Increase In Virus Concentration Research Articles

SARS-CoV-2 virus label-free electrochemical nanohybrid MIP-aptasensor based on Ni3(BTC)2 MOF as a high-performance surface substrate.

A dual recognition biosensor was developed via introducing aptamer strings and molecular imprinting polymer (MIP) for the selective detection of intact SARS-CoV-2 virus based on screen printed carbon electrode (SPCE) modified with nickel-benzene tricarboxylic acid-metal–organic framework (Ni3(BTC)2 MOF) synthesized by in situ growth method, SARS-CoV-2 S protein-specific amino-aptamer and electropolymerization of dopamine (ePDA). The proposed biosensor showed an excellent linear relationship between charge transfer resistance (Rct) and increase in virus concentration in the range 10 to 108 plaque-forming units/mL (PFU/mL) with a low detection limit of 3.3 ± 0.04 PFU/mL and response time of 20 min. Compared with single-element sensors (aptamer or MIP), it showed higher selectivity for the SARS-CoV-2 virus and facilitated detection in real samples.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05357-8.

The Effects of Cytopathic and Non-cytopathic Biotypes of Bovine Viral Diarrhea Virus on Sperm Vitality and Viability of Holstein Dairy Bulls in Vitro

BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the most important pathogens. OBJECTIVES: The aim of this study was to investigate the effects of cytopathic (CP) and non-cytopathic (NCP) biotypes of BVDV on vital status, membrane integrity, and motility of sperm cells in Holstein dairy bulls in vitro. METHODS: BVDV-free frozen semen samples were counted after thawing and centrifuged to separate live sperms. A sample containing 105 spermatozoa/mL was prepared. CP and NCP BVDV with 3 different doses of 105 (high dose), 104 (medium dose), and 103 (low dose) tissue culture infectious dose (TCID) 50/mL were challenged to sperm cells. After 2 hours of incubation at 38.5°C, eosin-nigrosine staining and hypoosmotic swelling (HOS) test were performed to assess the sperm viability and plasma membrane integrity. Computer assisted semen analysis (CASA) was used to evaluate the sperm motility parameters. The obtained data were analyzed using GLM method in SAS software. RESULTS: The percentage of live spermatozoa in the control group was 72±3.60%. However, it decreased significantly with the increase of virus concentration in both groups (p ≤0.05). Sperm integrity in the control group showed that the quality of semen was 65± 3.21. But the effect of virus biotypes resulted in a significant decrease in both high (105) and low (103) concentrations (p ≤0.05). BVDV biotypes are able to reduce different sperm movements as their concentration-increases. CONCLUSIONS: We concluded that CP and NCP biotypes of BVDV had a significant effect (p ≤0.05) on survival, plasma membrane integrity, and motility of sperm cells in vitro.

Low Host Abundance and High Temperature Determine Switching from Lytic to Lysogenic Cycles in Planktonic Microbial Communities in a Tropical Sea (Red Sea).

The lytic and lysogenic life cycles of marine phages are influenced by environmental conditions such as solar radiation, temperature, and host abundance. Temperature can regulate phage infection, but its role is difficult to discern in oligotrophic waters where there is typically low host abundance and high temperatures. Here, we study the temporal variability of viral dynamics and the occurrence of lysogeny using mitomycin C in a eutrophic coastal lagoon in the oligotrophic Red Sea, which showed strong seasonality in terms of temperature (22.1–33.3 °C) and large phytoplankton blooms. Viral abundances ranged from 2.2 × 106 to 1.5 × 107 viruses mL−1 and were closely related to chlorophyll a (chl a) concentration. Observed high virus-to-bacterium ratio (VBR) (4–79; 16 ± 4 (SE)) suggests that phages exerted a tight control of their hosts as indicated by the significant decrease in bacterial abundance with increasing virus concentration. Heterotrophic bacterial abundance also showed a significant decrease with increasing temperature. However, viral abundance was not related to temperature changes and the interaction of water temperature, suggesting an indirect effect of temperature on decreased host abundance, which was observed at the end of the summertime. From the estimated burst size (BS), we observed lysogeny (undetectable to 29.1%) at low percentages of 5.0% ± 1.2 (SE) in half of the incubations with mitomycin C, while it increased to 23.9% ± 2.8 (SE) when the host abundance decreased. The results suggest that lytic phages predominate, switching to a moderate proportion of temperate phages when the host abundance reduces.

Fully Packaged Portable Thin Film Biosensor for the Direct Detection of Highly Pathogenic Viruses from On-Site Samples.

