1
Detection and quantification of infectious avian influenza A (H5N1) 1
virus in environmental water using real-time RT-PCR 2
3
4
C. I. Dovas1*
, M. Papanastassopoulou1, M. P. Georgiadis
2, E. Chatzinasiou
1, V. I. 5
Maliogka1,3
, G. K. Georgiades4 6
7
Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, Aristotle 8
University of Thessaloniki, 54124 Thessaloniki, Greece1; Laboratory of Animal Production 9
Economics, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 10
Thessaloniki, Greece2; Plant Pathology laboratory, School of Agriculture, Aristotle University of 11
Thessaloniki, 54124 Thessaloniki, Greece3; and Ministry of Rural Development and Food, CTVI 12
/ IIPD, Department of Avian Diseases, 26th October Street 80 GR-54627 Thessaloniki, Greece4 13
14
15
* Corresponding author: Tel.: +302310999870; fax: +302310999959. E-mail address: 16
[email protected] (C. I. Dovas). 17
18
19
Abstract 20
21
Routes of avian influenza virus (AIV) dispersal among aquatic birds involve 22
direct (bird-to-bird) and indirect (water-borne) transmission. The environmental 23
persistence of H5N1 in natural water reservoirs can be assessed by virus isolation in 24
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01929-09 AEM Accepts, published online ahead of print on 29 January 2010
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embryonated chicken eggs. Here we describe the development and evaluation of a real-1
time quantitative RT-PCR (qRT-PCR) for the detection of H5N1 AIV in environmental 2
water. It is based on virus particle adsorption onto formalin-fixed erythrocytes followed 3
by qRT-PCR detection. The number of hemagglutinin RNA copies detected from 4
erythrocyte-adsorbed H5N1 HPAIV particles correlated highly with the infectious dose 5
of the virus that was determined in three different types of artificially inoculated 6
environmental water over a 17-day incubation period. The advantages of the method 7
include detection and quantification of infectious H5N1 AIVs with high sensitivity, wide 8
dynamic range and reproducibility, as well as increased biosecurity. The lowest 9
concentration of H5N1 virus that could be reproducibly detected was 0.91 of the 50% egg 10
infectious dose per ml. In addition, a virus with high virion stability (Tobacco mosaic 11
virus) was used as an internal control to accurately monitor the efficiency of RNA 12
purification, cDNA synthesis and PCR amplification for each individual sample. This 13
detection system could be useful for rapid high-throughput monitoring of the presence of 14
H5N1 AIVs in environmental water and in studies designed to explore their viability and 15
epidemiology in different waterfowl ecosystems. The proposed method may also be 16
adapted for the detection of other AIVs and to the assessment of their prevalence and 17
distribution in environmental reservoirs. 18
19
Keywords: formalin-fixed erythrocytes, H5N1 HPAIV, real-time RT-PCR, 20
environmental water. 21
22
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INTRODUCTION 1
2
Wild aquatic birds of the orders Anseriformes (ducks, geese, swans) and 3
Charadriiformes (gulls, terns, shore birds) are the natural reservoirs of avian influenza 4
viruses (AIVs) and play an important role in their ecology and propagation (30, 37). The 5
epidemiology of AIVs is best understood in ducks where predictable temporal and spatial 6
patterns of infection have been reported (29). Most AIVs replicate preferentially in the 7
gastrointestinal tract of ducks, are excreted at high concentrations in feces, and are 8
transmitted via the fecal-oral route, sometimes involving contaminated water on shared 9
aquatic habitats (11). 10
The transmission and persistence of AIVs in wild bird populations remain poorly 11
understood because they depend both on the ecology of the host (e.g. population density, 12
migration patterns) and on the environment (e.g. water characteristics may influence AIV 13
persistence). Routes of AIV dispersal among aquatic birds involve direct (bird-to-bird) 14
and indirect (water-borne) transmission (24). Indeed, the viruses can be transmitted 15
without the occurrence of direct contact between birds, and this can provide a natural 16
mechanism to maintain influenza viruses in avian species (31, 32, 14, 4, 5). This water-17
borne indirect transmission may play an important role in the epidemiology of these 18
infections or even be the main determinant of the disease dynamics in wild birds 19
highlighting the potential importance of the persistence of viral particles in water (26, 20
24). However, the importance of water-borne transmission in natural ecosystems is not 21
adequately evaluated, especially in the case of the emerged H5N1 highly pathogenic 22
avian influenza viruses (HPAIVs). In contrast to wild-type AIVs, the H5N1 HPAIVs are 23
shed at greater titers and for longer durations from the trachea and upper respiratory tract 24
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than by the cloacal route (34, 2). Although there is a possibility that H5N1 respiratory 1
infections do not result in the same environmental contamination levels as fecal shedding, 2
this factor may be offset by the possibility that transmission of H5N1 may require a low 3
infectious dose (3). 4
Highly pathogenic H5N1 viruses have been responsible for outbreaks in poultry 5
over the last 13 years, providing the longest recorded event of HPAI circulation in 6
poultry. Due to its continuous circulation over this period, this virus lineage has 7
undergone extensive genotype alterations, through reassortment, to produce numerous 8
H5N1 genotypes that evolved into 10 distinct H5 hemagglutinin lineages (World Health 9
Organization/World Organization for Animal Health/Food and Agriculture Organization 10
H5N1 Evolution Working Group, 2008). The ecological success of this virus in many 11
avian species allowed its worldwide geographical expansion, which necessitated an 12
intense virological monitoring in wild bird populations (12, 19, 8, 20, 33). Although 13
H5N1 AIVs have the ability to persist for extended periods of time in water in a wide 14
range of temperature and salinity levels (4), the impact of environmental contamination 15
on the transmission between birds and the persistence of the infection in aquatic habitats, 16
are unknown. 17
Concentrations of influenza viruses excreted by infected birds in natural water 18
reservoirs are often below the detection threshold of most commonly used diagnostic 19
methods. The most sensitive detection methods are virus isolation in embryonated 20
specific-pathogen-free (SPF) chicken eggs and RT-PCR (15). Isolation in SPF eggs is 21
highly sensitive in detecting infectious virus particles. However, this method is quite 22
labor- and time-consuming and, especially in the case of H5N1, it requires the 23
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maintenance of a high biosecurity level in order to prevent exposure of humans to the 1
virus, because it involves replication of the virus in high titers. On the other hand, real-2
time RT-PCR is a sensitive and reliable method for the detection and quantification of 3
AIVs, which does not involve replication of infectious virus. However, the degree of 4
correlation between the number of virus RNA copies detected by RT-PCR and virus 5
infectivity in the respective samples has not been reported. 6
