Inactivation of Coxsackievirus by Chlorine, Silver, and Solar Disinfection for Safe Global Water

Transcription

1 Inactivation of Coxsackievirus by Chlorine, Silver, and Solar Disinfection for Safe Global Water Theresa Vonder Haar Bernardo Vázquez, Martin Page, Joanna Shisler, Benito Mariñas University of Illinois at Urbana-Champaign April 11, 2011 Cincinnati, OH Savfe Water

2 Outline Motivation Background Methodology CDC Disinfection Kinetics Aimee Gall Future Work

3 Motivation

4 Viruses are emerging pathogens in both developed and developing regions. Motivation Combined chlorine is becoming increasingly prevalent. Due to the formation of regulated disinfection byproducts, free chlorine use is decreasing, while UV and combined chlorine use is increasing. Water sources are frequently contaminated with human and animal waste. Silver has potential for use in developing countries, particularly point-of-use approaches. Low risk to human health Proven antimicrobial agent Solar is commonly used throughout the developing world. Simple and inexpensive technology Sun is readily available

5 Motivation Many viruses including dsdna adenoviruses, and ssrna enteroviruses (coxsackie) and noroviruses have greater resistance than (oo)cysts to UV inactivation and are resistant to combined chlorine. These point-of-use technologies have great potential for developing regions. The inactivation kinetics of such viruses have not been fully characterized. Mechanisms of viral inactivation are relatively unknown.

6 Global Water Facts Nearly 1 billion people lack access to improved drinking water Improved Drinking Water Coverage in Rural Areas in 2006 (UNICEF/WHO, 2008)

7 Global Water Facts Almost 2.4 billion people lack access to proper sanitation Basic Sanitation Coverage in Rural Areas in 2006 (UNICEF/WHO, 2008) 4,000 children die daily due to diseases from inadequate sanitation and unclean water Diarrhea and related diseases account for more than 1.5 million deaths of children every year

8 Personal Experience As undergrad at The Ohio State University, involved in Engineers for Community Service Honduras Teaching assistant for a senior design course in Civil and Environmental Engineering (CEE 449) Mexico Mexico Mexico, Kenya, and Ethiopia

9 2009: Veracruz Mexico Collaboration with Universidad de las Américas- Puebla, Mexico and CEDICAM 2010 & 2011: Oaxaca

10 Ethiopia Working with Addis Ababa University and NGO Relief Society of Tigray (REST) Wukro Aimee Gall

11 Background

12 RNA Viruses Rock & Yoo, UIUC PATH 433, 2010

13 Picornaviridae Family Genus: Aphtho-, Cardio-, Entero-, Erbo-, Hepato-, Kobu-, Parecho-, Rhino-, Tescho-virus CVB5 -Fields Virology

14 Coxsackievirus: Prevalence First discovered in Coxsackie, New York Sources: Most common nonpolio enterovirus Domestic wastewater and contaminated surface and groundwater, drinking water, and recreational waters Outbreaks in US, Taiwan, Brazil, and many other places Transferred via fecal-oral route Has shown resistance to free chlorine -Ramsingh, Group B Coxsackieviruses. Current Topics in Microbiology and Immunology, Romero, Group B Coxsackieviruses. Current Topics in Microbiology and Immunology, 2008

15 Coxsackievirus: Virulence Health Effects: Median Infection Dose, N 50 = 100 Hand, foot, and mouth disease; myocarditis; aseptic meningitis; gastrointestinal illnesses; fever; miscarriage CVB is most common viral agent linked with heart disease and causes more than 50% of all cases of viral myocarditis Echovirus, CVA & B been reported to cause fatal neonatal enteroviral outbreaks Enterovirus cause million illnesses/year in the US (most under age 10) Mortality Ratio = 1% -MWH: Water Treatment Principles and Design, Mena, et. al

16 Coxsackievirus: Structure nm, non-enveloped, icosahedral capsid Single stranded (ss) RNA genome, ~7.5 kilobase Capsid made up of 60 copies of each of four proteins: VP1, VP2, VP3 & VP4 VP1, VP2 and VP3 are the 3 major coat proteins: each contain ~270 amino acid residues and form the outer surface of the virus. VP4 contains only about 70 amino acids and is on the inside of the protein capsid and is in contact with the packaged RNA There is a star-shaped plateau at the 5-fold axis of symmetry, within a deep depression, canyonbinding site. -He, et al. Nature Structural Biology, Xiao, et al. Structure, Vol. 13, Oberste, Group B Coxsackieviruses. Current Topics in Microbiology and Immunology, 2008

17 Adenovirus Type 2 Water-borne pathogen found globally Double-stranded DNA virus nm, non-enveloped, icosahedral capsid Symptoms: fever, rhinitis (nasal inflammation), tonsillitis, cough, conjunctivitis (eye inflammation) s/1993/illpres/genes-in-pieces.html

