Irene Xagoraraki, Phd, Associate Professor 1

Home , Alphapapillomavirus, Erythroparvovirus, Mamastrovirus, Ortervirales, Salivirus

Early Detection of Viral Outbreaks

Presented at: Borchardt Conference, University of Michigan February 2020

Irene Xagoraraki, PhD, Associate Professor 1. Background

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 2 Viral Disease Exposure

1. Background XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 3 Proposed water-based surveillance system

Sampling and characterizing community wastewater, livestock manure, and wildlife waste represents a snapshot of the status of community human and animal health

1. Background XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 4 Proposed water-based surveillance system

RURAL COMMUNITIES URBAN COMMUNITIES

Composite sampling of surface Composite sampling of water and point sources at centralized wastewater critical locations and critical utility influent at time points in time intervals

Microbial pollution loading Sewer system modeling and source tracking modeling (data: population biomarkers, (data: land-use, water quality, water quality, sewer Viral abundance Viral abundance hydrological, weather, species- dimensions, detention time, and diversity specific pollution markers hydrological data) and diversity

Biostatistics modeling (data: public health records, disease incubation time and shedding rates)

Current status of endemic disease and early signals for detection of emerging disease

1. Background XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 5 Virus Transmission Route Detected in Virus Transmission Route Detected in Wastewater Wastewater or Human or Human Human to Zoonotic Water Water Excrement Human to Zoonotic Water Water Excrement human borne Related human borne Related

Adenoviruses ✓ ✓ ✓ Coronaviruses ✓ ✓ ✓ Astroviruses ✓ ✓ ✓ Ebola virus ✓ ✓ ✓ Enteroviruses ✓ ✓ ✓ ✓ Influenza ✓ ✓ ✓ Hepatitis A ✓ ✓ ✓ Herpesvirus ✓ ✓ Hepatitis E ✓ ✓ ✓ ✓ Papillomavirus ✓ ✓ Noroviruses ✓ ✓ ✓ Parechovirus ✓ ✓ Rotaviruses ✓ ✓ ✓ ✓ Arbovirus ✓ Aichi virus ✓ ✓ ✓ Hepatitis B ✓ Polyomaviruses ✓ ✓ ✓ Hepatitis C ✓ Salivirus ✓ ✓ ✓ HIV ✓ Sapovirus ✓ ✓ ✓ Rabies ✓ Torque Teno ✓ ✓ ✓ Rubella ✓ Dengue ✓ ✓ ✓ Smallpox ✓ West Nile ✓ ✓ ✓ Varicella ✓ Zika ✓ ✓ ✓ Crimean-Congo ✓ ✓ Yellow fever ✓ ✓ ✓ Marburg virus ✓ ✓ Chikungunya ✓ ✓ Arenaviruses ✓ ✓ Rift Valley fever ✓ ✓

1. Background XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 6 2. Approach

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 7 Measurements of Viral Concentrations in Wastewater: Temporal Distribution of Sample Collection: Weekly sampling Spatial Distribution of Sample Collection: Three interceptors servicing Detroit area municipalities Sample Processing and Analysis: Elution, DNA/RNA extraction, Shotgun sequencing, qPCR Shotgun qPCR sequencing Estimation of Detention Time Collection of Public in Sewer System Health Records Sewer System Parameters: Alignment with Confirmed weekly interceptor and sewer Reference Sequence Viral Diversity cases of viral Viral Outbreak diameter, length , material, disease per county Databases Identification Early Detection flow rate, gravity/pump and municipality RefSeq, Swiss-Prot, (Viral-ID) Model: (Viral-ED) Model: operation Custom sequence • Metagenomics • Biostatistics databases for viral • Temporal and • Temporal and pathogens Spatial spatial Estimation of Serviced Correlations Viral Disease correlations Population Characteristics Measurements of Biomarkers in wastewater: Incubation times, creatinine, HIAA Shedding rates

Identification of Potential Causes of Viral Disease Early Detection of Viral Outbreaks in Urban Areas

2. Approach XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 8 Detroit Wastewater Treatment Plant Detroit Water and Sewerage Department (DWSD) Largest single-site sewage treatment plant in the United States

Photo: Detroit Water and Sewerage Department

2. Approach XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 9 Study Area Three largest counties in Michigan (U.S. Census Bureau, 2010)

Oakland Macomb 1,202,362 840,978 State of Michigan (Troy) (Warren) Population 9,883,640 Major City (Detroit)

Wayne 1,820,584 (Detroit)

2. Approach XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 10 Schematic: Detroit Water and Sewerage Department Influent wastewater was collected from three sampling stations located within the Detroit wastewater treatment plant. Each station pulls raw sewage from one of the three main sewers (interceptors): North Interceptor—East Arm (NI-EA), Detroit River (DRI), and Oakwood- Northwest-Wayne County (O-NWI).

