Viruses in the defense: usefulness of antiviral sensitivity determination in the

Clinical Laboratory Robert Snoeck

Rega Institute for Medical Research K.U.Leuven, Belgium

February 3, 2014

Herpesviruses

• Human herpesviruses are ubiquitous

• Primary infection is usually asymptomatic

• All human herpesviruses establish latency, which persists for life

• Infection is characterized by intermittent reactivation

• Eradication of herpesviruses from their latent state has not yet been achieved NO CURE

Classification of Herpesviridae

Subfamily Biological properties

α-Herpesvirinae - Herpes simplex type 1 (HSV-1) - Herpes simplex type 2 (HSV-2) - Varicella-zoster virus (VZV)

Fats growing, cytolitic. Latent in neurons

β-Herpesvirinae - Human cytomegalovirus (HCMV) - Human herpesvirus 6 (HHV-6) - Human herpesvirus 7 (HHV-7)

Slow growing, cytomegalic. Latent in mononuclear cells

γ-Herpesvirinae - Epstein-Barr virus (EBV) - Human herpesvirus 8 (HHV-8 or KSHV)

Lymphoproliferative. Latent in lymphocytes

Clinical syndromes associated with human herpesviruses

HSV-1 HSV-2 VZV CMV EBV HHV-6 HHV-7 KSHV

Gingivostomatitis + + - - - - - -

Genital lesions + + - - - - - -

Keratoconjuntivitis + + + - - - - -

Cutaneous lesions + + + - - - - +

Neonatal infection + + + + - - - -

Retinitis + + + + - - - -

Esophagitis + + + + - - - -

Pneumonitis + + + + + + - -

Hepatitis + + + + + + - -

Meningitis - + + - - + - -

Encephalitis + + + + + + - -

Myelitis + + + + + + - -

Mononucleosis - - - + + + - +?

Hemolytic anemia - - + + + - - -

Leukopenia - - + + + + - -

Trombocytopenia - - + + + + - -

• Nearly all members of the herpesvirus family can cause serious acute and chronic neurological disease of the CNS

Herpesviruses

• All HHV infections can result in severe disease among immunocompromised patients (IC), including:

- AIDS patients - Solid organ transplant (SOT) recipients - Hematopoietic stem cell transplant (HSCT) recipients - Patients with primary immune deficiency (severe combined immune deficiency, SCID) - Patients undergoing chemotherapy and/or radiotherapy

Immunocompromised (IC) patients

• more susceptible to viral infection and disease • at higher risk to develop persistent infection • more likely to have multiple infections • may develop unusual clinical manifestations which are

not seen in immunocompetent patients

• Among IC patients, herpesviruses are the most frequent cause of viral infections

Phases of opportunistic infections among allogenic recipients

Viral infections in immunocompromised patients

• Herpesviruses: - CMV, HSV, VZV, EBV, HHV-6 • Respiratory viruses: RSV, influenza viruses and parainfluenza viruses • Adenoviruses

• Polyomaviruses: BK virus and JC virus • Human metapneumovirus

In addition to the direct consequences of virus infection, there are also indirect effects

Management of herpesvirus infections in immunocompromised patients

• The availability of active antivirals against herpesviruses allowed the design of well defined prophylactic as well as therapeutic attitudes

• Prevention of HCMV disease:

- prophylaxis (all patients at risk receive antiviral therapy for a defined period of time, often 3 months)

advantage: reduce onset of CMV disease & indirect effects of CMV

drawback: delayed-onset of CMV disease (occurs in 15-38% of CMV D+/R- SOT recipients who received 3 months of prophylaxis)

- pre-emptive therapy (antiviral treatment is given only to patients who develop evidence of CMV viremia)

Factors that influence the success of the antiviral treatment

• Underlying disease

• Severity of immunosuppression

• Concentration of antiviral agents

• Susceptibility of the patient’s viral strain to the administered antiviral drugs

