Capsid Damage Research Articles

Susceptibility of enveloped and non-enveloped viruses to ultraviolet light-emitting diode (UV-LED) irradiation and implications for virus inactivation mechanisms

UV-LED treatment at 265 nm was more efficient than UV-LED treatment at 280 nm in inactivating both enveloped and non-enveloped viruses. Capsid damage is not important in virus inactivation by UV-LEDs for both enveloped and non-enveloped viruses.

Development of a stable lyophilized adeno-associated virus gene therapy formulation

Adeno-associated viruses (AAV) are among the most actively investigated vectors for gene therapy. Supply of early clinical studies with frozen drug product (DP) can accelerate timelines and minimize degradation risks. In the long-term, logistical challenges of frozen DP may limit patient access. In this work, we developed a lyophilized (freeze-dried) formulation of AAV. The mass concentration of AAV is typically low, and AAV also requires a minimum ionic strength to inhibit aggregation. These factors result in a low collapse temperature, which is limiting to lyophilization. Mannitol crystallization was found to cause extensive degradation and potency loss of AAV during the freezing step. With further development, we determined that AAV could be lyophilized in a sucrose and citrate formulation with a more desirable high glass transition temperature of the dried cake. An optimal residual moisture range (1–3%) was found to be critical to maintaining AAV8 stability. Glycerol was found to protect AAV8 from over-drying by preventing capsid damage and genome DNA release. A lyophilized formulation was identified that maintained potency for 24 months at 2–8 °C, indicating the feasibility of a dried formulation for AAV gene therapy.

Sunlight Inactivation of Human Norovirus and Bacteriophage MS2 Using a Genome-Wide PCR-Based Approach and Enzyme Pretreatment.

Human norovirus (hNoV) is an important etiology of gastrointestinal illness and can be transmitted via ingestion of contaminated water. Currently impractical to culture, hNoV detection is reliant on real-time polymerase chain reaction (RT-PCR)-based methods. This approach cannot distinguish between infective and inactivated viruses because intact regions of the RNA genome can amplify even if the damage is present in other regions of the genome or because intact genetic material is not contained within an infectious virion. Herein, we employ a multiple long-amplicon RT-qPCR extrapolation approach to assay genome-wide damage and an enzymatic pretreatment to study the impact of simulated sunlight on the infectivity of hNoV in clear, sensitizer-free water. Using MS2 coliphage as an internal control, the genome-wide damage extrapolation approach, previously successfully applied for UV-254 inactivation, vastly overestimated sunlight inactivation, suggesting key differences in photoinactivation under different spectral conditions. hNoV genomic RNA was more susceptible to simulated sunlight degradation per base compared to MS2 genomic RNA, while enzymatic pretreatment indicated that hNoV experienced more capsid damage than MS2. This work provides practical and mechanistic insight into the endogenous sunlight inactivation of single-stranded RNA bacteriophage MS2, a widely used surrogate, and hNoV GII.4 Sydney, an important health-relevant virus, in clear sensitizer-free water.

Virucidal Action Mechanism of Alcohol and Divalent Cations Against Human Adenovirus.

Hygiene and disinfection practices play an important role at preventing spread of viral infections in household, industrial and clinical settings. Although formulations based on >70% ethanol are virucidal, there is a currently a need to reformulate products with much lower alcohol concentrations. It has been reported that zinc can increase the virucidal activity of alcohols, although the reasons for such potentiation is unclear. One approach in developing virucidal formulations is to understand the mechanisms of action of active ingredients and formulation excipients. Here, we investigated the virucidal activity of alcohol (40% w/v) and zinc sulfate (0.1% w/v) combinations and their impact on a human adenovirus (HAdV) using, nucleic acid integrity assays, atomic force microscopy (AFM) and transmission electron microscopy (TEM). We observed no difference in virucidal activity (5 log10 reduction in 60 min) against between an ethanol only based formulation and a formulation combining ethanol and zinc salt. Furthermore, TEM imaging showed that the ethanol only formulation produced gross capsid damage, whilst zinc-based formulation or formulation combining both ethanol and zinc did not affect HAdV DNA. Unexpectedly, the addition of nickel salt (5 mM NiCl2) to the ethanol-zinc formulation contributed to a weakening of the capsid and alteration of the capsid mechanics exemplified by AFM imaging, together with structural capsid damage. The addition of zinc sulfate to the ethanol formulation did not add the formulation efficacy, but the unexpected mechanistic synergy between NiCl2 and the ethanol formulation opens an interesting perspective for the possible potentiation of an alcohol-based formulation. Furthermore, we show that AFM can be an important tool for understanding the mechanistic impact of virucidal formulation.

