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					Concentration and detection of hepatitis A virus and rotavirus in spring water
                   samples by reverse transcription-PCR
                 Journal of Virological Methods 123 (2005) 163-169

         Julie Brassard, Karine Seyer, Alain Houde, Carol Simard, Yvon-Louis Trottier

                               Presentation by Kristen M. Castro
                                           Micro 405
                                      November 8th, 2006
                               What is
   belong to the Reoviridae family.
   Seven major groups, groups A, B,C infect humans,
   group A most common and widespread
   cause vomiting and diarrhea 4-8 days, low grade fever
   common cause of severe diarrhea in children,
   kills around 600,000 children per year
   vaccines have been shown to be safe and effective in 2006
   genome consists of 11dsRNA segments surrounded by there-layered
    icosahedral protein capsid
   cause acute gastroenteritis AKA "Infantile diarrhea", "winter diarrhea",
    "stomach flu", "acute nonbacterial infectious gastroenteritis", and
    "acute viral gastroenteritis"
    infective dose is presumed to be 10-100 infectious viral particles
   infection can be acquired through contaminated hands, objects, or
    What is Rotavirus

    Asymptomatic rotavirus is documented and may play a role in endemic
    incubation period from 1-3 days
    Temporary lactose intolerance may occur
    people recover but severe diarrhea without fluid and electrolyte
     replacement could result in death
    Childhood death from rotavirus is low in the U.S.
    100 cases/year, and reaches over 500,000 cases/year worldwide
    Association with other enteric pathogens may play a role in the
    viruses are transmitted via fecal-oral route
    usually through contaminates drinking water or food like raw shellfish,
     fresh fruit, vegetables, and ready to eat food
Why Do This Experiment?

   Rotavirus are stable under extreme conditions like pH, temperature and
   resistant to disinfectants or wastewater treatments
   contribute to their existence in the environment
   contamination of wastewater, recreational water, drinking water,
    irrigation water, ground or subsurface water, is reportedly the primary
    source for gastro-enteritis or hepatitis outbreaks
   microbiological quality of water is based on quantitative methods for
    fecal bacterial like E. coli
   these indicators cant be used to predict contamination or presence of
    enteric viruses in the water
   water treatment systems are more or less adequate in detection and
    elimination of resistant enteric viruses
   must develop a method to detect enteric viruses and detect them at a
    low concentration
Materials and Methods
Cell culture and virus

   Grown FRhK-4 and MA-104 cells in Eagles minimum essential
   Incubate cells w/o CO2 and grown to confluence
   human rotavirus strain Wa put into MA-104 cells, HAV strain HM-
    175 put into FRhK-4 cells
   cells were frozen and thawed 3 times and clarified using low speed
    centrifugation then divided into aliquots and stored at -70ºC
Materials and Methods
Viral titration

   Tissue culture infectious dose (TCID) 50% method used to determine
    the titer of stock suspension and viral dilutions
   96 well cell culture plates seeded with 2.0x10ˆ4 FRhK (HAV) per well
    and incubated for 24hrs at 37°C with 5% CO2
   cell infection performed by serial dilutions of HAV strain HM-175
    were made in EMEM w/ supplements and 2% foetal bovine serum
   well plates washed with phosphate buffer saline (PBS)
   50 uL viral dilution placed in 4 wells of a microplate w/ 175uL of
    maintenance medium
   plates incubated at 37°C w/ 5% CO2 and observed after 3-8 days
Materials and Methods
Concentration and elution

   Spring water samples were inoculated with 1.0x10ˆ3 to 1.0x10ˆ-3
    TCID50% of HAV and rotaviruses (conc. and elution step Fig 1)
   100mL of viral dilution filtered through a positively charged Zeta Plus
    60S filter to absorb virus
   to elute the virus from filter, 5mL of different eluents were used
   to the 5mL of eluate w/ viral particles, pH adjusted to 7.0-7.4, w/ 1N
   re-concentration to 150 uL with Microsep 100
   retained concentrate was used for RNA extraction
Flow Chart of Concentration and Elution
Fig 1.
Materials and Methods
Extraction of viral RNA from water concentrates

