Supplementary Materials for

JC polyomavirus mutants escape antibody-mediated neutralization

Upasana Ray, Paola Cinque, Simonetta Gerevini, Valeria Longo, Adriano Lazzarin,

Sven Schippling, Roland Martin, Christopher B. Buck,* Diana V. Pastrana*

*Corresponding author. E-mail: buckc@mail.nih.gov (C.B.B.); pastrand@mail.nih.gov (D.V.P.)

Published 23 September 2015, Sci. Transl. Med. 7, 306ra151 (2015)

DOI: 10.1126/scitranslmed.aab1720

This PDF file includes:

Text

Fig. S1. Transducibility of various cell lines.

Fig. S2. An example of luminometry results for a pilot JCV neutralization assay

using ART cells.

Fig. S3. Neutralization assay validation.

Fig. S4. PML patient neutralization serology (an expansion of Fig. 2).

Fig. S5. Serological analysis of mice after a booster dose of JCV VLPs.

Fig. S6. JCV-neutralizing titers after vaccination of PML patient 5228 (an

alternative version of Fig. 4).

Table S1. Characteristics of JCV pseudovirus stocks.

Table S2. Patient characteristics.

Table S3. PML patient neutralization serology (source data).

Table S4. Neutralization serology of patient 5228 (source data).

Table S5. Viremia of patient 5228 (source data).

References (40–48)

www.sciencetranslationalmedicine.org/cgi/content/full/7/306/306ra151/DC1

Supplementary Materials

Pseudovirus production

JCV isolates are traditionally classified into seven genotypes (40). A codon-modified

(41) expression plasmid encoding the VP1 protein of a JCV genotype 2A primary isolate derived

from the urine of a healthy subject was initially generated for production of a model wt JCV

pseudovirus (Table S1). An expression plasmid representing the VP1 of a phylogenetically

divergent urine-derived genotype 3B primary isolate was also constructed. Characteristic PML

mutations were introduced into the 2A or 3B background using PCR-based mutagenesis. In

some instances, VP1 mutations representing PML patient-derived isolates were introduced into

the background of the lab-adapted JCV isolate Mad1 (genotype 1A) using an expression plasmid

generously provided by Walter Atwood (14). All pseudoviruses employed the VP2 and VP3

minor capsid proteins of JCV 313B (accession AAK28470), a genotype 3B isolate that happens

to encode a VP2 similar to the average consensus sequence of all known JCV VP2 proteins. The

expression plasmids were used to produce reporter pseudovirions in 293TT cells (35) according

to previously reported methods (21, 29). Detailed production protocols and plasmid maps are

posted on our lab website, http://home.ccr.cancer.gov/Lco/

The VP1 sequences of patient CSF JCV isolates were determined by sequencing PCR

products captured by standard plasmid cloning. One to ten clones were subjected to Sanger

sequencing. In some cases the patient presented with a mixture of apparently wild-type VP1 and

sequences with established PML-associated mutations (11, 12). In instances where only

sequences with PML-associated mutations were observed, a wild-type sequence was inferred by

reverting the known PML-associated mutation to the amino acid residue typically observed in

wild-type strains.

Pseudovirions were produced with a mixture of two separate reporter plasmids, phGluc

(Gaussia luciferase (Gluc) under control of EF1α promoter) and pCGluc (Gluc under control of

CMV immediate early promoter). Two different promoters were used based on the guess that

one or the other promoter might perform better in the different cell lines used in the study.

Neuraminidase V and RNase were used during the pseudovirion harvest. After clarification of

the cell lysate, the pelleted cell debris was washed with DPBS containing 0.8 M NaCl with 1%

Triton X-100, with the goal of extracting any entrapped pseudovirions. VLPs were produced

using similar methods, except the cells were transfected only with the relevant VP1 expression

plasmid and the cell lysate was supplemented with Benzonase endonuclease (Sigma). After

Optiprep gradient purification of pseudovirions or VLPs, VP1 content was assessed by

comparison to BSA standards in SDS-PAGE gels stained with SYPRO Ruby dye (Invitrogen) or

by western blot comparisons using a blend of sera from JCV VLP-immunized mice (see below).