Agroforestry systems can mitigate the severity of cocoa swollen shoot virus disease

Currently, the only effective treatment for cocoa (Theobroma cacao L.) infected with the cocoa swollen shoot virus disease (CSSVD) is to cut and replant infected trees. Hence, the development of preventive control measures and strategies to mitigate the severity of the disease are of utmost importance. While past research has mainly focused on resistance breeding, mild strain cross protection and vector control, diversification measures such as agroforestry have received relatively less attention, despite their potential to mitigate CSSVD severity. Therefore, we studied the effects of shade on CSSVD symptom severity, capsid damage and cocoa yield along a gradient of increasing shade tree abundance in smallholder cocoa farms in Ghana. Furthermore, we measured photosynthetic active radiation and assessed soil fertility in order to elaborate on potential causal factors for possible shade effects on CSSVD symptom severity. Both CSSVD symptom severity and cocoa yields followed quadratic curves, and were found to be lowest and highest in plots with 54% and 39% shade, respectively. The simulated optimal shade levels for CSSVD symptom severity and cocoa yield overlapped between 45%–53%, indicating that agroforestry systems with around 50% shade cover may be an optimal coping strategy to balance CSSVD symptom severity versus reduced cocoa yield until diseased cocoa is replaced with more resistant varieties. Furthermore, our results suggest that rather than soil fertility, high-light and possibly also soil moisture stress may have been responsible for the shade effects on CSSVD symptom severity.

Bactericidal and virucidal mechanisms in the alkaline disinfection of compost using calcium lime and ash

In the present study, the bactericidal and virucidal mechanisms in the alkaline disinfection of compost with calcium lime and ash were investigated. Two indicator microorganisms, Escherichia coli and MS2 coliphage, were used as surrogates for enteric pathogens. The alkaline-treated compost with calcium oxide (CaO) or ash resulted primarily in damage to the outer membrane and enzyme activities of E. coli. The alkaline treatment of compost also led to the infectivity loss of the coliphage because of the partial capsid damage and RNA exteriorization due to a raised pH, which is proportional to the amount of alkaline agents added. These results indicate that the alkaline treatment of compost using calcium oxide and ash is effective and can contribute to the safe usage of compost from a mixing type dry toilet.

Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus

Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay.

Inactivation of bacteriophages by high levels of dissolved CO 2

We developed a system with high levels of dissolved CO 2 for water disinfection. Bacteriophages MS2, Qβ and ΦX174 were selected as the inactivation targets. A relatively mild inactivation effect was observed on MS2 and Qβ at different initial concentrations of dissolved CO 2 at 0.3 MPa in 20–30 min. When the pressure was increased to 0.6 MPa, the inactivation of MS2 and Qβ was differentially improved. However, this system was less effective for the inactivation of ΦX174. The capsid surface property is a probable reason for the low inactivation of ΦX174. The pH was not a key factor in the inactivation of bacteriophages; moreover, the results obtained using alternative gases (pressurized air and O 2) indicated that only CO 2 inactivated these bacteriophages. A comparison between the results of real time polymerase chain reaction (RT-PCR) and plaque assay showed that some RNA moved out from the capsid after treatment. Capsid damage by CO 2 expansion was the likely mechanism of inactivation with our method.

