Concentration Of Virus-like Particles Research Articles

A Hydrodynamic Approach to the Study of HIV Virus-Like Particle (VLP) Tangential Flow Filtration.

Emerging as a promising pathway to HIV vaccines, Virus-Like Particles (VLPs) have drawn considerable attention in recent years. A challenge of working with HIV VLPs in biopharmaceutical processes is their low rigidity, and factors such as shear stress, osmotic pressure and pH variation have to be reduced during their production. In this context, the purification and concentration of VLPs are often achieved by means of Tangential Flow Filtration (TFF) involving ultrafiltration hollow fiber modules. Despite the urgent need for robust upscaling strategies and further process cost reduction, very little attention has been dedicated to the identification of the mechanisms limiting the performance of HIV VLP TFF processes. In this work, for the first time, a hydrodynamic approach based on particle friction was successfully developed as a methodology for both the optimization and the upscaling of HIV VLP TFF. Friction forces acting on near-membrane HIV VLPs are estimated, and the plausibility of the derived static coefficients of friction is discussed. The particle friction-based model seems to be very suitable for the fitting of experimental data related to HIV VLP TFF as well as for upscaling projections. According to our predictions, there is still considerable room for improvement of HIV VLP TFF, and operating this process at slightly higher flow velocities may dramatically enhance the efficiency of VLP purification and concentration. This work offers substantial guidance to membrane scientists during the design of upscaling strategies for HIV VLP TFF.

Severe acute respiratory syndrome coronavirus 2 virus-like particles induce dendritic cell maturation and modulate T cell immunity.

Dendritic cells (DCs) are professional antigen-presenting cells that play an important role in both innate and acquired immune responses against pathogens. However, the role of DCs in coronavirus disease 2019 (COVID-19) is unclear. Virus-like particles (VLPs) that structurally mimic the original virus are one of the candidates COVID-19 vaccines. In the present study, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VLPs were used as an alternative to live virus to evaluate the interaction of the virus with DCs. The results revealed that SARS-CoV-2 VLPs induced DC maturation by augmenting cell surface molecule expression (CD80, CD86, and major histocompatibility complex class II (MHC-II)) and inflammatory cytokine production (tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-12p70) in DCs via the mitogen-activated protein kinase and nuclear factor-κB signaling pathways. In addition, mature DCs induced by SARS-CoV-2 VLPs promoted T cell proliferation, which was dependent on VLPs concentration. Our results suggest that SARS-CoV-2 VLPs regulate the immune response by interacting with DCs. These findings will improve the understanding of SARS-CoV-2 pathogenesis and SARS-CoV-2 vaccine development.

External quality assessment of molecular testing of 9 viral encephalitis-related viruses in China

BackgroundEastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), Hendra virus (HeV), Nipah virus (NiV), Yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and Tick-borne encephalitis virus (TBEV) have been detected in travelers returning to China and potentially pose a serious threat to public health. Real-time reverse transcription polymerase chain reaction (rRT-PCR) plays an important role in the detection of these viruses. Although these viruses are not mainly prevalent in China, occasionally imported cases have been reported with the increase in population mobility and entry-exit activities. Therefore, it is necessary to monitor the ability of major domestic laboratories to detect and identify exotic arbovirus infections in travelers. MethodsAn external quality assessment program for the molecular detection of EEEV, VEEV, WEEV, SLEV, WNV, YFV, TBEV, HeV and NiV was organized. The assessment panel included 26 negative and positive samples with different concentrations of virus-like particles and distributed to 31 laboratories to evaluate the accuracy of virus detection. ResultsAt the laboratory level, 87.5% (7/8, EEEV), 85.7% (12/14, WEEV), 100% (13/13, VEEV), 87.5% (7/8, HeV), 76.5% (13/17, NiV), 92.6% (25/27, YFV), 81.3% (13/16, WNV), 100% (5/5, SLEV) and 75.0% (6/8, TBEV) of the participants were considered “competent”. Of all the results, the false-positive and false-negative rates were 0.3% and 0.7%, respectively. The sensitivity of most detection assays (15/17, 88.2%) was more than 90%. In addition, we observed significantly different cycle threshold values when using primer-probe sets in different target regions to detect EEEV and SLEV. ConclusionsMost laboratories have reliable virus detection capabilities. However, laboratory testing capabilities need to be improved to avoid cross-contamination and to better manage undetected false-negative samples.

