Virus Recovery Methods Research Articles

Influence of membrane pore-size on the recovery of endogenous viruses from wastewater using an adsorption-extraction method

The ongoing COVID-19 pandemic has emphasized the significance of wastewater surveillance in monitoring and tracking the spread of infectious diseases, including SARS-CoV-2. The wastewater surveillance approach detects genetic fragments from viruses in wastewater, which could provide an early warning of outbreaks in communities. In this study, we determined the concentrations of four types of endogenous viruses, including non-enveloped DNA (crAssphage and human adenovirus 40/41), non-enveloped RNA (enterovirus), and enveloped RNA (SARS-CoV-2) viruses, from wastewater samples using the adsorption-extraction (AE) method with electronegative HA membranes of different pore sizes (0.22, 0.45, and 0.80 µm). Our findings showed that the membrane with a pore size of 0.80 µm performed comparably to the membrane with a pore size of 0.45 µm for virus detection/quantitation (repeated measurement one-way ANOVA; p > 0.05). We also determined the recovery efficiencies of indigenous crAssphage and pepper mild mottle virus, which showed recovery efficiencies ranging from 50% to 94% and from 20% to 62%, respectively. Our results suggest that the use of larger pore size membranes may be beneficial for processing larger sample volumes, particularly for environmental waters containing low concentrations of viruses. This study offers valuable insights into the application of the AE method for virus recovery from wastewater, which is essential for monitoring and tracking infectious diseases in communities.

Multiple Novel Human Norovirus Recombinants Identified in Wastewater in Pretoria, South Africa by Next-Generation Sequencing.

The genogroup II genotype 4 (GII.4) noroviruses are a major cause of viral gastroenteritis. Since the emergence of the Sydney_2012 variant, no novel norovirus GII.4 variants have been reported. The high diversity of noroviruses and periodic emergence of novel strains necessitates continuous global surveillance. The aim of this study was to assess the diversity of noroviruses in selected wastewater samples from Pretoria, South Africa (SA) using amplicon-based next-generation sequencing (NGS). Between June 2018 and August 2020, 200 raw sewage and final effluent samples were collected fortnightly from two wastewater treatment plants in Pretoria. Viruses were recovered using skimmed milk flocculation and glass wool adsorption-elution virus recovery methods and screened for noroviruses using a one-step real-time reverse-transcription PCR (RT-PCR). The norovirus BC genotyping region (570-579 bp) was amplified from detected norovirus strains and subjected to Illumina MiSeq NGS. Noroviruses were detected in 81% (162/200) of samples. The majority (89%, 89/100) of raw sewage samples were positive for at least one norovirus, compared with 73% (73/100) of final effluent samples. Overall, a total of 89 different GI and GII RdRp-capsid combinations were identified, including 51 putative novel recombinants, 34 previously reported RdRp-capsid combinations, one emerging novel recombinant and three Sanger-sequencing confirmed novel recombinants.

Capturing Coronaviruses: Comparison of Recovery Methods for Infectious Coronaviruses From Environmental Surfaces

