Effectiveness of a home-based balance-training program in reducing sports-related injuries among healthy adolescents: a cluster randomized controlled trial

Background

Sport is the leading cause of injury requiring medical attention among adolescents. We studied the effectiveness of a home-based balance-training program using a wobble board in improving static and dynamic balance and reducing sports-related injuries among healthy adolescents.

Methods

In this cluster randomized controlled trial, we randomly selected 10 of 15 high schools in Calgary to participate in the fall of 2001. We then recruited students from physical education classes and randomly assigned them, by school, to either the intervention (n = 66) or the control (n = 61) group. Students in the intervention group participated in a daily 6-week and then a weekly 6-month home-based balance-training program using a wobble board. Students at the control schools received testing only. The primary outcome measures were timed static and dynamic balance, 20-m shuttle run and vertical jump, which were measured at baseline and biweekly for 6 weeks. Self-reported injury data were collected over the 6-month follow-up period.

Results

At 6 weeks, improvements in static and dynamic balance were observed in the intervention group but not in the control group (difference in static balance 20.7 seconds, 95% confidence interval [CI] 10.8 to 30.6 seconds; difference in dynamic balance 2.3 seconds, 95% CI 0.7 to 4.0 seconds). There was evidence of a protective effect of balance training in over 6 months (relative risk of injury 0.2, 95% CI 0.05 to 0.88). The number needed to treat to avoid 1 injury over 6 months was 8 (95% CI 4 to 35).

Interpretation

Balance training using a wobble board is effective in improving static and dynamic balance and reducing sports-related injuries among healthy adolescents.Adolescents commonly participate in sports.^1,2 In a survey of adolescents in Alberta, 59% reported that they took part in sports more than 5 hours per week (unpublished data). In North America, sport is the leading cause of injury requiring medical attention and visits to an emergency department among adolescents.^3,4 In Alberta 26% of youths aged 15–19 years in a survey reported sustaining a sports-related injury requiring medical attention.⁵ The impact may be lifelong, as there is evidence that knee and ankle injuries may result in an increased risk of osteoarthritis later in life.^6,7,8 In addition, each year 8% of adolescents drop out of sports activities because of injury.⁹ The reduction in physical activity resulting from sports-related injuries could have significant long-term effects on morbidity and mortality.^10,11Proprioceptive balance training is used in rehabilitation following sports-related injuries and is becoming recognized as an important element in injury prevention in sports.^{12,13,14,15,16,17,18,19} Running, jumping or pivoting on one leg relies on a sense of joint position and muscular control for joint stability. There is evidence that static balance improves following proprioceptive balance training using a wobble board.^20,21,22,23 However, most of these studies did not examine the effect of dynamic proprioceptive balance training, which may improve postural control in athletic situations and prevent some injuries.There is evidence from randomized trials that multifaceted prevention programs, including proprioceptive balance training using a wobble board, are effective in reducing injuries to the lower extremities in specific sports.^{12,13,14,15,16,17,18,19} However, the programs in these trials were multifaceted (i.e., included warm-up, flexibility, jump training, strength training, rehabilitation and sport-specific technical components), and balance was not measured. The effectiveness of balance training alone on balance ability and prevention of injury remains unclear. Moreover, the use of these techniques in adolescents and non-elite athletes has not been studied.The objectives of our study were to determine the effectiveness of a proprioceptive home-based balance-training program in improving static and dynamic balance in adolescents and to examine the effectiveness of this training program on reducing sports-related injury among adolescents.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Vaccine protection by live, attenuated simian immunodeficiency virus in the absence of high-titer antibody responses and high-frequency cellular immune responses measurable in the periphery

