Infection Retention Research Articles

Research progress on environmental stability of SARS-CoV-2 and influenza viruses.

We reviewed research on SARS-CoV-2 and influenza virus detection on surfaces, their persistence under various conditions, and response to disinfectants. Viral contamination in community and healthcare settings was analyzed, emphasizing survival on surfaces influenced by temperature, pH, and material. Findings showed higher concentrations enhance survivability at room temperature, whereas stability increases at 4°C. Both viruses decline in low pH and high heat, with influenza affected by salinity. On various material surfaces, SARS-CoV-2 and influenza viruses demonstrate considerable variations in survival durations, and SARS-CoV-2 is more stable than influenza virus. On the skin, both virus types can persist for ≥2 h. Next, we delineated the virucidal efficacy of disinfectants against SARS-CoV-2 and influenza viruses. In daily life, exposure to ethanol (70%), isopropanol (70%), bleach (10%), or hydrogen peroxide (1-3%) for 15-30 min can effectively inactive various SARS-CoV-2 variants. Povidone-iodine (1 mg/mL, 1 min) or cetylpyridinium chloride (0.1 mg/mL, 2 min) may be used to inactive different SARS-CoV-2 variants in the mouth. Chlorine disinfectants (500 mg/L) or ultraviolet light (222 nm) can effectively inhibit different SARS-CoV-2 variants in public spaces. In conclusion, our study provides a scientific basis and practical guidance for reduction of viral persistence (retention of infectivity) on surfaces and environmental cleanliness.

Efficient prion disease transmission through common environmental materials

Prion diseases are a group of fatal neurodegenerative diseases associated with a protein-based infectious agent, termed prion. Compelling evidence suggests that natural transmission of prion diseases is mediated by environmental contamination with infectious prions. We hypothesized that several natural and man-made materials, commonly found in the environments of wild and captive animals, can bind prions and may act as vectors for disease transmission. To test our hypothesis, we exposed surfaces composed of various common environmental materials (i.e. wood, rocks, plastic, glass, cement, stainless steel, aluminum, and brass) to hamster-adapted 263K scrapie prions and studied their attachment and retention of infectivity in vitro and in vivo Our results indicated that these surfaces, with the sole exception of brass, efficiently bind, retain, and release prions. Prion replication was studied in vitro using the protein misfolding cyclic amplification technology, and infectivity of surface-bound prions was analyzed by intracerebrally challenging hamsters with contaminated implants. Our results revealed that virtually all prion-contaminated materials transmitted the disease at high rates. To investigate a more natural form of exposure to environmental contamination, we simply housed animals with large contaminated spheres made of the different materials under study. Strikingly, most of the hamsters developed classical clinical signs of prion disease and typical disease-associated brain changes. Our findings suggest that prion contamination of surfaces commonly present in the environment can be a source of disease transmission, thus expanding our understanding of the mechanisms for prion spreading in nature.

Assaying the Stability and Inactivation of AAV Serotype 1 Vectors.

Adeno-associated virus (AAV) vectors are a commonplace tool for gene delivery ranging from cell culture to human gene therapy. One feature that makes AAV a desirable vector is its stability, in regard to both the duration of transgene expression and retention of infectivity as a viral particle. This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4°C decreased 20% over 7 weeks. Over 10 freeze-thaw cycles, the resulting transduction efficiency became variable at 60-120% of a single thaw. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, it was found that an AAV1 vector can be reconstituted after 6 days of storage at room temperature. The stability of AAV is a desired feature, but effective decontamination procedures must be available for safety and experimental integrity. Multiple disinfectants commonly used in the laboratory for ability to inactivate an AAV1 vector were tested, and it was found that autoclaving, 0.25% peracetic acid, iodine, or 10% Clorox bleach completely prevented AAV-mediated transgene expression. These data suggest that peracetic acid should be used for inactivating AAV1 vectors on metal-based surfaces or instruments in order to avoid inadvertent transgene expression in human cells or cross-contamination of instruments.

