- Email: [email protected]
Use of a two-step ultrafiltration procedure to concentrate viral hemorrhagic septicemia virus (VHSV) in seawater
Journal of Virological Methods 224 (2015) 30–34
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Use of a two-step ultrafiltration procedure to concentrate viral hemorrhagic septicemia virus (VHSV) in seawater Soo-Jin Kim, Jong-Oh Kim, Wi-Sik Kim, Myung-Joo Oh ∗ Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea
a b s t r a c t Article history: Received 15 April 2015 Received in revised form 14 August 2015 Accepted 14 August 2015 Available online 20 August 2015 Keywords: VHSV Hollow-fiber ultrafiltration Centricon Seawater concentration qRT-PCR Cell culture
Viral hemorrhagic septicemia virus (VHSV) has been reported to be stable in both fresh as well as seawater, suggesting that VHSV exists in natural aquatic environments and might have an effect on the wild and cultured fish. However, VHSV is below the detectable limits of laboratory tests in natural seawater. In this study, a two-step ultrafiltration (UF) procedure was used to concentration of VHSV in seawater, providing samples that were tested for infectivity by cell culture and the presence of VHSV by quantitative reverse transcriptase PCR (qRT-PCR) methods. Overall, VHSV was approximately concentrated 100–1000 times in 1, 5 and 10 L, seawater volumes respectively: from 2.81 × 106 to 6.53 × 107 /mL and 103.3 to 103.8 TCID50 /mL prior to the UF procedure, to 3.78 × 108 , 1.16 × 1011 , and 9.12 × 1010 /mL after the procedure. This is the first report of concentrating VHSV using an UF method that was specifically designed for seawater samples. In addition, the two-step UF procedure appears to be compatible with viral cell culture and qRT-PCR methods. © 2015 Elsevier B.V. All rights reserved.
Viral hemorrhagic septicemia virus (VHSV) infection can cause mortality rates as high as 90% (Olsen, 1998; Snow et al., 1999), posing serious economic losses to the aquaculture industry (Hill, 1992). VHSV belongs to the family Rhabdoviridae, genus Novirhabdovirus. It has an extremely broad host range and has been detected in >80 teleost fish species (Schönherz et al., 2013). Several studies have reported the stability of VHSV in fresh- and seawater (Parry and Dixon, 1997; Mori et al., 2002; Hawley and Garver, 2008), suggesting that the VHSV exists in natural aquatic environments and might have a detrimental effect on the wild and cultured fish. It is essential to concentrate VHSV, since the virus is present in very low levels in the aquatic environmental conditions, and cannot be detected directly. Selecting a suitable filtration and concentration method is of utmost importance to estimate the distribution of VHSV in fresh- and seawater. Several methods have been developed to concentrate microbial pathogens in water, including ultrafiltration (UF) (Kimura and Yoshimizu, 1991; Winona et al., 2001; Morales-morales et al., 2003), polyethylene glycol precipitation (Batts and Winton, 1989; Zhang and Congleton, 1994), and adsorption-elution of viruses with an electropositive or electronegative filter (Lipp et al., 2001; Fong and Lipp, 2005). UF is a size-exclusion-based method of concentration, and is more reliable and consistent when applied to water sources than
∗ Corresponding author. Tel.: +82 61 659 7173; fax: +82 61 659 6947. E-mail address: [email protected] (M.-J. Oh). http://dx.doi.org/10.1016/j.jviromet.2015.08.006 0166-0934/© 2015 Elsevier B.V. All rights reserved.
the electropositive and electronegative filtration method, which is based on a filter-adsorbing virus (Paul et al., 1991; Winona et al., 2001; Ikner et al., 2011). UF has been used in various approaches since the 1970s for concentrating microbes (particularly viruses) in water (Belfort et al., 1974). When the viruses are smaller than the pore size, they are carried along with the fluid that passes through the membrane pores; when the pore size is smaller than the virus particles, the larger virus particles are trapped in the ultrafiltration. UF is unaffected by complex chemical constituents detected in natural water. Water samples are circulated through a column until the volume decreases to a desired level. Previous studies have demonstrated that UF is effective for simultaneously recovering viruses from water (Morales-morales et al., 2003; Hill et al., 2005). Recovery rates of concentrated viruses have also been determined previously (Katayama et al., 2002; Haramoto et al., 2007), and the recovery is associated with conventional viral isolation in cell culture and molecular methods, such as polymerase chain reaction (PCR) and quantitative reverse transcriptase PCR (qRT-PCR). Cell culture is essential for confirming an infectious virus. PCR and qRTPCR are the most widely used methods for detecting viruses due to their high sensitivity and specificity. However, they have the disadvantage of being unable to differentiate between non-infectious and infectious viruses (Fong and Lipp, 2005), whereas cell culture is optimized to detect infectious VHSV. Using the UF concentration procedure, it might be able to concentrate VHSV in seawater, and the existence of VHSV in natural water sources could be investigated without the requirement for host species. Therefore, in this