An attenuated derivative of simian immunodeficiency virus strain 239 deleted of V1-V2 sequences in the envelope gene (SIV239ΔV1-V2) was used for vaccine/challenge experiments in rhesus monkeys. Peak levels of viral RNA in plasma of 10⁴ to 10^6.5 copies/ml in the weeks immediately following inoculation of SIV239ΔV1-V2 were 10- to 1,000-fold lower than those observed with parental SIV239 (∼10^7.3 copies/ml). Viral loads consistently remained below 200 copies/ml after 8 weeks of infection by the attenuated SIV239ΔV1-V2 strain. Viral localization experiments revealed large numbers of infected cells within organized lymphoid nodules of the colonic gut-associated lymphoid tissue at 14 days; double-labeling experiments indicated that 93.5% of the virally infected cells at this site were positive for the macrophage marker CD68. Cellular and humoral immune responses measured principally by gamma interferon enzyme-linked immunospot and neutralization assays were variable in the five vaccinated monkeys. One monkey had responses in these assays comparable to or only slightly less than those observed in monkeys infected with parental, wild-type SIV239. Four of the vaccinated monkeys, however, had low, marginal, or undetectable responses in these same assays. These five vaccinated monkeys and three naïve control monkeys were subsequently challenged intravenously with wild-type SIV239. Three of the five vaccinated monkeys, including the one with strong anti-SIV immune responses, were strongly protected against the challenge on the basis of viral load measurements. Surprisingly, two of the vaccinated monkeys were strongly protected against SIV239 challenge despite the presence of cellular anti-SIV responses of low-frequency and low-titer anti-SIV antibody responses. These results indicate that high-titer anti-SIV antibody responses and high-frequency anti-SIV cellular immune responses measurable by standard assays from the peripheral blood are not needed to achieve strong vaccine protection, even against a difficult, neutralization-resistant strain such as SIV239.The characteristics of human immunodeficiency virus type 1 (HIV-1) infection suggest major difficulty for the development of a preventive vaccine (19, 23). Pessimism regarding the prospects for a vaccine is derived at least in part from the ability of HIV-1 to continually replicate in the face of apparently strong host immune responses, resistance to antibody-mediated neutralization, and the extensive sequence diversity in field strains of the virus. Lack of knowledge regarding the key components of a protective immune response also remains a major scientific obstacle. Vaccine/challenge experiments with macaque monkeys have been used to evaluate the properties and relative effectiveness of different vaccine approaches and to gauge the formidable nature of these difficulties.One lesson that has been learned from vaccine/challenge experiments with macaque monkeys is the importance of challenge strain on outcome. Vaccinated monkeys that have been challenged with strains of simian immunodeficiency virus (SIV) with an HIV-1 envelope (SHIV) have almost invariably exhibited strong, long-term protection against disease, irrespective of the nature of the vaccine. Even peptide immunogens have protected against SHIV-induced disease (6, 12, 38). Vaccine approaches that have protected against SHIV challenge include DNA (5, 13), recombinant poxvirus (4), recombinant adenovirus (57), other viral recombinants (18, 55), prime and boost protocols (3, 53, 65), and purified protein (10, 64). Vaccine protection against pathogenic SIV strains such as SIV239, SIV251, and SIV-E660 has been much more difficult to achieve (2, 11, 27, 63). The identical replication-defective gag-recombinant adenovirus that provided strong protection against SHIV challenge (57) provided little or no protection against SIV239 challenge (11). Disappointing levels of protection against SIV have often been observed in the face of apparently robust vaccine-induced immune responses (see, for example, Vogel et al. [63] and Casimiro et al. [11]). Some partial vaccine protections against these SIV strains have been achieved by recombinant poxvirus (7, 50), replication-competent recombinant adenovirus (51), replication-defective adenovirus (66), recombinant poliovirus (15), recombinant Venezuelan equine encephalitis virus (18), and recombinant Sendai virus (44).Differences between the biological properties of the SIV strains and those of the SHIV strains used for the above-mentioned studies provide clues as to what may be responsible for the differences in outcome. These SIV strains are difficult to neutralize (26, 34), use CCR5 as a coreceptor for entry into cells (21, 52), and induce a chronic, progressive disease course (17), and this course is independent of the infectious dose (17). The SHIV strains used for the above-mentioned studies are easier to neutralize, use CXCR4 for entry, and induce an acute decline in CD4 counts, and the disease course is dose dependent (29, 30, 48, 54). These SIV strains, like HIV-1 in humans, exhibit a marked preference for CD4⁺ CCR5⁺ memory cells, in contrast to the acutely pathogenic SHIV strains which principally target naïve cells (48).Live, attenuated strains of SIV have provided the strongest vaccine protection by far against SIV challenge. Although clinical use of a live, attenuated HIV vaccine is not being considered, understanding the basis of the strong protection afforded by live, attenuated SIV strains remains an important research objective for the insights that can be provided. Most of the attenuated SIV strains that have been used lack a functional nef gene (16, 31, 58, 67). Shacklett et al. (56) used an attenuated SIV strain with modifications in the gp41 transmembrane protein for protection. Here, we describe strong vaccine protection by a replication-competent SIV strain lacking 100 amino acids from the essential gp120 envelope protein in the absence of overtly robust immune responses.     