Aqueous two‐phase systems and monolithic chromatography as alternative technological platforms for virus and virus‐like particle purification

AbstractThere is a rising interest in viral and non‐viral gene delivery vehicles for human gene therapy and vaccination applications, and these vehicles include nanoparticulate virus, virus‐like particles (VLP) and plasmids. This communication debates the importance of designing innovative platforms for virus and VLP concentration and purification, envisaging downstream process integration and intensification. Two alternative separation platforms are suggested and discussed: affinity‐aqueous two‐phase systems and monolithic chromatography. These can integrate clarification, concentration, and purification downstream unit operations, significantly decreasing industrial manufacturing costs. Additionally, high recovery yields are expected after process optimization,which is achieved through manipulation of the physicochemical parameters governing partition coefficients. Such cutting‐edge technologies encompass several advantages at industrial scale, particularly in continuous operations, for example ease of scalability, continuous operation potential, easy process integration, low toxicity, and high biocompatibility. Together with innovative processing conditions, they can effectively overcome the major bottlenecks of conventional virus and VLP downstream processing, contributing to more efficient, sustainable, and cost effective industrial systems. © 2020 Society of Chemical Industry (SCI)

Quantification of the HIV-1 virus-like particle production process by super-resolution imaging: From VLP budding to nanoparticle analysis.

Virus-like particles (VLPs) offer great promise in the field of nanomedicine. Enveloped VLPs are a class of these nanoparticles and their production process occurs by a budding process, which is known to be the most critical step at intracellular level. In this study, we developed a novel imaging method based on super-resolution fluorescence microscopy (SRFM) to assess the generation of VLPs in living cells. This methodology was applied to study the production of Gag VLPs in three animal cell platforms of reference: HEK 293-transient gene expression (TGE), High Five-baculovirus expression vector system (BEVS) and Sf9-BEVS. Quantification of the number of VLP assembly sites per cell ranged from 500 to 3,000 in the different systems evaluated. Although the BEVS was superior in terms of Gag polyprotein expression, the HEK 293-TGE platform was more efficient regarding the assembly of Gag as VLPs. This was translated into higher levels of non-assembled Gag monomer in BEVS harvested supernatants. Furthermore, the presence of contaminating nanoparticles was evidenced in all three systems, specifically in High Five cells. The SRFM-based method here developed was also successfully applied to measure the concentration of VLPs in crude supernatants. The lipid membrane of VLPs and the presence of nucleic acids alongside these nanoparticles could also be detected using common staining procedures. Overall, a complete picture of the VLP production process was achieved in these three production platforms. The robustness and sensitivity of this new approach broaden the applicability of SRFM toward the development of new detection, diagnosis and quantification methods based on confocal microscopy in living systems.

Process monitoring of virus-like particle reassembly by diafiltration with UV/Vis spectroscopy and light scattering.

Virus‐like particles (VLPs) have shown great potential as biopharmaceuticals in the market and in clinics. Nonenveloped, in vivo assembled VLPs are typically disassembled and reassembled in vitro to improve particle stability, homogeneity, and immunogenicity. At the industrial scale, cross‐flow filtration (CFF) is the method of choice for performing reassembly by diafiltration. Here, we developed an experimental CFF setup with an on‐line measurement loop for the implementation of process analytical technology (PAT). The measurement loop included an ultraviolet and visible (UV/Vis) spectrometer as well as a light scattering photometer. These sensors allowed for monitoring protein concentration, protein tertiary structure, and protein quaternary structure. The experimental setup was tested with three Hepatitis B core Antigen (HBcAg) variants. With each variant, three reassembly processes were performed at different transmembrane pressures (TMPs). While light scattering provided information on the assembly progress, UV/Vis allowed for monitoring the protein concentration and the rate of VLP assembly based on the microenvironment of Tyrosine‐132. VLP formation was verified by off‐line dynamic light scattering (DLS) and transmission electron microscopy (TEM). Furthermore, the experimental results provided evidence of aggregate‐related assembly inhibition and showed that off‐line size‐exclusion chromatography does not provide a complete picture of the particle content. Finally, a Partial‐Least Squares (PLS) model was calibrated to predict VLP concentrations in the process solution. Q2 values of 0.947–0.984 were reached for the three HBcAg variants. In summary, the proposed experimental setup provides a powerful platform for developing and monitoring VLP reassembly steps by CFF.