   Virus concentrate incubated at 37°C w/ 1%SDS and 100ug of
    proteinase K for 1 hour
   450uL of RLT buffer added to the concentrate and reheated at 56°C
    for 2 min, then 5 min room temp incubation
   500uL of absolute ethyl alcohol added to the concentrate and vortexed
    for 15 mins
   suspension transferred to spin column in 700uL aliquots until lysate
    was loaded
   before elution step, column was washed 2 times
   RNA eluted 2 times w/ 30 and 20 uL of sterile RNAse free water
   viral RNA concentrated with SpeedVac
   viral RNA resuspended in a final volume of 5uL and kept at -70°C
    until use
    Materials and Methods
    Viral RNA extracted from inoculated water samples and heated at
     98°C for 5 mins then chilling on ice
    2 RT-PCR systems for the detection of the virus and 1 multiples RT-
     PCR were used for analyses
    Table 1 for sequences and localization's of oligonucleotides
    RT_PCR for each amplification system performed in 20uL reaction
     mixture with 2uL of extracted RNA using the Quiagen One Step RT-
    amplification conditions used for the 226bp HAV fragment using prot.
     1 and prot. 2 primers: 30 mins @ 50°C for reverse transcription step,
     95°C for 15 mins for initial denaturation, 35 cycles for 45sec @ 94°C
     for denaturation, 45sec @ 47°C for annealing, 1 min @ 72°C for
     extension and final extension
    transcription and amplification conditions for RT-PCR were the same
     for rotavirus and multiplex HAV-rotavirus same as above, annealing
     temperatures (43°C) and final extension (5 mins) were different
    rotavirus, Rota-1 and End-9 primers were used for amplification of
     268bp fragment
Materials and Methods
RT-PCR Cont.

   UV light was used for amplified products after electrophoresis on a 2%
    agarose gel w/ EtBr
Primers and Probes Used
Materials and Methods
Southern Blot

   RT-PCR products confirmed by Southern blot hybridization using
    internal oligoneucleotide probes (table 1)
   PCR products denatured and transferred from agarose gel to positively
    charged nylon membrane
   amplified DNA was crosslinked to membrane by 3 min exposure to
    UV light
   membrane was pre hybridized for 30 mins @ 55°C in hybridization
    solution w. 5x SSC0.1% N-laurylsarcosine, 0.02% sodium dodecyl
    sulfate (SDS), and 1% protein blocking reagent
   membrane was hybridized overnight in 50 pmol of labeled
    oligoneucleotide probe per milliliter
   membrane washed twice @ room temp for 5 mins in 2x SSC w/ 0.1%
    SDS and 2 times for 15 mins @ 55°C in 0.5x SSC w/ 0.1% SDS
   membrane incubated in blocking sln
Materials and Methods
Southern Blot Cont.

   anti-DIG-peroxidase concentration of 75U/ml added to blocking sln
   membrane incubated 30 mins and washed 5 times in PBS
   positive result characterized by blue precipitate when peroxidase
    substrate was added to membrane
Titration of HAV and rotavirus

   Before inoculation, water samples viral titer of HAV HM-175 and
    rotavirus Wa stock suspensions determined by TCID 50% method
   viral titers of HAV and rotavirus were 4.0x10ˆ7 TCID50%/ml and
    1.25x10ˆ6 TCID50%/ml respectively
   titers of viral dilutions to inoculate spring water samples were
    determined and corresponded to estimated values
   titrations were performed in triplicate
Viral concentration and RNA extraction

   Adsorption of viral particles to the membrane due to electrostatic
    interactions between the viral capsid and the membrane
   larger filtration is possible and eliminate potential inhibitors of RT-
    PCR reaction increasing the detection of lower levels of viral particles
    in the water sample
   to increase yield of purified RNA of rotavirus the viral load was
    incubated prior to extraction step with 1% SDS and 100ug/ml of
    proteinase K
   due to a double capsid RNA rotavirus extraction is more difficult than
   low yields of rotavirus were observed
   there was no beneficial effects or detriments on the RNA yield for
Detection limit of RT-PCR from experiments with artificially inoculated
spring water samples