In this study we describe the development of a novel real-time quantitative assay 7
for the H5 hemagglutinin (HA) gene, which is fully controlled in order to monitor the 8
efficiency of RNA purification, cDNA synthesis, PCR amplification and signal detection 9
for each individual sample. The described method can detect the virus in large quantities 10
of water and can quantify infectious viruses. The methodology is based on virus 11
adsorption onto formalin-fixed erythrocytes followed by detection and quantification by 12
real-time RT-PCR. The main advantage of this method is its high sensitivity in detecting 13
infectious virus, while involving smaller risks to the operators as no replication of the 14
virus is involved. 15
16
17
MATERIALS AND METHODS 18
19
H5N1 HPAIV isolates, virus propagation and titration. Fourteen H5N1 HPAIV 20
isolates were used in the described experiments. All isolates had been recovered from 21
affected birds during the virus incursion in Greece, in February and March 2006. Two 22
isolates, A/red-breasted goose/Greece/167/06 from Skyros Island and A/mute 23
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swan/Greece/436/06 from the Veria region, were propagated in 10 days-old SPF 1
embryonated chicken eggs (ECEs). Infective amnio-allantoic fluid (AAF) was harvested 2
at 96 h post inoculation (PI) and titrated as the 50% egg infectious dose (EID50)/ml using 3
ECEs (36), and as the 50% tissue culture infectious dose (TCID50)/ml using cell 4
suspensions prepared as primary cultures of chicken embryo fibroblasts (CEFs) (4). The 5
virus titer was calculated by the Reed-Muench method (22). Twelve isolates (188/06, 6
222/06, 230/06, 262/06, 266/06, 288/06, 346/06, 350/06, 432/06, 435/06, 492/06 and 7
391/06) recovered from 11 swans and a cormorant respectively, were propagated in ECEs 8
and the AAF was tested using the real-time RT-PCR described below. All experiments 9
subject to handling of material containing infectious HPAIV were conducted in biosafety 10
level 3 (BSL-3) facilities at the Faculty of Veterinary Medicine, Aristotle University of 11
Thessaloniki. 12
Adsorption of water-diluted AIV to formalin-fixed chicken erythrocytes. Twenty 13
four µl from a 50 mM stock solution of a neuraminidase inhibitor (4-guanidino-14
Neu5Ac2en) and 200 µl from a 10% (v/v) suspension of formalin-fixed chicken red blood 15
cells (CRBCs) were added to 50ml of either environmental or sterile double distilled 16
water (ddw) inoculated with infective AAF. The 4-guanidino-Neu5Ac2en stock solution 17
was prepared from RelenzaTM
(GlaxoSmithKline, Evreux, France) Rotadisks (5 mg of 18
zanamivir with lactose) by dissolving 5-mg blister capsules in 285 µl of PBS each and 19
was stored at -20 °C. The CRBCs were formalin-fixed as previously described (15). After 20
fixation, the erythrocyte solution was washed 8 times and could be stored up to 6 months 21
at 4°C in PBS at 10% (v/v) suspension containing antibiotics and antimycotics to the 22
following final concentrations: 200 units/ml Penicillin G, 200 µg/ml Streptomycin sulfate 23
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and 0.5 µg/ml Amphotericin. The tubes containing AIV inoculated water with CRBCs 1
were constantly mixed at room temperature using a rotator at 2 rpm for 90 min to allow 2
virus adsorption to CRBCs. The CRBCs with the adsorbed virus were pelleted by 3
centrifugation (20 min at 1800 g). The pellet was resuspended with 1.5 ml PBS, 4
transferred in a 2 ml tube and pelleted again by centrifugation (3 min at 6000 g). The 5
CRBCs were washed again with 1.5 ml PBS in the same tube and finally the pellet was 6
resuspended in ddw to a final volume of 140 µl. RNA extraction and qRT-PCR followed 7
as described below. 8
Preparation of H5 HA RNA standard and TMV internal control. Generation of 9
in vitro-transcribed H5 RNA was prepared as previously described (23). Briefly, a 557bp 10
HA amplicon was produced by RT-PCR using RNA extracted from A/mute 11
swan/Greece/436/06 and the primers H5-F1157 (GGGAGTGGGTACGCTGCAGA) and 12
H5-R1692 (Table 1). The amplicon was cloned into the pGEM®-T Easy Vector System I 13
(Promega, Mannheim, Germany). In vitro transcription reactions were performed using 14
1µg linearized plasmid and T7 RNA Polymerase (MBI Fermentas, St. Leon-Rot, 15
Germany). The mixture was treated with 5U RNase-free DNase I (Ambion, Austin, 16
USA). In vitro transcripts were recovered by phenol/chloroform extraction and ethanol 17
precipitation and quantified by spectrophotometry. RNA was also extracted from 18
allantoic fluid containing each of the two (A/red-brested-goose/Greece/167/06 and 19
A/mute swan/Greece/436/06) viruses with known virus titer. Dilutions of these RNAs 20
along with titrated virus stocks from allantoic fluid were used to determine the 21
amplification efficiency, variability, dynamic range of quantification and detection limit 22
of the qRT-PCR assays. TMV was purified from infected Nicotiana tabacum plants by 23
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two cycles of differential centrifugation and precipitation with PEG using the method of 1
the Association of Applied Biologists for the purification of Tomato mosaic virus 2
(http://www.dpvweb.net/dpv/showdpv.php?dpvno=156) based on Gooding and Hebert 3
(10). The RNA titer in the purified TMV suspension was estimated by Poisson endpoint 4
dilution analysis (25) using the optimized TMV specific real-time RT-PCR described 5
below. The suspension was diluted with 10 mM phosphate buffer (pH 7.4) to obtain a 6
solution of approximately 1.5x106 TMV RNA copies/µl and stored in aliquots at -70 ºC. 7
RNA extraction. The QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) was 8
used to extract total RNA directly from 140 µl virus-infected water samples or from 9
CRBCs with adsorbed virus particles. In the latter case, the 140 µl of resuspended CRBC 10
mixture were directly added to AVL-carrier RNA buffer, incubated for 10 min at room 11
temperature and additionally clarified by centrifugation at 12,000 g for 3 min. 12
Subsequently the manufacturer’s protocol was followed, modified by the addition of a 13
second wash with 300 µl AW2 buffer. RNA was eluted in 60 µl of AVE buffer. In order 14
to minimize the risk of generating false negative and falsely low quantitative results, 15
every sample was spiked with TMV particles. Every sample had 0.2 µl of TMV purified 16
particles added to the AVL buffer at the extraction stage, equivalent to approximately 17
3x105 copies of TMV RNA. 18
Design of primers and hydrolysis probes. All available different genotypes of 19
complete HA nucleotide sequences from H5N1 isolates since 2005 were downloaded 20
from the NCBI Influenza Virus Resource Database 21
(http://www.ncbi.nlm.nih.gov/genomes/FLU) and aligned (clustal X, version 2.0). The 22
final data set was comprised of 343 European/African and 697 Asian HA sequences. An 23
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influenza virus H5 gene-specific PCR primer set and a hydrolysis probe were designed 1
from conserved genomic regions tolerating one or two degenerated sites per 2
oligonucleotide in order to accommodate the variability revealed by the alignment. The 3
primers and probe were designed so that each oligonucleotide shared over 96% identity 4
with the targeted HA sequences allowing only for one base mismatch with a small 5
percentage of them. In some cases where two mismatches had to be tolerated, these were 6
located in the 5′-terminus of the oligonucleotide. The successful binding of the primers 7
and probe in the cases of target mismatches was assessed by calculating the base 8
mismatch DNA thermodynamics using the OligoAnalyzer 3.1 software 9
(http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/) developed by IDT 10
(Integrated DNA Technologies, Coralville, IA), and the DINAMelt web server 11
(http://dinamelt.bioinfo.rpi.edu/refs.php) (18). The sequences and details of primers and 12
probes are listed in Table 1. Similarly, two specific primers and a TaqMan probe were 13
designed from highly conserved regions of the RNA dependent RNA polymerase (RdRp) 14
gene of the Tobacco mosaic virus, used in the assay as an internal control (TMV, Acc. 15
No. AF165190). The H5N1 and TMV specific TaqMan probes were 5′ labeled with FAM 16
(6-carboxyfluoresin) and HEX (hexachloro-carbonylfluorescein) reporter dyes 17
respectively, and 3′ labeled with the quenching dye BHQ-1. Influenza Matrix-specific 18
primers (Table 1) were designed using a data set from 611 full length non-identical 19
matrix sequences from all available environmental and animal isolates, retrieved from the 20
Influenza Virus Resource at the National Center for Biotechnology Information 21
(http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). 22
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Quantitative real-time RT-PCR (qRT-PCR). The reverse transcription (RT) 1
reaction mixture contained 1Χ first strand buffer, 0.01 M DTT, 0.5 mM of each dNTP, 2
1.4 µM random hexamers, 50 units M-MLV reverse transcriptase, 13 units RNase 3
Inhibitor, 12 µl of sample RNA and RNase free water up to a final reaction volume of 20 4
µl (all reagents from Invitrogen, The Netherlands). The reaction mixture was then 5
incubated with the following thermal conditions: 25 ºC for 10 min, 37 ºC for 40 min, 42 6
ºC for 10 min, 95 ºC for 10 min and then cooled down to 4 ºC. Five µl of cDNA was 7
added to a separate real-time qPCR 50 µl-reaction mixture. The PCR reactions were 8
optimized for standard cycling conditions with an initial denaturation step at 95 ºC (3 9
min), followed by 45 cycles of denaturation at 95 ºC (30 s) and annealing at 60 ºC (1 min) 10
using the Mx3005P QPCR system (Stratagene Co., La Jolla, CA). Optimal multiplex 11
reaction conditions for qPCR were determined as follows; 2.5 units of Platinum® Taq 12
DNA polymerase (Invitrogen, The Netherlands), 5 µl PCR buffer (10Χ), 400 µM each 13
dATP, dCTP, dGTP, and dTTP, 6 mM MgCl2, the H5-HA and TMV specific primers and 14
probes in concentrations described in Table 1, and nuclease free water up to 50 µl. For 15
each dye, the analysis of fluorescence data was conducted using the MxPro-Mx3005P 16
software (Version 4.00; Stratagene, La Jolla, CA), the option for baseline correction was 17
an adaptive baseline and the threshold fluorescence levels used to derive threshold cycle 18
(Ct) values were determined automatically. The TMV Ct value for each sample was 19
required to lie within two standard deviations of the mean of the TMV Ct values. 20
Samples with a TMV Ct value that deviated from the mean by more than two standard 21
deviations were retested. To generate a standard curve for the correlation of Ct values 22
with virus infectivity and evaluate inter- and intra-assay variability of the qRT-PCR 23
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employed, different log dilutions of a known titer (TCID50/ml and ECD50/ml) of H5N1 1
virus in ddw were prepared and tested with qRT-PCR. RNA was extracted either directly 2
from the inoculated water or from H5N1 AIV particles after adsorption to CRBCs. 3
The specificity of H5-HA specific primers and probe in the multiplex real-time RT-4
PCR assay was additionally evaluated by testing RNA extracts of isolates from different 5
influenza subtypes (Table 2). An Influenza A Matrix-specific specific RT-PCR assay was 6
used for verifying the PCR positivity of all influenza isolates. Herein, the matrix specific 7
primers and probe were used in concentrations described in Table 1 by applying the same 8
cycling and reaction conditions described for the multiplex real-time PCR. 9
Sequencing. To confirm the specificity of the amplification, PCR products were 10
purified and sequenced using a Big Dye Terminator v3.0 Ready Reaction Cycler 11
Sequencer Kit (Applied Biosystems) according to the manufacturer’s instructions. 12
Water experiment: comparison of virus titration using CEFs, and qRT-PCR. To 13
compare microtiter endpoint titration and qRT-PCR results, environmental water samples 14
were inoculated with H5N1 virus and subsequently tested in parallel using CEFs and 15
qRT-PCR assays over a period of 17 days. Samples of environmental water were 16
collected from three different areas in the Evros river Delta wetland in north-eastern 17
Greece. The collection sites were areas where waterfowl are often observed (feeding, 18
roosting, swimming, etc.) and among which there exists variation in habitats, salinity and 19
pH. The water samples were transported at 4 oC, clarified by centrifugation (5 min at 20
3000 g) and sterilized by passing through a 0.22 micron filter. All three types of water (a: 21
river water, b: lagoon water and c: stable brackish water from the bank of a flooded zone) 22
along with ddw were inoculated with A/mute swan/Greece/436/06(H5N1) AIV to a final 23
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concentration of 103.7
to 105.7
TCID50/ml. Two ml aliquots of each inoculated water type 1
were placed at 4 oC. Starting at post inoculation day (PID) 0, aliquots were titrated twice 2
a week using CEFs and qRT-PCR. Quantification of total H5 HA gene copies was 3
performed using RNA directly extracted from 140 µl of inoculated water and qRT-PCR. 4
Infectivity (TCID50/ml) was quantified with microtiter endpoint titration using CEFs 5
beginning from inoculated water diluted 1:2 with MEM. Quantification of infectious 6
virus by qRT-PCR was performed after dilution of 140 µl of inoculated water in 50 ml of 7
respective water type followed by treatment with formalin-fixed CRBCs. The 8
corresponding titers determined by qRT-PCR were multiplied by the factor of dilution 9
(357) for final comparison with the titers obtained from the undiluted samples using 10
CEFs. 11
Statistical analysis. Probit regression was used to model the relation between the 12
number of RNA copies present in the RT reaction and the production of a positive test 13
result by qRT-PCR. For this purpose, eight replicates were tested at each of the following 14
4 concentrations: 108, 72, 48 and 24 RNA copies per RT reaction. Using the resulting 15
model, we estimated the 95% detection limit of the method. Furthermore, the relationship 16
of the logarithm of titer results obtained by qRT-PCR using CRBCs and microtiter 17
endpoint titration was assessed using simple linear regression. Linear regression models 18
were also used to assess whether the decline of the logarithm of virus titers, using qRT-19
PCR with CRBCs over time, was influenced by the type of environmental water that was 20
used. Two linear regression models were fitted, one for each initial virus concentration. 21
In each model, the dependent variable was log virus titer determined by qRT-PCR with 22
CRBCs in all three environmental waters. Independent variables were time and two 23
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indicator (0-1) variables, describing the three levels of the variable “environmental water 1
used” (a: river water, b: lagoon water and c: brackish water). The model also included the 2
respective 2-way interactions, between time and the type of water variables. Therefore, 3
for each initial virus concentration a linear regression model was fitted, with the 4
following response function: 5
E{log10(virus titer)} = α + β1(time) + β2(water1) + β3(water2) + β4(water1 x time) + β5(water2 x 6
time) 7
In this model, the two indicator variables (water1) and (water2) describe the 3 levels of the 8
variable “environmental water”. Specifically, each of these two variables can take the 9
values 0 or 1. Using this scheme, “water a” could be described as: (water1) = 0 and 10
(water2) = 0, for “water b” we would have (water1) = 1 and (water2) = 0 and for “water c” 11
we would have (water1) = 0 and (water2) = 1. This way, the regression model outlined 12
above can describe 3 regression lines, one for each water: 13
For “water a”: (water1) = 0 and (water2) = 0, the response function would be: 14
E{log10(virus titer)} = α + β1(time) 15
For “water b”: (water1) = 1 and (water2) = 0, the response function would be: 16
E{log10(virus titer)} = α + β1(time) + β2 + β4(time) = (α + β2) + (β1+ β4)(time) 17
For “water c”: (water1) = 0 and (water2) = 1, the response function would be: 18
E{log10(virus titer)} = α + β1(time) + β3 + β5(time) = (α + β3) + (β1+ β5)(time) 19
By assessing the significance of appropriate regression coefficients or groups of 20
regression coefficients, we could assess whether the three lines (of virus infectivity 21
decline over time measured by qRT-PCR assay using CRBCs, in each of the three waters) 22
could be considered to be coincident, parallel or none of the two. A similar approach was 23
used to model the decline in the logarithm of virus titers both by qRT-PCR using CRBCs 24
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and microtiter endpoint titration, separately for each water and initial virus concentration 1
combination. Six multiple linear regression model were fitted, one for each combination 2
of initial virus concentration and type of water. The dependent variable in each of these 3
models was log virus titer, while the independent variables were time and type of test 4
(0=qRT-PCR with CRBCs, 1=microtiter endpoint titration). The 2-way interaction term 5
between time and test was included in the model. Based on the significance of 6
appropriate regression coefficients or groups of regression coefficients, in each of the 6 7
models we could assess whether the two lines (of virus infectivity decline over time 8
measured by each of the two assays) could be considered to be coincident or parallel, for 9
every possible combination of initial virus concentration and water. Whenever statistical 10
significance was assessed the level of significance was 0.05. Statistical analyses were 11
conducted using the software program STATA (StataCorp. 2007. Stata Statistical 12
Software: Release 10. College Station, TX: StataCorp LP). 13
14
RESULTS 15
16
Amplification efficiency, dynamic range and sensitivity of the qRT-PCR. All 17
qRT-PCR assays that were performed with serially diluted in vitro-transcribed RNA and 18
the viral RNA from the two H5N1 virus isolates gave amplification plots with the same 19
calibration curve slopes showing equal amplification efficiencies. The linear range of 20
detection was from 2 x 108 to 72 RNA copies per reverse transcription reaction, with an 21
amplification efficiency of 99.9%. A typical amplification plot of a 10-fold dilution series 22
of H5 transcribed RNA used to construct the standard curve is shown in Fig. 1a. The 23
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amplification efficiency and linearity of the standard curve in dilutions of low HA RNA 1
concentration was not affected by the presence of the TMV RNA internal control in each 2
reaction (Fig. 1). The 95% detection limit, as estimated by probit regression modeling 3
was 61.6 RNA copies per RT. When the experiment was repeated without using the TMV 4
primers and probe in the qRT-PCR, the lower detection limit of the assay was not 5
affected. The Ct values obtained from qRT-PCR, using CRBC-adsorbed H5N1 HPAIV 6
particles from 50 ml water samples, were associated with the titer in water determined as 7
TCID50/ml and EID50/ml with CEFs and ECEs respectively. A linear dynamic 8
quantification range of approximately 5Log10 (0.91-105 EID50/ml or 0.057-10
3.81 9
TCID50/ml) was achieved (Fig. 2). The lowest concentration of H5N1 virus (expressed as 10
infectious dose/ml) present in a water sample that could be reproducibly detected was 11
0.91 EID50/ml or 0.057 TCID50/ml. The estimated ratio of infectious particles (calculated 12
as EID) to HA RNA copies detected from CRBC-adsorbed AIV particles was 1:17.4. 13
Specificity. All 14 H5N1 isolates examined using the H5 specific 14
oligonucleotides were successfully detected. Amplicons derived from virus isolates 167 15
and 436 were sequenced and both sequences were confirmed as belonging to the H5 HA 16
gene revealing 100% identity with the TaqMan probe. All available full as well as partial 17
HA sequences that included the PCR targeted region were retrieved and aligned using the 18
GISAID/EpiFluDB platform (http://platform.gisaid.org/). The data sets comprised 604 19
European/African and 891 Asian H5N1 viruses isolated since 2005. Sequence 20
comparisons of the targeted regions indicated that the oligonucleotides H5-F1532, H5-21
R1692 and H5-P1646 were 100% complementary to a high percentage of 22
European/African (95.7%, 93.7% and 84.3%, respectively) and Asian (72.5%, 85.6% and 23
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81.9%, respectively) HA sequences from H5N1 AIVs circulating after 2005. The most 1
frequently encountered mismatches with the targeted sequences are indicated in Table 1. 2
The oligonucleotides H5-R1692 and H5-P1646 had only one base mismatch with the 3
sequences that were not 100% complementary. In the case of primer H5-F1532 two 4
mismatches located in the 5′-terminus were revealed with 11.1% of the Asian sequences. 5
All of them were from Indonesia and belonged to subclade 2.1.3 (World Health 6
Organization/World Organization for Animal Health/Food and Agriculture Organization 7
H5N1 Evolution Working Group, 2008). All three oligonucleotides were designed to 8
have high melting temperatures (Table 1). Calculation of base mismatch DNA 9
thermodynamics suggested that the single mismatches identified are not adequate to 10
prevent successful binding of these oligonucleotides under the annealing conditions of the 11
real-time PCR described in the materials and methods section (data not shown). 12
Comparison of sequence homology through BLAST searches (1) predicted that 13
the H5 and TMV primer/probe sets would be specific for their respective targets and 14
would not crossreact with different influenza HA subtypes or other microbial sequences. 15
For further assessment of the methods’ specificity, AIV-negative swabs (tracheal and 16
cloacal) from twenty uninfected wild birds collected in Greece during 2009 and ten 17
random samples of 50 ml environmental water treated with CRBCs were tested and all 18
gave negative results. All these samples were verified as negative by ECE virus isolation. 19
Additional evidence of specificity for the H5-HA specific primers and probe was 20
demonstrated after testing different influenza subtypes, including the H2, H1 and H6 that 21
are the closest genetically related to H5. All different subtypes, including the Novel 2009 22
influenza A (H1N1)v from a human clinical sample, didn’t give a fluorescent signal in 23
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the multiplex real-time RT-PCR (Table 1). All influenza subtypes were confirmed by the 1
matrix-specific real-time RT-PCR. 2
Reproducibility. The inter-assay coefficient of variation (CV% based on 3
calculated HA RNA concentration in copies per ml of water), was calculated from eight 4
consecutive qRT-PCR assays ran over a two week period, using ddw samples artificially 5
inoculated with H5N1 virus and stored at 4°C. The samples were extracted either directly 6
or after treatment with CRBCs. Similarly, the intra-assay coefficient of variation was 7
calculated from 6 repeats of qRT-PCR assays. The experimental intra- and inter-assay 8
variability values obtained using different virus loads are summarized in Table 3. 9
Performance of inoculated environmental water samples. To elucidate if there 10
was a correlation between the quantitative results of the qRT-PCR assays and the TC 11
titration results, a qRT-PCR assay using CRBCs, and a microtiter endpoint titration assay 12
were carried out on the same samples from three different types of environmental water 13
and ddw inoculated with H5N1. Linear regression analysis of the logarithm of TCID50 14
values for all samples tested over a period of 17 days post inoculation, measured by 15
microtiter endpoint titration and qRT-PCR assay using CRBCs, showed a significant 16
linear relation between the two sets of values: the 95% confidence interval for the beta 17
coefficient was (0.975, 1.141) and R2 was 0.935 (Fig. 3). The fact that this confidence 18
interval includes 1, shows that the respective null hypothesis of the regression coefficient 19
β=1 could not be rejected at the 5% level of significance. This test would be equivalent to 20
testing that the slope of the regression line is 45o. 21
Figure 4 depicts a substantial drop in virus infectivity over time in the 3 22
environmental waters, observed using both methods (microtiter endpoint titration and 23
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qRT-PCR using CRBCs). In contrast, no apparent decrease of total HA copies over time 1
was detected when the quantification was performed by qRT-PCR without using CRBCs. 2
In ddw, virus infectivity, as determined by any of the three testing methods, remained 3
relatively constant during the 17-day period. 4
Linear regression modeling was used in order to compare the rate of decline of 5
virus infectivity by qRT-PCR using CRBCs, over time, in the three different 6
environmental waters that were tested. For an initial virus concentration of 105.7
the 7
regression lines describing the decline in virus titers in the 3 waters could not be 8
considered either coincident or parallel. However, water (a) and water (c) could be 9
modeled by one regression line, while the regression line for water (b) would have the 10
same intercept but a different (smaller in absolute value) slope. For initial virus 11
concentration of 103.7
the regression lines describing the decline in virus titers in the 3 12
waters could not be considered either coincident or parallel. However, water (a) and 13
water (b) could be modeled by one regression line, while the regression line for water (c) 14
would have a different intercept and a different slope. 15
Linear regression analysis of the decline of log virus titers, as determined by 16
microtiter endpoint titration and qRT-PCR using CRBCs, showed that the two regression 17
lines could be considered coincident, in all 6 fitted regression models, i.e. for all 6 18
combinations of initial virus concentration and type of water. 19
The mean Ct of the TMV internal control of each real-time PCR run ranged 20
between 32.12 and 33.38 cycles for all water samples tested. The standard deviation of 21
the TMV Ct values in each real-time PCR run ranged from 0.17 to 0.35 for the samples 22
extracted without CRBCs, and from 0.18 to 0.36 for the samples containing CRBCs. A 23
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mean 0.36 cycle delay for TMV was observed for the samples that contained the 1
recommended amount of CRBCs compared to the samples without CRBCs. Student's test 2
was used for statistical comparison of the respective Ct values within each real-time PCR 3
run and showed that they were not significantly different among the two sample groups 4
(P>0.05). In an experiment where additional samples were extracted using a 4fold 5
quantity of CRBCs (0.8 ml from a 10% suspension) a mean 1.78-cycle delay of the TMV 6
internal control was observed, compared to the samples containing the recommended 7
amount of CRBCs. In this case a significant difference (P=0.001) was found in the Ct 8
values among the two sample groups indicating the presence of RT-PCR inhibitors due to 9
the higher amount of CRBCs. Approximately 4% of the samples tested using the 10
recommended amount of CRBCs exhibited a delayed TMV Ct, indicating a possible 11
inhibition of the RT-PCR. These AIV quantification results were excluded from the 12
analysis and the extraction and quantification of the sample were repeated in a subsequent 13
run. 14
15
16
DISCUSSION 17
18
Understanding the epidemiology of H5N1 is important for the prevention of 19
human, wildlife and domestic animal disease caused by the virus. Environmental 20
persistence or inactivation of the virus in the various aquatic habitats (lakes, rivers, 21
ponds) of wild bird populations may play an important role in its transmission within and 22
among populations and its maintenance in the environment over time. 23
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A simple technique for the detection and isolation of H5N1 AIV from large 1
volumes of natural water has been described recently (15). In that study, particles of a 2
nonpathogenic reverse-genetic virus carrying the HA and neuraminidase (NA) genes of a 3
H5N1 virus were isolated from water using fixed CRBCs and ECEs. In the present study, 4
we further evaluated the use of CRBC-adsorbed H5N1 HPAIV particles for quantifying 5
infectious viruses in environmental water using real-time RT-PCR. 6
Environmental water sampling may be valuable for the assessment of the 7
presence, the diversity and the dissemination of influenza viruses in defined geographical 8
locations, since it can complement bird sampling, in comparison to which it is much 9
easier and faster (16). 10
RT-PCR is a culture-independent method that has been used for the detection of 11
influenza viruses in environmental reservoirs (14, 40, 16). However, the presence of 12
infectious particles was not proven in most cases because no isolation of infectious virus 13
in ECEs was performed. An assumption was made that the amplification of influenza 14
virus sequences resulted from RNA extracted from intact virus particles because it was 15
assumed that naked RNA genomes are usually degraded rapidly in environmental waters 16
(35, 16). Although real-time PCR results have been correlated with infectious doses of 17
H5 and H7 AIVs from samples obtained from clinically affected birds (17), the different 18
rates of virus inactivation under the various aquatic environmental conditions prohibit the 19
correlation of detected RNA copies with infectious virus for each sample. This was 20
demonstrated in our experiment using environmental water artificially inoculated with 21
H5N1 virus. No correlation of the number of detected RNA copies with the virus titer 22
over the 17 day incubation period was apparent when RNA was directly extracted from 23
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the water (Fig 4). However, this was not the case when CRBCs were used. We assume 1
that CRBC adsorbed virus particles are intact and therefore the correlation of virus titer 2
with infectivity can be configured. In the case of direct RNA extraction from water, 3
particles that are defective due to degradation of the viral envelope are expected to appear 4
in the course of time. Although these particles are not infectious, their RNA is still 5
protected from degradation within the matrix protein and the core and consequently it 6
will be detected by qRT-PCR. 7
At high ambient temperatures, NA promotes influenza virus release from cells. In 8
our experiment we used zanamivir as a NA inhibitor to avoid AIV release from CRBCs 9
during the virus adsorption stage. All H5N1 adsorption experiments in this work were 10
conducted at room temperature using zanamivir. However, no difference in H5N1 11
recovery was found with incubation at 4º C when omitting zanamivir (data not shown). 12
Our results show that real-time PCR using fixed CRBCs is a feasible alternative to 13
traditional virus detection methods from water samples for several reasons. 14
Conventionally, AI virus isolation from water is performed by cultivating virus 15
containing samples in ECE, which is labor intensive and takes at least 7 days. A large 16
number of ECEs is required to obtain virus titration. That system uses ECE as a growth 17
medium and it is expected that the degree of adaptation of individual viral strains to eggs 18
and the possible presence of bacterial or dual virus contamination, or inhibitors from the 19
water can result in failure of detection or misinterpretation of the actual infectious virus 20
titer in vivo. Moreover, increased biosecurity facilities are needed if the presence of 21
H5N1 HPAIV isolates is examined since replication of virus in ECEs is involved. 22
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Compared to the traditional ECE isolation methods, the method described is rapid, 1
reduces the handling of infectious material, is not vulnerable to inhibition, can directly 2
identify and quantify H5 AI infectious virus and is expected to discriminate the H5 viral 3
subtype in cases of mixed AIV infections. Real-time PCR using fixed CRBCs is a 4
sensitive method. In the present study, it was shown that the method was able to 5
reproducibly detect H5N1 HPAIV in water at a concentration as low as 0.91 EID50/ml or 6
0.057 TCID50/ml. Considering that the titration assays with CEFs and ECEs when applied 7
directly in water samples have minimal detection limits of 15.85 TCID50 and 3.98 EID50, 8
respectively, the present qRT-PCR using CRBC-adsorbed AIV particles is 280 times 9
more sensitive than the former method and 4.37-fold more sensitive than the latter. Most 10
importantly, the number of RNA copies detected using CRBC-adsorbed H5N1 HPAIV 11
particles correlates with the virus TCID50 titre determined in three different (in terms of 12
physicochemical properties and impact on virus inactivation rate) types of environmental 13
water, which may be indicative of the possibility of infectivity and virus transmission 14
through water. It was estimated that there were approximately 17.4 HA RNA copies per 15
infectious virus particle and this, presumably reflects a ratio of defective to infectious 16
viral genomes in the mature particles. 17
Good reproducibility is an essential requirement of quantitative assays and the 18
very low inter- and intra-assay variability of this qRT-PCR (Table 3) is comparable with 19
other protocols (9, 7). The assay’s sensitivity or linearity at low concentrations of target 20
HA nucleic acid was not impaired by the simultaneous detection of the TMV internal 21
control indicating that the reaction conditions ensure that the competition for the reagents 22
in this qRT-PCR does not affect its accuracy. This was accomplished by using high 23
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concentrations of Taq DNA polymerase, dNTPs, and Mg2+
as proposed for multiplex RT-1
PCR formats (21, 6). A two-step qRT-PCR protocol (i.e., a reverse transcription reaction 2
using random hexamers, followed by a real-time PCR detection) was selected as an initial 3
approach for the quantification of infectious H5N1 adsorbed in CRBCs and proved to be 4
robust and highly sensitive. The separate RT option allows for different real-time PCR 5
protocols (i.e., matrix gene- or subtype-specific) to be applied using the same cDNAs, 6
which can be better preserved compared to RNA. However, for large scale application a 7
validated one-step qRT-PCR would be more convenient especially in case fast results for 8
a specific influenza subtype are needed. 9
Inclusion of an internal control in each sample from the lysis step onwards is very 10
important for the accuracy of qRT-PCR results because each sample is monitored for the 11
efficiency of nucleic acid purification as well as the presence of PCR inhibitors. Several 12
investigators developed and reported qRT-PCR assays using external reference spikes 13
that involved encapsidated RNAs of animal viruses (39, 9) or naked RNAs (7, 23). The 14
use of intact virions rather than purified naked RNA as internal control is preferable since 15
it can additionally monitor the efficiency of the viral lysis step itself. However, the risk of 16
infection has to be considered when human or animal pathogenic viruses are involved, 17
rendering their applicability problematic for routine analysis in most laboratories. 18
Increased stability of internal controls is an important physical property that 19
affects the long term reproducibility of qRT-PCR applications. In the presented qRT-PCR 20
assay whole TMV particles were evaluated for their use as an internal control. TMV has 21
an extraordinarily stable virion with a 85-90°C thermal inactivation point and 3000 days 22
longevity in vitro (http://www.ncbi.nlm.nih.gov/ICTVdb) and thus can provide an ideal 23
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internal control showing excellent integrity and reproducibility in qRT-PCR applications 1
over long time periods in a laboratory. Furthermore, TMV is a plant virus, non-hazardous 2
to humans or animals and easy to culture and purify in large quantities. 3
Real-time PCR has been applied in the screening of the H5 AIVs in field samples 4
and is a reliable alternative to virus isolation in ECE. More specifically, North American 5
H5 AIVs were successfully detected and quantified from swab samples by targeting 152 6
bp in the 5´ region of the HA gene (28, 17). The H5 primers and probe targeting the 152 7
bp region were modified based on Eurasian H5 sequences from isolates obtained in the 8
period from 1995 to 2005, and used successfully to detect Eurasian avian lineage H5N1 9
AIVs (13, 27). These oligonucleotide sequences used in the currently recommended real-10
time PCR protocols require continual re-evaluation as nucleotide changes accumulate 11
over time particularly in the HA gene due to the evolution of the H5N1 lineage (World 12
Health Organization/World Organization for Animal Health/Food and Agriculture 13
Organization H5N1 Evolution Working Group, 2008). In the present study, an influenza 14
virus H5 HA gene-specific PCR primer set and hydrolysis probe were designed from 15
genomic regions conserved in H5N1 AIVs isolated since 2005 and presented high 16
specificity for the amplification of subtype H5N1. The targeted genomic region (182 bp) 17
is similar to that identified previously (28) with the exception of the region targeted by 18
primer H5-R1692. However, because of the large sequence variation of the H5 HA genes, 19
the probes and primers were primarily targeted to European/African and Asian H5N1 20
AIVs. A high number of available different genotypes of H5N1 HA sequences were used 21
for the design of these oligonucleotides. In silico analysis and examination of 1495 HA 22
sequences available, suggested that the genomic regions targeted are suitable for 23
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detecting European, African and Eurasian H5N1 AIVs circulating after 2005. 1
Nevertheless, slight modification of the primer sequences depending on the evolution and 2
dispersal of diverse H5N1 viruses may be required, e.g. for some isolates belonging to 3
subclade 2.1.3, but we do not recommend any increase in their degeneracy since it would 4
affect the characteristics of the quantitative PCR. 5
In conclusion, a qRT-PCR method using CRBCs provides a feasible alternative to 6
virus isolation in ECEs. The use of erythrocytes facilitates the inexpensive and effective 7
concentration of influenza virus particles from large volumes of water. Our results 8
indicate that the method developed offers several important advantages that include 9
detection and quantification of infectious H5N1 AIVs, with high sensitivity, wide 10
dynamic range and reproducibility. In addition, a large number of water samples can be 11
processed, results can be obtained rapidly (6 hrs) and there is no requirement for BSL-3 12
facilities since no replication of infectious virus is involved. Finally, the inclusion of 13
TMV as internal control minimises the risk of generating false negative and falsely-low 14
titre results. 15
We believe that this assay represents a good system for rapid high-throughput 16
monitoring of the presence of H5 HA subtype in environmental water and could be 17
valuable in studies designed to explore the viability and epidemiology of H5N1 AI virus 18
in different waterfowl ecosystems. This methodology may also be applied to the detection 19
of other influenza subtypes and to the assessment of their prevalence and distribution in 20
environmental reservoirs. Additional applications could involve its use for fast initial 21
evaluation of decontamination protocols and health risk assessments. This could have a 22
high impact on decontamination efforts, which is an extremely timely and important topic 23
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for public health. However, in all cases, the positive results should be confirmed and 1
validated by methods that employ propagation of the virus. 2
3
ACKNOWLEDGMENTS 4
5
This work was carried out for the INN-FLU project (“Influence of viral proteins of avian 6
influenza virus on the innate immune response of birds”). This project has received 7
research funding from the European Community’s Sixth Framework Programme, project 8
number: SSPE-CT-2006-044372. The work and information presented herein reflects 9
only the views of the authors and the European Community is not liable for any use that 10
may be made of the information contained herein. 11
The authors thank Mrs. Eleni Makrigianni, from the Management Body of the Evros 12
Delta National Park, for providing the environmental water samples. They also wish to 13
thank Dr. P. Loukopoulos and Prof. O. Papadopoulos for their constructive comments on 14
the manuscript. 15
16
17
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38. World Health Organization/World Organization for Animal Health/Food and 1
Agriculture Organization H5N1 Evolution Working Group. Toward a unified 2
nomenclature system for highly pathogenic avian influenza virus (H5N1) [conference 3
summary]. Emerg Infect Dis [serial on the Internet]. 2008 Jul [date cited]. Available 4
from http://www.cdc.gov/EID/content/14/7/e1.htm 5
39. Young, N. J., C. J. Thomas, M. E. Collins, and J. Brownlie. 2006. Real-time RT-6
PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA 7
reference. J. Virol. Methods 138: 218–222. 8
40. Zhang, G., D. Shoham, D. Gilichinsky, S. Davydov, J. D. Castello, and S. O. 9
Rogers. 2006. Evidence of influenza A virus RNA in Siberian lake ice. J. Virol. 80: 10
12229–12235. 11
12
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FIGURE LEGENDS 1
2
FIG. 1. Amplification plots of H5 HA RNA and TMV detection respectively. (A) FAM 3
fluorescent signals and (B) corresponding standard curve generated from a ten-fold 4
dilution series of H5 HA RNA (the 95% confidence limits for the standard curve are 5
shown as hashed lines). From left to right, the curves represent RNA from 2 x 108 to 200 6
copies per reverse transcription reaction performed in four replicates. (C) HEX 7
fluorescent signals generated from the internal TMV control demonstrating that all 8
samples have a satisfactory TMV Ct, i.e. within two S.D. of the mean, irrespective of the 9
concentration of the HA RNA target. 10
11
FIG. 2. Analysis of the qRT-PCR linearity using CRBC-adsorbed virus particles from 50 12
ml samples of serially diluted distilled water, inoculated with an infectious dose of H5N1 13
HPAIV. The standard curve was generated from Ct values plotted against the 14
corresponding titers expressed as the 50% tissue culture infectious dose (TCID50)/ml or 15
the 50% egg infectious dose (EID50)/ml. 16
17
FIG. 3. Linear regression line of the quantified infectivity of H5N1 HPAIV, in artificially 18
inoculated water samples, as determined using chicken embryo fibroblasts (CEFs) and 19
qRT-PCR with CRBC-adsorbed virus particles In both cases titration is expressed as the 20
log 50% tissue culture infectious dose (TCID50)/ml. 21
22
FIG. 4. Monitoring of H5N1 persistence in artificially inoculated water samples over a 23
17 day period at 4 ºC; a) river water (salinity 0.95 ppt, pH 7.95), b) lagoon water (salinity 24
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43.04 ppt, pH 8.53), c) brackish water (salinity 6.19 ppt, pH 8.72) and d) double distilled 1
water. The open triangles and black circles indicate water with initial H5N1 concentration 2
of 105.7
and 103.7
TCID50/ml respectively. The dashed lines indicate log virus titers 3
determined using chicken embryo fibroblasts, the solid lines indicate log virus titers 4
determined by qRT-PCR using CRBC adsorbed virions and the dotted lines indicate total 5
log HA copies detected by qRT-PCR without using CRBCs. 6
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TABLE 1. Specific primers and probes used in real-time PCR for the detection of avian 1