18 Coxsackie Adenovirus Receptor (CAR) CVBs (and Ad2) use CAR to recognize host cells. CAR is a membrane protein with two extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmic domain. CAR binds with the distal end of domain D1 in the canyon of CVB3. Binds with fiber knob of Ad. In CVBs, CAR can function for both attachment and infection. For adenovirus the major function of CAR is to mediate initial attachment of virus to the cell surface -He, et al. Nature Structural Biology 8, Hafenstein, et al. Journal of Virology, Vol. 81,

19 Replication cycle Attachment to receptor Entry/Uncoating -RNA genome uncoated Structural changes in capsid +RNA is translated in cytoplasm to make proteins for replication and new virus production After sufficient amount of capsid proteins produced, encapsidation occurs Assemble infectious virus particle with new +RNA Release via lysis (cell death) Rock & Yoo, UIUC PATH 433, 2010

20 Viral Replication in a Cell Rock & Yoo, UIUC PATH 433, 2010

21 Experimental Methodology

22 Experimental Methodology: Cell Culture CVB5 Buffalo Green Monkey Kidney (BGMK) Cells

23 Experimental Methodology: Free Chlorine Disinfection Batch reactors C o, Disinfectant Decay & Disinfection Constant mixing and temperature ph controlled via carbonate system ph and [Cl 2 ] monitored throughout experiment [Cl 2 ] determined by DPD Colorimetric Method

24 Experimental Methodology: Silver Disinfection Batch reactors Constant mixing and temperature ph controlled via carbonate system Silver concentration measured with a Hach Spec Silver stock

25 Experimental Methodology: Solar Disinfection 1000W Xenon arc lamp Magnetic Stirring Water-jacketed Reactor Series of filters to simulate conditions at equatorial latitudes

26 Samples Experimental Methodology: Viability Assessment (Cell monolayer, 3-5 days) Serial Dilutions Inoculate ~80 min Count plaques 3-4 days Incubate at 37ºC (Nutrient mixtures) Soft Agar Overlay Method

27 Results

28 Inactivation with Free and CombinedChlorine

29 Inactivation of Coxsackievirus B5 with Free Chlorine: ph Effect

30 Inactivation of Coxsackievirus B5 with Free Chlorine: Temperature Effect ph , 1.7C ph , 2C ph , 14C ph , 30C ph , 30C N/No CT (mg min/l min)

31 Inactivation of Coxsackievirus B5 and Adenovirus 2 and with Free Chlorine

32 Inactivation of Coxsackievirus B5 with Combined Chlorine 1 N/No ph , 7C ph , 14C ph , 14C ph , 30C CT (mg min/l)

33 Inactivation of Coxsackievirus B5 with Combined Chlorine Compared to Free Chlorine

34 Inactivation with Silver

35 Inactivation of Coxsackievirus B5 with Silver: Effect of ph and Temperature 1 CVB5 (0.26 mg/l, 20 C, ph 8.0) N/No CVB5 (0.23 mg/l, 30 C, ph 8.0) CVB5 (0.25 mg/l, 20 C, ph 9.1) CVB5 (0.22 mg/l, 31 C, ph 9.0) CT (mg/l min)

36 Inactivation of Coxsackievirus B5 with Silver: Effect of Concentration 1 CVB5 (0.89 mg/l, 20 C, ph 7.8) 0.1 CVB5 (1.92 mg/l, 20 C, ph 7.8) CVB5 (4.66 mg/l, 20 C, ph 7.8) N/No CT (mg/l min)

37 Inactivation with Solar

38 Inactivation of Coxsackievirus B5 with Solar N/No Time of continuous sunlight (hours)

39 Summary Free Chlorine CVB5 relatively resistant Combined Chlorine Much less effective than free chlorine Silver Increasing ph and temperature show increase in disinfection kinetics Solar Very low effectiveness for viral inactivation

40 Future Work

41 Future Work Continue disinfection experiments Free chlorine Combined chlorine Simultaneous disinfection Solar + Silver Solar + Chlorine N/No Stability of CVB5 ph 10, 30C ph C ph 9, 40C time (hours)

42 Motivation: Virus detection: viable vs. non-viable Sensor development Mechanistic Study Molecular mechanisms governing virus inactivation in drinking water are not well known Understand how disinfectants target virus Initially, focus on methods to determine how attachment is affected Ad2 and CVB5 both bind to CAR Yoo, UIUC PATH 433, 2010

43 Produce CAR protein Mechanistic Study: Next Steps Perfrom Enzyme Linked Immunosorption Asssay (ELISA) and Sandwich ELISA Analyze: Attachment Affinity for attachment = virus = antibody Photo Credit: Martin Page

44 Safe Global Water is a Collaborative Effort For project success, a true collaboration is required Global partnership Engineers, natural scientists, social scientists, economists Benito Marinas, 2011

45 Acknowledgements WaterCAMPWS, a Science and Technology Center of the National Science Foundation National Science Foundation Shaoying Qi Mariñas Research Group

46 THANK YOU!