NI-EA

O-NWI DRI

2. Approach XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 11 DWSD Service Area Interceptor sewage area map . DWSD service municipalities in Wayne, Oakland, and Macomb counties in Michigan NI-EA

. Service municipalities are based on the 2018 Great DRI Lakes Water Authority sewer map for DWSD O-NWI DRI, NI-EA . Color code represent DRI main interceptor sewage NI-EA areas All O-NWI

2. Approach XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 12 DWSD Service Area Interceptor service population and daily flowrate

**Estimated Estimated Average *Estimated Service . Estimated flowrates and Average Total Flow2 Dry Weather Flow2 Interceptor Population1 population based on interceptor (MGD) (MGD) DRI 1,168,777 538 369 service areas and DWSD district NI-EA 1,347,858 604 424 flowrate and population estimates O-NWI 1,010,239 505.6 346.3 . All interceptors have gravity flow *Estimates based on 2010 Census . DRI (Wayne county) receives the greatest **DWSD flowrates based on 2003 Greater Detroit Regional Sewer portion of industrial waste System (GDRSS) Model simulations . NI-EA (Macomb county) has the largest proportion of municipal waste and the lowest proportion of stormwater

1. Jones, B., Cushingberry, G Jr., Ayers J., Benson, S., Castaneda-Lopez, R., Leland, G., Sheffield, M., & Spivey, A. Detroit Water and Sewerage Department. (2015). Rehabilitation of the Rectangular Primary Clarifiers, Electrical/Mechanical Buildings and Pipe Gallery. City of Detroit. 2. Detroit Water and Sewerage Department. (2003). Collection System: DWSD Wholesale Sewer Rates 201. City of Detroit. 2. Approach XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 13 3. Methods

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 14 Sample Collection

Figure 3. Virus sampler setup in O- NWI station: filter cartridge, virus filter house, vacuum pump, battery, Wastewater sampling Figure 1. Onsite water quality testing Figure 2. meter from NI-EA interceptor

3. Methods XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 15 Collection of untreated Sample Collection wastewater

SY1 (6 events): Nov. 2017 – Feb. 2018 O-NWI NI-EA DRI

SY2 2 (9 events): Oct. 2018 – Mar. 2019

Samples were collected in 9 Filter samples 9 Grab samples triplicates from each sampling station. 24-44L average/filter 1L/sample Total samples per event: 9 = 3 replicates per interceptor Viral Load and pH, Temperature, Nitrate, Diversity Dissolved Oxygen, *3 interceptors) Conductivity, Biomarkers Electropositive NanoCeram column filters for virus sampling

3. Methods XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 16 SAMPLING YEAR 1 (SY 1) SAMPLING YEAR 2 (SY 2)

Sampling Sampling Location Number of Sampling Sampling Location Number of Date (Interceptor) Replicates Date (Interceptor) Replicates NI-EA 3 10/17/2018 DRI 3 O-NWI 3 11/17/2017 NI-EA 3 DRI 3 O-NWI 3 10/31/2018 NI-EA 3 DRI 3 O-NWI 2 12/1/2017 NI-EA 3 DRI 3 O-NWI 3 11/28/2018 NI-EA 3 DRI 3 O-NWI 3 12/14/2017 NI-EA 3 DRI 3 O-NWI 3 12/12/2018 NI-EA 3 DRI 3 O-NWI 3 DRI 2 1/19/2018 NI-EA 3 1/17/2019 NI-EA 3 O-NWI 3 O-NWI 3 DRI 3 2/7/2019 NI-EA 3 2/2/2018 NI-EA 3 2/14/2019 NI-EA 3 O-NWI 3 DRI 3 2/28/2019 DRI 3 NI-EA 3 2/16/2018 NI-EA 3 DRI 3 3/14/2019 O-NWI 3 NI-EA 3 Total: 54 Total: 58