Coinfecting Prion Strains Compete for a Limiting Cellular Resource

Prion strain interference can influence the emergence of a dominant strain from a mixture; however, the mechanisms underlying prion strain interference are poorly understood. In our model of strain interference, inoculation of the sciatic nerve with the drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent prior to superinfection with the hyper (HY) strain of TME can completely block HY TME from causing disease. We show here that the deposition of PrP^Sc, in the absence of neuronal loss or spongiform change, in the central nervous system corresponds with the ability of DY TME to block HY TME infection. This suggests that DY TME agent-induced damage is not responsible for strain interference but rather prions compete for a cellular resource. We show that protein misfolding cyclic amplification (PMCA) of DY and HY TME maintains the strain-specific properties of PrP^Sc and replicates infectious agent and that DY TME can interfere, or completely block, the emergence of HY TME. DY PrP^Sc does not convert all of the available PrP^C to PrP^Sc in PMCA, suggesting the mechanism of prion strain interference is due to the sequestering of PrP^C and/or other cellular components required for prion conversion. The emergence of HY TME in PMCA was controlled by the initial ratio of the TME agents. A higher ratio of DY to HY TME agent is required for complete blockage of HY TME in PMCA compared to several previous in vivo studies, suggesting that HY TME persists in animals coinfected with the two strains. This was confirmed by PMCA detection of HY PrP^Sc in animals where DY TME had completely blocked HY TME from causing disease.Prions are infectious agents of animals, including humans, which are comprised of PrP^Sc, a misfolded isoform of the noninfectious host encoded protein PrP^C (17, 24, 50, 63). Prion diseases of humans are unique neurodegenerative disorders in that they can have either a sporadic, familial, or infectious etiology. Prions cause disease in economically important domestic and wild animal species such as bovine spongiform encephalopathy in cattle and chronic wasting disease in wild and captive cervids (20, 62). Prion diseases can be zoonotic as illustrated by the transmission of bovine spongiform encephalopathy to humans that resulted in the emergence of variant Creutzfeldt-Jacob disease (14, 19, 22, 23, 46, 61, 68). Prion diseases are inevitably fatal and there are currently no effective treatments (21).Prion strains are defined by a characteristic set of features that breed true upon experimental passage (33, 34). Strain-specific differences have been identified in incubation period, clinical signs, agent distribution, overdominance, host range, neuropathology, and biochemical properties of PrP^Sc (5, 10, 11, 13, 28, 34, 42, 44). Strain-specific conformations of PrP^Sc are hypothesized to encode prion strain diversity; however, it is not understood how these differences result in the distinct strain properties (11, 19, 40, 47, 59, 66).Prion strain interference may be involved in the emergence of a dominant strain from a mixture as could occur during prion adaptation to a new host species or during prion evolution (4, 36, 43, 48, 56). In the natural prion diseases, there are examples where an individual host may be infected with more than one prion strain (15, 25, 55, 57, 58). Experimentally, coinfection or superinfection of prion strains can result in interference where a blocking, long incubation period strain extends the incubation period or completely blocks a superinfecting, short incubation period strain from causing disease (26, 27). Prion interference has been described in experimental studies of mice and hamsters infected with a wide variety of prion strains and routes of inoculation, suggesting it may be a common property of prion disease (3, 27, 52, 53, 60).It has been proposed that prion strains compete for a shared “replication site”; however, mechanistic details are not known, and it is unclear whether the blocking strain destroys or occupies the replication sites required for the superinfecting strain (28). The transport to and relative onset of replication of interfering strains in a common population of neurons is an important factor that can determine which strain will emerge (8). In the present study, we sought to determine whether the blocking strain disables transport and spread of the superinfecting strain or whether prion interference is due to competition for a cellular resource.     