Low antiviral drug levels

Low immune status • Drug induced • T cell depletion (e.g. haploidentical donor trasnplants) • Cord blood transplantation

Subclinical CMV load

Prolonged drug administration (before

and/or after transplant)

Resistance

+

+

+

+

Antiviral agents for the management of HSV and VZV infections

• Acyclovir, ACV (Zovirax®)

• Valacyclovir, VACV (Valtrex®)

• Penciclovir, PCV (Vectavir®, Denavir®)

• Famciclovir, FAM (Famvir®)

• Brivudin, BVDU (Zostex®)

• Foscarnet, PFA (Foscavir®)

Target: Viral DNA polymerase

Viral DNA polymerase

Viral TK

Alteration in substrate binding or phosphate transfer sites

ACV ACV ACV P P ACV P P P

Alteration in catalytic site or relative increase in exonuxlease activity

P

ACV resistance due to mutations in the TK gene

Treatment options: - 1st line: PFA - 2nd line: CDV - Additional agents: imiquimod (HSV)

ACV resistance due to mutations in the DNA pol

gene

Treatment options: - 1st line: CDV - Additional agents: imiquimod (HSV)

Mechanisms of drug-resistance in HSV and VZV

Antiviral agents for the management of CMV infections

• Ganciclovir, GCV (Cytovene®, Cymevene® )

• Valganciclovir, VGCV (Valcyte®)

• Cidofovir, CDV (Vistide®)

• Foscarnet, PFA (Foscavir®)

• Fomivirsen (Vitravene®)

Target: Viral DNA polymerase

Viral DNA polymerase

Protein kinase UL97

Alteration in substrate binding or phosphate transfer sites

GCV GCV GCV P P GCV P P P

Alteration in catalytic site or relative increase in exonuxlease activity

P

GCV resistance due to mutations in the UL97 gene

Treatment options: - 1st line: PFA - 2nd line: CDV

GCV resistance due to mutations in the DNA pol

gene

Treatment options: - 1st line: PFA - Additional agents: Maribavir Letermovir

Mechanisms of drug-resistance in HCMV

Cross-resistance with CDV

Prevalence of herpesviruses drug-resistance

• In immunocompetent patients:

- extremely low for HSV (0.1 to 0.7%).

- CNS: higher incidence of HSV ACVr isolates

• In immunocompromised patients:

- HSV:

4.3 to 14 % among all immunocompromised groups (up to 36% in HSCT patients)

- CMV:

Transplant recipients: 5% to 10%

HIV patients

- Before HAART: ~8% incidence after 3 months, 11% after 6 months and 27.5% by 9 months of treatment

- After HAART: ~5%

Herpesviruses drug-resistance

• Virologists have to provide clinicians with fast and reproducible drug resistant testing to:

- determine viral drug resistance as reason for failure of therapy

- optimize antiviral therapy

- avoid drug toxicity

- improve patient care

- reduce costs of antiviral treatment

A Reference and Service Center created in 2009 by the funding received from the Belgian National Cancer Plan (Federal Public Service “PUBLIC HEALTH, FOOD CHAIN SAFETY and ENVIRONMENT” Our aim: the diagnosis and typing of drug-resistant herpesviruses in immunocompromised patients that fail antiviral therapy and improvement of the scientific background

Antiviral resistance tests available

Workflow for HSV Workflow for HCMV and VZV

Antiviral resistance tests available

Workflow for HHV-6 Workflow for other viruses - Adenoviruses (Ad) - Polyomaviruses (i.e. BK virus) - Parainfluenza virus (PI) - EBV - KSHV