3. Methods XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 17 Sample Processing Virus elution from filters

Figure 4. Pressure vessel setup Figure 5. pH adjustment Figure 6. Membrane filtration with beef extract 3. Methods XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 18 Wastewater Sampling for Viral Analysis Onsite virus enrichment/concentration: VIRADEL electropositive filtration Wastewater Sampling for Biomarker Analysis Virus elution and secondary concentration 1 L grab samples

Nucleic acid extraction

viruses RNA pH adjusted to 2 viruses RNA

DNA viruses DNA Isotope DNA viruses DNA creatinine Creatinine and 5-HIAA and 5-HIAA pure standards standards Reverse Transcription Reverse Transcription (virus specific primer) random amplification Mixed equally Solid Phase Extraction (SPE) (universal primer) for cDNA in methanol

Method Optimization Standards (10, 100, 250, selection of qPCR primers/probes, 500, 1000 and 2500ppb) HPLC preparation Illumina whole genome creation of standard curves, inhibition • mobile phase A: 0.3% folic acid shotgun sequencing control, limit of detection detrmination • mobile phase B: pure methanol

Metagenomic Analysis quality analysis, assembly, Quantitative polymerase chain reaction alignment, taxonomic HPLC analysis qPCR classification

Creatinine and 5-HIAA Concentrations Viral Concentrations Viral Diversity

3. Methods XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 19 4. Results – Biomarkers

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 20 Average biomarkers in the interceptors over 2 years (11/18/2017 to 3/14/19)

. Creatinine is breakdown substance of muscle tissues . 5-hydroxyindoleacetic acid (5-HIAA) is serotonin metabolite . Both of these compounds have been known to be excreted by humans

4. Results - biomarkers XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 21 5. Results – Viral Diversity

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 22 Data used in the following clinical cases graphs are extracted from Michigan Department of Health and Human services weekly and annual disease surveillance reports.

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 23 Norovirus Encephalitis, California Flu Like Disease Encephalitis, Powassan Influenza Encephalitis, St. Louis Meningitis - Aseptic Hepatitis A Acute Flaccid Myelitis Hepatitis B, Acute Encephalitis, Post Chickenpox Hepatitis B, Chronic Reported Conditions per Encephalitis, Post Other Hepatitis B, Perinatal Encephalitis, Primary Total Hepatitis B County per Week Gastrointestinal Illness Hepatitis C, Acute Hantavirus Hepatitis C, Chronic Hemolytic Uremic Syndrome Hepatitis C, Perinatal Kawasaki Total Hepatitis C Novel Coronavirus Hepatitis E Rabies Total Hepatitis Chickenpox (Varicella) Zika Shingles West Nile Virus Varicella Zooster Chikungunya Measles Dengue Fever Mumps Encephalitis, Eastern Equine

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 24 Quality Illumina Whole Genome Shotgun Sequencing Analysis Viral Diversity

Construction of whole Metagenomic Analysis of Viromes (RNA viruses) genomes

Trimmed sequence reads with a BLAST Trim Illumina adapters, alignment Quality Analysis quality of ≥ 20 (nucleotide called remove low quality bases against viral with 99% accuracy or more) databases Assembled consensus sequences De novo Assembly (IDBA-UD) Taxonomic (contigs) classification Alignment threshold tBLASTx alignment against virus database (RefSeq) Suggested viral sequences E-value 10-3 LCA algorithm for long reads; Diversity of viruses in wastewater Thresholds: bit score 50, contigs Taxonomic analysis and classification (MEGAN6 using NCBI taxonomy) (representative at family level) within top 10% of alignment Extraction of contigs assigned to human-associated viral families virus root

BLASTx against all UniProtKB (Swiss-Prot) human-associated viral Alignment threshold E-value 10-5 Putative human viral sequences proteins

LCA algorithm for long reads; Taxonomic analysis and classification of human-associated viral signals Thresholds: bit score 50, contigs (MEGAN6 using NCBI taxonomy) within top 10% of alignment

Human Virus Diversity and Abundance in Wastewater Samples

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 25 Viruses Not Assigned

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0% O-NWI NI-EA DRI O-NWI NI-EA DRI O-NWI NI-EA DRI O-NWI NI-EA DRI O-NWI NI-EA DRI O-NWI NI-EA DRI 17-Nov-17 1-Dec-17 14-Dec-17 19-Jan-18 2-Feb-18 16-Feb-18

