Concentration and detection of hepatitis A virus and rotavirus in spring water

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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
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What is Rotavirus?

• 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 utensils

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What is RotavirusCont.

• Asymptomatic rotavirus is documented and may play
  a role in endemic disease.
• 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 severity
• 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

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Rotavirus
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Why Do This Experiment?

• Rotavirus are stable under extreme conditions
  like pH, temperature and moisture
• 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

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Materials and MethodsCell culture and virus

• Grown FRhK-4 and MA-104 cells in Eagles minimum
  essential medium
• 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

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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 2.0x104
  FRhK (HAV) per well and incubated for 24hrs at
  37C 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 37C w/ 5 CO2 and observed
  after 3-8 days

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Materials and MethodsConcentration and elution

• Spring water samples were inoculated with
  1.0x103 to 1.0x10-3 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 HCL
• re-concentration to 150 uL with Microsep 100
• retained concentrate was used for RNA extraction

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Flow Chart of Concentration and Elution Fig 1.
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Materials and MethodsExtraction of viral RNA
from water concentrates

• Virus concentrate incubated at 37C w/ 1SDS and
  100ug of proteinase K for 1 hour
• 450uL of RLT buffer added to the concentrate and
  reheated at 56C 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 -70C until use

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Materials and MethodsRT-PCR

• Viral RNA extracted from inoculated water samples
  and heated at 98C 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-PCR
• amplification conditions used for the 226bp HAV
  fragment using prot. 1 and prot. 2 primers 30
  mins _at_ 50C for reverse transcription step, 95C
  for 15 mins for initial denaturation, 35 cycles
  for 45sec _at_ 94C for denaturation, 45sec _at_ 47C
  for annealing, 1 min _at_ 72C 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 (43C) and final extension (5 mins)
  were different
• rotavirus, Rota-1 and End-9 primers were used for
  amplification of 268bp fragment

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Materials and MethodsRT-PCR Cont.

• UV light was used for amplified products after
  electrophoresis on a 2 agarose gel w/ EtBr

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Primers and Probes Used
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Materials and MethodsSouthern 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 _at_ 55C 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 _at_ room temp for 5 mins in
  2x SSC w/ 0.1 SDS and 2 times for 15 mins _at_ 55C
  in 0.5x SSC w/ 0.1 SDS
• membrane incubated in blocking sln

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Materials and MethodsSouthern 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

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ResultsTitration 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 4.0x107
  TCID50/ml and 1.25x106 TCID50/ml respectively
• titers of viral dilutions to inoculate spring
  water samples were determined and corresponded to
  estimated values
• titrations were performed in triplicate

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ResultsViral 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 others
• low yields of rotavirus were observed
• there was no beneficial effects or detriments on
  the RNA yield for HAV

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ResultsDetection 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 (10-3 TCID50/ml)
  and at least 10 fold more sensitive for HAV
  (10-1 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

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MMolecular Ladder NNegative Control PPositive
Control
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MMolecular Ladder NNegative Control PPositive
Control (HAV and Rotavirus) RPositive Rotavirus
Control
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ResultsConfirmation 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

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Discussion

• 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 Report)
• 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
  supply
• spring water and mineral water even though
  treated for the removal of unwanted chemical and
  microbiological components can not be labeled as
  natural

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DiscussionCont.

• 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

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Discussion Cont.

• 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

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DiscussionCont.

• 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 viruses
• 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

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DiscussionCont.

• 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

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