Inactivation of MS2 Coliphage by Ferrous Ion and Zero-Valent Iron Nanoparticles

This study demonstrates the inactivation of MS2 coliphage (MS2) by nano particulate zerovalent iron (nZVI) and ferrous ion (Fe[II]) in aqueous solution. For nZVI, the inactivation efficiency of MS2 under air-saturated conditions was greater than that observed under deaerated conditions, indicating that reactions associated with the oxidation of nZVI were mainly responsible for the MS2 inactivation. Under air-saturated conditions, the inactivation efficiency increased with decreasing pH for both nZVI and Fe(II), associated with the pH-dependent stability of Fe(II). Although the Fe(II) released from nZVI appeared to contribute significantly to the virucidal activity of nZVI, several findings suggest that the nZVI surfaces interacted directly with the MS2 phages, leading to their inactivation. First, the addition of 1,10-phenanthroline (a strong Fe(II)-chelating agent) failed to completely block the inactivation of MS2 by nZVI. Second, under deaerated conditions, a linear dose-log inactivation curve was still observed for nZVI. Finally, ELISA analysis indicated that nZVI caused more capsid damage than Fe(II).

Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR

The advent of quantitative PCR has improved the detection of human viral pathogens in the environment. However, a serious limitation of this method may arise from the inability to discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard. To assess whether propidium monoazide (PMA) pre-treatment is a good approach to inhibiting DNA amplification from non-infectious viruses, bacteriophage T4 survival was measured using cell culture titration and real-time PCR with and without PMA pre-treatment. Heat (85 °C) and proteolysis methods were carried out. After these inactivation treatments, the results indicated that the PMA pre-treatment approach is not appropriate for differentiating infectious viruses. However, when a heat treatment at 110 °C was undertaken, PMA pre-treatment did allow differentiation of non-infectious from infectious viruses. In this case, effective binding of PMA to bacteriophage T4 DNA could be taken to indicate capsid damage. Therefore, PMA pre-treatment may be appropriate for assessing effective disinfection treatments and for a more reliable understanding of the factors that contribute to viral inactivation through capsid damage monitoring. The PMA-PCR approach could be useful as a rapid and inexpensive analytical tool for screening and evaluation of the efficacy of disinfectants.

Assessment of UV-B damage in cyanophage PP

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 58:323-328 (2010) - DOI: https://doi.org/10.3354/ame01386 Assessment of UV-B damage in cyanophage PP Ming-Jun Liao1,2,3, Kai Cheng2, Jiao-Yan Yang2, Yi-Jun Zhao2,*, Zheng-Li Shi1,** 1State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China 2Hubei Key Laboratory of Urban Water Environmental Ecology, Central China Normal University, Wuhan 430079, PR China 3Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China Corresponding authors. Email: *zhaoyj@mail.ccnu.edu.cn; **zlshi@wh.iov.cn ABSTRACT: Cyanophage PP is a short-tailed, icosahedral-shaped, double-stranded DNA virus and can be frequently detected with high abundance and activity in many eutrophic lakes in China. Solar radiation is one of the main factors affecting cyanophage infectivity. In this study, cyanophage PP was treated with different intensities of UV-B radiation, and the accumulation of cyclobutane pyrimidine dimers (CPDs), viral particle destruction and viral attachment ability were analyzed. The viral infectivity decay rate under white and red light was also studied using plaque-forming assays. The results indicate that (1) the level of CPDs induced by UV-B was significantly correlated with radiation intensity, while exposure dose had little effect on the DNA damage level, (2) the level of CPDs was significantly correlated with both the decay rate and the repair rate in cyanophage PP, and (3) UV-B could result in the destruction of viral particles and the decrease in viral attachment. This work suggests that the secondary and tertiary structure of the DNA may influence the formation of CPDs, and that capsid damage caused by UV-B radiation may play an important role in viral decay and survival. KEY WORDS: Cyanophage PP · UV-B · Cyclobutane pyrimidine dimers · CPDs · Capsid damage Full text in pdf format PreviousCite this article as: Liao MJ, Cheng K, Yang JY, Zhao YJ, Shi ZL (2010) Assessment of UV-B damage in cyanophage PP. Aquat Microb Ecol 58:323-328. https://doi.org/10.3354/ame01386 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 58, No. 3. Online publication date: February 11, 2010 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2010 Inter-Research.