Country City Hospital Number of samples

Belgium

Aalst OLV Ziekenhuis Aalst-Asse-Ninove 12

Antwerpen

ZNA Middelheim 22

ZNA Jan Palfijn 1

ZNA Stuivenberg 4

UZ Antwerpen 24

Bonheiden Imeldaziekenhuis 1

Brugge AZ Sint Jan Brugge-Oostende 41

Brussels

Brugmann Ziekenhuis - Brussel 3

Hôpital Erasme 38

CHU Saint Luc 10

CHU Saint Pierre 132

UZ Brussel 9

CHIREC 4

Ghent UZ Gent 40

Hasselt Jessa Ziekenhuis 19

Leuven UZ Leuven 108

Liège CHU de Liège - Sart Tilman 24

CHR de la Citadelle 19

Roeselare-Menen H.-Hartziekenhuis Roeselare-Menen 1

Yvoir CHU Mont-Godinne 7

519

Number of samples per hospital analyzed by RegaVir (January 2009- January 2014)

Country City Hospital Number of samples

France

Paris Hôpiral de la Salpêtrière 6

Hôpital Necker-Enfants malades 3

Lille CHU Lille 2

Lyon Centre de Biologie et Pathologie Est, Hospices Civils de Lyon

30

Suresnes Hôpital Foch 1

Luxembourg Luxembourg

Centre Hôspitalier de Luxembourg 9

Centre Hôspitalier de Kirchberg 1

The Nederlands Amsterdam Academisch Medisch Centrum Amsterdam

10

USA West Virginia West Virginia University 3

65

Number of samples per hospital analyzed by RegaVir (January 2009- January 2014)

Total number of samples encoded: 519 (national) + 65 (international) = 584

Total number of analysis: 631

Some of the samples analyzed for more than one virus!

Number of tests per virus performed by RegaVir (January 2009- January 2014)

HSV

-1

HSV

-2VZV

HCM

V

HHV-6

EBV

KSH

V

Par

ainflu

enza

viru

s

Aden

oviru

s

Poly

omav

irus

0

100

200

300

18

113

2360

85

305N

um

ber

of

tests

12 4 4 7

Types of samples analyzed by RegaVir received by RegaVir (January 2009- January 2014)

HSV-1 HSV-2 VZV HCMV HHV-6

Blood 3 3 5 225 12

Serum 1 - 1 7 -

Plasma 1 1 2 26 -

CSF 9 9 15 20 4

Eye fluid 1 - - 2 -

Urine - - - 8 -

Swab from lesion 71 66 33 1 (mouth) -

Biopsy 4 2 1 10 4

Mouth rinse 7 - - -

Respiratory

sample 14 1 2 6 2

Vaginal fluid 1 1 - - -

Bone marrow - - - - 1

Not available 1 2 1 - -

Total 113 85 60 305 23

Types of samples analyzed by RegaVir received by RegaVir (January 2009- January 2014)

EBV HHV-8

(KSHV)

Parainfluenza

virus Adenovirus Polyomavirus

Blood 10 4 - 2 2

Serum - - - - 1

Plasma 1 1 - - 1

CSF 1 1 - 1 -

Urine 1 1 - - 2

Biopsy 3 3 - - -

Bone marrow 1 1 - - -

BAL 1 1 - - 1

Virus isolate - - 4 1 -

Total 18 12 4 4 7

Number of patients for whom 1, 2, 3, or >3 samples were analyzed

→ Virus compartmentalization → Evolution of viral population

1 2 3 >3

0

50

100

150

200

35

163

22

60

Samples analyzed per patient

Nu

mb

er

of

pati

en

ts

Type of analysis performed by RegaVir

Virus Only

Phenotyping

Only

genotyping

Phenotyping

+

Genotyping

Negative

sample*

Total number

of tests

performed

HSV-1 10 21 66 16 113

HSV-2 5 25 49 6 85

VZV - 38 3 19 60

HCMV - 259 - 46 305

HHV-6 - 7 - 15 23

EBV 13** 5 18

KSHV 3** 9 12

Adenovirus 1 2 - 1 4

BK Polyomavirus - 4 - 3 7

Parainfluenza

virus 4 - - - 4

631

* A sample was considered negative when it was not amplifiable by polymerase chain reaction (PCR) and/or when the virus could not be isolated in cell culture. ** No genotyping was performed; only viral detection was carried out for -herpesviruses (i.e. EBV and HHV-8).

Proportion of HSV-1 samples showing drug-resistance as determined phenotypically and/or genotypically

Neg

ativ

e

wild

-typ

e

TK m

utants

DNA p

ol muta

nts

Double

muta

nts

0

20

40

60

HSV-1 drug-resistant viruses: 64.6% (62/96)

62

34

169

2

51

Nu

mb

er

of

sam

ple

s

Proportion of HSV-2 samples showing drug-resistance as determined phenotypically and/or genotypically

Neg

ativ

e

wild

-typ

e

TK m

utants

DNA p

ol muta

nts

Double

muta

nts

0

20

40

60

HSV-2 drug-resistant viruses: 78.2% (61/78)

61

17

612 10

39

Nu

mb

er

of

sam

ple

s

Proportion of VZV samples showing drug-resistance as determined phenotypically and/or genotypically

Neg

ativ

e

wild

-typ

e

TK m

utants

DNA p

ol muta

nts

Double

muta

nts

0

10

20

30

40

50

VZV drug-resistant viruses: 26.8% (11/41)

11

30

19

4 07N

um

ber

of

sam

ple

s

Proportion of HCMV samples showing drug-resistance as determined genotypically

Neg

ativ

e

wild

-typ

e

UL97

muta

nts

UL54

muta

nts

Double

muta

nts

Unkn

own s

ignifi

cance

0

50

100

150

200

HCMV drug-resistant viruses: 29.3% (72/246)

71

174

46 49

176 10

Nu

mb

er

of

sam

ple

s

• Two UL97 mutants resistant to maribavir • Maribavir + ganciclovir = antagonistim → no combination

Proportion of HHV-6 samples showing drug-resistance as determined genotypically

Neg

ativ

e

wild

-typ

e

U69

muta

nts

DNA p

ol muta

nts

Double

muta

nts

0

5

10

15

20

HHV-6 drug-resistant viruses: 42.9% (3/7)

34

15

3

00

Nu

mb

er

of

sam

ple

s

Immune privileged sites

• Sites able to tolerate the introduction of antigens without eliciting an inflammatory immune response

• However, immune priviliged sites are neither isolated nor passive in its interactions with the immune system (complex barriers separate immune-priviliged sites from the circulation)

• Immune privilige is thought to be an evolutionary adaptation to protect vital structures from the potentially damaging effects of the inflammatory immune response.

• They include:

- CNS: brain + spinal cord

- Eye

- Placenta and fetus

- Testes

Herpetic Encephalitis

Herpesvirus infections in the central nervous system (CNS)

• Cerebrospinal fluid (CSF) analysis: diagnosis by PCR

• Clinical presentation and magnetic resonance imaging (MRI): help to complete diagnosis

• In cases of immunosuppression, the clinical manisfestations may be atypical and the diagnosis therefore especially challenging

• The most significant CNS manifestations are due to HSV and CMV

Herpesvirus infections in the CNS

• Risk of development of drug-resistance in both immunocompetent and immunocompromised patients

• CNS: immune priviliged site; environment with reduced

immune surveillance

• Blood brain barrier and pharmacokinetic of antiviral drugs that may favor selection of drug-resistance

Characterization of HSV-1 isolates recovered from the CSF of patients suffering from herpetic encephalitis

• Patient 1 (immunocompetent): 72 years old man with HSV-1 encephlitis under acyclovir therapy. He died under ACV treatment.