S.-J. Kim et al. / Journal of Virological Methods 224 (2015) 30–34
study, the two-step UF procedure was improved to concentrate VHSV in seawater for TCID50 and qRT-PCR analysis. The VHSV strain (FYeosu 05, genotype IVa) used in this study was isolated in 2005, from VHSV-infected olive flounder at Yeosu (Kim et al., 2009). VHSV was propagated in the fathead minnow (FHM) cell line in 75 cm2 tissue culture flasks, cultured in Dulbecco’s minimum essential medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco), 150 IU/mL penicillin G and 100 g/mL streptomycin (Gibco), maintained at 18 ± 1.0 ◦ C. The cultured VHSV was spiked into sterilized seawater using 103.3 –103.8 50% tissue culture infectious dose (TCID50 )/mL, in 1-, 5-, and 10-L seawater, respectively. The seawater samples containing VHSV were then passed through 47-mm diameter GF/F filters (Whatman, Maidstone, Kent, UK) using a vacuum pump (Gast Manufacturing, Inc., Benton Harbor, MI, USA), to remove the large particles and bacteria. Hollow fiber UF was set up as described by Oh et al. (2000), as per the diagram shown in Fig. 1. The hollow fiber ultrafilter module was polyacrylonitrile, 100,000 molecular weight cut-off (MWCO)
31
(HUF1010 PN10B, Synopex Inc., Seoul, Korea) having a surface area of 0.12 m2 , containing a gear-type pump (Iwaki, Tokyo, Japan) and valves to control transmembrane pressure and flow rate. The ultrafilter module was disinfected before and after each experiment by circulating 1.0 N NaOH for 20 min. Following this, the ultrafilter module was flushed with sterile ultrapure water and sterile seawater for 20 min. Seawater was circulated continuously through the UF. Large volumes (5- and 10-L) of seawater were sampled and reduced to 1-L. The samples were further reduced to 100 mL to analyze genome number and viral infectivity. The wastewater was randomly collected to evaluate the losses of VHSV. Secondary concentration of VHSV was achieved using Centricon plus-15 (Amicon, Mumbai, India) centrifugal ultrafilters (100,000 MWCO), which were double-loaded to concentrate the entire UF sample, as per the manufacturer’s instructions. Concentrated seawater was added to the Centricon filter and concentrated via centrifugation (3000 × g, 30 min, 4 ◦ C). The mean volume of the secondary concentrate was 0.6–1.0 mL.
Fig. 1. Schematic representation of the two-step ultrafiltration procedure. (A) The seawater sample containing VHSV was passed through a 47-mm GF/F filter using a vacuum pump, to remove the large particles and bacteria. (B) First-step UF procedure: Hollow-fiber ultrafilter module was 100,000 MWCO having surface area of 0.12 m2 , containing a gear-type pump and valves to control transmembrane pressure and flow rate. (C) Secondary-step UF procedure: Centrifugal ultrafilter module was 100,000 MWCO. Concentrated seawater (from the first UF procedure) was centrifuged 3000 × g for 30 min at 4 ◦ C.
32
S.-J. Kim et al. / Journal of Virological Methods 224 (2015) 30–34
The qRT-PCR procedure was performed as described by Kim et al. (2014). Viral RNA was extracted using RNAiso Plus (Takara Bio Inc, Shiga, Japan), following standard protocols. The dried RNA pellet was dissolved in RNase-DNase free water (Sigma-Aldrich, St.
Louis, MO, USA) and cDNA was synthesized using M-MLV Reverse Transcriptase (Bioneer, Daejeon, South Korea), as per the manufacturer’s protocol. Briefly, the mixture of extracted RNA and 10 pmol of qVN 310F primer (5 -ATCGAAGCCGGAATCCTTATGC-3 ) was incubated at 65 ◦ C for 10 min and cooled immediately on ice
Fig. 2. Concentration of VHSV using the two-step ultrafiltration procedure. VHSV detection levels generated by qRT-PCR (copy numbers, blue diamond symbols, primary axis) and TCID50 /mL (black triangle symbols, secondary axis) are shown. A, B and C represent the 1-, 5- and 10-L seawater concentrations. (a) Results of the copy numbers and infectivity before two-step UF procedure. (b) Results of the first UF procedure. (c) Results of the secondary UF procedure.