97. Stability and Inactivation of AAV Serotype 1 Vectors

Adeno-associated viral (AAV) vectors have become a commonplace tool for gene delivery ranging from cell lines to human gene therapy. One feature that makes AAV desirable for gene delivery is its relative safety owing to no known disease associated with wild-type AAV. For this reason, it is often used with BSL1 practices negating the need for dedicated BSL2 facilities. Another useful property of AAV is its stability, both the duration of transgene expression and retention of infectivity as a viral particle. In the current study, we examine the stability of AAV serotype 1 vectors which are capable of transducing human cells and are routinely produced and used in our facility. First, we examined stability at 4° C for up to 7 weeks and found <10% decrease in transduction efficiency compared to a freshly-thawed aliquot. After 10 freeze-thaw cycles, 25% loss in transduction efficiency was observed. These data indicated that AAV serotype 1 is a highly stable viral particle with regards to retaining infectivity. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, we demonstrate that infectious AAV vector expressing Cre-recombinase can be reconstituted and used up to 7 days at room temp, although there was a clear decrease in transgene expression over time. The stability of AAV is a desired feature as a tool; however, it is important that the vector can be inactivated to avoid transgene delivery to end users or cross-contamination of non-disposable instruments. AAV decontamination procedures can vary. We tested multiple disinfectants commonly used in the laboratory for their ability to inactivate an AAV serotype 1 vector. The vector was added directly into solution for 5 or 30 minutes at a 1:10 ratio. It was then diluted, dialyzed, concentrated, and tested for transduction on primary neurons. Of the decontamination procedures tested, only autoclaving, 3.9% peracetic acid, iodine or 10% Clorox bleach completely prevented the subsequent detection of transgene expression. Although the nature of the transgene (e.g. proto oncogene or toxin) can elevate an AAV vector to BSL2, our data show that when working with AAV serotype 1 vectors, it's important that safety procedures for handling and decontaminating AAV are effective to avoid inadvertent transgene expression in human cells or cross contamination of AAV vectors used in non-disposable instruments. Other serotypes have not yet been tested.

Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in meat by using phages immobilized on modified cellulose membranes.

The ability of phages to specifically interact with and lyse their host bacteria makes them ideal antibacterial agents. The range of applications of bacteriophage can be extended by their immobilization on inert surfaces. A novel method for the oriented immobilization of bacteriophage has been developed. The method was based on charge differences between the bacteriophage head, which exhibits an overall net negative charge, and the tail fibers, which possess an overall net positive charge. Hence, the head would be more likely to attach to positively charged surfaces, leaving the tails free to capture and lyse bacteria. Cellulose membranes modified so that they had a positive surface charge were used as the support for phage immobilization. It was established that the number of infective phages immobilized on the positively charged cellulose membranes was significantly higher than that on unmodified membranes. Cocktails of phages active against Listeria or Escherichia coli immobilized on these membranes were shown to effectively control the growth of L. monocytogenes and E. coli O157:H7 in ready-to-eat and raw meat, respectively, under different storage temperatures and packaging conditions. The phage storage stability was investigated to further extend their industrial applications. It was shown that lyophilization can be used as a phage-drying method to maintain their infectivity on the newly developed bioactive materials. In conclusion, utilizing the charge difference between phage heads and tails provided a simple technique for oriented immobilization applicable to a wide range of phages and allowed the retention of infectivity.

Thrichomys laurentius (Rodentia; Echimyidae) as a Putative Reservoir of Leishmania infantum and L. braziliensis: Patterns of Experimental Infection