- CSF → DNA isolation → sequencing HSV TK → G59W mutation

• Patient 2 (immunocompetent): 45 years old man with HSV-1 encephlitis under acyclovir therapy. His treatment was changed to foscarnet and he survived

- CSF → DNA isolation → sequencing HSV TK → G59W mutation

Evolution of HSV-1 strain in the CSF of a 50-years-old woman suffering from herpetic encephalitis

23/02/2011

Mutations in HSV-1 TK

Adenosine (A) del. in a string of 4As (Nts 184-187)

01/03/2011

Adenosine (A) del. in a string of 4As (Nts 133-137)

Mixed populations of wild-type and two different drug-resistant viruses

Adenosine (A) del. in a string of 4As (Nts 184-187)

17/03/2011

Mixed populations of wild-type and drug-resistant virus

Pure population of Drug-resistant virus

Adenosine (A) del. in a string of 4As (Nts 133-137)

RV-171 RV-172 RV-178

Schematic illustration of herpes simplex virus (HSV) type 1 load in cerebrospinal fluid (CSF) throughout the course of disease.

FIGURE 1: Schematic illustration of herpes simplex virus (HSV) type 1 load in cerebrospinal fluid (CSF) throughout the course of

disease. Symptoms began approximately 4days prior to admission to our hospital. On the day of admission (day 1), triple antimicrobial

therapy was started and deescalated to acyclovir monotherapy when the positive HSV polymerase chain reaction result was obtained

on

day 2. At this point, viral load was 411,000 genome equivalents (geq)/ml, which increased to 1,420,000geq/ml on day 6, at which time

foscarnet was added to the therapeutic regime. By day 9, HSV DNA was no longer detectable in CSF. Treatment with acyclovir and

foscarnet was continued until day 19. [Color figure can be viewed in the online issue, which is available at

www.interscience.wiley.com.] ANN NEUROL 2010;67:830–833

Herpetic keratitis (HSV-1) in a renal transplant patient after 6 months valacyclovir therapy

Left eye

Right eye

10/01/14

TK: del. C Nts 548-553

RV-570

15/01/14

TK: del. C Nts 548-553 +

W259stop

RV-573

TK : T245M

RV-574

The patient had ocular VZV ; samples negative for VZV

Characterization of HSV-1 isolates recovered from patients suffering from herpetic keratitis

(Duan et al, 2008)

• 173 immunocompetent patients with herpetic keratitis (HK)

• 11 isolates (6.4%) were ACVr

- 10 mutations in the viral TK

- 5 isolates were cross-resistant to GCV

- 1 isolate was cross-resistant to GCV and PFA

COMPARTMENTALIZATION &

VIRAL HETEROGENICITY

Characterization of VZV isolates recovered from a patient suffering from skin lesions (herpes zoster) and associated

ocular disease

• Patient 3 (immunocompromised, HSCT recipient): 35 years old woman with skin lesions and associated ocular infection under acyclovir therapy. She was treated with foscarnet but developed blindness

- Skin lesions → VZV TK: wild-type

- CSF → VZV TK: stop mutation at codon 303

CMV compartmentalization in a case of congenital HCMV in a severe immune deficiency neonate - emergence of novel mutations

Blood

CSF

21/10

10/11

23/12 06/01

Death 2009

pUL97 wt / Pol wt

14/11

02/11

28/11

pUL97 wt / Pol wt

04/11

09/11

16/11

17/11

14/11

25/11

23/11

Sample negative

07/12

pUL97 wt / Pol ?

28/12

pUL97 wt / Pol ?

30/12

UL97 wt / Pol K513N

Sample negative

10/01

2010 11/01

pUL97 wt / Pol del C524?

20/01

18/01

20/01

25/01 01/02

pUL97 wt / Pol wt

15/02

pUL97 wt / Pol L501I

pUL97 wt / Pol wt

22/02

pUL97 wt / Pol L501I

01/03

GCVR / CDVR

GCVR / CDVR GCVR / CDVR Novel

Neonatal herpes with CNS disease: HSV-2 compartmentalization - combined therapy

13/06/11

Day of birth

20/06/11

HSV-2 vesicular lesions developed on the trunk

and face

ACV 60mg/kg/day i.v.

0 7 28

11/07/11

CSF remained positive for HSV-2. New skin lesions

39

22/07/11

PFA 40mg/kg 3x/day i.v.

