Concentration and detection of hepatitis A virus and rotavirus

Concentration and detection of hepatitis A virus and rotavirus in spring water samples by reverse transcription-PCR
By: Journal of Virological Methods 123 (2005) 163-169
Julie Brassard, Karine Seyer, Alain Houde, Carol Simard, Yvon-Louis Trottier
Presentation by Kristen M. Castro

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
* 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

Rotavirus 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

Materials and Methods Cell 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

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 HCL
* re-concentration to 150 uL with Microsep 100
* retained concentrate was used for RNA extraction

Flow Chart of Concentration and Elution

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
RT-PCR

* 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-PCR
* 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
* 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
* 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

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

Results
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

Results
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

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”
* 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 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

* 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

Concentration and detection of hepatitis A virus and rotavirus.ppt

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Infection

Infection

General points: A general listing of different viral diseases that affect different organs of the body.
Viruses infect specific Organisms Tissues. Effects can range from Unnoticeable Deadly

Routes of viral entry

Respiratory tract diseases
Virus induced respiratory infections kill about 1 million children each year as well as a significant number of older adults.

* Many of the viruses that infect only the respiratory tract do not induce a strong immune response and hence reinfection with the same or a similar strain is common.
* Children get about 6 colds a year and adults get 2-3 per year.
* Deaths from respiratory infections: approx. 4.4 x 106/yr for Bact. and viruses world wide.
* Influenza- 20,000 deaths/yr in US during epidemic years (most years), Spanish flu of 1918, 500,000 in US 20 x 106 worlwide, Hong Kong outbreak 68-69, 34,000 in US.
* Human Respiratory Syncytial Virus- 4,500 deaths/yr (children) in US.

Respiratory tract diseases
* Rhinitis (Common Cold)-watery nasal discharge and obstruction, sneezing, mild sore throat and coughing but little or no fever. Infection based in upper respiratory. Sometimes complicated by superinfection with bacteria.

* Pharyngitis (most are viral)-sore throat, malaise, fever, sometimes cough. Infection based in pharynx. Can be caused by many of the same agents that cause common colds. RSV and adenovirus are prominent causes in young children and Herpes type viruses in young adults.

* Laryngotracheobronchitis (Croup)-fever, barking or metallic cough, respiratory distress, sometimes complete laryngeal obstruction. Most common causes are influenza and parainfluenza virus.

Respiratory tract diseases

* Bronchiolitis-Rapid and labored breathing, persistent cough, wheezing, cyanosis, variable amount of atelectasis, marked emphysema. Major causes are Influenza, parainfluenza, and RSV especially in young children. RSV can develop virtually overnight causing SIDS in young children.

* Pneumonia-often develops following upper resp. tract infection. Febrile, some cough, degree of dyspnea, wheezing, moist rales. X-rays often show only scattered areas of consolidation and diffuse lesions unlike pronounced lesions and consolidated areas in bacterial pneumonia. RSV, Influenza, parainfluenza, adenoviruses are major causes. A major cause of death to older people and young children. RSV is the major respiratory pathogen in young children and kills 4,500 children a year in the U.S. alone.

Viral Gastroenteritis

* Inflammation of the stomach and intestines
* Usually manifested as diarrhea which is watery but not bloody in the case of viral infection.
* Fever and Vomiting common with some infectious agents.
* Diarrheal diseases kill 3 million children each year mostly in developing countries.
* Rotaviruses are the main culprits and are responsible for most of the deaths.
* Astroviruses and Caliciviruses (Norwalk virus) can also cause diarrhea.

Central Nervous system diseases
* Most result as rare complication of primary infections elsewhere in the body.
* Viruses may be
o neuroinvasive (able to enter the nervous system) and/or
o neurovirulent (cause nerve damage).
o e.g. Mumps is highly neuroinvasive but not very neurovirulent while herpes is just the opposite.
* Viruses cause disease in a variety of ways
o infection of a specific area (Ammon's horn with rabies, and temporal lobes with HSV)
o Broad infection.
* Some lyse neurons while some cause demyelination of axons.

Central Nervous system diseases
* Deaths - Measles which can cause a variety of brain infections kills about 1 million/yr worldwide, mostly in area without vaccination.

* Meningitis-Infection of meningeal cells. Viruses are major cause but viral infection is much less severe than bacterial with full recovery likely. Headache, Fever, and neck stiffness with/or without vomiting and photophobia are symptoms. Mumps and Enteroviruses are most common agents.

* Paralysis-Mostly from poliovirus in countries where the virus has not been eradicated. Poliomyelitis involving demyelination of nerve cells.

* Encephalitis-Fever, headache, neck stiffness, vomiting, alterations in state of consciousness indicating involvement of brain parenchymal tissue. Patient is lethargic and confused and seizures, or paralysis may develop before coma and death. Postinfectious Encephalitis- Occurs a few days after infection of children with measles, mumps, or varicella. Severe demyelination is observed and prognosis is grim.

Central Nervous system diseases
* Guillain-Barre syndrome- Demyelination is common finding. Most frequently caused by EBV infection and appearing 1-4 weeks after mononucleosis infection. Most recover completely but 15% have residual nerve damage.

* Reye's syndrome - Cerebral edema without inflammation is common symptom. 25% of cases are fatal. Follow influenza or chickenpox infection in children and seems to be an association between syndrome and aspirin administration.

* AIDS Dementia Complex - Results from destruction of nerve cells often late in AIDS syndrome. Direct destruction of nerve cells and demyelination are involved.

Skin Rashes
* Macular-Flat colored spots;
* Papular-Slightly raised containing no expressible fluid;
* Vesicles-blister with clear fluid from which virus can be isolated;
* Pustular- Blisters containing puss;
* Nodular- Generally warts.