The importance of the genus Thrichomys in the retention of infection and transmission of Leishmania species is supported by previous studies that describe an ancient interaction between caviomorphs and trypanosomatids and report the natural infection of Thrichomys spp. Moreover, these rodents are widely dispersed in Brazil and recognized as important hosts of other tripanosomatids. Our main purpose was to evaluate the putative role of Thrichomys laurentius in the retention of infection and amplification of the transmission cycle of Leishmania infantum and L. braziliensis. Male and female T. laurentius (n = 24) born in captivity were evaluated for the retention of infection with these Leishmania species and followed up by parasitological, serological, hematological, biochemical, histological, and molecular assays for 3, 6, 9, or 12 months post infection (mpi). T. laurentius showed its competence as maintenance host for the two inoculated Leishmania species. Four aspects should be highlighted: (i) re-isolation of parasites 12 mpi; (ii) the low parasitic burden displayed by T. laurentius tissues; (iii) the early onset and maintenance of humoral response, and (iv) the similar pattern of infection by the two Leishmania species. Both Leishmania species demonstrated the ability to invade and maintain itself in viscera and skin of T. laurentius, and no rodent displayed any lesion, histological changes, or clinical evidence of infection. We also wish to point out the irrelevance of the adjective dermotropic or viscerotropic to qualify L. braziliensis and L. infantum, respectively, when these species are hosted by nonhuman hosts. Our data suggest that T. laurentius may act at least as a maintenance host of both tested Leishmania species since it maintained long-lasting infections. Moreover, it cannot be discarded that Leishmania spp. infection in free-ranging T. laurentius could result in higher parasite burden due the more stressing conditions in the wild. Therefore the tissular parasitism of the skin, infectiveness to the vector, and amplification of the transmission cycle of both Leishmania species could be expected.

Carrier Cell-based Delivery of an Oncolytic Virus Circumvents Antiviral Immunity

Oncolytic viruses capable of tumor-selective replication and cytolysis have shown early promise as cancer therapeutics. However, the host immune system remains a significant obstacle to effective systemic administration of virus in a clinical setting. Here, we demonstrate the severe negative impact of the adaptive immune response on the systemic delivery of oncolytic vesicular stomatitis virus (VSV) in an immune-competent murine tumor model, an effect mediated primarily by the neutralization of injected virions by circulating antibodies. We show that this obstacle can be overcome by administering virus within carrier cells that conceal viral antigen during delivery. Infected cells were delivered to tumor beds and released virus to infect malignant cells while sparing normal tissues. Repeated administration of VSV in carrier cells to animals bearing metastatic tumors greatly improved therapeutic efficacy when compared with naked virion injection. Whole-body molecular imaging revealed that carrier cells derived from solid tumors accumulate primarily in the lungs following intravenous injection, whereas leukemic carriers disseminate extensively throughout the body. Furthermore, xenogeneic cells were equally effective at delivering virus as syngeneic cells. These findings emphasize the importance of establishing cell-based delivery platforms in order to maximize the efficacy of oncolytic therapeutics.

Overview of the Vaccinia Virus Expression System

The vaccinia virus expression system differs from others in that transcription occurs in the cytoplasm of mammalian cells rather than in the nucleus. As a vector, vaccinia virus has a number of useful characteristics, including a capacity that permits cloning large fragments of foreign DNA (20+ kbp) with retention of infectivity, a wide host range, a relatively high level of protein synthesis, and "appropriate" transport, secretion, processing, and posttranslational modifications as dictated by the primary structure of the expressed protein and the cell type used. This overview discusses the life cycle of the vaccinia virus along with effects of vaccinia infection. The vaccinia vector expression system is described along with specific steps for expressing genes using these vectors. Important safety considerations are also presented.

THE 'LIFE CYCLE' OF PEAR DECLINE PHYTOPLASMA IN THE VECTOR CACOPSYLLA PYRI

The ‘life cycle’ of pear decline phytoplasma in the vector Cacopsylla pyri has been studied. For two consecutive years, groups of insects caught monthly from March to October were used for transmission trials. The psylla was equally infective throughout the experimental period. In the second year, insects were fed on dormant plants from November to March and regularly tested. Retention of infectivity of the pear decline phytoplasma in the vector lasted through the winter. The overwintering adults of C. pyri did not transmit the phytoplasmas to plants in dormancy, but they did, without any previous recharge feeding, after bud break in the spring. Control of the overwintering adults of C. pyri appears to be of fundamental importance for preventing the spread of pear decline.

Transmission Characteristics of the European Stone Fruit Yellows Phytoplasma and its Vector Cacopsylla Pruni

A study was carried out on the transmission parameters of the European stone fruit yellows phytoplasma by the vector Cacopsylla pruni. In the greenhouse, using groups of psyllids, the minimum acquisition period was 2–4 days, the minimum latent period 2–3 weeks and the minimum inoculation period 1–2 days. The vectors retained infectivity until their death. Under natural conditions retention of infectivity in C. pruni lasts through the winter and the following spring, when the overwintering insects reach the stone fruit trees, they are already infected and infective. The research shows that the vector C. pruni transmits the European stone fruit yellows phytoplasma in a persistent manner.

PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo.

Replication-defective recombinant adenovirus (Ad) vectors are under development for a wide variety of gene therapy indications. A potential limiting factor associated with virus gene therapy requiring repeated treatment is the development of a humoral immune response to the vector by the host. In animal models, there is a dose-dependent rise in neutralizing antibodies after primary vector administration, which can preclude effective repeat administration. The strategy we have developed to circumvent the neutralization of adenovirus vectors by antibodies is to mask their surface by covalent attachment of the polymer polyethylene glycol (PEG). Covalent attachment of PEG to the surface of the adenovirus was achieved primarily by using activated PEG tresylmonomethoxypolyethylene glycol (TMPEG), which reacts preferentially with the epsilon-amino terminal of lysine residues. We show that the components of the capsid that elicit a neutralizing immune response, i.e., hexon, fiber, and penton base, are also the main targets for PEGylation. Several protocols for PEGylation of an adenovirus vector were evaluated with respect to retention of virus infectivity and masking from antibody neutralization. We show that covalent attachment of polymer to the surface of the adenovirus can be achieved with retention of infectivity. We show further that PEG-modified adenovirus can be protected from antibody neutralization in the lungs of mice with high antibody titers to adenovirus, suggesting that PEGylation will improve the ability to administer Ad vectors on a repeated basis.

Overview of the Vaccinia Virus Expression System

The vaccinia virus expression system differs from others in that transcription occurs in the cytoplasm of mammalian cells rather than in the nucleus. As a vector, vaccinia virus has a number of useful characteristics, including a capacity that permits cloning large fragments of foreign DNA (20+ kbp) with retention of infectivity, a wide host range, a relatively high level of protein synthesis, and "appropriate" transport, secretion, processing, and posttranslational modifications as dictated by the primary structure of the expressed protein and the cell type used. This overview discusses the life cycle of the vaccinia virus along with effects of vaccinia infection. The vaccinia vector expression system is described along with specific steps for expressing genes using these vectors. Important safety considerations are also presented.

Persistence of infectivity by germinated spores of vesicular-arbuscular mycorrhizal fungi in soil

Spores of Glomus caledonium and Acaulospora laevis , which had been germinated in untreated or steamed field soil without plants, retained their capacity to infect plants for periods up to 4 months. Retention of infectivity was greater for A. laevis than G. caledonium in either soil. Decreased infectivity appeared to be due to the loss of the capacity of the hyphae to form pre-penetration and penetration structures. For equivalent amounts of inoculum (the same number of germinated spores that were similar in size), A. laevis had a greater capacity than G. caledonium to colonize roots. Germination of the spores of either species prior to sowing increased the extent of colonization of young roots.

Estimation of the numbers and infectivity of Nippostrongylus larvae.

Experimental investigations of the subcutaneous infection of third-stage larvae of Nippostrongylus brasiliensis reveal discrepancies between the actual and intended inoculum, and also variability between replicate inocula. Emphasis is thus placed on the importance of obtaining accurate estimates of the level of confidence associated with inoculum size. Under specified laboratory conditions, the retention of infectivity of N. brasiliensis larvae was found to be age-dependent, with a maximum of between 120 and 156 days. The motility of the larvae was not found to be indicative of their ability to complete migration and to become established in the small intestine of the host following subcutaneous inoculation.

Purification of Rickettsia tsutsugamushi by Percoll density gradient centrifugation.

Purification of Rickettsia tsutsugamushi has been achieved by Percoll density gradient centrifugation. The microorganisms purified showed good retention of infectivity and intracellular morphology. Budding rickettsiae in the egressing stage and intracellular rickettsiae in the multiplying process were harvested separately and purified by this technique. In electron microscopic observations, the intracellular rickettsiae obtained were surrounded with double membrane-layers of cell wall and cell membrane, and the budding rickettsiae were enveloped with an additional outermost membrane which may have originated from host cell membrane obtained in the budding process.