12/08/11

HSV-2 DNA negative in blood and CSF

60

19/08/11

67

Appearance of new skin lesions. No signs of invasive

HSV-2 infection

PFA 40mg/kg 3x/day i.v.

25/08/11

74

Suppressive ACV therapy fpr 6 months

25/02/12

257 94

The patient was discharged from

hospital

Normal neurological development

RV-216 (skin): ACVR (*)

RV-217 (CSF): ACVR (*)

RV-218 (tracheal aspirate): wt RV-219 (blood): wt

13/07/11

11/08/11

RV-235 (CSF): negative RV-236 (blood): wt

22/08/11

RV-239 (skin): wt RV-240 (CSF): negative RV-241 (blood): negative

11

HSV-2 DNA in blood and CSF

* TK mutation: C deletion Nts 551-556

Multidrug-resistance HCMV infection in a 5-months old infant conferred by a virus bearing a multi-drug resistant DNA pol mutation

31/05

pUL97 M460V * Pol wild-type

Ganciclovir Foscavir

17/06

blood RV-511

RV-517

pUL97 M460V * Pol wt

CSF

19/06

RV-516

pUL97 M460V * Pol wild-type

19/06 04/07 11/07

RV-521 RV-520

pUL97 M460V * Pol 981-982 del*

22/07 25/07

RV-525 RV-523

29/07 01/08

RV-528 RV-526

01/08

RV-527

Foscavir + Cidofovir

pUL97 wild-type - Pol 981-982 del

Urine

31/07

RV-524

08/08

death

*Mixed populations of wild-type and mutant virus pUL97 M460V: ganciclovir-resistance DNA pol del 981-982: ganciclovir, cidofovir and foscavir resistance

VZV Compartmentalization in an HSCT recipient: emergence of novel mutations

16/09/2010 03/11/2010

Skin lesion

RV-130 RV-146 RV-147 RV-148 RV-149

TK mutations wt wt Sample negative wt wt

DNA pol mutations

wt S854C Sample negative S423N

P688S*

17/11/2010

blood CSF CSF Skin lesion

*Heterogeneous population of wt and mutant virus. • Novel mutations are underlined. • The S854C mutation can be linked to drug-resistance because of homology to known mutations in other herpesviruses (bold). • Novel mutations most probably linked to drug resistance because of their location in conserved regions of the viral enzyme are indicated in italics.

23/02*

TK: Adenosine (A) del. in a string of 4As (Nts 184-187)

01/03*

TK: Adenosine (A) del. in a string of 4As (Nts 133-137)

TK: Adenosine (A) del. in a string of 4As (Nts 184-187)

17/03*

TK: Wild-type TK: Adenosine (A) del. in a string of 4As (Nts 133-137)

RV-171 RV-178

25/02

Hospital admission 1st lumbar punction

(abnormal parameters)

2nd MRI: injury progression

Acyclovir: 10mg/kg/8h

02/03

Neurological deterioration 2nd lumbar punction (abnormal

parameters)

1st MRI

03/03

Acyclovir: 15mg/kg/8h

25/03 05/03

Foscavir: 40mg/kg/8h

04/04

Fever

Clinical improvement 3rd lumbar punction

(improvement)

Hospital discharge

TK: Wild-type

RV-172

DNA pol: Wild-type

DNA pol: N.A.

DNA pol: N.A.