The thin film transistor (TFT) is a promising biosensor system with great sensitivity, label-free detection, and a quick response time. However, even though the TFT sensor has such advantageous characteristics, the disadvantages hamper the TFT sensor's application in the clinical field. The TFT is susceptible to light, noise, vibration, and limited usage, and this significantly limits its on-site potential as a practical biosensor. Herein, we developed a fully packaged, portable TFT electrochemical biosensor into a chip form, providing both portability through minimizing the laboratory equipment size and multiple safe usages by protecting the semiconductor sensor. Additionally, a safe environment that serves as a miniature probe station minimizes the previously mentioned disadvantages, while providing the means to properly link the TFT biosensor with a portable analyzer. The biosensor was taken into a biosafety level 3 (BSL-3) laboratory setting to analyze highly pathogenic avian influenza virus (HPAIV) samples. This virus quickly accumulates within a host, and therefore, early stage detection is critical to deterring the further spread of the deadly disease to other areas. However, current on-site methods have poor limits of detection (105-106 EID50/mL), and because the virus has low concentration in its early stages, it cannot be detected easily. We have compared the sample measurements from our device with virus concentration data obtained from a RT-PCR (virus range: 100-104 EID50/mL) and have identified an increasing voltage signal which corresponds to increasing virus concentration.

Optimized production and purification of Coxsackievirus B1 vaccine and its preclinical evaluation in a mouse model

Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus. Safety and immunogenicity of the formalin inactivated CVB1 vaccine was characterized in a mouse model. Two of the developed methods were found to be optimal for virus purification: the first employed PEG-precipitation followed by gelatin-chromatography and sucrose cushion pelleting (three-step protocol), yielding 19-fold increase in virus concentration (0.06µg/cm2) as compared to gold standard method. The second method utilized tandem sucrose pelleting without a PEG precipitation step, yielding 83-fold increase in virus concentration (0.24µg/cm2), but it was more labor-intensive and cannot be efficiently scaled up. Both protocols provide radically higher virus yields compared with traditional virus purification protocols involving PEG-precipitation and sucrose gradient ultracentrifugation. Formalin inactivation of CVB1 produced a vaccine that induced a strong, virus-neutralizing antibody response in vaccinated mice, which protected against challenge with CVB1 virus. Altogether, these results provide valuable information for the development of new enterovirus vaccines.

Multifaceted Mechanisms of HIV Inhibition and Resistance to CCR5 Inhibitors PSC-RANTES and Maraviroc

Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated. In single-cycle assays, these HIV-1 clones displayed equal sensitivity to PSC-RANTES inhibition, suggesting effective receptor downregulation. Prolonged PSC-RANTES exposure resulted in desensitization of the receptor to internalization such that increasing virus concentration (substrate) could saturate the receptors and overcome PSC-RANTES inhibition. In contrast, resistance to MVC was observed with the MVC-resistant HIV-1 (R3 versus S2) in both multiple- and single-cycle assays and with altered virus concentrations, which is indicative of allosteric inhibition. MVC could also mediate inhibition and possibly resistance through competitive mechanisms.

Effects of fluctuations in river water level on virus removal by bank filtration and aquifer passage — A scenario analysis

Riverbank filtration is an effective process for removing pathogenic viruses from river water. Despite indications that changing hydraulic conditions during floods can affect the efficacy of riverbank filtration to remove viruses, the impact on advection and dispersion of viruses in the riverbank is not well understood. We investigated the effects of fluctuations in river water level on virus transport during riverbank filtration, considering 3-D transient groundwater flow and virus transport. Using constant removal rates from published field experiments with bacteriophages, removal of viruses with distance from the riverbank was simulated for coarse gravel, fine gravel and fine sandy gravel. Our simulations showed that, in comparison with steady flow conditions, fluctuations in river water level cause viruses to be transported further at higher concentrations into the riverbank. A 1-5 m increase in river water levels led to a 2- to 4-log (log10 reduction in concentration relative to the initial concentration in the river) increase in virus concentration and to up to 30% shorter travel times. For particular cases during the receding flood, changing groundwater flow conditions caused that pristine groundwater was carried from further inland and that simulated virus concentrations were more diluted in groundwater. Our study suggests that the adverse effect of water level fluctuations on virus transport should be considered in the simulation of safe setback distances for drinking water supplies.