Intracellular characterization of Gag VLP production by transient transfection of HEK 293 cells.

Transient transfection is a fast, flexible, and cost-effective approach to produce biological products. Despite the continued interest in transient transfection, little is known regarding the transfection process at the intracellular level, particularly for complex products, such as virus-like particles (VLPs). The kinetics of PEI-mediated transfection following an established in-house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement. For this purpose, DNA/PEI polyplexes' internalization in cells was tracked using Cy3 DNA staining. The production of a fluorescently labeled Gag polyprotein (a Gag-GFP fusion construct that forms fluorescent Gag-VLPs) was monitored by flow cytometry and confocal microscopy, and the VLP concentration in supernatants was measured by fluorometry. DNA/PEI polyplexes interact with the cell membrane immediately after polyplex addition to the cell culture. A linear increase in the number of cells expressing the protein is observed during the first 60 min of contact between the cells and polyplexes. No additional improvement in the number of cells expressing the protein (up to 60%) or VLP production (up to 1 × 1010 VLPs/mL) is observed with additional contact time between the cells and polyplexes. Polyplexes can be detected in the cytoplasm of transfected cells as early as 1.5 h post-transfection (hpt) and reach the nucleus approximately 4 hpt. GFP fluorescence is observed homogeneously in the cytoplasm of transfected cells 24 hpt, but generalized VLP budding is not observed by microscopy until 48 hpt. Although all cells have internalized a polyplex soon after transfection, only a fraction of cells (60%) express the fluorescent Gag protein. VLP production kinetics was also studied. Fluorescence in the supernatant (enveloped VLPs) is 40% less than total fluorescence, supernatant plus pellet (total Gag-GFP), indicating that there is a fraction of Gag that remains inside the cells. The maximum VLP concentration in the cell culture supernatant with cell viability >89% was observed at 72 hpt, which was determined to be the optimal harvest time. Biotechnol. Bioeng. 2017;114: 2507-2517. © 2017 Wiley Periodicals, Inc.

Viruses in the Oceanic Basement.

ABSTRACTMicrobial life has been detected well into the igneous crust of the seafloor (i.e., the oceanic basement), but there have been no reports confirming the presence of viruses in this habitat. To detect and characterize an ocean basement virome, geothermally heated fluid samples (ca. 60 to 65°C) were collected from 117 to 292 m deep into the ocean basement using seafloor observatories installed in two boreholes (Integrated Ocean Drilling Program [IODP] U1362A and U1362B) drilled in the eastern sediment-covered flank of the Juan de Fuca Ridge. Concentrations of virus-like particles in the fluid samples were on the order of 0.2 × 105 to 2 × 105 ml−1 (n = 8), higher than prokaryote-like cells in the same samples by a factor of 9 on average (range, 1.5 to 27). Electron microscopy revealed diverse viral morphotypes similar to those of viruses known to infect bacteria and thermophilic archaea. An analysis of virus-like sequences in basement microbial metagenomes suggests that those from archaeon-infecting viruses were the most common (63 to 80%). Complete genomes of a putative archaeon-infecting virus and a prophage within an archaeal scaffold were identified among the assembled sequences, and sequence analysis suggests that they represent lineages divergent from known thermophilic viruses. Of the clustered regularly interspaced short palindromic repeat (CRISPR)-containing scaffolds in the metagenomes for which a taxonomy could be inferred (163 out of 737), 51 to 55% appeared to be archaeal and 45 to 49% appeared to be bacterial. These results imply that the warmed, highly altered fluids in deeply buried ocean basement harbor a distinct assemblage of novel viruses, including many that infect archaea, and that these viruses are active participants in the ecology of the basement microbiome.

