Assessment of different factors on the influence of glass wool concentration for detection of main swine viruses in water samples

Virus strains

Different types of viruses were used for evaluating the effects of the water-concentration method developed in this study. Among these types of viruses, ASFV strain HuB-2 was isolated from the lung of a pig, and the evaluation based on ASFV was performed in the Animal Biosafety Level III Laboratory of Huazhong Agricultural University. Considering the biosecurity risk of using wildtype viral strains, we selected two attenuated vaccine strains for the assessment of the method on concentrating PRV (strain HB98; Keqian Bio., Wuhan, China) and PEDV (strain AJ1002; Keqian Bio., Wuhan, China). In addition, our previously collected Salmonella bacteriophage ph2-2 (Zhao et al., 2022) was also included for evaluation in this study. Initial viral solutions containing ASFV (188,456 copies/μl), PRV (291,288 copies/μl), and PEDV (322,130 copies/μl) were prepared.

Preparation of glass wool

Preparation of glass wool was optimized based on previous studies (Kiulia et al., 2010; Lambertini et al., 2008; Millen et al., 2012) to reduce the amount of water and processing reagents used. Briefly, glass wool (U-1339; Johns Manville, Denver, CO, USA) was soaked in double-distilled water for 15 min, then soaked in 0.5 M hydrochloric acid for 20 min, rinsed three times with double-distilled water, and soaked in 0.5 M sodium hydroxide for 20 min, then rinsed three times with double-distilled water. The processed glass wool was packed into filters, which were circular PVC containers with a diameter of 45 mm and a length of 105 mm, with interfaces on both sides to connect with pipelines. Approximately 60 g of dry processed glass wool were placed in each filter, and finally, the glass wool was stored in PBS (pH 6.7–7.0) at 4 °C for future use.

Primary concentration method

Before the experiment, the equipment is wiped and operating table surface with 0.5% sodium hypochlorite solution (Abd-Elmaksoud et al., 2014), and then wipe with water after 15 min. Use a peristaltic pump (YZ1515X; Runze, Shenzhen City, China) to extract the seeded virus water from the container and filter it through a glass wool filter element at different speeds, and let the peristaltic pump continue to work for 3 min after all the water has been filtered. Soak the glass wool filter element with 75 ml of 3% beef extract buffer (B8570; Solarbio, Beijing, China) solution containing 0.5 M glycine (1275KG2P5; BioFroxx, Hangzhou, China) and pH 9.0 for 20 min, then wash with 75 ml of beef extract buffer solution again. Collect the total 150 ml buffer solution in a clean container, adjust the eluent pH to neutral with 1M hydrochloric acid, and store at 4 °C; if over 48 h, it must be stored at −20 °C or lower temperature.

Assessment of the influence of different factors on primary concentration of PRV, ASFV and PEDV

To assess the influence of different factors on viral enrichment by primary concentration, a series water samples containing ASFV, PRV, and PEDV under the following conditions were prepared for primary concentration. Viral nucleic acids in water samples before and after concentration were detected using real-time fluorescence quantitative PCR (qPCR), and the results were compared.

(1) pH values: To test the influence of pH values on virus recovery, PBS (4,000 ml) containing different types of viruses (200 µl) at different pH values (6.0, 7.0, 8.0, 9.0) were prepared since the pH values of environmental waters generally ranged from 6.0 to 9.0.

(2) Water types: Water samples were prepared by seeding viruses (200 µl) in samples (4,000 ml) collected from different sources, including collected from different sources, including tap water (pH = 8.0, nephelometric turbidity unit (NTU) = 0, containing no organic matters and low concentrations of salt ions), urban inland lake water (pH = 9.0, NTU = 17, containing rich in organic matters and microorganisms), water from the mainstream of Yangtze River (pH = 7.9, NTU = 9.0, containing silts), water from suburban rivers (pH = 7.8, NTU = 25.0, receiving some domestic sewages from nearby villages), and PBS (pH = 7.4, NTU = 0, containing salt ions but no organic matters).

(3) Filtration speeds: PBS solutions (pH 7.4; 4,000 ml) mixed with 200 µl of different viruses were filtered at 500, 1,000, and 1,500 ml/min; at the same time, PBS solution without mixed virus was set as a negative control group.