Converging farmers' and scientists' perspectives on researchable constraints on organic cocoa production in Ghana: results of a diagnostic study

A diagnostic study was conducted to identify the major constraints on organic cocoa production at Brong-Densuso and surrounding communities in the Suhum-Kraboa-Coaltar District, Eastern Region, Ghana. The study followed a technographic study that highlighted cocoa as a public crop requiring broad techno-social innovations. In the technographic study, problems identified included low yields, persistent pest management constraints and a low adoption rate of technologies developed by the Cocoa Research Institute of Ghana. The diagnostic study adopted a Participatory Learning and Action Research approach to set up and implement fieldwork with relevant stakeholders leading to problem identification, prioritization, and collective design of an action plan (research agenda). Cocoa farmers within the study area are conscious of the environmental problems associated with the use of inorganic pesticides and the high cost of using them. Hence, they produce cocoa without applying any pesticides. Quite recently, however, their association with an organic marketing company led to a search for non-chemical pest and disease control measures and for ways to certify their cocoa beans as organic. A misconception as to what species of cocoa pests constitute ‘capsids’ was settled between farmers and scientists using a cage experiment on capsid damage. The farmers became convinced that the Cocoa Mosquito ( Helopeltis spp.) (Hemiptera: Miridae), which they had previously considered an important pest, was a capsid species that caused little or no damage to the beans inside the pods. After this clarification, damage caused by the Brown Capsid ( Sahlbergella singularis; Hemiptera: Miridae) and the Black Capsid ( Distantiella theobroma; Hemiptera: Miridae) emerged as the most serious production constraint, followed by Black Pod disease (caused by Phytophtora palmivora). The malfunctioning of tenure agreements and the mistrust between landlords, who are mainly absentee farmers, and their caretaker cocoa farmers pose a serious threat to pest management innovations, especially where pruning to control Black Pod disease and uprooting trees infected with Swollen Shoot disease are concerned. The key stakeholders involved in the study agreed on three innovative (organic) capsid control methods for further research: the use of sex pheromone traps, crude aqueous neem ( Azadirachta indica) seed extracts, and the use of ant ( Oecophylla longinoda) colonies as biological control agents, the latter being proposed by farmers. The paper reflects on the diagnostic study as a continuous process in response to a continually changing context even beyond the end of the diagnostic research phase.

In vitro transcription assay of ozonated T7 phage dna

It has been proven that ozone is able to modify the structure of DNA, since the two fundamental effects of ozone on the hereditary material are the degradation of some nitrogenated bases and the production of single- and double-strand breaks in DNA chains. The ability of ozone to inactivate viruses has been reported by many researchers. It seems that ozone reacts with DNA when the capsid is altered, but according to Roy et al. inactivation by low dosages of ozone cannot be attributed to the protein capsid damage affecting the capacity to adsorb and/or to inject DNA into the host cell. An alternative to inactivation of the phages by ozone, one can postulate that ozone-damaged DNA alters template activity. In order to assess the role of ozone-damaged DNA on DNA metabolism, the authors examined isolated T7 bacteriophage DNA template properties for in vitro transcription activity. The influence of an ozone treatment on the kinetics of synthesis, the initiation, and the elongation steps have been studied.

Temperature-induced structural stabilization of the encapsidated RNA of southern bean mosaic virus and its biologic significance

Virions of southern bean mosaic virus (SBMV) show two distinct sensitivity patterns upon heating. Infectivity loss and capsid denaturation occur concurrently if virions are exposed at 50–55° in 0.1 M sodium phosphate buffer, pH 7.5, or 0.1 M glycine-phosphate buffer, pH 9.0. Contrastingly, virions are inactivated without any detectable capsid damage at 65–70° in 0.1 M sodium phosphate buffer, pH 6.0, 0.1 M Tris-HCI buffer, pH 7.5, or 0.1 M glycine—NaOH buffer, pH 9.0. Heat treatment causes no genomic degradation in these two situations; the divergent sensitivity of virions is due, apparently, to a differential thermal tolerance of the capsid protein to the buffer components and/or pH. SBMV-RNA isolated from virions inactivated at 65° in 0.1 M Tris-HCl buffer, pH 7.5, possesses low infectivity (1–2%). Observations based upon sucrose gradient sedimentation, temperature: absorbance relationship, and sensitivity to ribonuclease T 1 suggest that such RNA is structurally more compact and stable relative to that of the RNA from the nonheated virions. Neither the capsid protein nor the genome protein plays a direct role in the temperature-induced structural stabilization of SBMV-RNA in situ. If treated with 8 M urea, 50% formamide or exposed at 55°, the infectivity of RNA from the heat-inactivated SBMV is restored and is comparable to that of the RNA isolated from the nonheated virions. The observed mode of SBMV thermal inactivation, i.e., stabilization of RNA structure in situ, is unique among the viruses. Furthermore, these results suggest that the ability to initiate infection depends upon the secondary structure of the SBMV genome.