Hemorrhagic fever
* A variety of diseases that share the common feature of widespread hemorrhages from the bodies epithelial tissue including internal mucosa such as the gastrointestinal tract and the skin.
* A variety of internal damage is often associated with the different diseases.
* Hanta virus for example causes severe renal necrosis.
* Other agents (Ebola and Yellow fever virus) cause severe liver damage.
* Many have high fatality rates.

Genitourinary infections
* Herpes simplex virus 2 and Papillomaviruses are the major viruses infecting the genital area.
* Sexual transmission is the main way of acquiring the agent.
* Herpes manifests as painful itchy ulcerated vesicular lesions occasionally accompanied by fever and malaise especially in woman. Spread to the central nervous system occurs in 10% of cases with mild meningitis resulting. Recurrences are common although generally less severe than the initial infection.
* Papillomaviruses: Genital warts. Warts appear as external condyloma and usually disappear without treatment within a couple of years. Certain types of HPV may progress over several years through stages of cervical intraepithelial neoplasia (CIN) to invasive squamous cell carcinoma.
* HIV: Deaths by HIV-about 1 million/yr worldwide, 20,000/yr in US although was much higher before triple drug therapy

Eye diseases
* A number of common childhood viral diseases can involve conjunctivitis
o Inflammation of the conjunctiva which is the transparent membrane covering the sclera i.e. white of the eye and the inner eyelid).
o Results in redness, discomfort and discharge and is commonly called pink eye. Some types are particularly dangerous, especially those involving the cornea (keratoconjunctivitis).
* HSV is the commonest infectious agent that causes blindness in the Western world.
* A number of eye diseases, e.g. cataracts, glaucoma, and retinopathy are associated with congenital rubella syndrome and cytomegalic inclusion disease of infants.

Viral arthritis
* Stiff and painful joints.
* Usually accompanied by fever and myositis (inflammation of muscle tissue causing pain tenderness and weakness)
* Can occur with or without rash.
* Rarely persists for more than a few weeks.
* Major causative agents are certain toga-, flavi-, and bunyaviruses (arboviruses).
* It has been hypothesized that rheumatoid arthritis may have a viral origin.

Viral Carditis
* Myocarditis = inflammation of the heart muscle
* Pericarditis = inflammation of pericardium membrane that encloses he heart
* Cardiomyopathy = diseases causing reduction in the force of the heart
* Associated with certain enteroviruses (family of picornaviruses), most notably coxsackie B virus.
* Infections often reoccur, leading to permanent myocardial damage, cardiomegaly (enlarged heart), or congestive cardiac failure.

Viral Hepatitis
* Inflammation of the liver with accompanying liver cell damage.
* 20-30 cases per 100,000 people per year in the U.S.
* Symptoms :jaundice often proceeded by flu-like illness.
* Liver failure can occurs.
* Can become chronic depending on the infectious agent and cirrhosis
* Transmission:
o parenteral (needle infection usually),
o perinatal (occurring just before or after birth),
o sexual, or enteric (via gut cells).
* 5 viruses known to infect the liver as the primary organ. Other viruses can also cause hepatitis e.g. Herpes viruses and some viruses causing hemorrhagic fever.
* Deaths- Hepatitis B is the most common killer killing 2 million/yr worldwide and 5000/yr in US.

Viral pancreatitis and diabetes
* Mumps infection can be complicated by severe pancreatitis (inflammation of the pancreas).
* Can also be caused by certain other enteroviruses.
* There is some evidence that viral infections triggers insulin-dependent juvenile diabetes mellitus.

Chronic fatigue syndrome
* Characterized by extreme fatigue.
* EBV, Coxsackie B, CMV, and HTLV are among the many viruses to be isolated from such patients
* Importantly, no cause-effect relationship has been established

Congenital and Perinatal Infections
* Include a variety of diseases acquired
o Prenatally,
o Intrapartum (during birth process) or
o Postnatally within the first few weeks.
* Can be particularly dangerous since the newborn is not protected by the mothers antibodies

Infection.ppt

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Bacterial Diseases

Bacterial Diseases

A. Airborne Bacterial Diseases
B. Foodborne & Waterborne Bacterial Diseases
C. Soilborne Bacterial Diseases
D. Arthropodborne Bacterial Diseases
E. Sexually Transmitted Bacterial Diseases
F. Miscellaneous Bacterial Diseases

V. A. Airborne Bacterial Diseases

1. Streptococcal Diseases
2. Diphtheria
3. Pertussis
4. Meningococcal Infections
5. Haemophilus influenzae Infections
6. Tuberculosis
7. Pneumococcal Pneumonia
8. Primary Atypical Pneumonia
9. Legionellosis

Foodborne & Waterborne Bacterial Diseases

1. Foodborne Intoxications vs Infections
2. Botulism
3. Staphylococcal Food Poisoning
4. Clostridial Food Poisoning
5. Typhoid Fever
6. Salmonellosis
7. Shigellosis
8. Cholera
9. Diseases associated with Escherichia coli
10. Camphylobacteriosis and Helicobacteriosis

Soilborne Bacterial Diseases

1. Anthrax
2. Tetanus
3. Gas Gangrene
4. Leptospirosis
5. Listeriosis

Arthropodborne Bacterial Diseases

1. Plague
2. Lyme Disease
3. Rocky Mt. Spotted Fever
4. Epidemic Typhus
5. Endemic Typhus

Sexually Transmitted Bacterial Diseases

1. Syphilis
2. Gonorrhea
3. Chlamydia
4. Chanchroid

Miscellaneous Bacterial Diseases

1. Leprosy
2. Staphylococcal Infections
3. Pseudomonas aeruginosa Infections

Bacterial Diseases .ppt

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