The emergence of novel, zoonotic betacoronavirus, SARS‐CoV‐2, demands quantification of infectious virus rather than viral RNA to provide more accurate assessment of transmission risk for COVID‐19. To maximize investigator safety, similar in morphology and inactivation profile, human betacoronavirus OC43 (OC43) was used as a surrogate to refine infectious virus recovery methods for SARS‐CoV‐2, a biosafety level (BSL)‐3 pathogen. Using OC43 and SARS‐CoV‐2, we examined the ability of InnovaPrep Mano Surface Samplers (MANOs), made of hydrophobic, open‐cell polymeric foam, to recover virus from large stainless‐steel surfaces hypothesizing that they would outperform hydrophilic cellulose sponges (sponges), used for environmental sampling.In triplicate, 1.0 x 105 TCID50 of OC43 and SARS‐CoV‐2 were applied to the inner surface of a biological safety cabinet (BSC) within the literature or vendor specified optimal surface area, 1267.36 cm2 (MANO) and 100 cm2 (sponges). The areas were sampled with eluant presoaked MANOs and sponges, and samples aliquoted and stored at ‐80ºC until batch titration by indirect immunofluorescence TCID50 assay, using human ileocecal colorectal adenocarcinoma cells (HRT‐18 CCL‐244, ATCC) for OC43 samples and crystal violet detection‐based TCID50 using African green monkey kidney epithelial cells (VeroE6 CRL‐1586, ATCC) for SARS‐CoV‐2. Tested eluants were a beef extract buffer, pH 7 (BEB7), BEB7 with 0.05% Tween‐20 (BEB7/T) and (MANOs only) phosphate buffered saline with 0.05% Tween‐20 (PBS/T), the vendor default. Additionally, an expanded surface area (0.77m2 for MANOs, 300cm2 for sponges) was tested with OC43 as described above, except with 1.0x104 TCID50 inoculum.For OC43, recovery for MANOs with BEB7 was 2.66x105 TCID50 compared to PBS/T’s 4.40x104 TCID50. BEB7 MANOs had a higher average percent recovery (102%) than BEB7 sponges (84%). By one‐way ANOVA (α=0.05), MANO BEB7’s and BEB7/T’s average recovery percentages were significantly greater than PBS/T’s. Samples taken from the larger surface areas for both samplers were below the assay’s limits of detection for BEB7; however, OC43 eluted with BEB7/T from sponges was detectable (3.47x102 TCID50, 32% recovery). For SARS‐CoV‐2, MANO BEB7’s average recovery percentage (27%) was again shown to be significantly greater than PBS/T’s (0.38%). However, decreased recovery of infectious virus was seen across both tools and all eluants, perhaps indicating a difference between SARS‐CoV‐2 and OC43 for BEB7.Use of BEB7 improved OC43 and SARS‐CoV‐2 recovery from optimal surface areas with MANOs. Surprisingly, when the surface area was enlarged, all samples except those from BEB7/T sponges were below the limit of detection. This might be explained by additional eluant loss on the larger surface during mechanical recovery or virus inactivation due to desiccation from BSC airflow or other environmental factors. Similar factors might explain the SARS‐CoV‐2 results. Quantification by RT‐qPCR or flow virometry, and planned experiments in BSL‐3 agricultural space, where the room is primary containment, may provide further insights.

Evaluation of Bead-Based Assays for the Isolation of Foodborne Viruses from Low-Moisture Foods

Foodborne viruses such as norovirus and hepatitis A virus (HAV) are highly transmissible, persistent in the environment, and resistant to many conventional inactivation methods. Foods can become contaminated with these viruses either at the source of harvest or during food handling and processing. Multiple lines of evidence suggest that foodborne viruses can survive desiccation and dry conditions. Several foodborne virus outbreaks have been linked to low-moisture foods (LMFs), indicating that these foods can be vehicles of virus transmission. However, the efficiencies of common virus extraction methodologies have not been examined with LMFs. We adapted the International Organization for Standardization (ISO) 15216-1:2017 method for virus recovery for use with chocolate, pistachios, and cornflakes. We also developed a magnetic bead assay for the recovery of HAV from LMFs and used the porcine gastric mucin-coated magnetic beads (PGM-MBs) to extract norovirus surrogates, feline calicivirus (FCV), and murine norovirus (MNV) from the same LMFs. The efficiency of virus recovery using the bead-based assay was then compared with that of the ISO 15216-1:2017 method. In chocolate and pistachios, the recovery rates with the PGM-MB method were 5.6- and 21.3-fold higher, respectively, for FCV and 1.65- and 18-fold higher, respectively, for MNV than those with the ISO 15216-1:2017 method. However, the PGM-MB method failed to recover MNV and FCV from cornflakes. The recovery rates for HAV in chocolate, pistachios, and corn flakes with the magnetic bead method were 11.5-, 3-, and 5.6-fold higher, respectively, than those with the ISO 15216-1:2017 method. Thus, depending upon the food matrix and the target virus, the bead-based assays can be used to efficiently and rapidly extract viruses from LMFs.

