Journal of Virological Methods xxx (2014) xxx– xxx
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Journal of Virological Methods
jou rn al hom ep age: www.elsev ier .com/ locate / jv i romet
simple and efficient method for detecting avian influenza virus inater samples
ongbo Zhanga,d, Quanjiao Chena, Ze Chena,b,c,∗
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, ChinaCollege of Life Science, Hunan Normal University, Changsha 410081, Hunan, ChinaShanghai Institute of Biological Products, Shanghai 200052, ChinaInstitute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
rticle history:eceived 26 December 2012eceived in revised form 15 January 2014ccepted 21 January 2014vailable online xxx
a b s t r a c t
Waterborne transmission plays an essential role in the transmission and spread of avian influenza viruses.The abundance of influenza viruses in environmental water is usually extremely low and viruses or viralgenomes can hardly be detected by conventional reverse transcription (RT-) PCR without concentration.In the present study, an electropositive filter membrane was used to concentrate influenza viruses fromwater sample, in addition, a glass fiber filter has been used prior to positive charged membrane for the
prefiltration. Unlike the traditional adsorption–elution method, Trizol-LS reagent was used to lyse theviruses attached directly to the electropositive filter membrane and the influenza virus genomic RNA wasextracted, followed by RT-PCR analysis. The method established in this study could improve the efficiencyof the conventional RT-PCR technique used to detect the M, NP, and HA genes of influenza virus in naturalwater samples. This method could also reduce the time taken for the traditional adsorption–elution
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everse transcription PCR concentration procedure
. Introduction
Migratory birds that carry avian influenza viruses might shediruses into the environment along their migration routes. Afterirds leave an area, the environmental persistence of viruses maylay an important ecological role in virus transmission (Lang et al.,008; Brown et al., 2009). The shedding of viruses into water could
nfect any other waterfowl that visit the same area by direct orndirect fecal-oral routes (Webster et al., 1992). It is accepted that
ater is an important component of the transmission cycle of aviannfluenza viruses (Ito et al., 1995; Khalenkov et al., 2008; Zhangt al., 2011a,b). Recently, the presence of avian influenza virusesn water samples from aquatic bird habitats was described. How-ver, there have been relatively few studies on the concentrationf avian influenza viruses from natural water samples (Khalenkovt al., 2008; Dovas et al., 2010; Deboosere et al., 2011). The titerf influenza viruses in water samples associated with infectedirds is generally too low to be detected by most standard reverse
Please cite this article in press as: Zhang, H., et al., A simple and efficient mMethods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.01.013
ranscription (RT-) PCR methods. Therefore, virus concentrationrocedures are required to detect viruses present at low titer inater samples prior to RT-PCR, and several concentration methods
∗ Corresponding author at: Wuhan Institute of Virology, Chinese Academy of Sci-nces, Wuhan 430071, Hubei, China. Tel.: +86 027 87198167; fax: +86 027 87198167.
have been described previously for viruses other than influenzavirus (Gilgen et al., 1997; Katayama et al., 2002; Moce-Llivina et al.,2002; Kittigul et al., 2005; Haramoto et al., 2007). In the conven-tional methods used for the concentration of viruses from watersamples, the first step involves filtration, which is followed byfurther concentration steps, including washing, elution, ultracen-trifugation, or precipitation. In the present study, to avoid recoverylosses during the secondary concentration step, which affect theultimate sensitivity, elution and lysis steps had been combinedtogether prior to RNA extraction. Water samples from DongtingLake were inoculated with known amounts of influenza virusesand filtered through positively charged membranes, before theviruses were lysed directly on the membranes without any addi-tional steps. The extracted RNA was used for reverse transcriptionand the HA, NP, and M gene fragments of the influenza viruses weredetected by PCR using specific primers. This method may increasethe efficiency of detecting influenza viruses in water samples andno complex equipment is involved. This method also requires lesstime than other methods.
2. Materials and methods
ethod for detecting avian influenza virus in water samples. J. Virol.
2.1. Virus strains
The viruses A/duck/Huan/9/2009(H4N2) (Zhang et al., 2012),A/environment/Hunan/1-8/2007 (H5N1), A/chicken/Hunan/
/2008 (H9N2) (Zhang et al., 2011b), and A/environment/Dongtingake/Hunan/3-9/2007 (H10N8) (Zhang et al., 2011a) werenoculated into the allantoic cavities of 10-day-old specific-athogen-free (SPF) embryonated chicken eggs (Merial Vital,eijing, China) and incubated at 37 ◦C for 24–72 h. After incu-ation, the allantoic fluid was harvested and centrifuged at2,000 rpm for 10 min at 4 ◦C. The confirmed influenza virus stockas divided into aliquots and stored at −80 ◦C until use. Fifty per-
ent egg infection dose (EID50) titers of each virus were determinedn SPF eggs by 10-fold serial dilution of viruses, and endpoints
ere calculated by Reed and Munch formula (Reed and Muench,938). The titer of the virus stocks is: H4N2: 108.5EID50/ml; H5N1:09.3EID50/ml; H9N2: 1010.3EID50/ml; H10N8: 109.3EID50/ml.
.2. Concentration and enrichment of influenza viruses fromater
To develop the method for concentrating influenza viruses fromater samples, water samples were taken from Dongting Lake,hina. The Dongting Lake wetland is an important habitat and anver-wintering area for East Asian migratory birds, which is locatedt 28◦30′–30◦20′ N and 111◦40′–113◦40′ E in the Northeastern partf Hunan Province, China. The pH value of the water samples is.95. The influenza virus stock was used to produce tenfold serialilutions (104–108 EID50) and 0.1 ml of each dilution was inocu-
ated into 1 L Dongting Lake water samples. Dongting Lake waterontained large amounts of microorganisms and other impurities,o direct filtration through the electropositive membrane wouldave blocked the filter membrane. Thus, sedimentation of theater samples had been done after the addition of influenza virus.
47-mm diameter glass fiber filter with a low adsorption rate washen placed on the surface of the electropositive membrane beforeltration. Finally, the water was filtered through a glass fiber filterpore size = 1.0 �m, APFB04700, Millipore, Billerica, USA) and thelectropositive membrane (pore size = 0.45 �m, INYC00010, Milli-ore, Billerica, USA) at a flow rate of 80–100 ml/min (Fig. 1). More
nformation about the glass fiber filter could be retrieved from thisinkage: http://www.millipore.com/catalogue/item/apfb04700.
ore information about the electropositive membrane coulde retrieved from this linkage: http://www.millipore.com/atalogue/item/inyc00010.
.3. Extraction of viral RNA genomes
After the filtration, the glass fiber filter membrane containeduch micro-organism and other foreign substance, which made
he RNA extract become difficult and the quality of the extractedNA was not good enough for the sequent experiment. So the glassber filter membrane was abandoned after the filtration.
The viral RNA was directly extracted from the positivelyharged membrane as described previously with some modifica-ion (Gentry-Shields and Stewart, 2013). The positively charged
embrane adsorbed influenza viruses from the water sample. Itas taken from the filter and placed in a 60-mm diameter dish
nd covered with 2 ml Trizol LS reagent (Gaithersburg, USA) for0 min. The lysate was divided equally and transferred to two 1.5 mlNAse-free Eppendorf tubes (Axygen, New York, USA), then mixedompletely with 400 �l chloroform. After incubation for 2–3 min,he viral genomic RNA was extracted according to the followingrotocol: centrifugation at 12,000 rpm for 10 min and collection ofhe supernatant; addition of an equal volume of isopropanol andncubation for 30 min at −20 ◦C; centrifugation at 12,000 rpm for
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0 min at 4 ◦C, before discarding the supernatant, followed by theddition of 1 ml 70% ethanol; final centrifugation at 12,000 rpm for0 min at 4 ◦C and discarding the supernatant. After 10-min air-rying completely, 13 �l RNAase-free water was used to dissolve
PRESSl Methods xxx (2014) xxx– xxx
the RNA precipitates in the two 1.5 ml Eppendorf tubes, beforereverse transcription was conducted.
2.4. Reverse-transcription PCR
Reverse transcription was carried out in a reaction mixture(25 �l) that contained 5 �l of 5× reaction buffer (Promega, Madi-son, USA), 4 �l dNTP mixture (2.5 mM each of four dNTPs, Promega),1 �l M-MLV reverse transcriptase (2000 U/�l, Promega), 1 �l RNaseinhibitor (40 U/�l, Promega), 1 �l Uni12 primer, and 13 �l of RNAextract (about 100 ng). The 13 �l of RNA was the maximum tem-plate volume according to the manufacturer’s protocol.
In this study, the primers M-229f and M-299r were used todetect the M genes of four influenza virus strains. The primers NP-1200f and NP-1529r were used to detect NP genes. The primersH4-8f and H4-777r, H5-155f and H5-699r, H9-151f and H9-638r,H10-521f and H10-932r were used to detect the HA genes of H4N2,H5N1, H9N2, and H10N8 influenza viruses, respectively. All of theprimer sequences have been reported previously (Lee et al., 2001),and all of the primers used in this study were synthesized by SangonBiotech (Shanghai, China) and diluted to the 20 �M for use.
The HA genes of H5N1, H9N2, and H10N8 were detected by PCRusing the following conditions: an initial denaturation step of 3 minat 95 ◦C, followed by 35 cycles at 95 ◦C (denaturation) for 30 s, 50 ◦C(annealing) for 40 s, 72 ◦C (extension) for 40 s, and a final exten-sion step at 72 ◦C for 10 min. The annealing temperature used todetect the HA gene of the H4N2 virus was 55 ◦C but the other con-ditions were the same. The annealing temperature used to detectthe NP and M genes from each virus strain was 52 ◦C but all otherconditions were the same (Lee et al., 2001). In addition, the PCRreaction volume is 25 �l containing 5 �l cDNA, 2 �l dNTP mixture(2.5 mM each), 2.5 �l 10× PCR Buffer (Mg2+ Plus), 0.2 �l TaKaRa TaqDNA polymerase 5 U/�l (Dalian, China), 0.5 �l the upper and lowerprimers each (20 �M each) and 14.3 �l ddH2O. One tube was testedfor each PCR.
2.5. Real-time PCR
The M gene fragment of the H10N8 virus was insertedinto the pMD18-T vector (TaKaRa, Dalian, China) and thisplasmid was used to construct the standard curve. A setof primers (5′-AAGACCAATCCTGTCACCTCTGA-3′ and 5′-CAAAGCGTCTACGCTGCAGTCC-3′) designed by Karlsson et al.(2007) was used in the real-time PCR analysis. An SYBR® PremixEx TaqTM kit (TaKaRa, Dalian, China) was used for real-time PCR,according to the manufacturer’s instructions. The real-time PCRreaction system is 20 �l containing 10 �l SYBR® Premix Ex Taq (TliRNaseH Plus) (2×), 0.4 �l PCR forward primer (10 �M), 0.4 �l PCRreverse primer (10 �M), 0.4 �l ROX reference dye II (50×), 2 �lcDNA and 6.8 �l ddH2O. The real-time PCR was performed using a7900HT Real-Time PCR System (Applied Biosystems, Foster City,USA) according to the following protocol: an initial denaturationstep at 95 ◦C for 30 s, followed by 40 cycles at 95 ◦C for 5 s, and60 ◦C for 30 s, followed by dissociation stage at 60 ◦C for 1 min, and95 ◦C for 15 s. All of the samples were analyzed in triplicate foreach reaction. The data were analyzed using 7900HT System SDSVersion 2.4 (Applied Biosystems, Foster City, USA).
3. Results
3.1. Virus concentration and RT-PCR
ethod for detecting avian influenza virus in water samples. J. Virol.
The H10N8 virus was tenfold serially diluted (104–108 EID50)and added to 1 L Dongting Lake water. The water samples were thenfiltered through the glass fiber filter and positively charged mem-brane at a flow rate of 80–100 ml/min. After filtration, the virus
H. Zhang et al. / Journal of Virological Methods xxx (2014) xxx– xxx 3
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borne transmission mechanism and the disease dynamics of avianinfluenza in aquatic birds. However, there are few previous reportsof these techniques (Khalenkov et al., 2008; Dovas et al., 2010;
Table 1Recovery of avian influenza virus RNA from water.
No. of copies of viral genome %Recovery(mean ± S.D.)
Fig. 1. Method used for the concentration and detectio
enomic RNA was extracted from the positively charged mem-rane. RNA was also extracted from water samples before filtrationnd used as the control for the RT-PCR analysis (Fig. 1).
The results demonstrated that this method improved the detec-ion efficiency of influenza virus genomes substantially in naturalater (Fig. 2). In the water samples before filter concentration,
he M, NP, and HA genes of the influenza virus genomes wereardly detected by RT-PCR. By contrast, the three gene seg-ents were all detected in water samples concentrated using
he electropositive membrane, which indicated that the use oflectropositive filter membrane to concentrate influenza virusesrom water samples can increase the RT-PCR detection efficiencyreatly (Fig. 2A). The length of PCR products by the NP and
gene assays was 330 and 229 base pairs (bps), respectively,hereas that of PCR products by the HA gene assay varied between
trains (or isolates). To demonstrate that the method estab-ished in this study was effective with various influenza virusesnd to validate the generality of this method, the method wasalidated using other virus strain subtypes stored in our lab-ratory: A/duck/Huan/9/2009 (H4N2), A/environment/Hunan/1-/2007 (H5N1), and A/chicken/Hunan/2/2008 (H9N2). Similaresults were obtained using these virus strains (Fig. 2B–D).
.2. Real-time PCR
A real-time PCR analysis was performed to confirm the con-entration efficiency of the method established in this study. The
gene of the H10N8 virus was cloned into the pMD18-T vector
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nd a standard curve of the influenza virus genome copy numberas generated by absolute quantification. The optical density of thelasmid DNA (pMD18-T-M) at OD260 and 280 nm was determinedy using the Nano-Drop 2000 spectrophotometer (Wilmington,
enomic RNA from influenza viruses in water samples.
USA). The concentration of plasmid DNA was calculated by the rela-tionship: 1OD 260 unit = 50 ng/�l DNA. The length of the plasmidDNA for standard curve is 3719 base pairs (bps). According to themolecular weight of the DNA, 4.03 ng of pMD18-T-M correspond-ing to the number of copies is 109copies. The standard curve isfive points. The range in the concentrations of the plasmid used todraw a standard curve in real-time PCR is from 109 to 105 copieswith ten fold serial dilutions. The real-time PCR analysis showedthat the highest recovery efficiency of the electropositive filter toinfluenza virus genomes in Dongting lake water samples was 10.3%(Table 1).
4. Discussion
The improvement of methods for detecting avian influenzaviruses in environmental waters will help to elucidate the water-
ethod for detecting avian influenza virus in water samples. J. Virol.
4 H. Zhang et al. / Journal of Virological Methods xxx (2014) xxx– xxx
Fig. 2. The effects of influenza virus concentration on amplification using RT-PCR. The virus stock was tenfold serially diluted and 0.1 ml of the diluted virus stock was addedt positR rmed
c virus;
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o 1 L of Dongting Lake water. The water containing the virus was filtered through aNA extracted from the membranes. b10−1, b10−2, b10−3, and b10−4: RT-PCR perfoontrol. M: DNA Marker. A: inoculated with H10N8 virus; B: inoculated with H4N2
eboosere et al., 2011). The use of positively charged membranesas been shown to be more efficient than negatively charged filters,VDF filters, and other filters (Gilgen et al., 1997; Katayama et al.,002; Moce-Llivina et al., 2002; Kittigul et al., 2005; Haramoto et al.,007). The adsorption–elution method based on positively chargedembranes is a very promising approach, which was assessed in
he present study. In order to develop a method for concentratingnfluenza viruses from water samples that was taken from Dongt-ng Lake. Unlike the traditional adsorption–elution method, the
ethod used in the present study combined elution and lysed stepogether and the viral RNA was directly extracted from the posi-ively charged membrane (Gentry-Shields and Stewart, 2013). Theesults showed that this method increased the RT-PCR detectionfficiency for influenza viral genomic RNA in large volumes of waterompared with extracting the viral RNA from water samples with-ut concentration (Fig. 2). The synthesized cDNA could be used toetect multiple genes from influenza viruses. The phylogeny andrigin of the viruses could be analyzed further using sequencingechniques.