Establishment of a pseudovirus-based JCV neutralization assay

To identify cell lines that support the infectious entry of PML mutant JCV isolates, we

compared the transducibility of a diverse range of cell lines with wt JCV-2A pseudovirus or with

a pseudovirus carrying a representative PML-associated mutation, S267F (2A-267F, see Table

S1). This mutant was chosen over other PML mutants because it happened to give relatively

high infectious yields in initial pseudovirus production experiments. Cell lines previously shown

to be readily transducible with a BK polyomavirus (BKV) pseudovirus or a pseudovirus based on

Merkel cell polyomavirus (MCV) were favored, based on the presumption that these lines lack

innate antiviral defenses against polyomavirus-mediated transduction (42).

The 293TT are an SV40 large T antigen (LT) stable cell line that is believed to be of

neuronal origin (35, 43). SVG cells were obtained from the ATCC. We presume, based on a

recent report, that the SVG culture is chronically infected with BKV (44). It is unclear what

effect this might have had on pseudovirus transduction efficiency. An SV40 LT transformed

human Schwann cell line, HSC, was generously provided by Ahmet Höke (45). The central

nervous system (CNS) tumor-derived cell lines SF-268, SF-295, SF-539, SNB-19, SNB-75, and

U251, as well as the ovarian tumor line NCI/ADR-RES, are part of the “NCI60” panel and were

obtained from the DCTD Tumor Repository, National Cancer Institute at Frederick, Maryland.

Early experiments suggested that three cell lines: NCI/ADR-RES (an ovarian tumor line

similar to OVCAR-8 (37, 38)), SF-539 (a gliosarcoma line (39)), and SNB-75 (a glioblastoma

line (46)) were each similarly transducible with both the 2A and 2A-267F pseudoviruses (Fig. S1

and data not shown). The three permissive cell lines were stably transfected with pTIH, which

encodes the cDNA of SV40 Large T antigen (LT), with the goal of amplifying transduced

reporter pseudogenomes. The SNB-75-LT (SNBT) cell line was eventually abandoned due to its

slow growth rate, which made it impractical for use in high-throughput screening.

Additional PML-mutant pseudoviruses were applied to NCI/ADR-RES-LT (ART) and

SF-539-LT (SFT) cell lines. Each of the pseudoviruses gave readily measurable transduction of

both cell lines, except the 3B-271K mutant, which gave very low Gluc luminometry values

(Table S1).

Neutralization assays were conducted essentially as previously described for BKV (29).

Fig. S2 shows an example of raw luminometric values for a neutralization assay using wt JCV-

2A and PML mutant 2A-267F pseudoviruses. 50% neutralizing titers (EC50) were calculated

using GraphPad Prism software to fit a sigmoidal dose-response curve, with top and bottom

constrained based on “no antibody” and “no virus” controls. In initial setup experiments

examining a set of six representative human sera, the 2A and 2A-267F pseudoviruses were

applied to ART cells at various VP1 doses spanning a five-fold range. EC50 titers for each of the

six human sera were similar with different pseudovirion doses (data not shown), suggesting that

the neutralization assay complies with the assumptions of the law of mass action (47).

Similar neutralizing EC50 titers (expressed as the inverse log10 of the calculated 50%

neutralizing dilution of serum) were observed for a panel of sera from healthy adults using either

ART or SFT cells (Fig. S3). Serum samples from mice primed with JCV VLPs likewise showed

comparable neutralizing activity on both ART and SFT cells (data not shown).

Sera

24 anonymized human serum samples used for initial validation of the neutralization

assay (Fig. S3) were provided by Eugene Major (NINDS) under the auspices of the second

meeting of the Standardization of JCV Serology Workshop. Serum samples were heat-

inactivated at 56°C for 30 minutes, followed by brief centrifugation to sediment any aggregated

material.

A previously described panel of 96 anonymized sera from healthy human subjects were

purchased from Equitech-Bio and Innovative Research (21). Ethical assurances are provided on

the suppliers’ websites. Serum IgG antibodies were purified out of the serum samples using

Melon Gel (Pierce) resin according to manufacturer’s instructions. Sera were first buffer-

exchanged into Melon Gel purification buffer using a Zeba (Pierce) 96-well spin desalting plate

(40K MWCO). Buffer-exchanged samples were then loaded onto a 96-well Melon Gel spin

plate. Finally, the Melon Gel-purified antibody samples were buffer exchanged into PBS using a

Zeba 96-well spin desalting plate.