Proportion of virus-associated contigs and contigs not assigned for cDNA sequence alignment against the Viral RefSeq Database

5. Results - diversity XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 26 Taxonomic analysis of sewage

samples with MEGAN - Genomic dsDNA viruses, no RNA stage Ortervirales ssDNA viruses ssRNA viruses group composition per sample unclassified archaeal viruses unclassified bacterial viruses unclassified RNA viruses unclassified viruses 100%

98%

96%

Single-stranded RNA (ssRNA) 94% genomes

92% Pathogens like Norovirus and Hepatitis A belong to this group CompositionPercent 90%

88%

86% DRI DRI DRI DRI DRI DRI NI-EA NI-EA NI-EA NI-EA NI-EA NI-EA O-NWI O-NWI O-NWI O-NWI O-NWI O-NWI 17-Nov-17 1-Dec-17 14-Dec-17 19-Jan-18 2-Feb-18 16-Feb-18

5. Results - diversity XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 27 Virus family diversity of virus-associated contigs obtained from alignment of cDNA sequences to the Viral RefSeq database.

5. Results - diversity XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 28 5. Results - diversity XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 29 Associated Disease Virus Family Virus Genus cDNA Reported in MDSS Adenoviridae Mastadenovirus + Y Astroviridae Mamastrovirus + Y Norovirus + Y Caliciviridae Sapovirus + Y Human viral pathogens detected in Coronaviridae Betacoronavirus + Y wastewater and their associated disease Flaviviridae Hepacivirus + Y Hepeviridae Orthohepevirus + Y reported in the Michigan Disease Lymphocryptovirus + NR Roseolovirus + NR Surveillance System (MDSS) Weekly Herpesviridae Simplexvirus + NR Surveillance Reports (WSR). Varicellovirus + Y Matonaviridae Rubivirus + Y Note: Positive detection is denoted as (+). Virus Papillomaviridae Alphapapillomavirus + NR Bocaparvovirus + Y presence is considered positive if detected in at least Parvoviridae Erythroparvovirus + NR one sample. Negative detection is denoted as (-). Cardiovirus + Y Enterovirus + Y Picornaviridae Hepatovirus + Y Parechovirus + Y Orthopoxvirus + NR Poxviridae Parapoxvirus + NR Deltaretrovirus + NR Retroviridae Gammaretrovirus + NR Lentivirus + Y Togaviridae Alphavirus + Y

5. Results - diversity XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 30 6. Results – Hepatitis A

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 31 Annual Hepatitis A Cases in Michigan

800 2017 2008-2018 700

600

500

400

Number of Cases 300

200

100

0 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 Year

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 32 Annual Hepatitis A Cases in Michigan

300

250

200

150

100

0 Wayne+Detroit Oakland Macomb 2017 2018

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 33 Hepatitis A Standard Curve

40 Limit of detection (LOD) for HAV concentrations = 100 copies/rxn 35

30 Note: • For statistical analysis non-detects are 25 set to half of the LOD (50 copies/rxn)

Cq 20 Method used to determine LOD: 15 y = -3.6139x + 45.025 • Serial dilations: 12.5, 25, 50, 100 R² = 0.9939 copies/rxn 10 • 10 replicates for each dilution • LOD is the lowest dilution in which 95% 5 of the replicates have a positive qPCR 0 response 0 2 4 6 8 10 12 Concentration (Log copies/rxn)

Positive Standard: Hepatitis A viral stock (ATCC) amplified, cloned into plasmid vector, transformed into TOP10 E.coli cell. Plasmid DNA carrying the segment of Hepatitis gene is purified, serial diluted, and used as standard

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 34 30

SY1 SY2 20

15 Number of Cases

0 7-Jul-18 7-Jan-17 3-Jun-17 7-Oct-17 1-Apr-17 6-Apr-19 8-Sep-18 2-Feb-19 9-Dec-17 1-Dec-18 5-Aug-17 28-Jul-18 15-Jul-17 3-Mar-18 5-May-18 20-Jan-18 12-Jan-19 28-Jan-17 16-Jun-18 24-Jun-17 28-Oct-17 20-Oct-18 14-Apr-18 22-Apr-17 27-Apr-19 16-Sep-17 29-Sep-18 10-Feb-18 23-Feb-19 18-Feb-17 30-Dec-17 22-Dec-18 26-Aug-17 18-Aug-18 18-Nov-17 10-Nov-18 11-Mar-17 24-Mar-18 16-Mar-19 13-May-17 26-May-18 Date of Onset (Week Ending)

Epidemic curve for confirmed hepatitis A cases in Macomb, Oakland, and Wayne counties from January 2017 through April 2019. Sampling year one (SY1) and sampling year two (SY2) are denoted by dashed lines.

6. Results – hepatitis A XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 35 Distribution of Hepatitis A Concentrations Between Interceptors

SY 1 *SY 2

O-NWI NI-EA DRI O-NWI NI-EA DRI 1.8E+08 1.2E+07

1.6E+08 1.0E+07 1.4E+08

1.2E+08 8.0E+06

1.0E+08 6.0E+06 8.0E+07

6.0E+07 4.0E+06

4.0E+07 2.0E+06 Wastewater Concentation (copies/L) 2.0E+07 Wastewater Concentration (copies/L)

0.0E+00 0.0E+00 17-Nov-17 1-Dec-17 14-Dec-17 19-Jan-18 2-Feb-18 16-Feb-18 31-Oct-18 28-Nov-18 12-Dec-18 17-Jan-19 Sampling Date Sampling Date *Graph only considers weeks that all interceptors were sampled

SY1: 162 qPCR analyses (54 samples analyzed in triplicate) SY2: 174 qPCR analyses (58 samples analyzed in triplicate)

6. Results – hepatitis A XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 36 Boxplots for concentrations of HAV in wastewater samples per interceptor during sampling years one (A) and two (C) along with average concentrations per sampling date during years one (B) and two (D). Note: Median concentrations are denoted with a horizontal line. Due to operational conditions during sampling year two (SY2), DRI and O-NWI sampling sites were not sampled during weeks where no data is reported.

6. Results – hepatitis A XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 37 Conceptual Temporal Correlation Model: Hepatitis A

Hepatitis A Virus infection cycle Incubation period ranges between 15-50 days Exposure Excretion in feces: 10-12 days

Median incubation period of 28 days

Days 1 7 14 21 28 35 42 49

Wastewater sampling on Fridays (SY1) and Wednesdays or Thursdays (SY2) for each 2 week window of reported sampling event. Estimated concentrations of clinical cases. Estimates number infected individuals from Day 1 of people infected during Day 1

Sampling and clinical data collection

Acheson, D.,&Fiore, A. E. (2004). Hepatitis A transmitted by food. Clinical infectious diseases, 38(5), 705-715. Centers for Disease Control and Prevention. (2015). Epidemiology and Prevention of Vaccine-Preventable Diseases. Retrieved from https://www.cdc.gov/vaccines/pubs/pinkbook/hepa.html

6. Results – hepatitis A XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 38 Data used: • Confirmed clinical cases for Hepatitis A: total of 2 weeks of reported cases starting one week after sampling • Average of qPCR data

6. Results – hepatitis A XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 39 7. Results – Herpesvirus

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 40 Illumina Whole Genome Shotgun Sequencing Viral Diversity (Herpesviruses) Metagenomic Analysis of Viromes

Trim adapters, remove low Quality Analysis quality bases and short reads

De bruijn assemblers De novo (IDBA-UD) Assembly

Annotation and Taxonomic BLAST against virus specific database Classification (RefSeq)

PYTHON code to extract all contigs of HHV

EXCEL analysis to filter contigs: • Selected contigs great with BitScore >200 & >200bp (Repeats) • Remove contigs that are assigned same name based on lowest E-Value (Non Repeats)

Herpesvirales Diversity

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 41 Average number of contigs in all samples (O-NWI, NI-EA and DRI for all six dates) of different HHVs detected in metagenomics analysis

200

180

160

140

120

100

Number of contigs 60

0 HHV1 HHV2 HHV3 HHV4A HHV4B HHV5 HHV6A HHV6B HHV7 HHV8

7. Results - herpesviruses XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 42 Prevalence of Human Herpesvirus (HHV) in USA