Enumerating Virus-Like Particles and Bacterial Populations in the Sinuses of Chronic Rhinosinusitis Patients Using Flow Cytometry

There is increasing evidence to suggest that the sinus microbiome plays a role in the pathogenesis of chronic rhinosinusitis (CRS). However, the concentration of these microorganisms within the sinuses is still unknown. We show that flow cytometry can be used to enumerate bacteria and virus-like particles (VLPs) in sinus flush samples of CRS patients. This was achieved through trialling 5 sample preparation techniques for flow cytometry. We found high concentrations of bacteria and VLPs in these samples. Untreated samples produced the highest average bacterial and VLP counts with 3.3 ± 0.74 x 107 bacteria ml-1 and 2.4 ± 1.23 x 109 VLP ml-1 of sinus flush (n = 9). These counts were significantly higher than most of the treated samples (p < 0.05). Results showed 103 and 104 times inter-patient variation for bacteria and VLP concentrations. This wide variation suggests that diagnosis and treatment need to be personalised and that utilising flow cytometry is useful and efficient for this. This study is the first to enumerate bacterial and VLP populations in the maxillary sinus of CRS patients. The relevance of enumeration is that with increasing antimicrobial resistance, antibiotics are becoming less effective at treating bacterial infections of the sinuses, so alternative therapies are needed. Phage therapy has been proposed as one such alternative, but for dosing, the abundance of bacteria is required. Knowledge of whether phages are normally present in the sinuses will assist in gauging the safety of applying phage therapy to sinuses. Our finding, that large numbers of VLP are frequently present in sinuses, indicates that phage therapy may represent a minimally disruptive intervention towards the nasal microbiome. We propose that flow cytometry can be used as a tool to assess microbial biomass dynamics in sinuses and other anatomical locations where infection can cause disease.

Generation of E. coli-derived virus-like particles of porcine circovirus type 2 and their use in an indirect IgG enzyme-linked immunosorbent assay.

Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4% were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4% (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes.

Evaluation of Bio-Layer Interferometric Biosensors for Label-Free Rapid Detection of Norovirus Using Virus Like Particles

This study evaluated the label-free bio-layer interferometric (BLI) biosensor for the detection of norovirus (NoV) using two types of virus like particles (VLPs) that represent human NoV GI.1 and GII.4. To construct biosensors for NoV GI.1 and GII.4 detection, the commercial AMC sensors, on which anti-mouse Fc-specific antibodies were preimmobilized on the surfaces, were further bound with the capture antibodies mAb3901 and mAb NS14, respectively, by using the Blitz system. The kinetics of immobilization of capture antibodies on the AMC sensors demonstrated that mAb3901 and mAb NS14 reached saturated binding phase almost at the same time (~415 s). The optimal concentration of capture antibodies for immobilization was 15 μg/mL for both mAb3901 and mAb NS14. The AMC sensors loaded more mAb NS14 than mAb3901 at the same binding condition. The biosensors constructed by immobilization of the capture antibodies at their optimal concentration showed tight binding interactions with their respective GI.1 VLPs and GII.4 VLPs, with the affinity constant of 6.01 × 10-7 M and 2.01 × 10-7 M, respectively. For both biosensors, the VLPs binding rates were linearly increased with the increase of VLP concentrations. These biosensors were able to detect GI.1 or GII.4 VLPs at the concentration of 5 μg/mL in PBS, and showed intense and stable binding interactions at VLP concentration of 10 μg/mL and above. The mAb NS14-immoblized biosensors for GII.4 VLP detection were more sensitive than the mAb3901-immoblized biosensors for GI.1 VLP detection. This detection technique was label-free, easy, rapid (2 min), and accurate, requiring a very small sample volume (4 μL).

External quality assessment for the molecular detection of MERS-CoV in China

BackgroundIn May 2015, an imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection occurred in China, so rapid and reliable diagnosis of suspected cases was necessary. ObjectivesAn external quality assessment (EQA) program for the molecular detection of MERS-CoV was organized by the National Center for Clinical Laboratories (NCCL). Study designMS2 virus-like particles (VLPs) encapsulating specific RNA sequences of MERS-CoV were prepared as positive specimens. The assessment panel, which comprised of three negative and seven positive samples with different concentrations of VLPs, was distributed to 56 laboratories from 16 provinces, municipalities, or autonomous regions for molecular detection. ResultsAmong the received data sets, three employed an in-house-developed real-time reverse-transcription polymerase chain reaction (rRT-PCR) assay and the others applied various commercial rRT-PCR kits. Overall, the majority of laboratories (46/56, 82.1%) could achieve 100% accuracy for MERS-CoV detection, but three laboratories (5.4%) still had room for improvement. Consequently, all negative samples were identified correctly, reaching 100% specificity. The false-negative rate was 3.1%, and most of the false-negative results were obtained from samples with relatively low concentration, indicating an urgent need to improve detection in weak-positive specimens. ConclusionsThe majority of participants possessed reliable diagnostic capacity for the detection of MERS-CoV. Moreover, EQA is indispensable because it can help enhance the diagnostic capability for the surveillance of MERS-CoV infections and allow comparison of the results among different laboratories.