Granulosis virus in the intestinal lumen of Neoaplectana carpocapsae: Retention of infectivity after treatment with formaldehyde or high pH

High pH values (>11.0) cause the dissolution of occlusion bodies of the granulosis virus (GV) of Pseudaletia unipuncta and subsequent inactivation of the virus within 24 hr. The GV is also inactivated within 48 hr by 0.04% formaldehyde. The GV is found in the intestinal lumen of infective third stage nematodes (dauer juveniles) of Neoaplectana carpocapsae when development occurs in GV-infected hosts. The GV in these dauer juveniles retains its infectivity even when the nematodes are placed into an alkaline solution with pH values of 11.1 or 12.1 or in 0.04% formaldehyde up to 336 hr. However, significant loss of infectivity of GV occurs when the nematodes are in formaldehyde but not at high pH values. The dauer juveniles are ensheathed by the second stage cuticle. This cuticle probably protects the GV in the intestinal lumen of the nematode from the high pH and formaldehyde.

Cell-Free Varicella-Zoster Virus in Cultured Human Melanoma Cells

Varicella-zoster virus (VZV) has been isolated and serially propagated in a continuous cell line derived from a human malignant melanoma tumour. Human melanoma cells (HMC) have been further evaluated as a substrate for the production of cell-free virus and compare favourably with human embryo cells. Within 60 h after inoculation with VZV-infected cells, HMC monolayers incubated at 32 degrees C exhibited advanced syncytial cytopathic effect, and the overlying culture medium contained greater than 10(2) p.f.u./ml. The cell pellet from a mechanically dispersed 150 cm2 monolayer yielded 10(5) p.f.u. after sonic disruption, while the medium ('scraping medium') in which the cells had been harvested contained up to one log more infectious virus than was found in the cells from the same monolayer. When infected cells were subjected to Dounce homogenization, most of the infectivity was found in the nuclear fraction. The concentration and purification of cell-free virus were also investigated. Concentration was carried out by three methods: ultracentrifugation, dialysis against hydrophilic compounds and liquid polymer phase separation. The first two procedures caused considerable loss of biological activity, whereas precipitation with 8% polyethylene glycol resulted in a 50-fold increase in titre. Purification of cell-free virus with retention of infectivity was achieved by rate zonal centrifugation in linear potassium tartrate gradients. Infectious virus was also recovered after sedimentation in combination equilibrium-viscosity gradients of potassium tartrate and glycerol, but not after centrifugation to equilibrium in caesium chloride gradients.

Evidence for Noncirculative Transmission of Pierce's Disease Bacterium by Sharpshooter Leafhoppers

Alexander H. Purcell and Allan FinlayDepartment of Entomological Sciences, University of California, Berkeley, 94720.The California Table Grape Commission and the Napa Valley Viticultural Research Fund supported this work in part.We thank Dennis Larsen for technical assistance.Accepted for publication 10 October 1978.ABSTRACTPURCELL, A. H., and A. H. FINLAY. 1979. Evidence for noncirculative transmission of Pierce's disease bacterium by sharpshooter leafhoppers.Phytopathology 69:393-395.Half of the leafhoppers (Graphocephala atropunctata) allowed acquisi- which was in close agreement with estimates for which no latent period wastion access on grapevines affected with Pierce's disease (PD) became assumed. Neither G. atropunctata nor Draeculacephala minerva retainedinfective within 2.0 hr, and there was no significant increase in acquisition infectivity after molting. The loss of infectivity after molting and lack of abeyond 24 hr. The median inoculation access period was 3.9 hr. Three of 34 latent period suggest a noncirculative mechanism of transmission of the P[)(9%) insects transmitted after I hr each for acquisition and for inoculation, bacterium by leafhoppers.Additional key words: Hordnia, Graphocephala, Draeculacephala, lucerne dwarf, alfalfa dwarf, almond leaf scorch, rickettsia-like bacteria, stylet-borne.Pierce's disease (PD) of grapevines usually is lethal to grapevines Princeton, NJ 08540) 25% WP in water at recommended rates and(Vitis vinifera); periodically it has caused serious losses to the held in a heated greenhouse. Symptoms of PD normally appearedCalifornia grape industry and it has precluded successful after 10-14 wk. Grapevines without symptoms of PD for 22-25 wkproduction of bunch grapes in the southeastern USA (5). Bacteria were recorded as free of PD.transmitted by xylem-feeding suctorial insects cause PD in grape Acquisition tests. Fifty to sixty adult and nymphal G.(2) and also are thought to cause alfalfa dwarf (7) and almond leaf atropunctata previously exposed to pretest plants for 4 days orscorch (9) diseases. longer were placed for 1, 3, 6, 12, 24, and 48-hr acquisition accessLong considered a virus disease, PD has been unusual in its periods (AAP) on PD source plants and then caged singly for a 4-vector-pathogen relationships in several respects: (i) a large but day inoculation access period (IAP) on test plants. Leafhoppersdistinct group of vectors (xylem-feeding suctorial insects such as allowed the I-hr AAP were immediately transferred to test plantssharpshooter leafhoppers of the subfamily Cicadellinae and for 1 hr and then to a second set of test plants for 4 days.spittlebugs [Cercopidae] [3]) (ii) a minimum or required latent Inoculation access period (IAP). Fifty adults and nymphs of G.period of less than 2 hr, and (iii) persistence of infectivity for long atropunctata with AAP of 2 days or more were transferred singly toperiods (perhaps permanently) in adult vectors (14). Although test plants for lAP of 1, 3, 6, 12, 24, 48, and 96 hr. One group wassharpshooter nymphs can transmit PD (14) apparently no studies transferred in the sequence of 1, 48, and 12 hr. Because ofof transtadial passage (retention of infectivity by vectors after cumulative mortality to the first group, a second group fed on themolting) have been made (12). The purpose of these studies was to same source plants was transferred in the sequence of 3, 24, 96, andinvestigate the pathway of transmission in the vector by evaluating 6 hr in order to allow all transfers to be made during daylight hours.the effects of molting and of varying acquisition and inoculation Adult D. minerva were tested for 6, 12, and 24-hr IAP.access times on transmission of the PD bacterium. A preliminary Effect of molting on vector transmission. First through fifthreport has been published (11). instar nymphs with an AAP of 48 hr or more were confinedindividually on test seedlings. Plants were inspected dailyMATERIALS AND METHODS (exceptions noted) and insects transferred approximately weekly oras soon as molting was noted. In one experiment, insects wereThe leafhopper Graphocephala atropunctata (Signoret) (= transferred to new plants every 2 days or as soon as a molt wasHordnia circellata Baker) (16) was collected from natural detected. After access to at least one test plant following molting,populations in Berkeley, CA. To test for infectivity with the PD test insects usually were transferred to a PD source for I day or in abacterium, G. atroptinctata used in acquisition tests first were few cases for 2 days and then continued on a sequence of test plants.pretested on rooted Pinot Noir cuttings for four days or longer (13).The results with naturally infective insects were not used in analyses RESULTSof acquisition data. Draeculacephala minerva Ball was colonizedon barley cultivar Atlas from the progeny of leafhoppers reared Effects of AAP and IAP. Graphocephala atropunctata. Thefrom eggs hatched in petri dishes on moist paper (4). percentage of insects that transmitted PD was correlatedGrapevines with advanced PD symptoms (6) were used as significantly (P 0.5) from eachother.

Survival and retention of infectivity of bacterial stalk rot pathogen of maize and its perpetuation on varied cropping pattern

The sterilized environment prompted the maize stalk rot pathogen Erwinia carotovora f. sp. zeae Sabet to retain its infectivity for a longer period than the unsterilized one. The survival as well as infectivity potency of the pathogen present in the soil having naturally-infested or artificially-inoculated host plant as debris was superior to that in the soil or even the soil containing maize stalk tissue.

Retention of Infectivity and Pathogenicity by Tuberculina maxima in Culture

(1970). Retention of Infectivity and Pathogenicity by Tuberculina Maxima in Culture. Mycologia: Vol. 62, No. 6, pp. 1209-1211.