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Na?ve and Formalin-Inactivated RSV-Immunized BALB/c Mice

We examined whether prophylactically administered anti-respiratory syncytial virus (anti-RSV) G monoclonal antibody (MAb) would decrease the pulmonary inflammation associated with primary RSV infection and formalin-inactivated RSV (FI-RSV)-enhanced disease in mice. MAb 131-2G administration 1 day prior to primary infection reduced the pulmonary inflammatory response and the level of RSV replication. Further, intact or F(ab′)₂ forms of MAb 131-2G administered 1 day prior to infection in FI-RSV-vaccinated mice reduced enhanced inflammation and disease. This study shows that an anti-RSV G protein MAb might provide prophylaxis against both primary infection and FI-RSV-associated enhanced disease. It is possible that antibodies with similar reactivities might prevent enhanced disease and improve the safety of nonlive virus vaccines.Respiratory syncytial virus (RSV) infection in infants and young children causes substantial bronchiolitis and pneumonia (11, 27, 28, 40) resulting in 40,000 to 125,000 hospitalizations in the United States each year (27). RSV is also a prominent cause of respiratory illness in older children; those of any age with compromised cardiac, pulmonary, or immune systems; and the elderly (6, 7, 11, 17, 18, 39). Despite extensive efforts toward vaccine development (3, 5, 8, 20, 30, 38), none is yet available. Currently, only preventive measures are available that focus on infection control to decrease transmission and prophylactic administration of a humanized IgG monoclonal antibody (MAb) directed against the F protein of RSV (palivizumab) that is recommended for high-risk infants and young children (4, 7, 17). To date, no treatment has been highly effective for active RSV infection (17, 21).The first candidate vaccine, a formalin-inactivated RSV (FI-RSV) vaccine developed in the 1960s, not only failed to protect against disease but led to severe RSV-associated lower respiratory tract infection in young vaccine recipients upon subsequent natural infection (8, 16). The experience with FI-RSV has limited nonlive RSV vaccine development for the RSV-naïve infant and young child. Understanding the factors contributing to disease pathogenesis and FI-RSV vaccine-enhanced disease may identify ways to prevent such a response and to help achieve a safe and effective vaccine.The RSV G, or attachment, protein has been implicated in the pathogenesis of disease after primary infection and FI-RSV-enhanced disease (2, 26, 31). The central conserved region of the G protein contains four evolutionarily conserved cysteines in a cysteine noose structure, within which lies a CX3C chemokine motif (9, 29, 34). The G protein CX3C motif is also immunoactive, as suggested by studies with the mouse model that show that G protein CX3C motif interaction with CX3CR1 alters pulmonary inflammation (41), RSV-specific T-cell responses (12), FI-RSV vaccine-enhanced disease, and expression of the neurokinin substance P (14) and also depresses respiratory rates (32). Recent studies demonstrated that therapeutic treatment with a murine anti-RSV G protein monoclonal antibody (MAb 131-2G) which blocks binding to CX3CR1 can reduce pulmonary inflammation associated with primary infection (13, 23). These findings led us to hypothesize that prophylactic administration of this anti-RSV G monoclonal antibody may also diminish pulmonary inflammation associated with RSV infection in naïve and in FI-RSV-vaccinated mice. In this study, we evaluate the impact of prophylactic administration of MAb 131-2G on the pulmonary inflammatory response to primary infection and to RSV challenge following FI-RSV immunization in mice.     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.Hantaviruses cause 2 human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (50). HPS and HFRS are multifactorial in nature and cause thrombocytopenia, immune and endothelial cell responses, and hypoxia, which contribute to disease (7, 11, 31, 42, 62). Although these syndromes sound quite different, they share common components which involve the ability of hantaviruses to infect endothelial cells and induce capillary permeability. Edema, which results from capillary leakage of fluid into tissues and organs, is a common finding in both HPS and HFRS patients (4, 7, 11, 31, 42, 62). In fact, both diseases can present with renal or pulmonary sequelae, and the renal or pulmonary focus of hantavirus diseases is likely to result from hantavirus infection of endothelial cells within vast glomerular and pulmonary capillary beds (4, 7, 11, 31, 42, 62). All hantaviruses predominantly infect endothelial cells which line capillaries (31, 42, 44, 61, 62), and endothelial cells have a primary role in maintaining fluid barrier functions of the vasculature (1, 12, 55). Although hantaviruses do not lyse endothelial cells (44, 61), this primary cellular target underlies hantavirus-induced changes in capillary integrity. As a result, understanding altered endothelial cell responses following hantavirus infection is fundamental to defining the mechanism of permeability induced by pathogenic hantaviruses (1, 12, 55).Pathogenic, but not nonpathogenic, hantaviruses use β₃ integrins on the surface of endothelial cells and platelets for attachment (19, 21, 23, 39, 46), and β₃ integrins play prominent roles in regulating vascular integrity (3, 6, 8, 24, 48). Pathogenic hantaviruses bind to basal, inactive conformations of β₃ integrins (35, 46, 53) and days after infection inhibit β₃ integrin-directed endothelial cell migration (20, 46). This may be the result of cell-associated virus (19, 20, 22) which keeps β₃ in an inactive state but could also occur through additional regulatory processes that have yet to be defined. Interestingly, the nonpathogenic hantaviruses Prospect Hill virus (PHV) and Tula virus (TULV) fail to alter β₃ integrin functions, and their entry is consistent with the use of discrete α₅β₁ integrins (21, 23, 36).On endothelial cells, α_vβ₃ integrins normally regulate permeabilizing effects of vascular endothelial growth factor receptor-2 (VEGFR2) (3, 24, 48, 51). VEGF was initially identified as an edema-causing vascular permeability factor (VPF) that is 50,000 times more potent than histamine in directing fluid across capillaries (12, 14). VEGF is responsible for disassembling adherens junctions between endothelial cells to permit cellular movement, wound repair, and angiogenesis (8, 10, 12, 13, 17, 26, 57). Extracellular domains of β₃ integrins and VEGFR2 reportedly form a coprecipitable complex (3), and knocking out β₃ causes capillary permeability that is augmented by VEGF addition (24, 47, 48). Pathogenic hantaviruses inhibit β₃ integrin functions days after infection and similarly enhance the permeability of endothelial cells in response to VEGF (22).Adherens junctions form the primary fluid barrier of endothelial cells, and VEGFR2 responses control adherens junction disassembly (10, 17, 34, 57, 63). Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific adherens junction protein and the primary determinant of paracellular permeability within the vascular endothelium (30, 33, 34). Activation of VEGFR2, another endothelial cell-specific protein, triggers signaling responses resulting in VE-cadherin disassembly and endocytosis, which increases the permeability of endothelial cell junctions (10, 12, 17, 34). VEGF is induced by hypoxic conditions and released by endothelial cells, platelets, and immune cells (2, 15, 38, 52). VEGF acts locally on endothelial cells through the autocrine or paracrine activation of VEGFR2, and the disassembly of endothelial cell adherens junctions increases the availability of nutrients to tissues and facilitates leukocyte trafficking and diapedesis (10, 12, 17, 55). The importance of endothelial cell barrier integrity is often in conflict with requirements for endothelial cells to move in order to permit angiogenesis and repair or cell and fluid egress, and as a result, VEGF-induced VE-cadherin responses are tightly controlled (10, 17, 18, 32, 33, 59). This limits capillary permeability while dynamically responding to a variety of endothelial cell-specific factors and conditions. However, if unregulated, this process can result in localized capillary permeability and edema (2, 9, 10, 12, 14, 17, 29, 60).Interestingly, tissue edema and hypoxia are common findings in both HPS and HFRS patients (11, 31, 62), and the ability of pathogenic hantaviruses to infect human endothelial cells provides a means for hantaviruses to directly alter normal VEGF-VE-cadherin regulation. In fact, the permeability of endothelial cells infected by pathogenic Andes virus (ANDV) or Hantaan virus (HTNV) is dramatically enhanced in response to VEGF addition (22). This response is absent from endothelial cells comparably infected with the nonpathogenic TULV and suggests that enhanced VEGF-induced endothelial cell permeability is a common underlying response of both HPS- and HFRS-causing hantaviruses (22). In these studies, we comparatively investigate responses of human endothelial cells infected with pathogenic ANDV and HTNV, as well as nonpathogenic TULV.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Virological Synapse-Mediated Spread of Human Immunodeficiency Virus Type 1 between T Cells Is Sensitive to Entry Inhibition

Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4⁺ T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.As with enveloped viruses from several viral families, the human immunodeficiency virus type 1 (HIV-1) can disseminate both by fluid-phase diffusion of viral particles and by directed cell-cell transfer (39). The primary target cell for HIV-1 replication in vivo is the CD4⁺ T-cell (13), which is infectible by CCR5-tropic (R5) and CXCR4-tropic (X4) viral variants (29). R5 HIV-1 is the major transmitted viral phenotype and dominates the global pandemic, whereas X4 virus is found later in infection in ca. 50% of infected individuals, and its presence indicates a poor disease progression prognosis (23). Cell-cell HIV-1 transfer between T cells is more efficient than diffusion-limited spread (8, 16, 32, 38), although recent estimates for the differential range from approximately 1 (42) to 4 (6) orders of magnitude. Two structures have been proposed to support contact-mediated intercellular movement of HIV-1 between T cells: membrane nanotubes (33, 43) and macromolecular adhesive contacts termed virological synapses (VS) (15, 17, 33). VS appear to be the dominant structure involved in T-cell-T-cell spread (33), and both X4 (17) and R5 HIV-1 (6, 15, 42) can spread between T cells via this mechanism.VS assembly and function are dependent on HIV-1 envelope glycoprotein (Env) engaging its primary cellular receptor CD4 (2, 6, 17). This interaction recruits more CD4 and coreceptor to the site of cell-cell contact in an actin-dependent manner (17). Adhesion molecules cluster at the intercellular junction and are thought to stabilize the VS (18). In parallel, viral Env and Gag are recruited to the interface by a microtubule-dependent mechanism (19), where polarized viral budding may release virions into the synaptic space across which the target cell is infected (17). The precise mechanism by which HIV-1 subsequently enters the target T-cell cytoplasm remains unclear: by fusion directly at the plasma membrane, fusion from within an endosomal compartment, or both (4, 6, 15, 25, 34).Viruses from diverse families including herpesviruses (9), poxviruses (22) and hepatitis C virus (44) evade neutralizing antibody attack by direct cell-cell spread, since the tight junctions across which the these viruses move are antibody impermeable. It has been speculated that transfer of HIV-1 across VS may promote evasion from immune or therapeutic intervention with the inference that the junctions formed in retroviral VS may be nonpermissive to antibody entry (39). However, available evidence regarding whether neutralizing antibodies (NAb) and other entry inhibitors can inhibit HIV-1 cell-cell spread is inconsistent (25). An early analysis suggested that HIV-1 T-cell-T-cell spread is relatively resistant to neutralizing monoclonal antibodies (NMAb) (12). A later study agreed with this conclusion by demonstrating a lack of permissivity of HIV-1 T-cell-T-cell spread, measured by transfer of viral Gag, to interference with viral fusion using a gp41-specific NMAb and a peptidic fusion inhibitor (6). In contrast, another analysis reported that anti-gp41-specific NMAb interfered effectively with HIV-1 spread between T cells (26). Inhibitors of the HIV-1 surface glycoprotein (gp120)-CD4 or gp120-CXCR4 interaction reduced X4 HIV-1 VS assembly and viral transfer if applied prior to mixing of infected and receptor-expressing target cells (17, 19), but the effect of these inhibitors has not been tested on preformed VS. Thus, the field is currently unclear on whether direct T-cell-T-cell infectious HIV-1 spread is susceptible or not to antibody and entry inhibitor-mediated disruption of VS assembly, and the related question, whether the VS is permeable to viral entry inhibitors, including NAb. Addressing these questions is of central importance to understanding HIV-1 pathogenesis and informing future drug and vaccine design.Since estimates reported in the literature of the relative efficiency of direct HIV-1 T-cell-T-cell spread compared to cell-free spread vary by approximately 3 orders of magnitude (6, 38, 42), and the evidence for the activity of viral entry inhibitors on cell-cell spread is conflicting, we set out to quantify the efficiency of infection across the T-cell VS and analyze the susceptibility of this structure to NAb and viral entry inhibitors. Assays reporting on events proximal to productive infection show that the R5 HIV-1 T-cell VS is approximately 1 order of magnitude more efficient than cell-free virus infection, and imaging analyses reveal that the VS assembled by HIV-1 is most likely permeable to inhibitors both during, and subsequent to, VS assembly. Thus, we conclude that the T-cell VS does not provide a privileged environment allowing HIV-1 escape from entry inhibition.     

Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability

A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.The production of biofuels from nonfood cellulosic biomass would benefit the economy, the environment, and national energy security (17, 32). The largest technological and economical obstacle is the release of soluble fermentable sugars at prices competitive with those from sugarcane or corn kernels (17, 31). One of the approaches is discovering new cellulases from cellulolytic microorganisms, followed by cellulase engineering for enhanced performance on pretreated solid substrates. However, cellulase engineering remains challenging because enzymatic cellulose hydrolysis is complicated, involving heterogeneous substrates (33, 37), different action mode cellulase components (18), synergy and/or competition among cellulase components (36, 37), and declining substrate reactivity over the course of conversion (11, 26). Directed enzyme evolution, independent of knowledge of the protein structure and the enzyme-substrate interactions (6, 34), has been conducted to generate endoglucanase mutants, such as enhanced activities on soluble substrates (14, 16, 22), prolonged thermostability (20), changed optimum pH (24, 28), or improved expression levels (21). Here, we cloned and characterized a family 5 glycoside hydrolase (Cel5A) from a cellulolytic bacterium, Clostridium phytofermentans ISDg (ATCC 700394) (29, 30), and engineered it for enhanced thermostability.     

Detection of Infectious Adenoviruses in Environmental Waters by Fluorescence-Activated Cell Sorting Assay

Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 10⁴ PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r² value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r² value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.Waterborne viral infection is one of the most important causes of human morbidity in the world. There are hundreds of different types of human viruses present in human sewage, which, if improperly treated, may become the source of contamination in drinking and recreational waters (6, 12, 19). Furthermore, as water scarcity intensifies in the nation, so has consideration of wastewater reuse as a valid and essential alternative for resolving water shortages (31).Currently, routine viral monitoring is not required for drinking or recreational waters, nor is it required for wastewater that is discharged into the environment. This lack of a monitoring effort is due largely to the lack of methods that can rapidly and sensitively detect infectious viruses in environmental samples. In the past 20 years, tremendous progress has been made in detection of viruses in the environment based on molecular technology (32, 33, 35). PCR and quantitative real-time PCR (qPCR) methods have improved both the speed and sensitivity of viral detection compared with detection by the traditional tissue culture method (2, 11, 17, 18). However, they provide little information on viral infectivity, which is crucial for human health risk assessment (22-24, 35). Our previous work using a real-time PCR assay to detect human adenoviruses (Ads) in sewage could not differentiate the infectious viruses in the secondary treated sewage from those killed by chlorination disinfection (15). In this research, we pursued an innovative approach to detecting infectious viruses in water using fluorescence-activated cell sorting (FACS). This method is rapid and sensitive, with an established record in microbiological research (29, 34, 39).FACS is a specialized type of flow cytometry which provides a method for counting and sorting a heterogeneous mixture of biological cells into two or more kinds, one cell at a time, based upon the specific light-scattering and fluorescent characteristics of each cell (4, 25, 34, 38). It is a useful method since it provides fast and quantitative recording of fluorescent signals from individual cells (14, 16, 34, 47). The FACS viral assay is based on the expression of viral protein inside the recipient cell during viral replication (16). Specific antibody labeled with fluorescence is bound to the target viral protein, which results in fluorescence emission from infected cells. Viral particles outside the cell will not be captured, because the size of virus is below the detection limit of flow cytometry. Therefore, detection of cells, which can be captured with fluorescently labeled viral antibody, is a definitive indication of the presence of infectious virus.This research used human Ads as the target for development of the FACS method. The rationale for this choice is as follows. (i) Ads are important human pathogens that may be transmitted by water consumption and water spray (aerosols) (26, 32). The health hazard associated with exposure to Ads has been demonstrated by epidemiological data and clinical research (1, 7, 9, 35, 40, 43). (ii) Ads are among the most prevalent human viruses identified in human sewage and are frequently detected in marine waters and the Great Lakes (17, 32, 33, 35). (iii) Ads are more resistant to UV disinfection than any other bacteria or viruses (3, 5, 10, 24, 41, 42, 44). Thus, they may survive wastewater treatment as increasing numbers of wastewater treatment facilities switch from chlorination to UV to avoid disinfection by-products. (iv) Some serotypes of Ads, including enteric Ad 40 and 41, are fastidious. They are difficult to detect by plaque assay, and a routine assay of infectivity takes 7 to 14 days (8, 20).In this study, recombinant Ad serotype 5 (rAd5) with the E1A gene (the first transcribed gene after infection) replaced by a green fluorescent protein (GFP) gene was first used to test for sensitivity and speed of the assay. Two other viral proteins were then used as targets for development of FACS assays using Ad serotype 2 (Ad2) and Ad41. This study demonstrated the feasibility, sensitivity, and reliability of the assay for detection of infectious Ads in environmental samples.