Characterization and quantitation of fluorescent Gag virus-like particles

Background Upon expression, the Gag polyprotein of HIV-1 spontaneously assembles giving rise to enveloped virus-like particles (VLPs). These particulate immunogens offer great promise as HIV-1 vaccines. In order to develop robust VLP manufacturing processes, the availability of simple, fast and reliable quantitation tools is crucial. Traditionally, commercial p24 ELISA kits are used to estimate Gag VLP concentrations. However, this quantitation technique is time-consuming, laborious, costly and prone to methodological variability. Reporter proteins are frequently used during process development to allow a straightforward monitoring and quantitation of labeled products. This alternative was evaluated in the present work by using a Gag-GFP fusion construct.

Characterization of Persistent Virus-Like Particles in Two Acetate-Fed Methanogenic Reactors

The objective of this study was to characterize the morphology, size-distribution, concentration and genome size of virus-like particles (VLPs) in two acetate-fed Methanosaeta-dominated reactors to better understand the possible correlation between viruses and archaeal hosts. The study reactors were dominated by a single genus of acetoclastic methanogen, Methanosaeta, which was present at 6 to 13 times higher than the combined bacterial populations consisting of Proteobacteria, Firmicutes, and Bacteroidetes. Epifluorescent microscopy showed VLPs concentration of 7.1 ± 1.5×107 VLPs/ml and 8.4 ± 4.3×107 VLPs/ml in the two laboratory reactors. Observations of no detectable import of VLPs with the reactor feed combined long operational time since the last inocula were introduced suggests that the VLP populations were actively propagating in the reactors. Transmission electron microscopy images showed VLPs with morphology consistent with Siphoviridae in both reactors, and VLPs with morphologies consistent with Myoviridae in one of the reactors. The morphology, size-distribution and genome size of VLPs were distinct between reactors suggesting that unique viral populations inhabited each reactor, though the hosts of these VLPs remain unclear.

Development of Bead-based Immunoassay to Quantify Neutralizing Antibody for Human Papillomavirus 16 and 18

Human papillomavirus (HPV) has drawn great attention globally because of its association with virtually all (99%) cases of cervical cancer. HPV virus-like particles (VLPs) have been implicated as an effective HPV vaccine candidate. In this study, we optimized the relevant parameters for bacterial production of high-risk HPV16 and HPV18 VLP L1 proteins. The combination of glutathione S-transferase fusion and late log phase culture induction enhanced the solubility and yield of HPV L1 proteins. For detection and quantification of HPV-16 and -18 antibodies, a Luminex-based competitive immunoassay was developed for use in vaccine clinical trials. The characteristics of the assay that were optimized included monoclonal antibody specificity, conjugation of VLP to microspheres, VLP concentration, antibody concentration, dilution of samples, and incubation time. No cross-reactivity occurred. This immunoassay was proven to be sensitive and accurate, and is potentially valuable for vaccine candidate evaluation and clinical use.