Risk of Viral Acute Gastrointestinal Illness from Nondisinfected Drinking Water Distribution Systems

Acute gastrointestinal illness (AGI) resulting from pathogens directly entering the piping of drinking water distribution systems is insufficiently understood. Here, we estimate AGI incidence from virus intrusions into the distribution systems of 14 nondisinfecting, groundwater-source, community water systems. Water samples for virus quantification were collected monthly at wells and households during four 12-week periods in 2006-2007. Ultraviolet (UV) disinfection was installed on the communities' wellheads during one study year; UV was absent the other year. UV was intended to eliminate virus contributions from the wells and without residual disinfectant present in these systems, any increase in virus concentration downstream at household taps represented virus contributions from the distribution system (Approach 1). During no-UV periods, distribution system viruses were estimated by the difference between well water and household tap virus concentrations (Approach 2). For both approaches, a Monte Carlo risk assessment framework was used to estimate AGI risk from distribution systems using study-specific exposure-response relationships. Depending on the exposure-response relationship selected, AGI risk from the distribution systems was 0.0180-0.0661 and 0.001-0.1047 episodes/person-year estimated by Approaches 1 and 2, respectively. These values represented 0.1-4.9% of AGI risk from all exposure routes, and 1.6-67.8% of risk related to drinking water exposure. Virus intrusions into nondisinfected drinking water distribution systems can contribute to sporadic AGI.

Integrating Silicon Nanowire Field Effect Transistor, Microfluidics and Air Sampling Techniques For Real-Time Monitoring Biological Aerosols

Numerous threats from biological aerosol exposures, such as those from H1N1 influenza, SARS, bird flu, and bioterrorism activities necessitate the development of a real-time bioaerosol sensing system, which however is a long-standing challenge in the field. Here, we developed a real-time monitoring system for airborne influenza H3N2 viruses by integrating electronically addressable silicon nanowire (SiNW) sensor devices, microfluidics and bioaerosol-to-hydrosol air sampling techniques. When airborne influenza H3N2 virus samples were collected and delivered to antibody-modified SiNW devices, discrete nanowire conductance changes were observed within seconds. In contrast, the conductance levels remained relatively unchanged when indoor air or clean air samples were delivered. A 10-fold increase in virus concentration was found to give rise to about 20-30% increase in the sensor response. The selectivity of the sensing device was successfully demonstrated using H1N1 viruses and house dust allergens. From the simulated aerosol release to the detection, we observed a time scale of 1-2 min. Quantitative polymerase chain reaction (qPCR) tests revealed that higher virus concentrations in the air samples generally corresponded to higher conductance levels in the SiNW devices. In addition, the display of detection data on remote platforms such as cell phone and computer was also successfully demonstrated with a wireless module. The work here is expected to lead to innovative methods for biological aerosol monitoring, and further improvements in each of the integrated elements could extend the system to real world applications.

Effects of calcium ion on adenovirus production with high densities of HEK293 cells

One of the major restrictions on the production of adenoviral vectors (AdV) is due to the decrease in virus concentration concomitant with an increase in cell concentration at infection (CCI) which is known as “cell density effect”, this could be because of the limited access to the nutrients or significant accumulation of toxic by-products. However, current strategies, such as developing robust serum-free medium and performing nutrient feeding, will partially address this issue. Therefore, the objective of this study was to further optimize serum-free culture medium by exploring the influence of calcium ion on virus production. Our studies showed that an optimal concentration of calcium ion significantly enhances AdV production, especially at a high CCI. During the virus infection process, a high concentration of calcium ion (≥ 1 mM) caused a reduction in virus infection efficiency, possibly as a result of cell aggregation. However, by optimizing the timing of the addition of calcium ion (i.e., 12 hours post-infection [hpi]), high virus infection efficiency could be maintained. The addition of 0.1 to 2 mM calcium ion at 12 hpi increased virus proliferation dose-dependently. Therefore, the optimal selection of both the concentration and the supplementation time of calcium ion during the process of AdV production could minimize the “cell density effect,” and a 2.6- and 3.2-fold increase in virus concentration could be achieved at CCI3 and CCI4, respectively.

Fluctuations of Prunus Necrotic Ringspot Virus (PNRSV) at Various Phenological Stages in Peach Cultivars.