Plasma (EDTA) samples from PML patients (Table S2) were collected under the

approval of the ethical review board of the San Raffaele Scientific Institute, Milan, Italy. All

time points for all PML patients tested seropositive in a JCV VP1 ELISA. PML patient plasma

samples and mouse serum samples were heat inactivated at 56ºC for 30 minutes and tested

without Melon Gel purification.

Patient 5228 clinical details

Patient 5228 showed altered gait in April 2012. On 4/24/2012, a CT scan revealed a

hypodense right temporal-parietal-occipital lesion that was initially interpreted to be of ischemic

origin. Progression of symptoms and of brain lesions by MRI was monitored during subsequent

weeks. On 5/24/2012, JCV DNA was detected (48) in CSF (16,650 copies/ml) and the patient

was diagnosed with PML and admitted at the Department of Infectious Diseases of San Raffaele

Hospital, Milan, Italy. The patient’s clinical condition deteriorated rapidly and she became

comatose. A 1250 mg induction dose of mefloquine was given on 6/2/2012 and 6/3/2012, then

500 mg twice a week from 6/6/2012 to 6/18/2012. Mirtazapine (15 mg) was given daily from

6/2/2012 to 6/10/2012.

MRI lesions were active until January 2013, but no activity was observed by March 2013

MRI (Fig. 5). On 9/12/2012 CSF JCV-DNA increased to 95,430 c/ml, but decreased to low level

on 2/22/2013 (2841 c/ml); no additional CSF examination was performed. Plasma JCV DNA

levels increased progressively and remained stable at high copy numbers until early October

2012, at which point levels began decreasing and ultimately became undetectable in February

2013 (Fig. 4). Fig. S5 shows the patient’s CD4 T cell counts alongside the same neutralization

information shown in Fig. 4. The patient survived but remained comatose. There was no longer

evidence of lesion activity at the last MRI in December 2013. JCV DNA remained undetectable

in plasma at last examination in May 2014.

Fig. S1. Transducibility of various cell lines. Pseudovirions representing wt JCV-2A or the

model PML mutant 2A-267F were applied to various cell lines. The two pseudovirus stocks had

roughly equivalent amounts of VP1 (within the general range of concentrations shown in Table

S1). After five days, the expression of Gluc reporter protein was measured in relative light units

(RLU) at a gain of 3800. The signal for the ART cell line is pegged at the luminometer’s

maximum measurable signal for all but the most dilute pseudovirus dose. The experiment was

conducted in the wake of our realization that the SNBT cell line was too slow-growing to be

practical for high-throughput neutralization assays. The goal of the experiment was to identify

an additional PML mutant-permissive cell line to use as a counterpoint to the more tractable

ART cell line. The results indicate that SF-539 cells support the infectivity of the 2A-267F

pseudovirus. Transfection was used to generate an SF-539 line that stably expresses SV40 large

T antigen (SFT cells, see Table S1 and Fig. S3).

2A

0.01 0.1 12

3

4

5

6

7

µl of pseudovirus stock

RLU

(Log

10)

293TT

ART

HSC

SF268

SF295

SF539

SNB19

SNB75

SVG

U251

SNBT

2A-267F

0.01 0.1 12

3

4

5

6

7

µl of pseudovirus stock

RLU

(Log

10)

293TT

ART

HSC

SF268

SF295

SF539

SNB19

SNB75

SVG

U251

SNBT

Fig. S2. An example of luminometry results for a pilot JCV neutralization assay using ART

cells. The data are from an early experiment performed during the initial development of the

JCV neutralization assay. A pool of sera from mice prime-boost immunized with 3B-267F VP1-

only virus-like particles (VLPs) was subjected to serial dilutions (x-axis). The diluted serum was

mixed with roughly equal doses of JCV-2A or 2A-267F pseudovirions and applied to ART cells.

Five days later, culture supernatants were subjected to Gluc assay, with a gain of 2800. Relative

light units (RLU) are indicated on the y-axis. Supernatants from five control wells to which

pseudoviruses were added in the absence of serum (no serum), or supernatants from five mock-

infected wells (no virus) are shown at arbitrary points to the left of the curves. Pooled pre-

immune sera from the mice, which were tested in a later experiment using different virus stocks,

showed luminometric values statistically identical to the relevant no serum control at a serum

dilution of 1:100 (i.e., the pre-immune sera were non-neutralizing). The results confirm that, on

ART cells, the 2A-267F PML mutant pseudovirus has infectivity roughly comparable to that of

the wild-type 2A virus. The infectivity of both pseudoviruses is completely inhibited by VP1-

specific antiserum.

0 2 4 6 8

0

5

10

Serum dilution (reciprocal log10)

RLU

x10

-4

2A-267F

2A

No Virus

2A no serum

2A-267F no serum

Fig. S3. Neutralization assay validation. Panel A: 24 human serum samples were titered for

neutralization of wt JCV- 2A pseudovirus using either ART or SFT cells. Similar neutralizing

EC50 values (defined as the inverse log10 of the calculated 50% neutralizing dilution) for each

serum sample were observed using either cell line. Panel B: the sera showed similar

neutralizing titers for 2A and 3B pseudoviruses (results using SFT cells shown, similar results

were observed using ART cells). The results show that ART and SFT cells give comparable

neutralization results for two different wt JCV genotypes.

A

B

FIGURE S1

Fig. S4. PML patient neutralization serology (an expansion of Fig. 2). Pre- and post-PML

plasma samples from six patients were tested for neutralization of wt (blue) and PML-mutant

(red) pseudoviruses indicated in each panel. Patients whose disease progressed (left column) are

indicated with (P), patients who survived (right column) are indicated with (S). PML-associated

mutations observed in each patient’s CSF are indicated. Y-axes indicate neutralizing EC50.

Error bars represent standard error of the mean for data from three independent experimental

replicates, two of which were performed with blinding. Arrows below x-axes indicate the date

of onset of PML symptoms (see Table S2). Date format is month/day/year.

Fig. S5. Serological analysis of mice after a booster dose of JCV VLPs. Groups of mice that

initially showed evidence neutralizing blind spots after an initial priming dose of JCV VLPs (see

Fig. 3) were given a booster dose of the vaccine. Four weeks after boosting, sera from the mice

were tested using ART cells for neutralization of a pseudovirus based on the JCV genotype

indicated above each table. Numerical values represent EC50 neutralizing titers for individual

animals. Pre-immune sera were non-neutralizing at a dilution of 1:100.

Fig. S6. JCV-neutralizing titers after vaccination of PML patient 5228 (an alternative version of

Fig. 4). Patient 5228 was administered JCV VLPs intramuscularly at the indicated time points

(downward purple triangles). Recombinant IL-7 was also administered subcutaneously at 10

µg/kg once a week for two cycles of three weeks (upward green triangles). The patient’s

neutralizing titer against her cognate PML-mutant JCV (red squares) or inferred wt JCV (blue

circles) was monitored over time. The patient’s CD4 T cell count (gray diamonds) was also

monitored over time.

Genotype Differences relative to 2A VP1 stock (ng/µl)

VP1 dose (ng/well)

ART RLU x 10-5

SFT RLU X 10-5

2A (wt) None, accession AAK97910 0.9 0.2 4.1 ± 1.3 2.8 ± 0.7

3B (wt) G134A, K164T, V321I, E332Q

3.9 0.4 5.1 ± 0.9 3.8 ± 0.4

3B-55F 3B+L55F 2.4 0.2 4.7 ± 0.6 8.1 ± 2.7

3B-265S 3B+L55F, N265S 2.4 0.2 2.5 ± 0.5 5.1 ± 1.2

2A-267F S267F, Q271H 6.0 1 3.2 ± 0.4 6.0 ± 1.0

3B-267F 3B+S267F, Q271H 3.0 0.6 0.6 ± 0.4 0.7 ± 0.1

2A-269F S269F 1.9 0.2 3.1 ± 0.7 5.9 ± 0.9

3B-271K† 3B+L55F, Q271K 1.8 nd nd nd

GCN1 Y346* 0.5 0.05 1.0 ± 0.8 0.1 ± 0.2

1A (Mad1)

K75R, T117S, V158L, R345K

nd (1:500 diln)

nd nd

5029w N74T, T128A, R345K 2.0 0.2 3.5 ± 1.6 nd

5029m 5029w+S269F 18 2.2 2.6 ± 0.9 nd

5031w 3B 3.9 0.4 5.1 ± 0.9 3.8 ± 0.4

5031ma 3B-55F nd (1:150 diln)

1.03 ± 0.2 nd

5031mb 3B+Q271H 2.4 0.2 4.7 ± 0.6 8.1 ± 2.7

5031mc 3B+L55F, Q271H nd (1:150 diln)

nd

5040w† 1A+T128S nd nd nd nd

5040m 5040w+H122R 2.0 0.2 4.0 ± 0.7 nd

5053w T128A, R345K nd (1:800 diln)