Detection of antibodies to HPV vaccine types using a multiplexed immunoassay

Measurement of HPV antibodies in unvaccinated individuals has been used as measure of lifetime exposure to HPV. With the implementation of HPV vaccines, reliable assays are needed to evaluate the impact of altered dosing schemes or of new vaccine formulations. An ideal assay platform would allow multiplex type‐specific detection with minimal sample requirement.We used the Meso Scale Discovery (MSD) electrochemiluminescence ECL based detection platform to develop a multiplex direct‐ virus‐like particles (VLP) assay. HPV 6, 11, 16, and 18 VLPs were produced in mammalian cell‐culture and purified using Optiprep™ gradient followed by agarose gel filtration. MSD prepared the plates in the 7‐spot/well format, using the purified VLPs (4 spots) and PBS pH 7.4 as blank (one spot). We used four different coating conditions, varying VLP concentrations with and without bovine serum albumin (BSA). Results were evaluated using a set of 17 sera, WHO International Standard for HPV 16, and an optimized ELISA protocol for MSD plates on the SI6000 imager. Three point titrations and the parallel line method was used to calculate antibody titers. Stability of the plates was evaluated by comparing results after varying times of storage.The use of BSA in spot‐coating allowed for increased stability of the VLPs on the plate as indicated by similar RLUs on plates tested 1 month apart. The intra and inter‐assay CVs between PLL values calculated for each type was less than 8% and 17% respectively. No cross‐reactivity was observed between HPV types. The MSD platform shows promise for simultaneous quantitation of the antibody responses to several HPV types in a high‐ throughput manner. Funding for this project was provided by the American Recovery and Reinvestment Act.The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency

A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles

Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis worldwide. NoV GII-4 VP1 protein was expressed in a recombinant baculovirus system using Sf9 insect cells. Several methods for purification and concentration of virus-like particles (VLPs) were evaluated. Electron microscopy (EM) and histo-blood group antigen (HBGA) binding assays showed that repeated sucrose gradient purification followed by ultrafiltration resulted in intact VLPs with excellent binding to H type 3 antigens. VLPs were stable for at least 12 months at 4°C, and up to 7 days at ambient temperature. These findings indicate that this method yielded stable and high-quality VLPs.

Viral dynamics and patterns of lysogeny in saline Antarctic lakes

Antarctic lakes are extreme ecosystems with microbially dominated food webs, in which viruses may be important in controlling community dynamics. A year long investigation of two Antarctic saline lakes (Ace and Pendant Lakes) revealed high concentrations of virus like particles (VLP) (0.20–1.26 × 108 ml−1), high VLP: bacteria ratios (maximum 70.6) and a seasonal pattern of lysogeny differing from that seen at lower latitudes. Highest rates of lysogeny (up to 32% in Pendant Lake and 71% in Ace Lake) occurred in winter and spring, with low or no lysogeny in summer. Rates of virus production (range 0.176–0.823 × 106 viruses ml−1 h−1) were comparable to lower latitude freshwater lakes. In Ace Lake VLP did not correlate with bacterial cell concentration or bacterial production but correlated positively with primary production, while in Pendant Lake VLP abundance correlated positively with both bacterial cell numbers and bacterial production but not with primary production. In terms of virus and bacterial dynamics the two saline Antarctic lakes studied appear distinct from other aquatic ecosystems investigated so far, in having very high viral to bacterial ratios (VBR) and a very high occurrence of lysogeny in winter.

Flow cytometric analysis of virus-like particles and heterotrophic bacteria within coral-associated reef water

Using flow cytometry, two distinct populations of virus-like particles (VLP) and heterotrophic bacteria were defined within the 12 cm water layer immediately overlying healthy, diseased and dead acroporid corals. Bacterial abundances were similar in overlying water for all coral types, however, VLP were 30% higher above diseased corals than healthy or dead corals. Mean virus to bacteria ratios (VBR) were up to 30% higher above diseased corals than above healthy or dead coral or in distant water. Concomitant with increasing VLP concentrations within 5 cm of coral surfaces, VBR distributions were generally highest above healthy and diseased coral and depressed above dead coral. These results suggest fundamental shifts in the VLP and bacterial community in water associated with diseased corals.

Optimization and Validation of a Multiplexed Luminex Assay To Quantify Antibodies to Neutralizing Epitopes on Human Papillomaviruses 6, 11, 16, and 18

A human papillomavirus (HPV) multiplexed competitive Luminex immunoassay first described by Opalka et al. (D. Opalka, C. E. Lachman, S. A. MacMullen, K. U. Jansen, J. F. Smith, N. Chirmule, and M. T. Esser, Clin. Diagn. Lab. Immunol. 10:108--15, 2003) was optimized and validated for use in epidemiology studies and vaccine clinical trials. Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (MAb) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, MAb concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines.