Virus Suspension Research Articles

Optical manipulation of a single human virus for study of viral-cell interactions.

Although Ashkin and Dziedzic first demonstrated optical trapping of individual tobacco mosaic viruses in suspension as early as 1987, this pioneering work has not been followed up only until recently. Using human immunodeficiency virus type 1 (HIV-1) as a model virus, we have recently demonstrated that a single HIV-1 virion can be stabled trapped, manipulated and measured in physiological media with high precision. The capability to optically trap a single virion in suspension not only allows us to determine, for the first time, the refractive index of a single virus with high precision, but also quantitate the heterogeneity among individual virions with single-molecule resolution, the results of which shed light on the molecular mechanisms of virion infectivity. Here we report the further development of a set of microscopic techniques to physically deliver a single HIV-1 virion to a single host cell in solution. Combined with simultaneous epifluorescence imaging, the attachment and dissociation events of individual manipulated virions on host cell surface can be measured and the results help us understand the role of diffusion in mediating viral attachment to host cells. The establishment of these techniques opens up new ways for investigation of a wide range of virion-cell interactions, and should be applicable for study of B cell interactions with particulate antigens such as viruses.

Antiviral activity of 7-keto-stigmasterol obtained from green Antarctic algae Prasiola crispa against equine herpesvirus 1

The aim of this investigation was to evaluate the in vitro cytotoxic effect and antiviral properties of 7-keto-stigmasterol from the crude extract of the green Antarctic alga Prasiola crispa in dichloromethane against the replication of equine herpesvirus 1 (EHV-1), a worldwide enzootic etiological agent of clinical signs such as abortion, as well as neurological and respiratory diseases. Extract samples were fractionated in silica gel of 70–230 and 230–400 mesh and identified with 1H and 13C nuclear magnetic resonance spectroscopy. The cytotoxic effect was assayed with 3-(4,5-dimethylthiazol-2yl)-2,5diphenyl tetrazolium bromide (MTT), neutral red, and violet crystal in rabbit kidney (RK-13) cells treated with 12.5, 25, 50, 100, and 200 μM of 7-keto-stigmasterol. The CC50 of MTT, neutral red, and violet crystal were 1884 ± 7.5, 799 ± 6.7, and 1002 ± 6.3 μM, respectively. To characterize the antiviral action of 7-keto-stigmasterol and acyclovir, RK-13 cells were inoculated with 200 plaque-forming units of EHV-1 and treated with 12.5, 25, and 50 μM of both compounds and the EC50 value (39 ± 6 μM) of 7-keto-stigmasterol protected 50 % of RK-13 cells, with a selectivity index of 47, 20, and 26 for MTT, neutral red, and violet crystal, respectively. However, acyclovir showed no antiviral activity on the EHV-1 variant studied. EHV-1 was inactivated by mixing a viral suspension with 1 × 104 plaque-forming units with different concentrations of 7-keto-stigmasterol and incubated at room temperature (24 °C) for 1 h; a control of untreated infected cells was performed under the same conditions. The samples were then diluted, and the residual infectivity was determined by a plaque assay. The percentages of EHV-1 inactivation with 7-keto-stigmasterol were 12.5 (51 %), 25 (67 %), 50 (91 %), and 100 μM (100 %). The inactivation of EHV-1 by direct interaction with 7-keto-stigmasterol probably interferes with EHV-1 attachment to cells with irreversible inactivation of virus infectivity.

Pulsed light inactivation of murine norovirus, Tulane virus, Escherichia coli O157:H7 and Salmonella in suspension and on berry surfaces

Pulsed light (PL) inactivation of two human norovirus (HuNoV) surrogates, murine norovirus (MNV-1) and Tulane virus (TV), and two bacterial pathogens, Escherichia coli O157:H7 and Salmonella, were evaluated. The viruses and bacteria were suspended in phosphate buffered saline (PBS) to final populations of ∼6 log PFU/mL and ∼6 log CFU/mL, respectively. Both viral and bacterial suspensions were then irradiated by PL for different durations and the reductions of each microorganisms were determined. MNV-1 and TV were significantly (P < 0.05) more resistant to PL treatment than Salmonella and E. coli O157:H7 in PBS suspension. MNV-1, Salmonella and E. coli O157:H7 were also inoculated on strawberries and blueberries and the PL inactivation of each microorganism was determined. Lower inactivation of each microorganism was achieved on berry surfaces than in PBS suspension. This study shows that PL can induce rapid inactivation of MNV-1, TV, Salmonella and E. coli O157:H7 in clear suspension with viruses more resistant to PL treatment than bacteria. The efficacy of PL treatment is substantially influenced by food surface structure.

Clinical and virological characteristics of calves experimentally infected with a Brazilian isolate of bovine viral diarrhea virus type 1a

ABSTRACT: To study the pathogenicity of the Brazilian bovine viral diarrhea virus (BVDV) type 1a 241.10 isolate, four calves were intranasally inoculated with a viral suspension containing 107.2 TCID50 mL-1. One calf was left uninoculated and kept in contact with the other calves to investigate viral transmissibility. After inoculation, the animals were monitored daily for clinical signs of infection. The presence of the virus in the blood and nasal secretions was confirmed by virus isolation in cell culture. White blood cells were quantified prior to and every 3 days after infection, and the presence of antibodies was checked every 7 days, starting at day 0 until day 42 post-inoculation (pi). After infection, nasal and ocular serous secretions were observed between days 1 and 5 pi, along with a mild cough from days 2 to 4 pi; however, no severe clinical signs were present. Body temperature was slightly elevated between days 4 and 6 pi. The control calf did not develop any of the signs observed in the infected animals. Cell culture-mediated virus isolation confirmed viremia between days 4 and 8 pi and the presence of the virus in the nasal secretions between days 1 and 10 pi. All infected animals showed a decrease in white blood cell count. Antibodies could be detected from day 14 pi, and these levels remained high until day 35 pi. The control calf had no viremia, viral presence in nasal secretions, or positive serology, indicating the absence of viral transmission. Thus, isolate BVDV 1a 241.10 has low pathogenicity and transmissibility but retains immunosuppressive capacity.

Ultrastructure of Zika virus particles in cell cultures.

Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient’s blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible.

Dialysis buffer with different ionic strength affects the antigenicity of cultured nervous necrosis virus (NNV) suspensions

Nervous necrosis virus (NNV) belongs to the genus Betanodavirus (Nodaviridae). It is highly pathogenic to various marine fishes. Here, we investigated the antigenicity changes of cultured NNV suspensions during 14days of dialyses using a dialysis tube at 1.4×104 molecular weight cut off (MWCO) in three different buffers (Dulbecco’s phosphate buffered saline (D-PBS), 15mM Tris–HCl (pH 8.0), and deionized water (DIW)). Total NNV antigen titers of cultured NNV suspension varied depending on different dialysis buffers. For example, total NNV antigen titer during D-PBS dialysis was increased once but then decreased. During Tris–HCl dialysis, it was relatively stable. During dialysis in DIW, total NNV antigen titer was increased gradually. These antigenicity changes in NNV suspension might be due to changes in the aggregation state of NNV particles and/or coat proteins (CPs). ELISA values of NNV suspension changed due to changing aggregates state of NNV antigens. NNV particles in suspension were aggregated at a certain level. These aggregates were progressive after D-PBS dialysis, but regressive after Tris–HCl dialysis. The purified NNV particles self-aggregated after dialysis in D-PBS or in Tris–HCl containing 600mM NaCl, but not after dialysis in Tris–HCl or DIW. Quantitative analysis is merited to determine NNV antigens in the highly purified NNV particles suspended in buffer at low salt condition.

Immunodetection of bacteriophages by a piezoelectric resonator with lateral electric field

It has been demonstrated that electroacoustic analysis with polyclonal antibodies can be used for bacteriophage detection. The frequency dependences of the real and imaginary parts of electrical impedance of a resonator with a viral suspension with antibodies were shown to be essentially different from the dependences of a resonator with control viral suspension without antibodies. It was shown that ΦAl-Sp59b bacteriophages were detected with the use of antibodies in the presence of foreign virus particles. The ΦAl-Sp59b bacteriophage content in the analyzed suspension was ~1010–106 phages/mL; the time of analysis was no more than 5 min. The optimally informative parameter for obtaining reliable information was the change in the real or imaginary part of electrical impedance at a fixed frequency near the resonance upon the addition of specific antibodies to the analyzed suspension. It was demonstrated that the interaction between bacteriophages and antibodies can be recorded, offering good prospects for the development of a biological sensor for liquid-phase identification and virus detection.

Stimulated low-frequency Raman scattering in a suspension of tobacco mosaic virus

The interaction of laser pulses with tobacco mosaic virus (TMV) in Tris-HCl pH7.5 buffer and in water has been investigated. Ruby laser pulses of 20 ns duration have been used for excitation. The spectrum of the light passing through the sample was registered with the help of a Fabry–Perot interferometer. In the case of TMV in water we observed in the spectrum only one line of the exciting laser light, but for TMV in Tris-HCl pH7.5 buffer a second line appeared, corresponding to stimulated low-frequency Raman scattering (SLFRS) on the breathing radial mode of TMV. The frequency shift of the SLFRS by 2 cm−1 (60 GHz), the conversion efficiency and the threshold are measured for the first time to the best of our knowledge.

Variable Genome Sequences of the Murine Pneumotropic Virus (Polyomaviridae) Regulatory Region Isolated from an Infected Mouse Tissue Viral Suspension.

The murine pneumotropic virus genome, isolated from an infected murine tissue homogenate, was sequenced to completion. The lungs, liver, spleen, and kidneys were the source of the tissue homogenate in order to mirror the heterogeneity of the virus population in vivo. The regulatory region sequence was found to be highly variable.

Atomic force microscopy-based single virus particle spectroscopy

Atomic force microscopy-based single virus particle force spectroscopy was developed using dielectrophoresis for fixing virions at the tip of an atomic force microscope (AFM) probe. Electron microscopic visualization was found to be necessary to prove the deposition of virus particles on the tip of the AFM probe, while fixation of single virions by incubating the tip with a virus suspension proved impossible. Force spectroscopy measurements were performed for the vaccinia virus, influenza virus, and bacteriophage AP22. ForceReader special software was designed for analyzing the force–distance curves.

In situ localization and tissue distribution of ostreid herpesvirus 1 proteins in infected Pacific oyster, Crassostrea gigas

Immunohistochemistry (IHC) assays were conducted on paraffin sections from experimentally infected spat and unchallenged spat produced in hatchery to determine the tissue distribution of three viral proteins within the Pacific oyster, Crassostrea gigas. Polyclonal antibodies were produced from recombinant proteins corresponding to two putative membrane proteins and one putative apoptosis inhibitor encoded by ORF 25, 72, and 87, respectively. Results were then compared to those obtained by in situ hybridization performed on the same individuals, and showed a substantial agreement according to Landis and Koch numeric scale. Positive signals were mainly observed in connective tissue of gills, mantle, adductor muscle, heart, digestive gland, labial palps, and gonads of infected spat. Positive signals were also reported in digestive epithelia. However, few positive signals were also observed in healthy appearing oysters (unchallenged spat) and could be due to virus persistence after a primary infection.Cellular localization of staining seemed to be linked to the function of the viral protein targeted. A nucleus staining was preferentially observed with antibodies targeting the putative apoptosis inhibitor protein whereas a cytoplasmic localization was obtained using antibodies recognizing putative membrane proteins. The detection of viral proteins was often associated with histopathological changes previously reported during OsHV-1 infection by histology and transmission electron microscopy. Within the 6h after viral suspension injection, positive signals were almost at the maximal level with the three antibodies and all studied organs appeared infected at 28h post viral injection. Connective tissue appeared to be a privileged site for OsHV-1 replication even if positive signals were observed in the epithelium cells of different organs which may be interpreted as a hypothetical portal of entry or release for the virus. IHC constitutes a suited method for analyzing the early infection stages of OsHV-1 infection and a useful tool to investigate interactions between OsHV-1 and its host at a protein level.

Effect of Holding Conditions on the Detection of Chikungunya and Venezuelan Equine Encephalitis Viruses in Mosquito Pools.

Emerging and re-emerging arboviruses continue to be a threat to global public health, and viral surveillance of mosquito populations is critical for mosquito control operations. Due to the tropical climate of many of the affected areas, it may be difficult to maintain a cold chain as the samples travel from collection sites to laboratories for testing. We determined how suboptimal holding temperatures affected the ability to detect viruses in pools of mosquitoes. Adult female Aedes albopictus and Ae. taeniorhynchus individuals were inoculated with chikungunya virus or Venezuelan equine encephalitis virus suspensions, respectively, and placed at 26°C for 8 days. One infected mosquito was then added to a vial of 24 negative mosquitoes and held at -80°C, -20°C, 4°C, 22°C, or 35°C for up to 14 days. Mosquito pools were analyzed for both infectious virus by plaque assay and for viral ribonucleic acid (RNA) with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). At higher temperatures, the amount of infectious virus decreased rapidly, but viruses in samples held at 4°C or lower remained relatively stable. In contrast, viral RNA was detectable from pools held at all temperatures and holding times by RT-qPCR. Cycle threshold (Ct) values increased as temperatures and holding times increased. These findings suggest that if viral RNA detection is the goal of surveillance efforts, then mosquito pools do not require storage at ≤4°C. This enhances the feasibility of field-based arbovirus surveillance programs in which maintaining a cold chain may not be a possibility.

Relationship Between Values of Fowlpox ELISA and the Presence of "Takes" After Vaccination.

Values from an ELISA for evaluating the immune response induced by a commercial vaccine against fowlpox virus and the lesion at the site of inoculation (i.e., swelling of the skin or a pox where the vaccine was applied) were compared. The ELISA was carried out with an antigen prepared by precipitation of a cell culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation. A 0.1 M acetate buffer (pH 5) was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well. Four experiments were conducted where the birds were bled once a week before and after vaccination and then were examined simultaneously for evidence of "takes." This study showed that there is a relationship between the ELISA values to the fowlpox vaccine that are considered positive and the presence of postvaccination lesions.

Resistance to OsHV-1 Infection in Crassostrea gigas Larvae

The ostreid herpesvirus (OsHV-1) is one of the major diseases that affect the Pacific oyster Crassostrea gigas. Selective breeding programs were recently shown to improve resistance easily to OsHV-1 infections in spat, juvenile and adult oysters. Nevertheless, this resistance has never been investigated in larvae, whereas this developmental stage has crucial importance for the production of commercial hatcheries, as well as explaining the abundance of spatfall. A first trial tested several viral suspensions at several concentrations using contaminated water with OsHV-1 in four-day-old and ten-day-old larvae that were produced from an unselected broodstock. In follow up on the results, one viral suspension at a final concentration of 10+6 OsHV-1 DNA copies per L was used to assess resistance to OsHV-1 infection in C. gigas larvae that were produced from selected and unselected broodstock. A second trial evaluated OsHV-1 resistance in larvae from both broodstocks in trials 2a, 2b and 2c with 4, 10 and 16-day-old larvae for 7 days, which corresponded to post D larvae, umbo larvae and eyed larvae, respectively. The mortality of unchallenged larvae for both stocks were low (<15%) at day 7 in trials 2a and 2b, whereas it ranged from 48 to 56% in trial 2c. More interestingly, selected larvae had significantly lower mortality than unselected larvae when exposed to OsHV-1 in all of the trials. Thus, the mortality was 11% and 49% for the selected larvae at day 7 post-exposure in trials 2a and 2c, respectively, in comparison with 84% and 97% for the unselected larvae. Although this difference in mortality was observed at day 5 in trial 2b, it was reduced at day 7, to 86% and 98% for the selected and unselected larvae, respectively. For the first time in the literature, the difference in mortality or the delayed onset of mortality between selected and unselected larvae have indicated a genetic resistance to OsHV-1 infection at the larval stage. Such finding should facilitate the selective breeding programs focusing on resistance to OsHV-1 infection by reducing the span of the genetic evaluation, and thus decreasing its cost

Design, RNA cleavage and antiviral activity of new artificial ribonucleases derived from mono-, di- and tripeptides connected by linkers of different hydrophobicity

A novel series of metal-free artificial ribonucleases (aRNases) was designed, synthesized and assessed in terms of ribonuclease activity and ability to inactivate influenza virus WSN/A33/H1N1 in vitro. The compounds were built of two short peptide fragments, which include Lys, Ser, Arg, Glu and imidazole residues in various combinations, connected by linkers of different hydrophobicity (1,12-diaminododecane or 4,9-dioxa-1,12-diaminododecane). These compounds efficiently cleaved different RNA substrates under physiological conditions at rates three to five times higher than that of artificial ribonucleases described earlier and displayed RNase A-like cleavage specificity. aRNases with the hydrophobic 1,12-diaminododecane linker displayed ribonuclease activity 3–40 times higher than aRNases with the 4,9-dioxa-1,12-diaminododecane linker. The assumed mechanism of RNA cleavage was typical for natural ribonucleases, that is, general acid-base catalysis via the formation of acid/base pairs by functional groups of amino acids present in the aRNases; the pH profile of cleavage confirmed this mechanism. The most active aRNases under study exhibited high antiviral activity and entirely inactivated influenza virus A/WSN/33/(H1N1) after a short incubation period of viral suspension under physiological conditions.

High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining.

The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed.

Khảo sát độc lực và tính gây bệnh của 1 chủng virus viêm gan vịt a genotype 3 trên phôi vịt

An examination on the virulence and the pathogenicity of a duck hepatitis A virus strain genotype 3 isolated in Hau Giang province was carried out by injecting 0.2 ml of virus suspension into allantoic cavity of 10-day-old duck embryos. The results showed that this virus was virulent and the ELD50 titer of the original virus suspension was 108,17/0.2ml.The earliest embryo death was recorded after 24 hours of inoculation and the latest death was 66 hours post inoculation. Most of embryos died after 30 to 45 hours of viral inoculation. Dead embryos had typical lesions of duck viral hepatitis infection, including stunting, edema, hemorrhagic skin, enlarged and yellowish liver with punctate or diffuse hemorrhages, discolored heart muscles. The embryos which died after 66 hours of inoculation had dark green liver and greenish allantoic fluid.

Effect of Viral Antigen Levels on the Serological Response and Efficiency of the Binary Ethylenimine-Inactivated Bluetongue Virus Serotype-16 Vaccine

Bluetongue (BT) is a serious hemorrhagic disease of ruminants caused by bluetongue virus (BTV). Inactive BTV vaccines have been successful in field trials in some areas, and inactivated vaccines are considered safer. However, information about the effect of the viral antigen level on the serological response and efficiency of the inactive BTV-16 vaccine is lacking. In the present study, the serological response and efficiency of the viral antigen concentration in the binary ethylenimine-inactivated Chinese BTV serotype-16 vaccine were investigated. The viral antigens in the viral suspension (VS) were quantified using a modified BTV AC-ELISA method. Four batches of vaccine containing 1, 5, 10, and 50 μg/ml of viral antigen were generated from the VS. Four groups of naive Chinese sheep were vaccinated with the different vaccine batches, and the serological response and vaccine efficiency were investigated before and after challenge infection. The vaccines containing 10 and 50 μg/ml of viral antigen induced significant ELISA and neutralizing antibody titers 14 days after vaccination, whereas the vaccines containing 1 and 5 μg/ml of viral antigen did not have these effects. A booster immunization at 21 days enhanced all groups’ antibody titers; however, the increased titer was related to the viral antigen level. In contrast to the serological response, the viral antigen level of the vaccines did not have a significant effect on the vaccine efficiency. With the exception of one sheep from the 5 μg/ml viral antigen group, all vaccinated sheep from the four antigen level groups showed strong resistance to infection based on their clinical symptoms, rectal temperatures and viremia. Collectively, these data suggested that viral antigen levels from 1 to 50 μg/ml had a significant effect on the serological response of the animals but a limited effect on the vaccine efficiency. The BTV-16 vaccine containing 1 μg/ml of viral antigen was sufficient to achieve high efficiency, but only the vaccines with more than 10 μg/ml of antigen induced a significant antibody response. To obtain a better serological response, we suggest the use of vaccines with more than 10 μg/ml of viral antigen. The findings in the study will be useful for BTV vaccine production.

Use of Ozone for Inactivation of Bacteria and Viruses in Cryostats

The biocidal efficacy of ozone has been investigated in cryostats at -20°C and +30°C and 30-120 min exposure time. Stainless steel coupons were inoculated with 50 μl bacterial or viral test suspension. The tests were performed using Staphylococcus aureus and Mycobacterium terrae as test bacteria and Polyoma virus (Simian virus 40, SV 40). The inoculated coupons were exposed to a combination of ozone and UV generated by UV lamps in the cryostats. To measure the level of disinfection achieved, the number of surviving bacteria recovered from the coupons and, respectively, the virus infectivity titer was determined. Reduction factors were calculated by dividing the initial titer by the titer of recovered viable bacteria or virus. Bactericidal efficacy at -20°C was demonstrated by inactivation of S. aureus by at least 5 log10 units in an automatic defrost/60 min ozone cycle in all test positions, which allowed to remove the +30°C/120 min test from the testing panel. In a 30 min treatment, a disinfection rate of 5 log10 units was achieved in some, but not all test positions. Mycobacterium terrae, the substitute for the pathogenic M. tuberculosis, was inactivated by >5.3 log10 units in all tested locations using a 120-minute ozone cycle at +30°C. The infective virus titer was reduced by at least 5 log10 units in the same ozone cycle on the pressure plate and chuck positions. Apart from this, significant reductions in viable cell and virus numbers have been shown for all disinfection protocols and test positions, thus demonstrating the principle efficacy of a combination of gaseous ozone and UV as a disinfectant in cryostats.

Detection and distribution of ostreid herpesvirus 1 in experimentally infected Pacific oyster spat

High mortality rates are reported in spat and larvae of Pacific oyster Crassostrea gigas and associated with ostreid herpesvirus 1 (OsHV-1) detection in France. Although the viral infection has been experimentally reproduced in oyster larvae and spat, little knowledge is currently available concerning the viral entry and its distribution in organs and tissues. This study compares OsHV-1 DNA and RNA detection and localization in experimentally infected oysters using two virus doses: a low dose that did not induce any mortality and a high dose inducing high mortality. Real time PCR demonstrated significant differences in terms of viral DNA amounts between the two virus doses. RNA transcripts were detected in oysters receiving the highest dose of viral suspension whereas no transcript was observed in oysters injected with the low dose. This study also allowed observing kinetics of viral DNA and RNA detection in different tissues of oyster spat. Finally, viral detection was significantly different in function of tissues (p<0.005), time (p<0.005) with an interaction between tissues and time (p<0.005) for each probe.