing of auditory pitch based solely on interaural timing differences ("dichotic pitch"), an acoustic phenomenon that is closely analogous to visual perception of stereoscopic depth based on retinal disparities. The present study extends this research with neuromagnetic recordings of auditory evoked fields (AEFs) elicited by dichotically-embedded pitches. Eight healthy adult subjects listened to control stimuli consisting of 500 ms segments of broadband acoustic noise presented identically to both ears via earphones, and dichotic pitch stimuli created by introducing a dichotic delay to a narrow frequency region of the same noise segments and resulting in a perception of a pitch lateralized to the left or right of auditory space. Auditory-evoked fields (AEFs) were recorded using a 151 channel whole-head MEG system. Comparison of control and dichotic-pitch AEFs showed reliable amplitude differences during a time window of 150-350 ms. AEFs over the left hemisphere showed larger effects for contralateral than ipsilateral pitches, while the right hemisphere showed no differences for differently lateralized sources. The results indicate a relatively late stage of neural processing of binaurally-derived cues for the perceptual segregation of concurrent sound sources and support a right-hemisphere dominance for the processing of sound-source localization.

Modulation of neuromagnetic oscillatory activity during the observation of oro-facial movements.

A number of MEG/EEG studies have shown modulation of endogenous sensorimotor (mu and beta) rhythms during the observation of hand movements. These modulations are similar to patterns that occur during execution of movement and it has been hypothesised that the neural substrates of these rhythms may play a role in action representation and understanding the actions of others. In this experiment we wished to determine whether similar responses would be obtained during the observation of oro-facial movements. Neuromagnetic recordings (151 channels, CTF Systems) were obtained from six healthy subjects while they (1) observed a video of an experimenter making oro-facial movements (2) imitated the same movements and (3) observed hand movements. Source scanning using synthetic aperture magnetometry (SAM) was used to find changes in source power between these active conditions compared to pre-stimulus control conditions where no movement occurred. SAM images were created with 5 mm resolution in the beta (15-35 Hz) and mu (8-15 Hz) bands and showed source power decreases over parietal, occipital and sensorimotor areas. Time-frequency analysis of virtual SAM sensors from sensorimotor areas showed event-related desynchronisation of mu and beta bands following the onset of movement in all three conditions. These data demonstrate comparable activations of visuomotor mechanisms during observation or imitation of mouth movements and during observation of hand movements. These results support the notion that sensorimotor mechanisms play a role in achieving a representation of the oro-facial gestures of others.

Two phases of HIV RNA decay in CSF during initial days of multidrug therapy.

BACKGROUND: Defining cellular and tissue sources of HIV-1 in CSF is important for understanding disease pathogenesis and optimal therapies for HIV infection in the brain. OBJECTIVE: To identify the time of maximal viral decay in CSF during the initial days of antiretroviral therapy. METHODS: Serial CSF and plasma data were available from four adults who underwent ultraintensive CSF sampling for 48 hours at baseline and again beginning 72 hours after starting antiretroviral therapy. Regression lines were generated using HIV-1 RNA data from 17 on-treatment serial CSF samples obtained at 3-hour intervals. Viral RNA was quantified by Nuclisens and Amplicor HIV-1 Monitor assays. RESULTS: Extrapolation of regression lines intersected baseline below actual baseline CSF HIV-1 RNA concentrations, indicating that virus decayed most rapidly on days 1 through 3 with half-lives of no more than 0.9 to 2.8 days. Half-lives on days 4 and 5 ranged from 1.3 to 4.9 days. Plasma data also showed early rapid decay. CONCLUSIONS: Multiple phases of viral decay suggest that virus in CSF originates from at least two sources during untreated, asymptomatic HIV-1 infection. The short half-life indicates that the primary source is CD4+ T cells. Sampling during days 1 through 3 and different stages of disease will better define sources of virus.

Growth characteristics of the veterinary vaccine candidate ChimeriVax-West Nile (WN) virus in Aedes and Culex mosquitoes.

In 1999 West Nile (WN) virus was introduced to North America where this flavivirus has spread rapidly among wildlife (especially birds) transmitted by various species of mosquitoes (Diptera: Culicidae). Increasing numbers of cases and deaths among humans, horses and other domestic animals require development of effective vaccines. 'ChimeriVax-West Nile(vet)' is being developed for use as a veterinary vaccine to protect against WN infection. This chimeric virus contains the pre-membrane (prM) and envelope (E) genes from the wild-type WN NY99 virus (isolated from a flamingo in New York zoo during the 1999 WN epidemic) in the backbone of yellow fever (YF) 17D vaccine virus. Replication kinetics of ChimeriVax-WN(vet) virus were evaluated in mosquito cell culture (Aedes albopictus C6/36), in WN vector mosquitoes [Culex tritaeniorhynchus Giles, Cx. nigripalpus Theobald and Cx. quinquefasciatus Say (Diptera: Culicidae)] and in YF vectors [Aedes aegypti (L) and Ae. albopictus (Skuse)], to determine whether these mosquitoes become infected through feeding on a viraemic vaccine, and their potential infectivity to transmit the virus. Growth of ChimeriVax-WN(vet) virus was found to be restricted in mosquitoes, compared to WN virus in Ae. albopictus C6/36 cells. When inoculated intrathoracically, ChimeriVax-WN(vet) and YF 17D viruses did not replicate in Cx. tritaeniorhynchus or Cx. nigripalpus; replication was very restricted compared to the wild-type WN virus in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. When fed on hanging drops with ChimeriVax-WN(vet) virus (7.7 log10 PFU/mL), none of the Culex mosquitoes became infected; one Ae. albopictus and 10% of the Ae. aegypti became infected, but the titre was very low and virus did not disseminate to head tissue. ChimeriVax-WN(vet) virus had a replication profile similar to that of the attenuated vaccine virus YF 17D, which is not transmitted by mosquitoes. These results suggest that the natural mosquito vectors of WN and YF viruses, which may incidentally take a bloodmeal from a vaccinated host, will not become infected with ChimeriVax-WN(vet) virus.

Non-identical neural mechanisms for two types of mental transformation: event-related potentials during mental rotation and mental paper folding.

Reaction times, accuracy and 128-channel event-related potentials (ERPs) were measured from 14 normal, right-handed subjects while they performed two different parity-judgment tasks that require transformations of mental images: a relatively simple task requiring a single transformation (mental letter rotation), and a more complex task involving a coordinated sequence of transformations (mental paper folding). Reaction times increased monotonically with larger angular displacements from the upright (for mental rotation) and with number of squares carried (for mental paper folding). Both the tasks resulted in amplitude modulation of an approximately 420-700 ms latency ERP component at parietal electrodes. Scalp topographies indicated that right parietal cortex was activated during mental rotation, but bilateral parietal regions were activated during mental paper folding. Our results support the notion of a right hemispheric superiority for tasks involving simple, single mental rotations, but indicate greater involvement of the left hemisphere when a more complex sequence of transformations are required. This task-dependent lability of hemispheric function may account for some of the inconsistent results reported by previous neuroimaging and electrophysiological studies.

High-density EEG mapping during general anaesthesia with xenon and propofol: a pilot study.

Anaesthetic-induced spatial inhomogeneities of the electrencephalogram(EEG) using "high density" electrode mapping have not previously been reported. We measured the scalp EEG with a dense electrode (128-channel) montage during the course of light general anaesthesia with xenon and then propofol in normal human subjects. EEG was measured during induction and recovery of general anaesthesia in five normal subjects, and we obtained analysable data from three of these subjects. EEG topographies were plotted on a realistic head surface. Scalp fields were spatially de-blurred using a realistic head model and projected onto an averaged cortical surface Both xenon and propofol elicited large increases in midline frontal theta-band EEG power. Propofol reliably elicited orbitofrontal delta activity. Xenon, but not propofol, caused large increases in delta over the posterior cortex. Increased gamma power was observed for both anaesthetic agents at midline electrodes over the posterior cortex, but not anteriorly. Anaesthesia-induced delta and theta waves were differentially distributed along the anterior-posterior axis of the brain in a manner that corresponds well to the anatomy of putative neuronal generators. The distribution of anaesthetic-induced changes in fast gamma-band power seems to reflect functional differences between the posterior and anterior aspects of the cerebral cortex. These preliminary observations were consistent within our small sample, indicating that larger studies of anaesthetic effects using high-density recordings are warranted.

Turn that frown upside down: ERP effects of thatcherization of misorientated faces.

When inverted, thatcherized faces appear normal. This may be due to a decrease in configural and an increase in featural processing. It is not known whether this processing is continuous or reflects two distinct processing systems. Using event-related potentials (ERPs), we investigated the Thatcher effect on thatcherized and normal faces at varying orientations. The ERPs paralleled the perceptual illusion. The effect of thatcherization on upright faces was visible in P1 and N170 ERP components, possibly reflecting attentional engagement due to unpleasantness of thatcherized faces. Effects were also found over two later components, the P250 component, which has been related to configural recognition, and a late parietal component possibly reflecting featural processing. The effect of thatcherization on the two later components decreased gradually (for the P250 component) and abruptly (for the late parietal component) as the faces were rotated away from the upright.

Perceptual and motor mechanisms for mental rotation of human hands.

We measured brain potentials from human subjects performing a mental rotation task requiring right-left judgments of misoriented hands, and a control task requiring palm-back judgments of the same stimuli. High-density, 128-channel event-related potentials (ERPs) were recorded from 16 normal, right-handed subjects. There was a main effect of task at five different latencies: 148 ms (occipital), 180 ms (parietal), 388 ms (vertex), 556 ms (central-parietal), and 900 ms (vertex). Source estimations derived from topographic data indicate that frontal brain regions were strongly activated after 300 ms in the control task, but not until about 900 ms in the rotation task. We conclude that the neural computations underlying mental hand rotation may be recruited from relatively early stages of visuo-perceptual analysis; these early computations influence subsequent processing within a parietal-prefrontal system for the integration of perception with action.

Evidence of a source of HIV type 1 within the central nervous system by ultraintensive sampling of cerebrospinal fluid and plasma.

Defining the source of HIV-1 RNA in cerebrospinal fluid (CSF) will facilitate studies of treatment efficacy in the brain. Four antiretroviral drug-naive adults underwent two 48-hr ultraintensive CSF sampling procedures, once at baseline and again beginning on day 4 after initiating three-drug therapy with stavudine, lamivudine, and nelfinavir. At baseline, constant CSF HIV-1 RNA concentrations were maintained by daily entry of at least 10(4) to 10(6) HIV-1 RNA copies into CSF. Change from baseline to day 5 ranged from -0.38 to -1.18 log(10) HIV-1 RNA copies/ml in CSF, and from -0.80 to -1.33 log(10) HIV-1 RNA copies/ml in plasma, with no correlation between CSF and plasma changes. There was no evidence of genotypic or phenotypic viral resistance in either CSF or plasma. With regard to pharmacokinetics, mean CSF-to-plasma area-under-the-curve (AUC) ratios were 38.9% for stavudine and 15.3% for lamivudine. Nelfinavir and its active M8 metabolite could not be accurately quantified in CSF, although plasma M8 peak level and AUC(0-8hr) correlated with CSF HIV-1 RNA decline. This study supports the utility of ultraintensive CSF sampling for studying HIV-1 pathogenesis and therapy in the CNS, and provides strong evidence that HIV-1 RNA in CSF arises, at least in part, from a source other than plasma.

Bcl-xL inhibits cytochrome c release but not mitochondrial depolarization during the activation of multiple death pathways by tumor necrosis factor-alpha.

Cells can respond differently to anti-CD95 antibody treatment. Type I cells show strong activation of caspase-8 and directly activate caspase-3. Type II cells weakly activate caspase-8 and must amplify their death signal through the mitochondria. These cells can be rescued by Bcl-x(L). Here we show that tumor necrosis factor-alpha induces both Type I and II pathways, which can be inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) and Bcl-x(L) in a cooperative fashion. Death induced in the presence of Z-VAD-fmk was associated with a partial inhibition of caspase-8, whereas no effects on cytochrome c release, DEVDase activity, and intranucleosomal DNA cleavage were observed. Thus, Z-VAD-fmk is likely weakening the death-inducing signaling complex-mediated activation of caspase-8 and diverting cells to a Type II pathway. Bcl-x(L) cooperates with Z-VAD-fmk by blocking the Type II pathway at the level of cytochrome c release. Surprisingly, although Bcl-x(L) was able to block cytochrome c release, it was unable to block mitochondrial depolarization, suggesting that these are separate events. This suggests that mitochondria occupy two places in apoptotic signaling, as initiators of apoptosis through the release of cytochrome c as well as a target for effector caspases.