Fluctuations in Prunus necrotic ringspot virus (PNRSV) concentration were researched in single plants of six peach (Prunus persicae) cultivars-Kurakata, Red Haven, Nectar Red, Start Delicious, Meadowlark, and Loadel-by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) of dormant buds (May, June), flowers (September), new sprouts (November), and mature leaves (January) (Southern Hemisphere). The optimum extract dilution (sample weight per buffer volume) to detect the virus was also quantified. The average absorbance patterns of the six cultivars show a steady increase in virus concentration, ranging from A 405nm 0.61 in May to A 405nm 0.86 in July for dormant buds, to A 405nm 1.22 in September in flowers, to 1.53 in November in new sprouts, where the highest concentration was found. Virus concentrations in mature leaves drop to values similar to those of noninfected plants in January ( A405nm 0.12). The yearly average (six noninfected peach trees) ranged from A405nm 0.04 to A405nm 0.08. This drop coincides with an increase in summer temperature and attenuates foliation symptoms caused by PNRSV. Analysis of dormants buds, flowers, or new sprouts with 5-cm-long leaves was reliable to differentiate infected from noninfected plants. Cluster analysis of absorbance profiles for single plants of cvs. Loadel and Meadowlark, however, showed a comparatively low profile, with a drop at flowering time (A405nm 0.20 in September) close to the average of healthy controls. The difference between infected and healthy plants did not become apparent in all cultivars from the analysis of plants at a given phenological stage, for example by the analysis of flower only, the material most preferred to diagnose the virus. Therefore, plants should be analyzed during flowering and sprouting or flowering and dormancy (dormant buds).

Astrocytes Infected with Replication-Defective Adenovirus Containing a Secreted Form of CNTF or NT3 Show Enhanced Support of Neuronal Populationsin Vitro

Neurotrophic factors have been shown to ameliorate neuronal death in severalin vitroandin vivomodels of neurodegenerative disease. However, delivery of polypeptide growth factors to compromised neurons in the CNS is problematic as the blood–brain barrier prevents systemic delivery, and chronic in-dwelling cannulae are required for intraparenchymal delivery. To circumvent these problems and specifically target neurotrophic factors to the environment surrounding degenerating neurons in the CNS, we have generated replication-defective adenovirus (Ad) vectors that contain a secretable form of ciliary neurotrophic factor (sCNTF) or neurotrophin-3 (NT-3). In this study, we demonstrate that sCNTF/Ad and NT-3/Ad can efficiently infect primary astrocytes, resulting in gene transcription and the production of functional protein. Using Northern blot analysis, dose-dependent expression of sCNTF or NT-3 mRNA was detected 7 days after infection. The levels of mRNA expressed in transgenic astrocytes was dependent on virus titer and increased with increasing virus concentration. sCNTF or NT-3 protein was also detected in astrocyte supernatants by immunoblot analysis and 2-site ELISA. ELISA indicated that astrocytes infected with sCNTF/Ad or NT-3/Ad secreted neurotrophic factors at a rate of approximately 120 pg/106cells/h and 350 pg/106cells/h, respectively. To test for secretion of bioactive sCNTF or NT-3 protein, E8 chick ciliary ganglion or nodose ganglion neurons were grown in medium conditioned by control astrocytes or astrocytes treated with sCNTF/Ad or NT-3/Ad, showing a robust and dose-dependent increase in neuronal survival when compared to control supernatant. In addition, motor neurons plated onto astrocyte monolayers pretreated with sCNTF/Ad showed a two- to fourfold increase in ChAT activity when compared to those grown on astrocytes pretreated with Lac-Z/Ad. This study demonstrates that, using replication-defective adenovirus, primary astrocytes can be efficiently engineered to secrete bioactive sCNTF or NT-3, resulting in enhanced survival of responsive peripheral and central neuronal populations.

Modeling AIDS vaccines: the cellular level

This paper discusses current strategies for the development of AIDS vaccines which allow immunization to disturb the natural course of HIV at different detailed stages of its life cycle. Mathematical models describing the main biological phenomena (i.e. virus and vaccine induced T4 cell growth; virus and vaccine induced activation of latently infected T4 cells; incremental changes in immune response as infection progresses; antibody dependent enhancement and neutralization of infection) and allowing for different vaccination strategies serve as a background for computer simulations. The mathematical models reproduce updated information on the behavior of immune cells, antibody concentrations and free viruses. The results point to some controversial outcomes of an AIDS vaccine such as an early increase in virus concentration among vaccinated when compared to nonvaccinated individuals.

Swedish Culex torrentium and Cx. pipiens (Diptera: Culicidae) as Experimental Vectors of Ockelbo Virus

The ability of the sibling species Culex pipiens (L.) and Culex torrentium (Martini) from central Sweden to transmit Ockelbo (OCK) virus was determined. Both species became infected after ingesting OCK virus from a viremic chicken; they transmitted this virus to chickens after 21-28 d of extrinsic incubation. In Cx. torrentium, infection rates were 90% or higher, and all 10 refeeding mosquitoes transmitted virus after feeding on chickens with a viremia of at least 10(3.0) plaque-forming units (PFU)/ml. In contrast, only 1 of 28 (4%) of the Cx. pipiens that ingested blood containing 10(3.0-3.9) PFU/ml became infected, and none of 16 refeeding mosquitoes transmitted virus. However, 98 of 184 (53%) of the Cx. pipiens that ingested a blood meal containing at least 10(6.0) PFU/ml became infected. Transmission rates in Cx. pipiens increased with increasing virus concentration in the blood meal to a maximum of 37% in mosquitoes that ingested greater than 10(8.0) PFU/ml. Based on these data, both Cx. pipiens and Cx. torrentium are capable of transmitting OCK virus in an enzootic cycle involving birds as hosts. However, Cx. torrentium appears to be physiologically a more efficient vector than Cx. pipiens.