2.3 ± 0.8 nd

5053m 5053w+L55F nd (1:400 diln)

1.7 ± 0.7 nd

5058w† 2A?+T117S, V158L nd nd nd nd

5058m 5058w+S269F nd nd nd nd

5147w 5053w[F171S?, T232N?, L252del?]

nd (1:800 diln)

2.3 ± 0.8 nd

5147m 5053w+S269F 18 2.2 2.1 ± 0.5 nd

5228w 5053w nd (1:800 diln)

2.3 ± 0.8 nd

5228m 5053w+S269F 18 2.2 2.1 ± 0.5 nd

Table S1. Characteristics of JCV pseudovirus stocks. Daggers indicate that the pseudovirus was

either not generated or had unusably low titer. Question marks indicate incomplete or uncertain

sequencing. Variations with incomplete sequencing support (question marks) were not

incorporated into the pseudovirus. Luminometry was performed with a gain of 2800.

Background luminometric values typically ranged between 300 and 400 RLUs.

Table S2. Patient characteristics. The table lists characteristics of PML patients with archived

serum samples available from prior to PML diagnosis and with CSF VP1 sequence information

available. CD4 count at the time of PML diagnosis is listed in the far right column.

ID# Gender Age at PML

PML diagnosis

Underlying disease

VP1 mutation

Date of death

CD4+ cells/µL

5029 M 36 6/1/96 HIV S269F 10/30/96 14

5031 M 34 1/1/97 HIV

L55F Q271H

4/24/97 20

5040 M 34 1/7/97 HIV H122R, N265T, S269F

2/21/06 122

5053 M 35 4/23/97 HIV L55F 53

5058 F 32 7/1/97 HIV S269F 162

5147 M 33 1/15/05 HIV S269F 5/15/05 7

5228 F 74 5/24/12

idiopathic lymphopenia

S269F

298

Serum collection

date

Mean LogEC50

SEM Mean LogEC50

SEM

5029w 5029(P)269F

6/13/95 4.0 0.4 2.4 0.3

8/23/95 3.7 0.3 2.5 0.2

7/23/96 3.3 0.5 1.8 0.3

5031w 5031(P)55F 271H

11/2/95 4.1 0.1 2.3 0.2

1/30/97 4.6 0.1 2.5 0.2

4/15/97 4.6 0.1 2.3 0.1

5040w 5040(S) 122R 265T 269F

8/29/96 2.9 0.2 2.3 0.1

1/9/97 3.8 0.5 2.3 0.1

7/9/04 4.8 0.3 4.1 0.2

1/12/05 4.7 0.1 4.3 0.2

5053w 5053(S) 55F

2/14/96 2.3 0.2 1.8 0.4

1/27/98 5.9 0.1 3.6 0.2

4/9/09 5.1 0.1 3.7 0.3

5058w 5058(S) 269F

6/5/97 2.4 0.1 1.7 0.0

7/11/97 3.2 0.0 2.3 0.1

3/24/09 4.5 0.1 2.7 0.1

5147w 5147(P) 269F

4/30/04 4.6 0.2 1.8 0.5

2/22/05 4.6 0.2 2.1 0.1

3/7/05 4.6 0.2 1.8 0.1

Table S3. PML patient neutralization serology (source data). The table list neutralizing titers

(LogEC50) used for construction of Fig. 2. SEM denotes standard error o f the mean.

5228w 5228m

Weeks after PML diagnosis

Mean SD Mean SD

1.3 5.6 0.4 3.6 0.3

2.3 5 0.3 2.9 0.2

3.3 5.2 0.2 3.6 0.3

4.3 5.4 0.0 3.8 0.6

4.9 5.2 0.1 3.3 0.1

11.0 5.4 0.4 3.9 0.2

16.3 6.2 0.5 4.0 0.0

20.6 7.4 0.8 5.3 0.1

24.3 7.1 0.4 5.0 0.1

29.0 6.7 0.1 5.4 0.2

42.0 6.9 0.1 5.2 0.4

55.0 7.0 0.5 5.2 0.0

Table S4. Neutralization serology of patient 5228 (source data). The table lists the neutralization

titers (LogEC50) of sera from patient 5228. These values have been used in Fig. 4. SD means

standard deviation.