Mechanisms of Poliovirus Inactivation by the Direct and Indirect Effects of Ionizing Radiation

This study was designed to measure the effects of ionizing radiation on poliovirus particles when given under conditions where either direct (in broth) or indirect (in water) effects were predominant. Under direct conditions, inactivation of poliovirus was found to be due primarily to RNA damage, although capsid damage could account for about one-third of the viral inactivation. RNA damage did not appear to be due to strand breakage and therefore was probably caused primarily by base damage or crosslink formation. Capsid damage under direct irradiation conditions did not result in significant alterations of either the sedimentation coefficients or the isoelectric points of the poliovirus particles or detectable modification of the sizes of the viral proteins. It did, however, cause loss of ability to bind to host cells. Under indirect conditions no more than 25% of viral inactivation appeared to be due to RNA damage. However, the sedimentation coefficients and isoelectric points of the viral particles were greatly altered, and their abilities to bind to cells were lost at about three-fourths the rate of loss of infectivity. Capsid damage in this case did result in changes in the sizes of capsid proteins. Therefore, the majority of the radiation inactivation under indirect conditions appeared to be due to protein damage.

Biology of Reduviidae of Cocoa Farms in Ghana

In the cocoa farms the members of family Reduviidae are distributed in a variety of habitats associated with particular food, defense and breeding requirements for various species. In general the ectrichodiines are exclusively litter dwellers and reduviines occur only under the loose bark of primary forest trees. The members. of subfamily Harpactorinae are associated with herbs, shrubs and the foliage of cocoa trees. A group of harpactorines comprising Authentac, Margasus, Nagusta and Odontogonus has been found to be completely arboreal. The food and feeding behavior of the cocoa farm Reduviidae were explored and data on the insects preyed upon by the Reduviinae obtained for the first time. Iff one locality at Tafo, one species of this subfamily utilized 24 different species of ants belonging to four subfamilies. As a preliminary to feeding, the reduviids were generally observed to exhibit grooming behavior which at times was repeated once the bug had finished feeding. Coloration, mimicry and scent glands were considered to contribute greatly to the primary defense of the Reduviidae found in the cocoa farms. The majority of Reduviidae living in cocoa canopy or concealed situations such as leaf litter tended to be dark-colored, usually matching their surroundings. The reduviids at shrub level are conspicuously colored and their adults exhibit a behavior pattern highly suggestive of certain wasps. Excepting the Emesinae and Tribelocephalinae, where the scent glands are completely absent, repugnatorial glands producing generally a pungent secretion were found in all the reduviidae occurring in cocoa farms. A unique system of glands situated at the base of dorsoabdominal hairs was discovered for the first time. This system produces secretions which assist in sticking camouflage material to the back of reduviine larvae. Early instars of some species of Harpactorinae were observed to mimic smaller species of ants such as Pheidole and Anochetus. Courtship, mating and oviposition in Reduviidae were studied and the significance of the precopulatory riding position, parental care and laying of eggs singly or in groups is discussed. Cocoa capsids are attacked by a number of parasites and predators including ants, reduviids (assassin bugs), mantids, long-horned grasshoppers and crickets. Williams (1954) showed that predators are a significant factor in the natural control of capsid numbers. He furnished quantitative data which suggested that the assassin bugs and mantids together account for more than one and a half times the capsid mortality caused by ant predation. Leston (1969), however, considered that ants afford considerable protection to cocoa trees against capsid damage. Marchart (1969), on the other hand, presented experimental evidence in support of his contention that ants are ineffectual against capsids. One point which is very clear from the work of these authors is that each predatory species individually causes an appreciable mortality of the capsids. Smith (1968) has stressed that it is these partial controls that are so important in integrated control programs. 1 Present address: Abidjan, P.O. Box 1604, Ivory Coast.