Many researchers have reported methods for concentratingiruses from water samples. However, the adsorption–elutionethod for concentrating viruses from water is considered to be
he most practical. The filter membranes used include electropos-tive membranes, electronegative membranes, PVDF membranes,
Please cite this article in press as: Zhang, H., et al., A simple and efficient mMethods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.01.013
nd cellulose nitrate membranes (Katayama et al., 2002; Moce-livina et al., 2002; Haramoto et al., 2007; Gibbons et al., 2010).owever, electropositive membranes have a high adsorption rate
ively charged membrane. c10−1, c10−2, c10−3, and c10−4: RT-PCR performed usingusing RNA extracted from water before filtering. PC: positive control. NC: negative
C: inoculated with H5N1 virus; D: inoculated with H9N2 virus.
for viruses in water samples so they are suitable for a wide pH rangeand no addition of ions to adjust the pH value is required, whichmeans that this method has many applications. In many previousstudies, the most common method used involves passing watersamples through an electropositive membrane, followed by elut-ing the virus from the membrane with a small volume of elutionbuffer and secondary concentration, generally with a precipitationor ultracentrifugation method (Gilgen et al., 1997; Katayama et al.,2002; Moce-Llivina et al., 2002). It should be noted that standardelution buffers require a relatively extreme pH value but the enve-lope of the influenza virus is sensitive to the pH value (Stallknechtet al., 1990a,b). Influenza viruses are also sensitive to temperature,so longer secondary enrichment durations yield lower numbersof influenza virus particles from water samples (Stallknecht et al.,1990a; Swayne and Beck, 2004). Furthermore, the buffer used toelute virus particles attached to filter membranes might inhibit thereverse transcription and PCR amplification (Abbaszadegan et al.,1993).
Several groups have also reported methods for detecting virusesfrom water samples or environment samples, and the most com-mon methods are RT-nested PCR and real-time PCR (Brooks et al.,2005; Kittigul et al., 2005). However, RT-nested PCR is highly sen-sitive, which can result in false positives, while real-time PCRrequires expensive equipment and reagents, as well as highly
ethod for detecting avian influenza virus in water samples. J. Virol.
experimental technology. By contrast, the method established inthe present study could detect the viral genomes of influenza virusin water samples by conventional RT-PCR. In order to evaluate the
oncentration and recovery efficiency of the established method,he real-time PCR were introduced in the present study, but it wasot a necessary step.
The method in this study was based on the characteristics of thelectropositive membrane, which can adsorb viruses from wateramples in an efficient manner. If water samples are filtered usinglectropositive membranes, the virus will be adsorbed onto the fil-er membranes. The direct extraction of viral genomic RNA fromlectropositive membranes before RT-PCR was performed insteadf a regular elution method and other secondary enrichment steps,hich reduced the detection time and simplified the procedure.
valuated by the real-time PCR, the recovery of viruses observed inhis study could be a little low (<10%). In our opinion, the reason
ight include the following three aspects: (1) the micro-organismnd other impurity that attached on the glass fiber filter membranebsorb part of the inoculated influenza virus; (2) the glass fiber fil-er membrane might absorb part inoculated virus directly, we couldot exclude this possibility, although there is no report about usinglass fiber filter membrane to concentrate virus from water sam-le; (3) the adsorption rate of the electropositive membranes to the
nfluenza virus might be low in natural water. However, the methodas still more sensitive than detecting influenza virus from water
amples without prior concentration. The method established inhis study requires simple equipment and it is widely applicable,hile it can increase the detection efficiency of influenza viruses
rom water samples. Thus, this method could be used to monitornfluenza viruses in natural water bodies. The influenza virus HAubtypes present in water could be determined rapidly using thisethod with appropriate specific primers.In conclusion, the method established in the present study is a
apid assay for avian influenza virus detection in water samples and useful approach for the effective monitoring of the epidemiologyf influenza viruses in natural water bodies. The sensitivity andfficiency of this method means it can be adapted readily to theetection of other viruses in water samples.
cknowledgements
This study was supported by the following research funds:ational 973 Project (2010CB530301); National Natural Scienceoundation of China (no. 81071346 and no. 81172738).
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