S.-J. Kim et al. / Journal of Virological Methods 224 (2015) 30–34
for cDNA synthesis. The mixture was then mixed with 4 L 5× MMLV Reverse Transcriptase reaction buffer, 2 L 100 mM DTT, 1 mM dNTP (each), 0.5 L RNase inhibitor (40 IU/L), 0.5 L M-MLV RTase (10 IU/L), and incubated at 37 ◦ C for 60 min. The qRT-PCR was carried out in an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer). The reaction conditions stated in the manufacturer’s instructions were followed. Briefly, a 10 min pre-denaturation cycle at 95 ◦ C, 40 cycles denaturation for 20 s at 95 ◦ C, and a 40 s annealing at 58 ◦ C were used. The qRT-PCR reaction specifications were analyzed by melting curve analysis, and the baseline was determined automatically by Exicycler Analysis software (Bioneer). The TCID50 of viral infectivity was determined using 96-well micro-plates seeded with FHM cells cultured at 18 ± 1.0 ◦ C for 2 weeks. The cytopathic effects (CPE) were evaluated to determine the TCID50 . The TCID50 of VHSV infectivity was estimated using the arithmetical method of Reed and Muench (1938). Seawater samples containing VHSV were initially concentrated to 150 mL by the first-step of UF procedure. The larger volumes (5 L and 10 L) of seawater were concentrated to desired levels within 4 h, and the smaller volume (1 L) was concentrated to 150 mL in 40–60 min, in this study. The second procedure using the Centricon plus-15 further concentrated the samples to 0.6–1.0 mL. Overall, the concentration rate was approximately 1:1000, 1:5000, and 1:10,000 in 1-, 5- and 10-L seawater, respectively. Concentration techniques, including UF, have been used to isolate and concentrate various parasites, bacteria, and viruses from water sources (Yoshimizu et al., 1991; Oh et al., 2000; Katayama et al., 2002; Morales-morales et al., 2003; Hill et al., 2005; Grant et al., 2011). The sampled water that has passed through a filter has increased concentrations of the target pathogens, and studies have shown that UF concentration is effective for recovering diverse waterborne microbes, such as viruses, bacteria, and parasites (Hill et al., 2005, 2007; Morales-morales et al., 2003). The two-step UF procedure used in this study was a modification of the research reported by Oh et al. (2000) for evaluating and utilizing a UF procedure to concentrate VHSV. Briefly, the first step was a hollow-fiber UF, and the second step was conducted using Centricon centrifugal UF filter. The processing resulted in a sample of desired volume for analysis, and samples were concentrated 1000, 5000 and 10,000 times from 1, 5 and 10 L of seawater, respectively. VHSV from the concentrated seawater samples was evaluated by cell culture and qRT-PCR to estimate the concentrations of VHSV in seawater. The viral genome copy numbers of VHSV and the viral infectivity by the log TCID50 /mL value and log viral copy numbers calculated by qRT-PCR are shown in Figure 2. The presence of VHSV genome and VHSV infectivity was 2.81 × 106 –6.53 × 107 /mL and 103.3 –103.8 TCID50 /mL respectively, in 1, 5 and 10 L of seawater prior to UF procedure. The copy numbers during the first UF procedure were calculated as 8.99 × 104 –2.92 × 106 (Fig. 2A), 3.19 × 106 –2.4 × 107 (Fig. 2B), and 1.78 × 107 –1.04 × 109 /mL (Fig. 2C) in 1-, 5-, and 10-L, respectively. The VHSV infectivity was shown to be 102.3 –103.6 (Fig. 2A), 102.8 – 104.8 (Fig. 2B), and 102.8 –103.8 (Fig. 2C) TCID50 /mL after the first UF procedure, in 1-, 5-, and 10L respectively. There was a slight decrease in the copy numbers and infectivity at the beginning of the UF procedure, which then increased gradually during the UF procedure. These results show that VHSV copy number and infectivity were maintained during the first UF procedure, suggesting the stability of VHSV during this process. The copy numbers after the second UF procedure were 3.78 × 108 , 1.16 × 1011 , and 9.12 × 1010/ mL in 1-, 5- and 10-L, respectively. Infectivity was 104.8 and 105.05 TCID50 /mL in 5- and 10L, respectively. The copy numbers and infectivity increased with decreasing volume of seawater. This results show that copy numbers were much higher than the VHSV titer under the conditions tested, which correlates with the VHSV density in concentrated seawater. It is also in agreement with the results of a previous study,
33
described by Kim et al. (2014). The qRT-PCR results showed slightly different copy numbers when compared to the volume of seawater. However, in this study, the VHSV concentration rate showed that, on an average, the presence of VHSV in seawater had increased 100–10,000 times, and the infectivity increased approximately100 times. This is the first report of concentrating VHSV using a UF procedure that was specifically designed for seawater samples. The lower volume used should help increase the efficacy of this detection method by allowing assays of very small volumes. In addition, the two-step UF procedure appears to be compatible with virus cell culture and qRT-PCR methods. Acknowledgement This research was a part of the project titled ‘Fish Vaccine Research Center’, funded by the Ministry of Ocean and Fisheries, Korea. References Batts, W.N., Winton, J.R., 1989. Enhanced detection of infectious hematopoietic necrosis virus and other fish viruses by pretreatment of cell monolayers with polyethylene glycol. J. Aquat. Anim. Health 1, 284–290. Belfort, G., Rotem, Y., Katzenelson, E., 1974. Virus concentration using hollow fiber membranes. Water Res. 9, 79–85. Fong, T.T., Lipp, E.K., 2005. Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools. Microbiol. Mol. Biol. Rev. 69, 357–371. Grant, A.A.M., Jakob, E., Richard, J., Garver, K.A., 2011. Concentration of infectious aquatic rhabdoviruses from freshwater and seawater using ultrafiltration. J. Aquat. Anim. Health 23, 218–223. Haramoto, E., Katayama, H., Oguma, K., Ohgaki, S., 2007. Recovery of naked viral genomes in water by virus concentration methods. J. Virol. Methods 142, 169–173. Hawley, L.M., Garver, K.A., 2008. Stability of viral hemorrhagic septicemia virus (VHSV) in freshwater and seawater at various temperatures. Dis. Aquat. Organ. 82, 171–178. Hill, V.R., Polaczyk, A.L., Hahn, D.H., Narayanan, J.K., Cromeans, T.L., Roberts, J.M., Amburgey, J.E., 2005. Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants. Appl. Environ. Microbiol. 71, 6878–6884. Hill, V.R., Kahler, A.M., Narayanan, J.K., Johnson, T.B., Hahn, D.H., Cromeans, T.L., 2007. Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-Liter tap water samples. Appl. Environ. Microbiol. 73, 4218–4225. Ikner, L.A., Soto-Beltran, M., Bright, K.R., 2011. New method using a positively charged microporous filter and ultrafiltration for concentration of viruses from tap water. Appl. Environ. Microbiol. 77, 3500–3506. Katayama, H., Shimasaki, A., Ohgaki, A., 2002. Development of a virus concentration method and its application to detection of enterovirus and Norwalk virus from coastal seawater. Appl. Environ. Microbiol. 68, 1033–1039. Kim, J.O., Kim, W.S., Kim, S.W., Han, H.J., Kim, J.W., Park, M.A., Oh, M.J., 2014. Development and application of quantitative detection method for viral hemorrhagic septicemia virus (VHSV) genogroup Iva. Viruses 6, 2204–2213. Kim, W.S., Kim, S.R., Kim, D.W., Kim, D.W., Park, M.A., Kitamura, S.I., Kim, H.Y., Kim, D.H., Han, H.J., Jung, S.J., Oh, M.J., 2009. An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed olive flounder Paralichthys olivaceus in Korea. Aquaculture 296, 165–168. Kimura, T., Yoshimizu, M., 1991. Viral diseases of fish in Japan. Annu. Rev. Fish Dis. 1, 67–82. Lipp, E.K., Lukasik, J., Rose, J.B., 2001. Human enteric viruses and parasites in the marine environment. Method. Microbiol. 30, 559–588. Morales-morales, H., Vidal, H.A.G., Olszewski, J., Rock, C.M., Dasgupta, D., Oshima, K.H., Smith, G.B., 2003. Optimization of a reusable hollow-fiber ultrafilter for simultaneous concentration of enteric bacteria, protozoa, and viruses from water. Appl. Environ. Microbiol. 69, 4098–4102. Mori, K.I., Linda, H., Nishizawa, T., Arimota, M., Nakajima, K., Muroga, K., 2002. Properties of viral hemorrhagic septicemia virus (VHSV) isolated from Japanese flounder Paralichthys olivaceus. Fish Pathol. 37, 169–174. Oh, M.J., Kim, S.R., Jung, S.J., Kim, H.R., Kim, H.Y., Yeo, I.K., 2000. A simple method for the concentration of fish pathogenic virus in seawater. J. Fish Pathol. 13, 61–66. Olsen, N.J., 1998. Sanitation of viral haemorrhagic septicaemia (VHS). J. Appl. Ichthyol. 14, 173–177. Parry, L., Dixon, P.F., 1997. Stability of nine viral haemorrhagic septicaemia virus (VHSV) isolates in seawater. Bull. Eur. Assoc. Fish. Pat. 17, 31–36. Paul, J.H., Jiang, S.C., Ross, J.B., 1991. Concentration of viruses and dissolved DNA from aquatic environment by vortex flow filtration. Appl. Environ. Microbiol. 57, 1297–2204.
34
S.-J. Kim et al. / Journal of Virological Methods 224 (2015) 30–34
Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent end points. Am. J. Hyg. 27, 493–497. Schönherz, A.A., Lorenzen, N., Katja, E.J., 2013. Inter-species transmission of viral hemorrhagic septicemia virus (VHSV) from turbot (Scophthalmus maximus) to rainbow trout (Onchorhynchus mykiss). J. Gen. Virol. 94, 869–875. Snow, M., Cunningham, C.O., Melvin, W.T., Kurath, G., 1999. Analysis of the nucleoprotein gene identifies distinct lineages of viral haemorrhagic septicaemia virus within the European marine environment. Virus Res. 63, 35–44.
Winona, L.J., Ommani, A.W., Olszewski, J., Nuzzo, J.B., Oshima, K.H.L.J., 2001. Efficient and predictable recovery of viruses from water by small scale ultrafiltration systems. Can J. Microbiol. 47, 1033–1041. Yoshimizu, M., Sami, M., Kohara, M., Yamazaki, T., Kimura, T., 1991. Detection of IHNV in hatchery water by molecular filtration method and effectiveness of U.V. irradiation of IHNV infectivity. Nippon. Suisan. Gakk. 57, 555–560. Zhang, Y., Congleton, J.L., 1994. Detection of infectious hematopoietic necrosis (IHN) virus in rearing units for Steelhead before and during IHN epizootics. J. Aquat. Anim. Health 6, 281–287.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Use of a two-step ultrafiltration procedure to concentrate viral hemorrhagic septicemia virus (VHSV) in seawater Soo-Jin Kim, Jong-Oh Kim, Wi-Sik Kim, Myung-Joo Oh ∗ Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea
a b s t r a c t Article history: Received 15 April 2015 Received in revised form 14 August 2015 Accepted 14 August 2015 Available online 20 August 2015 Keywords: VHSV Hollow-fiber ultrafiltration Centricon Seawater concentration qRT-PCR Cell culture
Viral hemorrhagic septicemia virus (VHSV) has been reported to be stable in both fresh as well as seawater, suggesting that VHSV exists in natural aquatic environments and might have an effect on the wild and cultured fish. However, VHSV is below the detectable limits of laboratory tests in natural seawater. In this study, a two-step ultrafiltration (UF) procedure was used to concentration of VHSV in seawater, providing samples that were tested for infectivity by cell culture and the presence of VHSV by quantitative reverse transcriptase PCR (qRT-PCR) methods. Overall, VHSV was approximately concentrated 100–1000 times in 1, 5 and 10 L, seawater volumes respectively: from 2.81 × 106 to 6.53 × 107 /mL and 103.3 to 103.8 TCID50 /mL prior to the UF procedure, to 3.78 × 108 , 1.16 × 1011 , and 9.12 × 1010 /mL after the procedure. This is the first report of concentrating VHSV using an UF method that was specifically designed for seawater samples. In addition, the two-step UF procedure appears to be compatible with viral cell culture and qRT-PCR methods. © 2015 Elsevier B.V. All rights reserved.
Viral hemorrhagic septicemia virus (VHSV) infection can cause mortality rates as high as 90% (Olsen, 1998; Snow et al., 1999), posing serious economic losses to the aquaculture industry (Hill, 1992). VHSV belongs to the family Rhabdoviridae, genus Novirhabdovirus. It has an extremely broad host range and has been detected in >80 teleost fish species (Schönherz et al., 2013). Several studies have reported the stability of VHSV in fresh- and seawater (Parry and Dixon, 1997; Mori et al., 2002; Hawley and Garver, 2008), suggesting that the VHSV exists in natural aquatic environments and might have a detrimental effect on the wild and cultured fish. It is essential to concentrate VHSV, since the virus is present in very low levels in the aquatic environmental conditions, and cannot be detected directly. Selecting a suitable filtration and concentration method is of utmost importance to estimate the distribution of VHSV in fresh- and seawater. Several methods have been developed to concentrate microbial pathogens in water, including ultrafiltration (UF) (Kimura and Yoshimizu, 1991; Winona et al., 2001; Morales-morales et al., 2003), polyethylene glycol precipitation (Batts and Winton, 1989; Zhang and Congleton, 1994), and adsorption-elution of viruses with an electropositive or electronegative filter (Lipp et al., 2001; Fong and Lipp, 2005). UF is a size-exclusion-based method of concentration, and is more reliable and consistent when applied to water sources than
∗ Corresponding author. Tel.: +82 61 659 7173; fax: +82 61 659 6947. E-mail address: [email protected] (M.-J. Oh). http://dx.doi.org/10.1016/j.jviromet.2015.08.006 0166-0934/© 2015 Elsevier B.V. All rights reserved.
the electropositive and electronegative filtration method, which is based on a filter-adsorbing virus (Paul et al., 1991; Winona et al., 2001; Ikner et al., 2011). UF has been used in various approaches since the 1970s for concentrating microbes (particularly viruses) in water (Belfort et al., 1974). When the viruses are smaller than the pore size, they are carried along with the fluid that passes through the membrane pores; when the pore size is smaller than the virus particles, the larger virus particles are trapped in the ultrafiltration. UF is unaffected by complex chemical constituents detected in natural water. Water samples are circulated through a column until the volume decreases to a desired level. Previous studies have demonstrated that UF is effective for simultaneously recovering viruses from water (Morales-morales et al., 2003; Hill et al., 2005). Recovery rates of concentrated viruses have also been determined previously (Katayama et al., 2002; Haramoto et al., 2007), and the recovery is associated with conventional viral isolation in cell culture and molecular methods, such as polymerase chain reaction (PCR) and quantitative reverse transcriptase PCR (qRT-PCR). Cell culture is essential for confirming an infectious virus. PCR and qRTPCR are the most widely used methods for detecting viruses due to their high sensitivity and specificity. However, they have the disadvantage of being unable to differentiate between non-infectious and infectious viruses (Fong and Lipp, 2005), whereas cell culture is optimized to detect infectious VHSV. Using the UF concentration procedure, it might be able to concentrate VHSV in seawater, and the existence of VHSV in natural water sources could be investigated without the requirement for host species. Therefore, in this
S.-J. Kim et al. / Journal of Virological Methods 224 (2015) 30–34
study, the two-step UF procedure was improved to concentrate VHSV in seawater for TCID50 and qRT-PCR analysis. The VHSV strain (FYeosu 05, genotype IVa) used in this study was isolated in 2005, from VHSV-infected olive flounder at Yeosu (Kim et al., 2009). VHSV was propagated in the fathead minnow (FHM) cell line in 75 cm2 tissue culture flasks, cultured in Dulbecco’s minimum essential medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco), 150 IU/mL penicillin G and 100 g/mL streptomycin (Gibco), maintained at 18 ± 1.0 ◦ C. The cultured VHSV was spiked into sterilized seawater using 103.3 –103.8 50% tissue culture infectious dose (TCID50 )/mL, in 1-, 5-, and 10-L seawater, respectively. The seawater samples containing VHSV were then passed through 47-mm diameter GF/F filters (Whatman, Maidstone, Kent, UK) using a vacuum pump (Gast Manufacturing, Inc., Benton Harbor, MI, USA), to remove the large particles and bacteria. Hollow fiber UF was set up as described by Oh et al. (2000), as per the diagram shown in Fig. 1. The hollow fiber ultrafilter module was polyacrylonitrile, 100,000 molecular weight cut-off (MWCO)
31
(HUF1010 PN10B, Synopex Inc., Seoul, Korea) having a surface area of 0.12 m2 , containing a gear-type pump (Iwaki, Tokyo, Japan) and valves to control transmembrane pressure and flow rate. The ultrafilter module was disinfected before and after each experiment by circulating 1.0 N NaOH for 20 min. Following this, the ultrafilter module was flushed with sterile ultrapure water and sterile seawater for 20 min. Seawater was circulated continuously through the UF. Large volumes (5- and 10-L) of seawater were sampled and reduced to 1-L. The samples were further reduced to 100 mL to analyze genome number and viral infectivity. The wastewater was randomly collected to evaluate the losses of VHSV. Secondary concentration of VHSV was achieved using Centricon plus-15 (Amicon, Mumbai, India) centrifugal ultrafilters (100,000 MWCO), which were double-loaded to concentrate the entire UF sample, as per the manufacturer’s instructions. Concentrated seawater was added to the Centricon filter and concentrated via centrifugation (3000 × g, 30 min, 4 ◦ C). The mean volume of the secondary concentrate was 0.6–1.0 mL.
Fig. 1. Schematic representation of the two-step ultrafiltration procedure. (A) The seawater sample containing VHSV was passed through a 47-mm GF/F filter using a vacuum pump, to remove the large particles and bacteria. (B) First-step UF procedure: Hollow-fiber ultrafilter module was 100,000 MWCO having surface area of 0.12 m2 , containing a gear-type pump and valves to control transmembrane pressure and flow rate. (C) Secondary-step UF procedure: Centrifugal ultrafilter module was 100,000 MWCO. Concentrated seawater (from the first UF procedure) was centrifuged 3000 × g for 30 min at 4 ◦ C.
32
S.-J. Kim et al. / Journal of Virological Methods 224 (2015) 30–34
The qRT-PCR procedure was performed as described by Kim et al. (2014). Viral RNA was extracted using RNAiso Plus (Takara Bio Inc, Shiga, Japan), following standard protocols. The dried RNA pellet was dissolved in RNase-DNase free water (Sigma-Aldrich, St.
Louis, MO, USA) and cDNA was synthesized using M-MLV Reverse Transcriptase (Bioneer, Daejeon, South Korea), as per the manufacturer’s protocol. Briefly, the mixture of extracted RNA and 10 pmol of qVN 310F primer (5 -ATCGAAGCCGGAATCCTTATGC-3 ) was incubated at 65 ◦ C for 10 min and cooled immediately on ice
Fig. 2. Concentration of VHSV using the two-step ultrafiltration procedure. VHSV detection levels generated by qRT-PCR (copy numbers, blue diamond symbols, primary axis) and TCID50 /mL (black triangle symbols, secondary axis) are shown. A, B and C represent the 1-, 5- and 10-L seawater concentrations. (a) Results of the copy numbers and infectivity before two-step UF procedure. (b) Results of the first UF procedure. (c) Results of the secondary UF procedure.
S.-J. Kim et al. / Journal of Virological Methods 224 (2015) 30–34
for cDNA synthesis. The mixture was then mixed with 4 L 5× MMLV Reverse Transcriptase reaction buffer, 2 L 100 mM DTT, 1 mM dNTP (each), 0.5 L RNase inhibitor (40 IU/L), 0.5 L M-MLV RTase (10 IU/L), and incubated at 37 ◦ C for 60 min. The qRT-PCR was carried out in an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer). The reaction conditions stated in the manufacturer’s instructions were followed. Briefly, a 10 min pre-denaturation cycle at 95 ◦ C, 40 cycles denaturation for 20 s at 95 ◦ C, and a 40 s annealing at 58 ◦ C were used. The qRT-PCR reaction specifications were analyzed by melting curve analysis, and the baseline was determined automatically by Exicycler Analysis software (Bioneer). The TCID50 of viral infectivity was determined using 96-well micro-plates seeded with FHM cells cultured at 18 ± 1.0 ◦ C for 2 weeks. The cytopathic effects (CPE) were evaluated to determine the TCID50 . The TCID50 of VHSV infectivity was estimated using the arithmetical method of Reed and Muench (1938). Seawater samples containing VHSV were initially concentrated to 150 mL by the first-step of UF procedure. The larger volumes (5 L and 10 L) of seawater were concentrated to desired levels within 4 h, and the smaller volume (1 L) was concentrated to 150 mL in 40–60 min, in this study. The second procedure using the Centricon plus-15 further concentrated the samples to 0.6–1.0 mL. Overall, the concentration rate was approximately 1:1000, 1:5000, and 1:10,000 in 1-, 5- and 10-L seawater, respectively. Concentration techniques, including UF, have been used to isolate and concentrate various parasites, bacteria, and viruses from water sources (Yoshimizu et al., 1991; Oh et al., 2000; Katayama et al., 2002; Morales-morales et al., 2003; Hill et al., 2005; Grant et al., 2011). The sampled water that has passed through a filter has increased concentrations of the target pathogens, and studies have shown that UF concentration is effective for recovering diverse waterborne microbes, such as viruses, bacteria, and parasites (Hill et al., 2005, 2007; Morales-morales et al., 2003). The two-step UF procedure used in this study was a modification of the research reported by Oh et al. (2000) for evaluating and utilizing a UF procedure to concentrate VHSV. Briefly, the first step was a hollow-fiber UF, and the second step was conducted using Centricon centrifugal UF filter. The processing resulted in a sample of desired volume for analysis, and samples were concentrated 1000, 5000 and 10,000 times from 1, 5 and 10 L of seawater, respectively. VHSV from the concentrated seawater samples was evaluated by cell culture and qRT-PCR to estimate the concentrations of VHSV in seawater. The viral genome copy numbers of VHSV and the viral infectivity by the log TCID50 /mL value and log viral copy numbers calculated by qRT-PCR are shown in Figure 2. The presence of VHSV genome and VHSV infectivity was 2.81 × 106 –6.53 × 107 /mL and 103.3 –103.8 TCID50 /mL respectively, in 1, 5 and 10 L of seawater prior to UF procedure. The copy numbers during the first UF procedure were calculated as 8.99 × 104 –2.92 × 106 (Fig. 2A), 3.19 × 106 –2.4 × 107 (Fig. 2B), and 1.78 × 107 –1.04 × 109 /mL (Fig. 2C) in 1-, 5-, and 10-L, respectively. The VHSV infectivity was shown to be 102.3 –103.6 (Fig. 2A), 102.8 – 104.8 (Fig. 2B), and 102.8 –103.8 (Fig. 2C) TCID50 /mL after the first UF procedure, in 1-, 5-, and 10L respectively. There was a slight decrease in the copy numbers and infectivity at the beginning of the UF procedure, which then increased gradually during the UF procedure. These results show that VHSV copy number and infectivity were maintained during the first UF procedure, suggesting the stability of VHSV during this process. The copy numbers after the second UF procedure were 3.78 × 108 , 1.16 × 1011 , and 9.12 × 1010/ mL in 1-, 5- and 10-L, respectively. Infectivity was 104.8 and 105.05 TCID50 /mL in 5- and 10L, respectively. The copy numbers and infectivity increased with decreasing volume of seawater. This results show that copy numbers were much higher than the VHSV titer under the conditions tested, which correlates with the VHSV density in concentrated seawater. It is also in agreement with the results of a previous study,
33
described by Kim et al. (2014). The qRT-PCR results showed slightly different copy numbers when compared to the volume of seawater. However, in this study, the VHSV concentration rate showed that, on an average, the presence of VHSV in seawater had increased 100–10,000 times, and the infectivity increased approximately100 times. This is the first report of concentrating VHSV using a UF procedure that was specifically designed for seawater samples. The lower volume used should help increase the efficacy of this detection method by allowing assays of very small volumes. In addition, the two-step UF procedure appears to be compatible with virus cell culture and qRT-PCR methods. Acknowledgement This research was a part of the project titled ‘Fish Vaccine Research Center’, funded by the Ministry of Ocean and Fisheries, Korea. References Batts, W.N., Winton, J.R., 1989. Enhanced detection of infectious hematopoietic necrosis virus and other fish viruses by pretreatment of cell monolayers with polyethylene glycol. J. Aquat. Anim. Health 1, 284–290. Belfort, G., Rotem, Y., Katzenelson, E., 1974. Virus concentration using hollow fiber membranes. Water Res. 9, 79–85. Fong, T.T., Lipp, E.K., 2005. Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools. Microbiol. Mol. Biol. Rev. 69, 357–371. Grant, A.A.M., Jakob, E., Richard, J., Garver, K.A., 2011. Concentration of infectious aquatic rhabdoviruses from freshwater and seawater using ultrafiltration. J. Aquat. Anim. Health 23, 218–223. Haramoto, E., Katayama, H., Oguma, K., Ohgaki, S., 2007. Recovery of naked viral genomes in water by virus concentration methods. J. Virol. Methods 142, 169–173. Hawley, L.M., Garver, K.A., 2008. Stability of viral hemorrhagic septicemia virus (VHSV) in freshwater and seawater at various temperatures. Dis. Aquat. Organ. 82, 171–178. Hill, V.R., Polaczyk, A.L., Hahn, D.H., Narayanan, J.K., Cromeans, T.L., Roberts, J.M., Amburgey, J.E., 2005. Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants. Appl. Environ. Microbiol. 71, 6878–6884. Hill, V.R., Kahler, A.M., Narayanan, J.K., Johnson, T.B., Hahn, D.H., Cromeans, T.L., 2007. Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-Liter tap water samples. Appl. Environ. Microbiol. 73, 4218–4225. Ikner, L.A., Soto-Beltran, M., Bright, K.R., 2011. New method using a positively charged microporous filter and ultrafiltration for concentration of viruses from tap water. Appl. Environ. Microbiol. 77, 3500–3506. Katayama, H., Shimasaki, A., Ohgaki, A., 2002. Development of a virus concentration method and its application to detection of enterovirus and Norwalk virus from coastal seawater. Appl. Environ. Microbiol. 68, 1033–1039. Kim, J.O., Kim, W.S., Kim, S.W., Han, H.J., Kim, J.W., Park, M.A., Oh, M.J., 2014. Development and application of quantitative detection method for viral hemorrhagic septicemia virus (VHSV) genogroup Iva. Viruses 6, 2204–2213. Kim, W.S., Kim, S.R., Kim, D.W., Kim, D.W., Park, M.A., Kitamura, S.I., Kim, H.Y., Kim, D.H., Han, H.J., Jung, S.J., Oh, M.J., 2009. An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed olive flounder Paralichthys olivaceus in Korea. Aquaculture 296, 165–168. Kimura, T., Yoshimizu, M., 1991. Viral diseases of fish in Japan. Annu. Rev. Fish Dis. 1, 67–82. Lipp, E.K., Lukasik, J., Rose, J.B., 2001. Human enteric viruses and parasites in the marine environment. Method. Microbiol. 30, 559–588. Morales-morales, H., Vidal, H.A.G., Olszewski, J., Rock, C.M., Dasgupta, D., Oshima, K.H., Smith, G.B., 2003. Optimization of a reusable hollow-fiber ultrafilter for simultaneous concentration of enteric bacteria, protozoa, and viruses from water. Appl. Environ. Microbiol. 69, 4098–4102. Mori, K.I., Linda, H., Nishizawa, T., Arimota, M., Nakajima, K., Muroga, K., 2002. Properties of viral hemorrhagic septicemia virus (VHSV) isolated from Japanese flounder Paralichthys olivaceus. Fish Pathol. 37, 169–174. Oh, M.J., Kim, S.R., Jung, S.J., Kim, H.R., Kim, H.Y., Yeo, I.K., 2000. A simple method for the concentration of fish pathogenic virus in seawater. J. Fish Pathol. 13, 61–66. Olsen, N.J., 1998. Sanitation of viral haemorrhagic septicaemia (VHS). J. Appl. Ichthyol. 14, 173–177. Parry, L., Dixon, P.F., 1997. Stability of nine viral haemorrhagic septicaemia virus (VHSV) isolates in seawater. Bull. Eur. Assoc. Fish. Pat. 17, 31–36. Paul, J.H., Jiang, S.C., Ross, J.B., 1991. Concentration of viruses and dissolved DNA from aquatic environment by vortex flow filtration. Appl. Environ. Microbiol. 57, 1297–2204.
34
S.-J. Kim et al. / Journal of Virological Methods 224 (2015) 30–34
Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent end points. Am. J. Hyg. 27, 493–497. Schönherz, A.A., Lorenzen, N., Katja, E.J., 2013. Inter-species transmission of viral hemorrhagic septicemia virus (VHSV) from turbot (Scophthalmus maximus) to rainbow trout (Onchorhynchus mykiss). J. Gen. Virol. 94, 869–875. Snow, M., Cunningham, C.O., Melvin, W.T., Kurath, G., 1999. Analysis of the nucleoprotein gene identifies distinct lineages of viral haemorrhagic septicaemia virus within the European marine environment. Virus Res. 63, 35–44.
Winona, L.J., Ommani, A.W., Olszewski, J., Nuzzo, J.B., Oshima, K.H.L.J., 2001. Efficient and predictable recovery of viruses from water by small scale ultrafiltration systems. Can J. Microbiol. 47, 1033–1041. Yoshimizu, M., Sami, M., Kohara, M., Yamazaki, T., Kimura, T., 1991. Detection of IHNV in hatchery water by molecular filtration method and effectiveness of U.V. irradiation of IHNV infectivity. Nippon. Suisan. Gakk. 57, 555–560. Zhang, Y., Congleton, J.L., 1994. Detection of infectious hematopoietic necrosis (IHN) virus in rearing units for Steelhead before and during IHN epizootics. J. Aquat. Anim. Health 6, 281–287.