Comparative Recovery of Two Human Norovirus Surrogates, Feline Calicivirus and Murine Norovirus, with a Wet Vacuum System, Macrofoam-Tipped Swab, and Bottle Extraction Method from Carpets

Human noroviruses (HuNoV) are the leading cause of known foodborne illness in the United States, but direct detection during outbreak investigations is challenging. On the other hand, sampling both hard and soft environmental surfaces can be used to improve outbreak investigations. Currently, we lack virus recovery methods for soft surfaces, such as carpet, despite evidence suggesting that carpets contribute to HuNoV outbreaks. Our aim was to compare two recovery methods, wet vacuum and swabbing, for routine carpet sampling of HuNoV against a laboratory reference method known as bottle extraction (BE). Specifically, we compared the microbial vacuum (MVAC), macrofoam-tipped swab (MS), and BE by using HuNoV surrogates, feline calicivirus (FCV) and murine norovirus (MNV), inoculated on wool and nylon carpet carriers. The highest recovery of infectious FCV from wool was 5.51, 3.76, and 5.16 log PFU, whereas on nylon, recovery was 5.03, 3.62, and 4.75 log PFU by using BE, MS, and MVAC, respectively. On the other hand, the highest recovery of infectious MNV from wool was 6.10, 4.50, and 5.99 log PFU, while recovery on nylon was 6.07, 4.58, and 6.13 log PFU by using BE, MS, and MVAC, respectively. Significantly more PFU and genomic copies were recovered by using BE and MVAC compared with MS, while buffer type played a significant role in recovery of infectious FCV.

Adenovirus and rotavirus recovery from a treated effluent through an optimized skimmed-milk flocculation method.

Sewage treatment may be insufficient for the complete removal of enteric viruses, such as human adenoviruses (HAdV) and group A rotavirus (RVA). The differences in the efficiency of the treatment methodologies used may interfere with the detection of these viruses. The objective of this study was to optimize a skimmed-milk flocculation technique for the recovery of HAdV and RVA in the samples of treated effluent. The treated effluent collected at the wastewater treatment plant (WWTP) was processed via four protocols including modifications in the initial centrifugation step and the final concentration of skimmed-milk. The viral load and recovery rate were determined by quantitative PCR TaqMan® System. The highest recovery rates of HAdV, RVA, and bacteriophage PP7 (internal control process) were obtained when the concentration of skimmed-milk was doubled and no centrifugation step was used for the sample clarification. The optimized protocol was assessed in a field study conducted with 24 treated effluent samples collected bi-monthly during 2015. HAdV and RVA were detected in 50.0% (12/24) and 33.3% (08/24) of the samples tested, respectively, throughout the year, without seasonal variation (p > 0.05). This study corroborates the use of the organic flocculation method for virus recovery in environmental samples with the adaptation of the protocols to different aquatic matrices.

Evaluation of virus recovery methods and efficiency of tannin-derived coagulants in removing total coliforms, E. coli and enteric viruses in effluents of a domestic sewage treatment plant.

In the present study, nine coagulants having potential to be used for sewage treatment were compared to assess their efficiency in removing total coliform bacteria, Escherichia coli and adenovirus. The coagulants tested were metallic and organic and their efficiency was compared when treating samples of raw and treated sewage (activated sludge). Before the efficiency tests of the coagulants, viral concentration methods were compared. Coagulation tests were carried out by using the jar-test system and the doses used ranged from 100 ppm to 1,000 ppm. Viral DNA was extracted and subjected to real-time polymerase chain reaction (qPCR) using primers for the gene of AdV hexon. Aluminum sulfate (1,000 ppm) presented the best results for raw sewage among metal coagulants whereas Acquapol® C118 and WW (1,000 ppm) had the most satisfactory results among organic coagulants, both reducing up to 7 logs for coliforms and 4 logs for virus. For the treated effluent, FeCl2 (1,000 ppm) presented best results for metal coagulants, whereas, from organic coagulants, the best removal rates were for Acquapol® 893/11 (1,000 ppm), both reducing up to 3 logs for coliforms and 4 logs for virus.

Comparison of Surface Sampling Methods for Virus Recovery from Fomites

The role of fomites in infectious disease transmission relative to other exposure routes is difficult to discern due, in part, to the lack of information on the level and distribution of virus contamination on surfaces. Comparisons of studies intending to fill this gap are difficult because multiple different sampling methods are employed and authors rarely report their method's lower limit of detection. In the present study, we compare a subset of sampling methods identified from a literature review to demonstrate that sampling method significantly influences study outcomes. We then compare a subset of methods identified from the review to determine the most efficient methods for recovering virus from surfaces in a laboratory trial using MS2 bacteriophage as a model virus. Recoveries of infective MS2 and MS2 RNA are determined using both a plaque assay and quantitative reverse transcription-PCR, respectively. We conclude that the method that most effectively recovers virus from nonporous fomites uses polyester-tipped swabs prewetted in either one-quarter-strength Ringer's solution or saline solution. This method recovers a median fraction for infective MS2 of 0.40 and for MS2 RNA of 0.07. Use of the proposed method for virus recovery in future fomite sampling studies would provide opportunities to compare findings across multiple studies.

Detection of norovirus genotype I.3b and II.4 in bioaccumulated blue mussels using different virus recovery methods

Noroviruses (NoVs) are the most common non bacterial human pathogens associated with shellfish borne gastroenteritis. Norovirus detection is based on molecular procedures such as reverse transcriptase (RT)-PCR. A variety of methods have been developed to extract viral RNA from complex shellfish matrixes and to reduce the level of RT-PCR inhibitors. The present study had three objectives: 1) Determine the most appropriate sample treatment protocol for detection of NoVs in mussels, 2) Examine whether there is a variation of the binding affinity between a NoV GI and a GII strain to mussel digestive tissue and how this influences the detection sensitivity, 3) Establish an internal control for sample processing and virus detection. Three RNA extraction methods were evaluated on extracts from blue mussels ( Mytilus edulis) spiked with NoV GII.4. The most efficient RNA extraction method was subsequently used for evaluation of three virus recovery methods of blue mussels bio accumulated with NoV GI.3b and GII.4. Mengovirus was evaluated as an internal process control and TaqMan RT-PCRs were used for virus detection. Elution of the two viruses from shellfish tissue differed, indicating a difference in binding affinities. Only a method based upon Proteinase K digestion followed by NucliSens®easyMAG was able to detect both NoV GI.3b and GII.4 (3.0% and 3.5% recovery respectively). The results show that the processing method influences the possibility to detect different variants of NoV.

Development of plaque assays for adenoviruses 40 and 41

Enteric adenoviruses, important agents of infantile gastroenteritis, are difficult to culture with low titers and limited CPE. Consequently, few plaque assays have been reported and none are used routinely by investigators who may need reproducible quantitative assays for these viruses. CPE in A549 cells (an epithelial lung carcinoma cell line) was induced by isolates of human adenovirus (HAdV) serotypes 40 or 41 that were obtained by prior limited passage in primary cynmolgous monkey kidney (pCMK), human embryonic kidney (HEK), and Graham 293 cells. CPE with HAdV 40 (Dugan strain) and HAdV 41 (Tak strain) inoculated in A549 cells was also observed. Monolayers of A549 cells were inoculated with a low multiplicity of infection (MOI) of the archived stock isolates and harvested at days 10–14 with full CPE. Subsequent passages were harvested in as few as 7 days with 100% CPE to prepare virus stocks for plaque assay. Large individual plaques under agarose overlay were picked prior to staining and clonal stocks prepared. Titers of final stock preparations after six to eight passages in A549 cells were in the range of 5 × 10 7–1 × 10 8 PFU/ml, which provides adequate virus for quantitative recovery studies. The particle to infectivity (P:I) ratios of the early passages of virus stocks were in the range reported previously. The ratio of non-infectious to infectious particles decreased with successive passages of HAdVs 40 and 41 in A549 cells. The specificity of the assay was confirmed by neutralization of plaques with type-specific antisera. Furthermore, sequence analysis of the HAdVs 40 and 41 plaque forming stocks ruled out contamination with any other HAdVs. The plaque assay developed will be useful for evaluation of virus recovery methods from water, food or other environmental matrices, as well as determination of the efficacy of water treatment techniques for inactivation of these viruses.

Round robin investigation of glass wool method for poliovirus recovery from drinking water and sea water

This study was initiated by the AFNOR water microbiology working group to evaluate the performance of the glass wool method for virus recovery. Its reliability was tested with drinking and sea water by respectively nine and thirteen laboratories. In both trials, six were actively involved in water virology research, one was designated as a central laboratory, the others had no experience in virological practices. Analysis of reproducibility and repeatability according to NF-ISO 5725-2 were realized. For drinking waters (24 assays), the average recovery efficiency was 72%, mean standard deviations: repeatability 12.4%; reproducibility 33.6%; inter-laboratories 21%. For sea waters (39 assays), the average recovery efficiency was 75% and the mean standard deviations 6.9%, 17.9% and 11% respectively.

Round robin investigation of glass wool method for poliovirus recovery from drinking water and sea water

This study was initiated by the AFNOR water microbiology working group to evaluate the performance of the glass wool method for virus recovery. Its reliability was tested with drinking and sea water by respectively nine and thirteen laboratories. In both trials, six were actively involved in water virology research, one was designated as a central laboratory, the others had no experience in virological practices. Analysis of reproducibility and repeatability according to NF-ISO 5725-2 were realized. For drinking waters (24 assays), the average recovery efficiency was 72%, mean standard deviations: repeatability 12.4%; reproducibility 33.6%; inter-laboratories 21%. For sea waters (39 assays), the average recovery efficiency was 75% and the mean standard deviations 6.9%, 17.9% and 11% respectively.

A comparison of current methods of poliovirus concentration from tap water

The efficiency of concentration of poliovirus from Jerusalem tap water was investigated for several types of “electronegatively-charged” and “electropositively-charged” microporous filters. In addition, the efficiency of organic flocculation as a procedure for reconcentrating poliovirus from filter eluates was investigated. The Balston and Cox filters had similar recovery efficiencies from tap water, with recovery of approx. 90% of the input virus, both after filtration and after organic flocculation. Decreasing the concentration of beef extract in the eluent from 3 to 1% did not negatively influence virus recovery. However, recovery of low virus numbers from large volume samples by Cox filters was variable. Balston filters were used in a series of high volume experiments to test the efficacy of the tentative standard method for virus recovery using a proportioner pump with two additive pumps. The method was inefficient without a simple modification, the addition of a second mixing chamber, which increased the virus recovery to an acceptable level. The Zeta Plus electropositively-charged type of filter had a high efficiency of virus recovery in the eluate, but approximately half of the virus was lost during organic flocculation. This may indicate the need for modification of the organic flocculation method when used with filters.

A Method for Recovery of Viruses from Oysters and Hard and Soft Shell Clams

A method for recovery of small numbers of enteric viruses from oysters and hard and soft shell clams was developed. As few as 3 plaque forming units (PFU) of virus per 100 g of shellfish homogenate could be detected with an overall accuracy of ca. 60 percent in each of the three species tested.

Method for Recovery of Viruses from Milk and Milk Products

A modification of the plaque-forming unit assay was developed for recovery of viruses from milk and milk products. Viruses were assayed on primary cell cultures of Cercopithecus aethiops (African green) monkey kidneys under an overlay medium. The medium contained 0.95% clarified Ionagar no. 2, Eagle's minimum essential medium with nonessential amino acids in Hanks’ balanced salt solution without phenol red, 2% fetal bovine serum, 0.19% NaHCO3, 0.0015% neutral red dye, 0.51% MgCl2 · 6H2O, and 1% sterile homogenized bovine milk. Geometric means and 95% confidence limits expressed as plaque-forming units/0.5ml are presented for recoveries of Adenovirus 12, Herpes simplex virus, Influenza A virus, and New castle disease virus from Eagle's medium and sterile milk. Recoveries of Adenovirus 12, Herpes simplex virus, Simian virus 40, and Reovirus 1 from milk, ice eream mix, chocolate milk, chocolate drink, or heavy cream were within±threefold of viral plaque-forming units input, estimated by dilution. Elimination or reduction of bacterial and fungal contamination in the nonsterile milk or milk products was achieved by adding 1,000 units of Penicillin G per milliliter, 1,000μg of streptomycin sulfate per milliliter, 50μg of tetracycline hydrochloride per milliliter, and 0.5μg of Amphotericin B per milliliter at least 48 hours before assay.