Use of Rotavirus Virus-Like Particles as Surrogates To Evaluate Virus Persistence in Shellfish

Rotavirus virus-like particles (VLPs) and MS2 bacteriophages were bioaccumulated in bivalve mollusks to evaluate viral persistence in shellfish during depuration and relaying under natural conditions. Using this nonpathogenic surrogate virus, we were able to demonstrate that about 1 log₁₀ of VLPs was depurated after 1 week in warm seawater (22°C). Phage MS2 was depurated more rapidly (about 2 log₁₀ in 1 week) than were VLPs, as determined using a single-compartment model and linear regression analysis. After being relayed in the estuary under the influence of the tides, VLPs were detected in oysters for up to 82 days following seeding with high levels of VLPs (concentration range between 10¹⁰ and 10⁹ particles per g of pancreatic tissue) and for 37 days for lower contamination levels (10⁵ particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks.     

Impact of internal RNA on aggregation and electrokinetics of viruses: comparison between MS2 phage and corresponding virus-like particles

We compare for the first time the electrokinetic and aggregation properties of MS2 phage (pH 2.5 to 7, 1 to 100 mM NaNO(3) electrolyte concentration) with those of the corresponding virus-like particles (VLPs), which lack entirely the inner viral RNA component. In line with our previous work (J. Langlet, F. Gaboriaud, C. Gantzer, and J. F. L. Duval, Biophys. J. 94:3293-3312, 2008), it is found that modifying the content of RNA within the virus leads to very distinct electrohydrodynamic and aggregation profiles for MS2 and MS2 VLPs. Under the given pH and concentration conditions, MS2 VLPs exhibit electrophoretic mobility larger in magnitude than that of MS2, and both have similar isoelectric point (IEP) values (～4). The electrokinetic results reflect a greater permeability of MS2 VLPs to electroosmotic flow, developed within/around these soft particles during their migration under the action of the applied electrical field. Results also support the presence of some remaining negatively charged component within the VLPs. In addition, MS2 phage systematically forms aggregates at pH values below the IEP, regardless of the magnitude of the solution ionic strength, whereas MS2 VLPs aggregate under the strict condition where the pH is relatively equal to the IEP at sufficiently low salt concentrations (<10 mM). It is argued that the stability of VLPs against aggregation and the differences between electrokinetics of MS2 and corresponding VLPs conform to recently developed formalisms for the stability and electrohydrodynamics of soft multilayered particles. The differences between the surface properties of these two kinds of particles reported here suggest that VLPs may not be appropriate for predicting the behavior of pathogenic viruses in aqueous media.     

Evaluation of viral clearance in the production of HPV-16 L1 virus-like particles purified from insect cell cultures

Biopharmaceutical products produced from cell cultures have a potential for viral contamination from cell sources or from adventitious introduction during production. The objective of this study was to assess viral clearance in the production of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs). We selected Japanese encephalitis virus (JEV), bovine viral diarrhea virus (BVDV), and minute virus of mice (MVM) as relevant viruses to achieve the aim of this study. A downstream process for the production of purified HPV-16 L1 VLPs consisted of detergent lysis of harvested cells, sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. The capacity of each purification/treatment step to clear viruses was expressed as reduction factor by measuring the difference in log virus infectivity of sample pools before and after each process. As a result, detergent treatment (0.5% v/v, Nonidet P-40/phosphate-buffered saline) was effective for inactivating enveloped viruses such as JEV and BVDV, but no significant reduction (< 1.0 log(10)) was observed in the non-enveloped MVM. The CsCl equilibrium density centrifugation was fairly effective for separating all three relevant adventitious viruses with different CsCl buoyant density from that of HPV-16 L1 VLPs (JEV, BVDV, and MVM = 4.30, 3.10, > or = 4.40 log(10) reductions). Given the study conditions we used, overall cumulative reduction factors for clearance of JEV, BVDV, and MVM were > or = 10.50, > or = 9.20, and > or = 6.40 log(10) in 150 ml of starting cell cultures, respectively.     

Development of a microRNA delivery system based on bacteriophage MS2 virus-like particles

Recently, microRNA (miRNA)-mediated RNA interference has been developed as a useful tool in gene function analysis and gene therapy. A major obstacle in miRNA-mediated RNAi is cellular delivery, which requires an efficient and flexible delivery system. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for RNA and drug delivery. However, MS2 VLP-mediated miRNA delivery has not yet been reported. We therefore used an Escherichia coli expression system to produce the pre-miR 146a contained MS2 VLPs, and then conjugated these particles with HIV-1 Tat(47-57) peptide. The conjugated MS2 VLPs effectively transferred the packaged pre-miR146a RNA into various cells and tissues, with 0.92-14.76-fold higher expression of miR-146a in vitro and about two-fold higher expression in vivo, and subsequently suppressed its targeting gene. These findings suggest that MS2 VLPs can be used as a novel vehicle in miRNA delivery systems, and may have applications in gene therapy.     

Characterization of Chikungunya Virus-Like Particles

Chikungunya virus (CHIKV) is becoming a global concern due to the increasing number of outbreaks throughout the world and the absence of any CHIKV-specific vaccine or treatment. Virus-like particles (VLPs) are multistructured proteins that mimic the organization and conformation of native viruses but lack the viral genome. They are noninfectious and potentially safer vaccine candidates. Recent studies demonstrated that the yield of CHIKV VLPs varies depending on the strains, despite the 95% amino acid similarity of the strains. This might be due to the codon usage, since protein expression is differently controlled by different organisms. We optimized the region encoding CHIKV structural proteins, C-E3-E2-6k-E1, inserted it into a mammalian expression vector, and used the resulting construct to transfect 293 cells. We detected 50-kDa proteins corresponding to E1 and/or E2 in the cell lysate and the supernatant. Transmission electron microscopy revealed spherical particles with a 50- to 60-nm diameter in the supernatant that resembled the native CHIKV virions. The buoyant density of the VLPs was 1.23 g/mL, and the yield was 20 µg purified VLPs per 10⁸ cells. The VLPs aggregated when mixed with convalescent sera from chikungunya patients, indicating that their antigenicity is similar to that of native CHIKV. Antibodies elicited with the VLPs were capable of detecting native CHIKV, demonstrating that the VLPs retain immunogenicity similar to that of the native virion. These results indicated that CHIKV VLPs are morphologically, antigenically, and immunologically similar to the native CHIKV, suggesting that they have potential for use in chikungunya vaccines.     

Experimental Transmission of Infectious Pancreatic Necrosis Virus from the Blue Mussel,Mytilus edulis,to Cohabitating Atlantic Salmon (Salmo salar) Smolts

Integrated multitrophic aquaculture (IMTA) reduces the environmental impacts of commercial aquaculture systems by combining the cultivation of fed species with extractive species. Shellfish play a critical role in IMTA systems by filter-feeding particulate-bound organic nutrients. As bioaccumulating organisms, shellfish may also increase disease risk on farms by serving as reservoirs for important finfish pathogens such as infectious pancreatic necrosis virus (IPNV). The ability of the blue mussel (Mytilus edulis) to bioaccumulate and transmit IPNV to naive Atlantic salmon (Salmo salar) smolts was investigated. To determine the ability of mussels to filter and accumulate viable IPNV, mussels were held in water containing log 4.6 50% tissue culture infective dose(s) (TCID₅₀) of the West Buxton strain of IPNV ml⁻¹. Viable IPNV was detected in the digestive glands (DGs) of IPNV-exposed mussels as early as 2 h postexposure. The viral load in mussel DG tissue significantly increased with time and reached log 5.35 ± 0.25 TCID₅₀ g of DG tissue⁻¹ after 120 h of exposure. IPNV titers never reached levels that were significantly greater than that in the water. Viable IPNV was detected in mussel feces out to 7 days postdepuration, and the virus persisted in DG tissues for at least 18 days of depuration. To determine whether IPNV can be transmitted from mussels to Atlantic salmon, IPNV-exposed mussels were cohabitated with naive Atlantic salmon smolts. Transmission of IPNV did occur from mussels to smolts at a low frequency. The results demonstrate that a nonenveloped virus, such as IPNV, can accumulate in mussels and be transferred to naive fish.     

Cucumis sativus Cryptic Virus, a New Virus in Cucumber

Isometric virus-like particles (VLPs) have been purified from cucumber leaf tissue. Three dsRNA segments with estimated molecular weights of 1.8, 1.1 and 1.0 × 10⁶d have been isolated from VLPs occurring in CsCl density gradient fractions but were also readily detected in preparations from as little as 1 g of fresh leaf tissue. The VLPs resemble dsRNA containing cryptic viruses and have been named Cucumis sativus cryptic virus (CsCV).     

Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells.

Infection of cells by human papillomaviruses (HPVs) associated with malignant genital lesions has not been studied because of the lack of an in vitro system and the unavailability of virions. We have now used virus-like particles (VLPs) of HPV type 33 to analyze the initial events in the interaction of the HPV capsid with cell lines. Binding of VLPs to HeLa cells was observed in biochemical assays and by immunofluorescence. VLP binding was inhibited by antisera raised against VLPs but not by monoclonal antibodies recognizing either L1 or L2 epitopes accessible on VLPs. Under saturating conditions, approximately 2 x 10(4) VLPs were bound per cell, with a dissociation constant of about 100 pM. VLPs composed of L1 alone bound as well as VLPs composed of both capsid proteins, indicating that L2 is not required for initial binding. VLPs dissociated into capsomers did not bind, demonstrating that intercapsomer contacts are required. Neither capsomers nor simian virus 40 virions competed with VLP binding. Uptake of VLPs by small and smooth endocytic vesicles was demonstrated by immunoelectron microscopy. Cellular binding of VLPs was sensitive to trypsin but not to sialidase, N-glycosidase, or octyl-beta-D-glycopyranoside treatment, suggesting that a cell surface protein is involved in the VLP binding. Cell lines originating from a variety of tissues and organisms as distantly related as insects and humans bound VLPs with similar efficiency and specificity. Therefore, the putative receptor mediating VLP attachment should be highly conserved and cannot be responsible for the species and tissue specificity of HPVs.     

Immunogenicity and Protective Efficacy of Replication-Incompetent Influenza Virus-Like Particles

Despite the success of influenza virus vaccines in reducing severe illness, their efficacy is suboptimal. We describe here the immunogenicity and protective capacity of replication-incompetent influenza virus-like particles (VLPs) which were generated entirely from cDNAs and lacked either the entire NS gene (encoding both the NS1 and NS2 protein) or only the NS2 gene. In mammalian cells infected with NS gene-deficient VLPs, the nucleoprotein, but not other viral proteins including hemagglutinin (HA) and neuraminidase (NA), was detected. In contrast, cells infected with VLPs expressing NS1 but not NS2 (NS2 knockout) expressed multiple viral proteins, including HA and NA. When challenged with lethal doses of an antigenically homologous mouse-adapted influenza virus, 94% of mice vaccinated with the NS2-knockout VLPs survived, compared with less than 10% of those given the NS-deficient VLPs. These results demonstrate the potential of replication-incompetent NS2-knockout VLPs as novel influenza vaccines and perhaps also as vectors to express genes from entirely unrelated pathogens.     

Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.     

Evaluation of lactic acid bacterium fermentation products and food-grade chemicals to control Listeria monocytogenes in blue crab (Callinectes sapidus) meat.

Fresh blue crab (Callinectes sapidus) meat was obtained from retail markets in Florida and sampled for viable Listeria monocytogenes. The pathogen was found in crabmeat in three of four different lots tested by enrichment and at levels of 75 CFU/g in one of the same four lots by direct plating. Next, crabmeat was steam sterilized, inoculated with a three-strain mixture of L. monocytogenes (ca. 5.5 log10 CFU/g), washed with various lactic acid bacterium fermentation products (2,000 to 20,000 arbitrary units [AU]/ml of wash) or food-grade chemicals (0.25 to 4 M), and stored at 4 degrees C. Counts of the pathogen remained relatively constant in control samples during storage for 6 days, whereas in crabmeat washed with Perlac 1911 or MicroGard (10,000 to 20,000 AU), numbers initially decreased (0.5 to 1.0 log10 unit/g) but recovered to original levels within 6 days. Numbers of L. monocytogenes cells decreased 1.5 to 2.7 log10 units/g of crabmeat within 0.04 day when washed with 10,000 to 20,000 AU of Alta 2341, enterocin 1083, or Nisin per ml. Thereafter, counts increased 0.5 to 1.6 log10 units within 6 days. After washing with food-grade chemicals, modest reductions (0.4 to 0.8 log10 unit/g) were observed with sodium acetate (4 M), sodium diacetate (0.5 or 1 M), sodium lactate (1 M), or sodium nitrite (1.5 M). However, Listeria counts in crabmeat washed with 2 M sodium diacetate decreased 2.6 log10 units/g within 6 days. In addition, trisodium phosphate reduced L. monocytogenes counts from 1.7 (0.25 M) to > 4.6 (1 M) log10 units/g within 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of human parainfluenza viruses in the South Polar environment

The survival of human parainfluenza virus types 1, 2, and 3 was measured in both indoor and outdoor environments at South Pole Station, Antarctica, in an effort to determine the long-term survival of these viruses in this environment and to identify the possible source of respiratory tract illnesses which occurred in this isolated population in 1978 after 10 and 27 weeks of total social isolation. Viruses were applied to plastic petri plate surfaces which were then stored in indoor (21.4 degrees C; water vapor density, 1.50 g of water per m3) and outdoor environments (-22.4 to -33.2 degrees C; water vapor density, 0.706 and 0.247 g of water per m3). Parainfluenza virus type 1 at an initial titer of 3.75 log10 50% tissue culture infective doses per ml was inactivated after 4 days at room temperature and after 7 days outside. Parainfluenza virus type 2 and 3 at initial titers of 5.58 and 5.38 log10 50% tissue culture infective doses per ml were inactivated after 7 and 12 days, respectively, at room temperature and after 17 days of storage outside. Results indicate that the long-term survival of parainfluenza virus in either environment for up to 10 weeks is unlikely and probably did not provide the source of infectious virus responsible for the midisolation outbreaks of parainfluenza virus-related respiratory tract illnesses observed in this population during the 1978 winter season.     

Survival of human parainfluenza viruses in the South Polar environment.

The survival of human parainfluenza virus types 1, 2, and 3 was measured in both indoor and outdoor environments at South Pole Station, Antarctica, in an effort to determine the long-term survival of these viruses in this environment and to identify the possible source of respiratory tract illnesses which occurred in this isolated population in 1978 after 10 and 27 weeks of total social isolation. Viruses were applied to plastic petri plate surfaces which were then stored in indoor (21.4 degrees C; water vapor density, 1.50 g of water per m3) and outdoor environments (-22.4 to -33.2 degrees C; water vapor density, 0.706 and 0.247 g of water per m3). Parainfluenza virus type 1 at an initial titer of 3.75 log10 50% tissue culture infective doses per ml was inactivated after 4 days at room temperature and after 7 days outside. Parainfluenza virus type 2 and 3 at initial titers of 5.58 and 5.38 log10 50% tissue culture infective doses per ml were inactivated after 7 and 12 days, respectively, at room temperature and after 17 days of storage outside. Results indicate that the long-term survival of parainfluenza virus in either environment for up to 10 weeks is unlikely and probably did not provide the source of infectious virus responsible for the midisolation outbreaks of parainfluenza virus-related respiratory tract illnesses observed in this population during the 1978 winter season.     

牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析

为了获得预防牛病毒性腹泻病毒1型(bovine viral diarrhea virus 1,BVDV-1)感染的病毒样颗粒,扩增C-Ems-E1-E2编码区段并克隆至pFastBacDaul载体,转化大肠杆菌DH10Bac感受态细胞与Bacmid重组获得Bacmid-BVDV-1,转染至Sf9细胞,获得重组杆状病毒B...     

Survival of Murine Norovirus,Tulane Virus,and Hepatitis A Virus on Alfalfa Seeds and Sprouts during Storage and Germination

Human norovirus (huNoV) and hepatitis A virus (HAV) have been involved in several produce-associated outbreaks and identified as major food-borne viral etiologies. In this study, the survival of huNoV surrogates (murine norovirus [MNV] and Tulane virus [TV]) and HAV was investigated on alfalfa seeds during storage and postgermination. Alfalfa seeds were inoculated with MNV, TV, or HAV with titers of 6.46 ± 0.06 log PFU/g, 3.87 ± 0.38 log PFU/g, or 7.01 ± 0.07 log 50% tissue culture infectious doses (TCID₅₀)/g, respectively. Inoculated seeds were stored for up to 50 days at 22°C and sampled during that storage period on days 0, 2, 5, 10, and 15. Following storage, virus presence was monitored over a 1-week germination period. Viruses remained infectious after 50 days, with titers of 1.61 ± 0.19 log PFU/g, 0.85 ± 0.21 log PFU/g, and 3.43 ± 0.21 log TCID₅₀/g for MNV, TV, and HAV, respectively. HAV demonstrated greater persistence than MNV and TV, without a statistically significant reduction over 20 days (<1 log TCID₅₀/g); however, relatively high levels of genomic copies of all viruses persisted over the testing time period. Low titers of viruses were found on sprouts and were located in all tissues as well as in sprout-spent water sampled on days 1, 3, and 6 following seed planting. Results revealed the persistence of viruses in seeds for a prolonged period of time, and perhaps of greater importance these data suggest the ease of which virus may transfer from seeds to sprouts and spent water during germination. These findings highlight the importance of sanitation and prevention procedures before and during germination.     

Tetracycline-inducible packaging cell line for production of flavivirus replicon particles

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 10(10) VLPs per 10(6) transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

Potential transmission of human polyomaviruses through the gastrointestinal tract after exposure to virions or viral DNA

The mechanism of human-to-human transmission of the polyomaviruses JC virus (JCV) and BK virus (BKV) has not been firmly established with regard to possible human exposure. JCV and BKV have been found in sewage samples from different geographical areas in Europe, Africa, and the United States, with average concentrations of 10(2) to 10(3) JCV particles/ml and 10(1) to 10(2) BKV particles/ml. Selected polyomavirus-positive sewage samples were further characterized. The JCV and BKV present in these samples were identified by sequencing of the intergenic region (the region found between the T antigen and VP coding regions) of JCV and the VP1 region of BKV. The regulatory region of the JCV and BKV strains found in sewage samples presented archetypal or archetype-like genetic structures, as described for urine samples. The stability (the time required for a 90% reduction in the virus concentration) of the viral particles in sewage at 20 degrees C was estimated to be 26.7 days for JCV and 53.6 days for BKV. The presence of JCV in 50% of the shellfish samples analyzed confirmed the stability of these viral particles in the environment. BKV and JCV particles were also found to be stable at pH 5; however, treatment at a pH lower than 3 resulted in the detection of free viral DNA. Since most humans are infected with JCV and BKV, these data indicate that the ingestion of contaminated water or food could represent a possible portal of entrance of these viruses or polyomavirus DNA into the human population.