Multiplication of Velvet Tobacco Mottle Virus in Nicotiana clevelandii Protoplasts Is Resistant to -Amanitin

Summary Protoplasts from Nicotiana clevelandii were infected with velvet tobacco mottle virus (VTMoV) using inocula containing 2 µg/ml poly-l-ornithine (PLO), 8 µg/ml VTMoV and 0.02 m-potassium citrate pH 5.0. Typically about 47% of the inoculated protoplasts become infected. The amount of virus particles bound to inoculated protoplasts was increased by a decrease in pH, an increase in PLO concentration or an increase in virus concentration. The one-step growth curve of VTMoV in N. clevelandii protoplasts showed that, although large amounts of virus were synthesized, the multiplication was slower than that published for other viruses. Alpha-amanitin (50 µg/ml) had no effect on the rate of accumulation of virus coat protein or on the synthesis of either RNA species of VTMoV when added to cultures immediately after inoculation.

Binding of rabies virus to purified Torpedo acetylcholine receptor

The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by α-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

The Effect of the Date of Rindite Treatment on the Concentration of Potato Virus Y in Tubers

AbstractThe concentration of potato virus Y (PVY) in tubers after artificial break of dormancy by Rindite treatment at three different dates (September 8, November 10, and January 11) after harvest (August 25, 1983) was examined by enzyme‐linked immuno‐sorbent assay (ELISA). The increase of virus concentration was more rapid after the early Rindite treatment than after the later ones. The virus concentration observed after the second and the third treatment varied among the cultivars and reached maximum values considerably later than after the first treatment. There seems to be a direct effect of the Rindite treatment on PVY multiplication in tubers.

Production and field evaluation of codling moth granulosis virus for control of Cydia pomonella in the United Kingdom

SUMMARYCodling moth granulosis virus (Cp GV) was produced in larvae of Cydia pomonella reared on artificial diet. The average yield of virus (9 × 109capsules/larva) was increased by raising the larvae on diet containing methoprene, a juvenile hormone analogue.In field trials in 1978 and 1979, one or two high‐volume applications of Cp GV at 7 × 1010 capsules/litre achieved reductions in numbers of mature larvae and damaged fruit that were little different from those obtained by two applications of azinphos‐methyl. A field trial in 1980 showed that at concentrations of Cp GV ≥ 6 × 108 capsules/litre, damaged diminished slowly with increasing virus concentration. Following virus application in 1980, Cp GV infectivity was reduced by half in 3 days, but some activity persisted at least 4–8 wk after spraying. The results indicate that codling moth GV is an effective control agent particularly in reducing numbers of larvae and the more severe forms of fruit damage.

Laboratory infection of the sandfly Phlebotomus papatasi Scopoli (Diptera, Psychodidae) with three Phleboviruses

Membrane feeding and intrathoracic inoculation techniques were used to infect Phlebotomus papatasi sandflies with Phlebotomus fever group viruses. Sandflies were orally infected with Pacui virus, and were inoculated with Pacui, Sicilian sandfly fever and Naples sandfly fever viruses. Pacui virus did not multiply in these insects after oral infection, but an 8 × 10 4-fold increase in virus concentration occurred within four days of intrathoracic inoculation of this virus. The infection rate was 100%. Sicilian sandfly fever virus multiplied in P. papatasi after infection by inoculation, although the yield was lower, a 20-fold increase in the mean virus concentration occurring over eight days. The results for Naples sandfly fever were inconsistent. A 32-fold increase in mean virus concentration was recorded in a small percentage of insects 14 days after inoculation of the virus.

Factors affecting the reproducibility of the serum neutralization test for infectious bursal disease

An experiment is described in which the main factors contributing to the variability of the serum neutralization test for infectious bursal disease were studied. Significant differences were found between test operators and readers. Antibody levels of sera from vaccinated SPF chickens and the method of preparing dilutions did not affect the variability of the test, but there was a rendency towards a decrease in precision with increasing virus concentration.