Type of Herpesvirus Prevalenceβ Reference HHV-1/ HSV-1 Age adjusted prevalence is 48.1% (2015-2016) McQuillan et al 2018 HHV-2/ HSV-2 Age adjusted prevalence is 12.1% (2015-2016) McQuillan et al 2018 Gershon et al 2010 HHV-3/VZV 1 million cases of Herpes Zoster (HZ) each year in Ragozzino et al 1982, Jumaan et al 2003, Mullooly et al USA. 2005, Leung et al 2011 CDC Shingles Varicella incidenceγ reduced by 76-87% (correct) Shingles incidence increasing (1945-2006) HHV-4/EBV Age adjusted prevalence is 66.5% (2003-2010 USA) Dowd et al 2013 CDC Cytomegalovirus, Bate et al 2010, Al-Zafiri et al HHV-5 /HCMV/ CMV Age adjusted prevalence is 50.4% (1988-1994, 2012, Braun et al 1997 1999-2004) HHV-6 90%-100% seroprevalenceΔ by age 2-3 in USA Campadelli-Fiume et al 1999, Braun et al 1997 (1999) HHV-7 85% seroprevalenceΔ in USA population (1994) Ablashi et al 1994 HHV-8/KSHV 5-10% seroprevalenceΔ in USA Gao et al 1996, Martin et al 2007 Notes: β Prevalence is the number of cases of a disease present in a particular population in a given time γ Incidence is the number of new cases that develop over a given period of time Δ Seroprevalence indicates individual tested positive based on serology (blood serum samples)

7. Results - herpesviruses XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 43 HHVs Shedding Rates

Disease Main outcome References (Gianella et al 2016, Miller et al 2006, HIV positive patients HHV-5/CMV, HHV-4/ EBV, HHV8 and HHV-1 shed at Gianella et al 2013, Lisco et al 2011, higher rate in HIV infected patients compared to Gianella Morris et al 2013, Lucht et al 1995, Lucht et al 1993) healthy controls (Sarmento regina et al 2018, Braz-Silva et Solid organ transplant HHV-4/EBV and HHV-1 shed at higher rate in recipients al 2006, Sarmento et al 2018) recipients (SOTR) compared to healthy controls Cancerous patients HHV-4/EBV shed at higher rate in cancerous patients (Palmieri et al 2018) compared to healthy controls (Ong et al 2004, Cook et al 2003, Linssen Critically ill non- HHV-1, HHV-5/CMV and HHV-4/EBV shedding et al 2008, Van den Brink et al 2003, immunocompromised associated with high mortality and morbidity in CINI Smith et al 2010, Libert et al 2015) patients (CINI) patients

7. Results - herpesviruses XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 44 Std curves for Herpesviruses

HSV 8 std curve HSV 6 std curve 35 35 30 30 25 25 20 20 Cq y = -3.589x + 35.771 Cq y = -3.883x + 36.002 15 15 R² = 0.9993 R² = 0.9959 10 10 5 5 0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Log10(concentration) Log10(concentration)

Source of Herpesvirus standards: Human Herpesvirus 6B: Quantitative Genomic DNA from Human herpesvirus 6B (ATCC® VR-1467DQ™) Human Herpesvirus 8: Quantitative Synthetic Human herpesvirus 8 DNA (ATCC® VR-3261SD™) Note: ATCC = American Type Culture Collection

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 45 Average copies/L of HHV8

7. Results - herpesviruses XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 46 7. Results - herpesviruses XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 47 Distribution of HHV8 copies/L per interceptor

7. Results - herpesviruses XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 48 Metagenomics analysis for HIV-AIDS

7. Results - herpesviruses XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 49 8. Upcoming work

XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY Page 50 Consortium for Viral Infectious Disease Research in Latin America and the Caribbean

Blue dots represent cenotes

2/27/2020 MCCALL, C., AND XAGORARAKI, I. | MICHIGAN STATE UNIVERSITY 51 Acknowledgments

PhD Students: Camille McCall, Brijen Miyani, Huiyun Wu, Evan O’Brien Collaborators: Anil Gosine, Michael Jurban (Detroit Water and Sewerage Department/ Great Lakes Water Authority)

NSF 1752773: A Wastewater-Based Epidemiology System for Early Detection of Viral Outbreaks in Detroit, MI Project Duration: September 2017 – August 2019 Thank you

For more info please contact [email protected]

Irene Xagoraraki, PhD, Associate Professor