   2 RT-PCR methods were used for detection of HAV and rotavirus
   “multiplex” RT-PCR used to detect HAV and rotavirus simultaneously
   analytical sensitivity was evaluated by using known titers of HAV and
    rotavirus in artificially inoculated samples of bottled water
   compared to the “multiplex” RT-PCR, the analytical sensitivity of the
    RT-PCR performed in the single mode was found to be at least 100
    fold more sensitive for rotavirus (10ˆ-3 TCID50%/ml) and at least 10
    fold more sensitive for HAV (10ˆ-1 TCID50%/ml)
   “multiplex” RT-PCR offers a detection of both viruses from a single
    amplification step with analytical sensitivities of at least 1
    TCID50%/ml for HAV and at least 0.1 TCID50%/ml for rotaviruses
M=Molecular Ladder
N=Negative Control
P=Positive Control
M=Molecular Ladder
N=Negative Control
P=Positive Control (HAV and Rotavirus)
R=Positive Rotavirus Control
Confirmation of RT-PCR results by Southern blot hybridization

   RT-PCR amplified fragments confirmed by Southern blot
    hybridization using specific primers (table 1)
   hybridization of the digoxigenin-labeled probes matched location of
    RT-PCR amplicons on the agarose gel
    Due to viral outbreaks of HAV and rotavirus the confidence in the
     safety of the drinking water is disrupted
    bottle water per capita in the United States increased 9.4% in 1998 and
     grew by more than 50% since 1991 (source: Beverage Marketing
    increase is due to the false conception that bottled water (spring or
     treated) is pure and does not contain any micro-organisms
    pilot study in Quebec City area said 56% of consumers drink bottled
     water on a regular basis
    spring or mineral water is defined by the Health Canadas Food and
     Drug Administration “bottled water derived from an approved
     underground water source and not from a public community water
    spring water and mineral water even though treated for the removal of
     unwanted chemical and microbiological components can not be
     labeled as “natural”

   due to the packaging and distribution of bottled water that has not been
    treated for pathogenic microorganisms, there is a possibility of risk due
    to exposure from the presence of these microorganisms
   European study shows in 3 brands of bottled drinking water, detection
    of 53 noroviruses out of 159 samples tested (33 percent)
   RNA signals were detected after 1 year of storage
   it is beneficial, for the public, to make available a new test to detect
    enteric viruses in water
   quality controls are based on routine monitoring of fecal contamination
    with bacterial indicators like fecal coliforms, coliforms, and E. coli
   there is no correlation that can be established between the presence of
    bacterial indicators and the presence of enteric viruses
   due to low quantities of viral presence, methodologies must be
    designed to detect the viruses at low levels

   use of positively charged membranes have been integrated in efficient
    virus concentration systems such as noroviruses, rotaviruses, hepatitis
    A, poliovirus, and coxsackievirus
   the method in this study allows for detection of a viral particle
    (concentrate the total viral load) present in the water sample and obtain
    a detection limit of 0.001 TCID50%/ml for rotavirus and 0.1
    TCID50%/ml for HAV
   In contrast to immunocapture technology, a charged membrane enables
    the concentration of all viral particle types present in the sample
   RT-PCR in the study for detection of HAV and rotavirus gives a better
    analytical sensivity than the multiplex
   multiplex system has the advantage of detecting both viruses
    simultaneously at levels of 1 TCID50%/ml for HAV and 0.1
    TCID50%/ml for rotavirus

   multiplex approach is less expensive for detection of enteric viruses in
    large amounts of bottled water
   single RT-PCR approach is the method of choice for detection of
    single virus types in lower concentrations
   water samples can be processed in 8 hours from concentration step up
    to detection of targets on agarose gel
   the system could be expanded to include other waterborne enteric
   can be used to concentrate any vial particle from a water sample and
    eliminate time of interference in the molecular amplification process
   extracted RNAs can be used in different molecular detection systems
    like RT-PCR, PCR (DNA viruses), nucleic acid sequence based
    amplification (NASBA) and Quantitative Real-Time PCR

   Routinely monitoring of enteric viruses that contaminate spring or
    mineral bottled water, underground or subsurface water, should be
    considered by manufactures as an important monitor of bacterial
    indicators for protecting the consumer and the general population for
    protection against pathogens associated with the viruses
                                              Works Cited

1. Brassard, Julie,, “Concentration and detection of hepatitis A virus and rotavirus in spring water
   samples by reverse transcription PCR”, Journal of Virological Methods, 123 (2005) 163-169.

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