Viral samples

TK / DNA pol Genotyping

Evolution of HSV-1 strains in the CSF of a 50-years-old woman (treated with TNF-α inhibitors) suffering from HSV encephalitis: co-existence of viruses with ≠ genotypes - combined therapy

VZV encephalitis in a 40-years-old woman (lung transplant recipient): risk for co-infection with HSV-2

16/03/2012

VZV TK: F270V

23/03/2012

RV-334 RV-339

Acyclovir Foscavir Cidofovir

VZV +

VZV -

HSV-1 -

HSV-2 +

HCMV -

HHV-6 -

HSV-2 DNA pol: M789T

CSF samples Persistence of neurological problems

VZV encephalitis in a 69-years-old men (HSCT recipient): risk for co-infection with HSV-2

09/05/2012

VZV: wt

22/05/2012

RV-370

Acyclovir Foscavir

HSV-2 TK: del C Nts 816-819

CSF

VZV skin lesions

RV-371 VZV: wt Lesions healed

RV-374

Persistence of neurological problems

VZV -

HSV-1 -

HSV-2 +

HCMV -

HHV-6 -

Oral Skin

swab Skin

swab

Skin

swab

14/04/201

Right

hand

Left

foot Sole of

left foot

28/04/2011

Palm of

left hand

Left

hallux

Left

index

Oral

01/06/2011

RV-194 RV-195 RV-196 RV-197 RV-200 RV-201 RV-202 RV-203 RV-204 RV-205 RV-206

Pheno- typing

wt ACVR

PFAS

ACVr

PFAR

ACVr

PFAR

ACVr

PFAR

ACVr

PFAR

ACVr

PFAR

ACVr

PFAR

ACVr

PFAR

ACVR

PFAR

ACVr

PFAR

23/05/2011 03/06/2011

ACV 15

mg/kg/8h PFA 6g 2x/day

HSV-2 Heterogeneity - compartmentalization in a HSCT recipient with a primary infection

HSV-2 Heterogeneity – compartmentalization – emergence of novel mutations in a HSCT recipient with a primary infection

Isolate Mutant variants

RV-194 Wild-type

RV-196

A606V*

RV-197

V842M*

R964H*

*Mutations detected by Sanger sequencing are highlighted.

HSV-2 Heterogeneity & compartmentalization in a HSCT recipient – primary infection

Isolate Mutant variants Number of clones

isolated

RV-194 Wild-type 5/5 (100%)

RV-196

A606V* 0/21

T934A 21/21 (100%)

RV-197

K533E 2/21 (9.5%)

C625R 5/21 (23.8%)

R628C 5/21 (23.8%)

S725G 3/21 (14.3%)

V842M*

R964H* 5/21 (23.8%)

*Mutations detected by Sanger sequencing are highlighted.

HSV-2 Heterogeneity & compartmentalization in a HSCT recipient – primary infection

Isolate Mutant variants % mutant variants (deep-sequencing)

Number of clones isolated

RV-194 Wild-type Wild-type 5/5 (100%)

RV-196

A606V* 41.6 0/21

Y823C 35.2 0/21

V842M 1.0 0/21

T934A 12.6 21/21 (100%)

RV-197

K533E 3.4 2/21 (9.5%)

A606V 6.0 0/21

G617S 1.7 0/21

C625R 8.0 5/21 (23.8%)

R628C 1.8 5/21 (23.8%)

S725G 1.1 3/21 (14.3%)

A840T 1.5 0/21

V842M* 42.9 0/21

R964H* 27.1 5/21 (23.8%)

*Mutations detected by Sanger sequencing are highlighted.

Take-home message

• Herpesvirus infections in the CNS and cornea:

Risk of development of drug-resistance in both immunocompetent and immunocompromised patients

• Compartimentalization

• Viral heterogenicity

• New genotypic resistance mutations continue to be identified

• Multiple drug-resistance strains – Infection with different drug-resistant viruses

– Single mutation confering multiple drug-resistance

RegaVir

• Rega Institute

Laboratory Virology and Chemotherapy Sarah Gillemot

Anita Camps

Steven Carmans

Graciela Andrei

Robert Snoeck

Laboratory Immunobiology Pierre Fiten

Ghislain Opdenakker

• UZ Leuven

Laboratory Virology Katrien Lagrou

Marc Van Ranst

• This work was supported by the action 29 of the National Cancer Plan (Federal Public Service ”Public health, food chain safety, and environment”) for translational research