influenza H5 gene, Tobacco mosaic virus (TMV), and influenza A matrix gene 2
Specificity Primer/probe
designation Sequence 5′→3′ a Location b
Concentration
in PCR
Estimated
Tm (ºC) c
H5-F1532 ACGTATGACTAYCCGCAGTATTCAGAAGA 1532 – 1560 400 nM 67.9 – 69.3
H5-P1646 FAM-ATGATTGCCAGTGCTAGRGARCTCGC-BHQ1 1671 – 1646 300 nM 69.9 – 71.5 Avian H5
H5-R1692 AACGAYCCATTGGAGCACATCC 1713 – 1692 400 nM 66.2 – 68.1
TMV-F4675 AGTTGATCTCGAAACTTGGTGCT 4675 – 4697 100 nM 65.0
TMV-P4713 HEX-TGGGAACACTTGGAGGAGTTCAGAAGGTCT-BHQ1 4713 – 4742 150 nM 71.8 TMV
TMV-R4802 AGCGTCGTCCAACTGTGTGT 4783 – 4802 100 nM 66.6
InA/M-F AGACCRATCYTGTCACCTCTGAC 170 – 192 800 nM 65.2 – 67.9
InA/M-P FAM-TCACCGTGCCCAGTGAGCGAGGACTGC-BHQ1 222 – 248 200 nM 76.1 Influenza A
matrix gene InA/M-R AGGGCATTYTGRACAAAICGTCTACG 251 – 276 800 nM 67.2 – 69.2
a IUPAC ambiguity codes. R: A or G; Y: C or T ; I: inosine. The most frequently encountered 3
mismatches with the targeted sequences are indicated in bold. 4
b Primer and probe annealing positions corresponding to the HA or the matrix gene of A/mute 5
swan/Germany/R1349/07(H5N1) with acc. nos. AM749442 and AM913994 respectively, 6
and to the TMV genome based on acc. no. AF165190. 7
c The melting temperatures were estimated using the OligoAnalyzer 3.1 software 8
(http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/) 9
10
11
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TABLE 2. Influenza virus strains examined by H5- or matrix-specific real-time 1
RT-PCR in specificity test. 2
Influenza virus strain Subtype Matrix
RT-PCR
H5
RT-PCR
A/Thessaloniki/1322/2009 v a Η1N1 + -
A/dk/Alb/35/76 b Η1N1 + -
A/dk/Germ/1215/73 b Η2N3 + -
A/tky/England/69 b
Η3N2 + -
A/dk/Czech/58 b
Η4N6 + -
A/ck/Scot/59 b Η5N1 + +
A/Northern Shoveler/Greece/892/06 c Η6N8 + -
A/Afr starling/ England /983/79 b Η7N1 + -
A/tky/Ontario/6118/68 b
Η8N4 + -
A/tky/Wisc/1/66 b
Η9N2 + -
a Novel 2009 influenza A (H1N1)v RNA from human clinical sample obtained by the 3
National Influenza Centre for North Greece, B’ Laboratory of Microbiology, School of 4
Medicine, Aristotle University of Thessaloniki, Greece. 5
b AIVs purchased by the Veterinary Laboratories Agency New Haw, Addlestone, Surrey, UK. 6
c AIV isolated by the CTVI / IIPD, Department of Avian Diseases, Thessaloniki, Greece. 7
8
9
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TABLE 3. Inter- and intra- assay variability of the qRT-PCR method presented 1
based on calculated HA RNA concentration (in copies per ml of water), with or 2
without the application of formalin-fixed chicken red blood cells (CRBCs) 3
Coefficient of variation (%)
Variation RNA
copies/ml qRT-PCR
without CRBCs
qRT-PCR
with CRBCs
Intra-assay 1 x 109
13.42 NTa
1 x 108 18.16 NT
1 x 107 17.02 18.99
1 x 106 9.27 15.67
1 x 105 16.58 30.02
1 x 104 19.52 17.38
Inter-assay 1 x 107 20.22 13.37
1 x 105 21.81 12.28
a not tested 4
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FIG. 1. 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
(C)
(A)
Y = -3.324*LOG(X) + 46.24, Eff. = 99.9%R2 = 0.999
H5 amplification plot
TMV amplification plot
H5 standard curve (B)
(C)
(A)
Y = -3.324*LOG(X) + 46.24, Eff. = 99.9%R2 = 0.999
H5 amplification plot
TMV amplification plot
H5 standard curve (B)
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FIG. 2. 1
2
3
4
5
6
7
8
9
10
11
y = -3.533x + 36.74
R2= 0.999
20
25
30
35
40
45
-2 -1 0 1 2 3 4 5
0 1 2 3 4 5 6
Log10 TCID50/ml
Log10 EID50/ml
Ct
( dR
)
y = -3.533x + 36.74
R2= 0.999
20
25
30
35
40
45
-2 -1 0 1 2 3 4 5
0 1 2 3 4 5 6
Ct
( dR
)
y = -3.533x + 36.74
R2= 0.999
20
25
30
35
40
45
-2 -1 0 1 2 3 4 5
0 1 2 3 4 5 6
Log10 TCID50/ml
Log10 EID50/ml
Ct
( dR
)
y = -3.533x + 36.74
R2= 0.999
20
25
30
35
40
45
-2 -1 0 1 2 3 4 5
0 1 2 3 4 5 6
Ct
( dR
)
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FIG. 3. 1
2
3
4
5
6
7
8
9
10
11
y = - 0.533 + 1.058x
R2 = 0.9345
0
1
2
3
4
5
6
0 1 2 3 4 5 6
Log10 TCID50/ml (CEFs)
Lo
g10
TC
ID5
0/m
l (q
RT
-PC
R) y = - 0.533 + 1.058x
R2 = 0.9345
0
1
2
3
4
5
6
0 1 2 3 4 5 6
Log10 TCID50/ml (CEFs)
Lo
g10
TC
ID5
0/m
l (q
RT
-PC
R)
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FIG. 4. 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Day
(a)
Lo
g10
TC
ID5
0/m
l
Lo
g10
RN
A c
op
ies
/ml
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
Lo
g1
0T
CID
50/m
l
Day
(b)
Lo
g10
RN
A c
op
ies
/ml
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 172
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 172
3
4
5
6
7
8
9
(c)
Lo
g10
TC
ID5
0/m
l
Lo
g10
RN
A c
op
ies
/ml
Day
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9 (d)L
og
10 T
CID
50/m
l
Lo
g10
RN
A c
op
ies
/ml
Day
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
Day
(a)
Lo
g10
TC
ID5
0/m
l
Lo
g10
RN
A c
op
ies
/ml
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
Lo
g1
0T
CID
50/m
l
Day
(b)
Lo
g10
RN
A c
op
ies
/ml
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 172
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 172
3
4
5
6
7
8
9
(c)
Lo
g10
TC
ID5
0/m
l
Lo
g10
RN
A c
op
ies
/ml
Day
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9 (d)L
og
10 T
CID
50/m
l
Lo
g10
RN
A c
op
ies
/ml
Day
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
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(C)
(A)
Y = -3.324*LOG(X) + 46.24, Eff. = 99.9%R2 = 0.999
H5 amplification plot
TMV amplification plot
H5 standard curve (B)
(C)
(A)
Y = -3.324*LOG(X) + 46.24, Eff. = 99.9%R2 = 0.999
H5 amplification plot
TMV amplification plot
H5 standard curve (B)
FIG. 1.
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y = -3.533x + 36.74
R2= 0.999
20
25
30
35
40
45
-2 -1 0 1 2 3 4 5
0 1 2 3 4 5 6
Log10 TCID50/ml
Log10 EID50/ml
Ct
( dR
)
y = -3.533x + 36.74
R2= 0.999
20
25
30
35
40
45
-2 -1 0 1 2 3 4 5
0 1 2 3 4 5 6
Ct
( dR
)
y = -3.533x + 36.74
R2= 0.999
20
25
30
35
40
45
-2 -1 0 1 2 3 4 5
0 1 2 3 4 5 6
Log10 TCID50/ml
Log10 EID50/ml
Ct
( dR
)
y = -3.533x + 36.74
R2= 0.999
20
25
30
35
40
45
-2 -1 0 1 2 3 4 5
0 1 2 3 4 5 6
Ct
( dR
)
FIG. 2.
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FIG. 3.
y = - 0.533 + 1.058x
R2 = 0.9345
0
1
2
3
4
5
6
0 1 2 3 4 5 6
Log10 TCID50/ml (CEFs)
Lo
g10
TC
ID5
0/m
l (q
RT
-PC
R) y = - 0.533 + 1.058x
R2 = 0.9345
0
1
2
3
4
5
6
0 1 2 3 4 5 6
Log10 TCID50/ml (CEFs)
Lo
g10
TC
ID5
0/m
l (q
RT
-PC
R)
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FIG. 4.
Day
(a)
Lo
g10
TC
ID5
0/m
l
Lo
g10
RN
A c
op
ies
/ml
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
Lo
g1
0T
CID
50/m
l
Day
(b)
Lo
g10
RN
A c
op
ies
/ml
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 172
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 172
3
4
5
6
7
8
9
(c)
Lo
g10
TC
ID5
0/m
l
Lo
g10
RN
A c
op
ies
/ml
Day
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9 (d)
Lo
g10 T
CID
50/m
l
Lo
g10
RN
A c
op
ies
/ml
Day
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
Day
(a)
Lo
g10
TC
ID5
0/m
l
Lo
g10
RN
A c
op
ies
/ml
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
Lo
g1
0T
CID
50/m
l
Day
(b)
Lo
g10
RN
A c
op
ies
/ml
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 172
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 172
3
4
5
6
7
8
9
(c)
Lo
g10
TC
ID5
0/m
l
Lo
g10
RN
A c
op
ies
/ml
Day
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9 (d)
Lo
g10 T
CID
50/m
l
Lo
g10
RN
A c
op
ies
/ml
Day
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
2
3
4
5
6
7
8
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