(4) Filtration volumes: Three groups of PBS solutions (pH 7.4) solutions were set, with volumes of 4,000, 12,000, and 20,000 ml respectively; a certain volume of virus mixed solution was added (ensuring the initial virus concentration of the sample before filtration was consistent) and mixed evenly; at the same time, PBS solution without mixed virus was set as a negative control group.

(5) Temperatures: Three groups of PBS solutions were set, and the temperature of PBS was adjusted to 4 °C, 20 °C and 32 °C respectively. Then 200 μl of virus mixed solution was added to each group and mixed evenly; at the same time, a negative control group without mixed virus was set for each temperature.

Effects of glass wool enrichment on viral particles and nucleic acids

To access the effect of glass wool on the enrichment of viral particles and nucleic acids, three types of virus-associated-samples were prepared: (1) “viral particles”; this type of sample was prepared by removing free viral nucleic acids thoroughly through addition of Benzanase (Merck, Darmstadt, Germany) and incubated at 37 °C for 20 min (Berg et al., 2016). (2) “nucleic acid”; this type of sample was prepared by extracting viral nucleic acids using either a commercial viral DNA or RNA preparation kit (Vazyme, Nanjing, China). (3) “viral solution”; no treatment was given and there might be both viral particles and nucleic acids inside. Thereafter, each of the prepared samples was added into 4,000 ml PBS for primary concentration. Finally, viral DNA/RNA in the concentration products of virus-associated-samples were extracted and were quantified by qPCR.

Evaluation of the efficacy of glass wool on concentrating live virus

To assess the efficacy of glass wool on enriching living virus, two types of phage-associated samples were prepared: (1) “phage particles” which was prepared by removing the free nucleic acids using Benzanase (Merck, Darmstadt, Germany); and (2) “phage solution” (1 × 10¹¹ PFU/ml for live phages or for phage nucleic acids) did not receive any special treatment. After that, either phage particles or phage solutions (200 µl) were speeded into 4,000 ml PBS for primary concentration by glass wool. The concentration products of phage particles were incubated with its host bacterium (Salmonella Paratyphi strain 201107 (Zhao et al., 2022)) for titering, while the DNAs in the concentration products of phage solutions were quantified by qPCR.

Secondary concentration effectiveness evaluation

A total of 150 ml of negative beef extract powder eluents containing 200 μl of ASFV, PRV, PEDV, or Salmonella bacteriophage ph2-2 were for secondary concentration, respectively. Three methods were used for secondary concentration: skimmed milk method, PEG-NaCl method and ultracentrifugation. The skimmed milk method was to add 0.2‰ skimmed milk (CN7861; Coolaber, Beijing, China) powder to 40 ml of eluent and adjust the pH to 3.5. Shake it at 200 ×g for 2 h at room temperature and centrifuge it at 2,000 ×g for 30 min. The precipitate was resuspended in 0.01 M PBS and stored at −80 °C (Assis et al., 2017; Calgua et al., 2008). The PEG-NaCl method was to add 15% PEG8000 (1363GR; BioFroxx, Hangzhou, China) and 0.2 M NaCl to 40 ml of eluent. Shake it at 200 ×g for 2 h at 4 °C after PEG is dissolved. Stand it still at 4 °C overnight; the next day, eluent was centrifuged at 4,500 ×g for 45 min and the precipitate was resuspended in 0.01 M PBS and stored at −80 °C (Abd-Elmaksoud et al., 2014; Lambertini et al., 2008). The ultracentrifugation method was to add 5 ml of 30% sucrose to 30 ml of eluent, then centrifuged at 30,000 ×g and 4 °C for 2 h. The precipitation is redissolved with 0.01 M PBS and stored at −80 °C (Ammersbach & Bienzle, 2011).

Concentration of wastewaters collected from pig farms and detection of different types of viruses

Wastewater samples collected from 70 pig farms in 13 regions of Hubei Province (including Wuhan, Xiangyang, Yichang, Xiaogan, Huanggang, Xianning, Shiyan, Enshi, Jingmen, Jingzhou, Huangshi & Ezhou, Tianmen & Qianjiang & Xiantao, and Suizhou) between June 1 and December 31, 2021 were concentrated by the method developed in this study for detecting the contamination of ASFV, PRV, and PEDV. In each region, 4–6 commercial pig farms were selected. Due to the requirements of biosecurity control in pig farms, all samples were collected by farm workers and delivered to the pig farm wall, then they were carried to laboratory for primary concentration within 48 h after collection. Following this, secondary concentrations were conducted for qPCR detection of ASFV, PRV, and PEDV.

qPCR assays

Total DNAs and/or RNAs were extracted from 200 μL water samples. RNAs were transcribed into cDNA by a reverse transcription reagent kit (RRA036, Takara, Japan) immediately. The detection of viral nucleic acids was performed using qPCR (CF96X; Bio-rad, Hercules, CA, USA), following the primers (Table S1) and protocols described previously (Chen et al., 2023; Guo et al., 2016; Lin et al., 2020). The standard curve was established as follows: a slightly longer gene sequence than the target fragment was designed, usually around 300–600 bp, and a specific primer was designed for PCR amplification and Gel recovery. The recovered fragment was introduced into the pMD-19T vector in DH5α cells. After single colony identification and sequencing through PCR, successful colonies were selected for further cultivation and then the plasmid was extracted and the OD₂₆₀ was read to calculate the plasmid copy number concentration. Then, the copy number was diluted to 10⁻⁷ by 10⁻¹, 10⁻², 10⁻³, and the Ct value was detected by qPCR method for each gradient. The relationship between the Ct value and copy number was established and a standard curve was plotted.

Bacteriophage titering

Bacteriophage was titered as described previously (Zhao et al., 2022). Briefly, Salmonella phage ph2-2 was diluted with PBS (pH 7.4) from 10⁻¹, 10^-2, 10⁻³, to 10⁻⁷, a total of seven gradients or estimated gradients. Next, 300 μL of Salmonella bacterial solution was cultured for 12 h and 1 ml of diluted ph2-2 bacteriophage virus solution were mixed with 7 ml of melted 45 °C semi-solid agar medium. The mixed semi-solid medium was quickly poured into a prepared TSA agar plate and gently rotated on a laminar flow hood to distribute evenly. The agar was allowed to solidify and then incubated overnight at 37 °C. Afterwards, the transparent plaques were observed and counted.

Statistical analysis

Data were analyzed statistically using Prism software 8.0 (GraphPad, San Diego, CA, USA) and expressed as the mean ± the standard errors (SE). Comparisons among different groups were evaluated using multiple t-tests-one per row. For Figs. 1–3: *p ≤ 0.05, statistically different; **p ≤ 0.01, highly statistically different; ***p ≤ 0.001, significantly statistically different.

The influence of different factors on the enrichment of PRV, ASFV, and PEDV by glass wool

Overall, ASFV, PRV, and PEDV could be enriched by glass wool effectively at these pH values, and the highest recovery rate was observed for the enrichment of PRV, followed by ASFV and PEDV, respectively (Fig. 1A). No statistical difference was observed for the enrichment of PRV and ASFV in water samples with different pH values (Fig. 1A).

In different types of water samples, the recovery rates of PRV, ASFV, and PEDV ranged between 10.9% and 80.4% (tap water, 10.9%; water from urban inland lakes, 67.9%; water from Yangtze River, 49.5%; water from suburban rivers, 80.4%; PBS, 36.9%), 17.6% and 48.4% (tap water, 28.7%; water from urban inland lakes, 17.6%; water from Yangtze River, 43.2%; water from suburban rivers, 48.4%; PBS, 29.5%), 4.9% and 6.9% (tap water, 6.9%; water from urban inland lakes, 4.9%; water from Yangtze River, 5.9%; water from suburban rivers, 5.1%; PBS, 6.8%), respectively (Fig. 1B). Strikingly, different types of water samples posed a significant influence on the enrichment of PRV and ASFV (Figs. 1B and 1C). The viral recovery rates in water samples with volumes of 4,000, 12,000, and 20,000 ml were 30.6%, 38.9%, and 27.8% respectively for ASFV, and 59.1%, 53.0% and 45.9% respectively for PRV, as well as 10.9%, 6.2% and 9.2% respectively for PEDV. There was no difference on recovery rates of the three viruses from different water volumes (Fig. 1D).

Regarding the influence of different speeds for filtration (500, 1,000, 1,500 ml/min), no difference was observed on recovery rates of the three viruses, but the overall trend was increased slightly as the filtering speed increased (Fig. 1E). The recovery rates of PRV under above-mentioned speeds were 45%, 50.8%, and 58.9%, respectively; while those for ASFV were 13.5%, 20.2%, 21.0%, and 8.6%, 8.0%, 8.1% for PEDV, respectively. Next, we investigated the influence of different temperatures, the results revealed that although higher recovery rates were observed for the enrichment of the three viruses at 20 °C than those for the three viruses at 4 °C and 32 °C (PRV: 59.6% (20 °C) vs. 47.0% (4 °C) vs. 52.7% (32 °C); ASFV: 54.3% (20 °C) vs. 42.0% (4 °C) vs. 40.1% (32 °C); PEDV: 9.2% (20 °C) vs. 6.8% (4 °C) vs. 7.4% (32 °C), but no statistical difference was observed between the viral enrichment under different temperatures (Fig. 1F).

The efficacy of glass wool on concentrating viral particles and nucleic acids

Considering wastewater may harbor different types of virus-associated agents, including viral particles, viral nucleic acids, or viral particles plus nucleic acids released by dead viruses (marked as viral solutions), we therefore assessed the efficacy of glass wool on concentrating the above-mentioned different types of virus-associated agents. The results revealed that the recovery rates of virus solutions, viral particles, and nucleic acids in PBS of PRV were 70.8%, 55.4%, and 44.8%, respectively (Fig. 2A). For ASFV, the recovery rates for the three types of virus-associated agents in PBS were 28.3%, 24.9%, and 39.7%, respectively (Fig. 2A). However, those for PEDV were 3.9%, 3.5%, and 18.1%, respectively (Fig. 2A).

The efficacy of glass wool on concentrating viral particles and nucleic acids was also evaluated using Salmonella phage as a model. In PBS containing phage particles, the recovery rate quantified by phage titering was 8.0%, while that quantified by qPCR was 17.3% (Fig. 2B). The recovery rate yielded by glass wool concentrating live phages and that yielded by glass wool concentrating phage DNAs exhibited a statistical difference (P < 0.01). In PBS containing phage solutions, the recovery rate quantified by qPCR was 10.9%, which was lower than that of the phage particle group (17.3%, P < 0.05) (Fig. 2B).

The efficacy of different methods for secondary concentration

According to the quantify results achieved by qPCR detecting viral nucleic acids, a highest recovery rate of PRV was found in second concentration using the skimmed milk method (56%), followed by the PEG-NaCl method (30.1%) and the ultracentrifugation method (3.48%). The skimmed milk method had significantly higher concentration efficiency than the other two methods (Fig. 3A). However, the highest recovery rate of ASFV was observed in the PEG method (27.8%), followed by the skimmed milk method (15.4%) and the ultracentrifugation method (6.68%). The PEG-NaCl method also had significantly higher concentration efficiency than the other two methods (Fig. 3A). For secondary concentration of PEDV, a significantly higher recovery rate was observed in the ultracentrifugation method (20.9%) and the PEG-NaCl method (19.9%) compared to that in and the skimmed milk method (10.6%) (Fig. 3A). Salmonella phage was also used to assess the role of different methods for secondary concentration on living viruses. The results demonstrated that second concentration through both ultracentrifugation method (79.97 ± 32.27%) and PEG-NaCl method (45.85 ± 29.49%) recovered living viruses, but almost no living phages were recovered by the skimmed milk method (Fig. 3B).

Detection of ASFV, PRV, and PEDV in farm wastewater

To assess the contamination of ASFV, PRV, and PEDV in pig farm wastewater, water samples collected from 70 farms in Hubei Province were adjusted the pH values to range in 6.0–9.0, and set for primary and secondary concentrations followed by qPCR detecting the target agents. Among the 70 samples, only one sample (1.43%, 1/70) collected from a farm in Xiangyang was detected to be positive for ASFV (Table 1). The Ct value for this sample was 35.12. However, this sample was detected as a negative one before concentration. In addition, 18 samples (25.7%, 18/70) were detected to be positive for the gH gene of PRV but negative for the gE gene of PRV, suggesting PRV detected in these samples were vaccine strains. In contrast only one sample (Sample NO.66, CT value 37.4) was positive for PRV gH gene before concentration. Strikingly, all 70 samples were concentrated and detected to be negative for PEDV.