Weeks after PML diagnosis

Viremia

1.3 3.5

1.4 3.7

1.6 3.3

1.7 4.0

2.0 4.2

2.1 3.4

2.3 3.3

2.4 3.6

2.6 3.3

2.7 3.5

2.9 3.5

3.0 3.2

3.1 3.6

3.3 3.2

3.4 3.2

3.6 3.5

3.7 3.7

3.9 3.7

4.0 3.8

4.1 3.8

4.3 3.9

4.4 4.0

4.6 4.0

4.7 4.1

4.9 3.4

5.0 3.5

5.1 3.4

5.3 3.7

5.4 3.7

5.6 3.6

5.7 3.7

5.9 3.9

6.0 3.7

6.1 4.2

6.3 3.7

6.4 4.1

6.6 3.7

6.7 4.2

6.9 3.9

7.0 4.0

7.1 3.6

7.3 3.8

7.4 3.8

7.6 4.1

7.7 4.2

7.9 4.2

8.0 4.0

8.1 4.1

8.3 4.5

8.4 4.2

8.6 4.1

8.7 4.3

8.9 4.4

9.0 4.6

9.1 4.5

9.3 4.0

9.4 4.0

9.6 4.1

9.7 4.1

9.9 4.0

10.0 3.8

10.1 4.0

10.3 4.3

10.4 4.1

10.6 4.1

10.7 4.6

10.9 3.6

11.0 4.1

11.1 4.5

11.4 3.9

11.6 3.9

11.7 4.4

11.9 4.5

12.4 3.9

12.6 4.0

12.7 4.4

12.9 3.8

13.1 4.0

13.3 3.6

13.4 3.9

13.6 4.0

13.7 4.5

13.9 4.5

14.0 3.9

14.1 3.9

14.3 4.3

14.4 4.2

14.6 4.1

14.7 4.1

14.9 4.3

15.0 4.8

15.1 4.5

15.3 4.4

15.4 4.0

15.6 3.9

15.7 4.5

15.9 3.4

16.0 4.3

16.1 4.5

16.3 4.1

16.4 4.2

16.6 4.0

16.7 4.5

16.9 4.4

17.0 4.3

17.1 4.3

17.3 4.8

17.4 4.4

17.6 4.3

17.7 4.3

17.9 4.2

18.0 4.3

18.1 4.5

18.3 4.4

18.4 4.8

18.6 4.5

18.7 4.6

18.9 4.2

19.0 4.1

19.1 4.7

19.3 4.8

19.4 4.6

19.6 4.4

19.7 4.7

19.9 4.5

20.0 4.8

20.1 4.8

20.3 4.5

20.4 4.6

20.6 4.4

20.7 4.8

20.9 4.8

21.0 4.3

21.1 4.3

21.3 4.4

21.4 4.2

21.6 4.2

21.7 4.2

21.9 4.3

22.0 4.1

22.1 4.0

22.3 4.3

22.4 4.3

22.6 4.3

22.7 3.9

22.9 4.2

23.0 4.2

23.1 4.0

23.3 4.1

23.4 4.0

23.6 4.1

23.7 4.3

23.9 4.0

24.0 3.9

24.1 3.9

24.3 4.2

24.4 3.8

24.6 4.1

24.7 3.8

24.9 3.8

25.0 3.8

25.1 3.5

25.3 3.7

25.4 4.0

25.6 3.9

25.7 3.7

25.9 3.8

26.0 4.0

26.1 3.9

26.6 4.0

27.0 3.7

27.6 3.6

28.0 3.6

28.7 4.1

29.0 3.5

29.6 3.0

30.0 2.8

30.6 3.1

31.0 3.3

31.6 2.5

32.0 2.8

32.6 3.0

33.0 2.5

33.6 2.7

34.0 2.6

34.6 2.2

35.0 2.1

35.6 2.7

36.0 2.4

36.6 2.6

37.0 2.0

37.6 2.1

38.0 2.0

38.6 2.0

39.0 2.0

39.6 2.2

39.7 2.3

40.0 2.2

40.6 2.5

41.0 2.0

41.6 2.0

42.0 2.0

42.6 2.0

43.0 2.0

43.6 2.0

44.0 2.0

45.0 2.0

45.6 2.0

46.0 2.0

46.6 2.0

47.0 2.0

47.6 2.0

48.1 2.0

48.6 2.0

Table S5. Viremia of patient 5228 (source data). The table lists the viremia corresponding to

each time point tested. These values have been used in Fig. 4.