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Viral sequences detection by high-throughput sequencing in cerebrospinal fluid of individuals with and without central nervous system disease

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Viral sequences detection by high-throughput sequencing in cerebrospinal fluid of individuals with and without central nervous

system disease

SCHIBLER, Manuel, et al .

Abstract

: Meningitis, encephalitis, and myelitis are various forms of acute central nervous system (CNS) inflammation, which can coexist and lead to serious sequelae. Known aetiologies include infections and immune-mediated processes. Despite advances in clinical microbiology over the past decades, the cause of acute CNS inflammation remains unknown in approximately 50% of cases. High-throughput sequencing was performed to search for viral sequences in cerebrospinal fluid (CSF) samples collected from 26 patients considered to have acute CNS inflammation of unknown origin, and 10 patients with defined causes of CNS diseases. In order to better grasp the clinical significance of viral sequence data obtained in CSF, 30 patients without CNS disease who had a lumbar puncture performed during elective spinal anaesthesia were also analysed. One case of human astrovirus (HAstV)-MLB2-related meningitis and disseminated infection was identified. No other viral sequences that can easily be linked to CNS inflammation were detected. Viral sequences obtained in all patient groups are discussed. While some of them reflect harmless viral [...]

SCHIBLER, Manuel, et al . Viral sequences detection by high-throughput sequencing in

cerebrospinal fluid of individuals with and without central nervous system disease. Genes , 2019, vol. 10, no. 8, p. E625

PMID : 31431002

DOI : 10.3390/genes10080625

Available at:

http://archive-ouverte.unige.ch/unige:123149

Disclaimer: layout of this document may differ from the published version.

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r(RT-)PCR assays

Viruses Detected

Sequence (5' to 3') Final [uM]

Fwd/Rev/

Probe

r(RT- )PCR anneling tempera

ture °C

Reference

Fwd primer Probe Rev Primer

Torque Teno

virus GTTTTCYACGCCCGTCC

FAM - ACTCACCTHCGGCACCCGC–

BHQ1

CCTTGACTYCGGTGTGTA

A 0.9/0.9/0.2 60

Masouridi-Levrat S, et al. Bone Marrow Transplant. 2016;51:440–442.

Adenovirus GCCCCAGTGGTCTTACA TGCACATC

FAM -

TGCACCAGACCCGGGCTCAGGTACTCCG A –TAMRA

gvGCCACGGTGGGGTTTC

TAAACTT 0.9/0.9/0.2 60 adapted from Verheyen J, et al.Appl Environ Microbiol 2009; 75: 2798–2801.

Merkel cell polyomavirus

CCCTTTGGAGCAAATTC CA

FAM -

CAAAATATCCACAAGCTCAGAAGTGA – TAMRA

CTGACCTCATCAAACATA GAGAA

0.6/0.6/0.2

5 55

Arvia R. et al. Journal of Virological Methods 246 (2017) 15–20

Human pegivirus-

1 GGCGACCGGCCAAAA

FAM -

TGACCGGGATTTACGACCTACCAACCCT

– TAMRA CTTAAGACCCACCTATAG

TGGCTACC 0.9/0.9/0.2 55

Chivero ET et al. The Journal of general virology.

2014;95:1307-19.

Epstein-Barr virus

CGGAAGCCCTCTGGACT TC

FAM -

TGTACACGCACGAGAAATGCGCCT – TAMRA

CCCTGTTTATCCGATGGA

ATG 0.2/0.2/0.1 60 Kimura H et al. J Clin Microbiol 1999: 37:132-6 Human herpes

virus 7 Genesig™ Detection Kit, Human Herpesvirus 7 unknown 60 PrimerDesign, England, United Kingdom

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0 50 100 150

Control Patient

Group

Millions Paired Reads

Type

DNA RNA

(4)

Negative_ctrl . CSF16 CSF17 CSF20 CSF21 CSF22 CSF23 CSF24 CSF25 CSF26 CSF27 CSF28 CSF29 CSF32 CSF34 CSF35 CSF36 CSF37 CSF01 CSF02 CSF03 CSF05 CSF12 CSF13 CSF14 CSF18 CSF30 CSF31 CSF33 CSF06 CSF08 ..

Xenotropic MuLV-related virus Polyomavirus 6

Human pegivirus-1 Human papillomavirus Molluscum contagiosum Merkel cell polyomavirus Herpesvirus 7

Gemycircularvirus

2

2 2 2 2 2 2 2 2 2 2

✔ ✔

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

2 2

# 2

# 2

# 2

# 2

# 2

# 2

# 2

# 2

# 2

# 2

# 2

# 1 #

= 0 Read ≤ 10 Reads ≤ 1.0e02 Reads ≤ 1.0e03 Reads ≤ 1.0e04 Reads ≤ 1.0e05 Reads ≤ 1.0e06 Reads > 1.0e06 Reads

1 : Most likely due to index hopping 2 : Probable contaminations

✔ : Positive by r(RT-)PCR

# : Undetected by r(RT-)PCR Groups :

Elective cesarean section

Elective urologic or digestive surgery Elective orthopedic surgery

#

(5)

.. ...1 ...2 ...3 ...4 ...5 P01 P03 P05 P07 P08 P15 P18 P30 P34 P09 P10 P11 P12 P14 P17 P20 P21 P27 P32 P36 P38 P13 P16 P24 P25 P28

Polyomavirus 6

Human papillomavirus Merkel cell polyomavirus Megavirus

Epstein-Barr virus Gemycircularvirus Bocavirus

Astrovirus MLB2 Anelloviridae

2

2 2 2 2 2 2 2 2 2 2 2 2 2 2

2

# 2

#

2

# 2

# 2

# 2

# 2

# 2

#

2

#

2

⌘

#

1

✔

✔ ✔ ✔ ✔ 1 ✔ ✔ ✔

= 0 Read ≤ 10 Reads ≤ 1.0e02 Reads ≤ 1.0e03 Reads ≤ 1.0e04 Reads ≤ 1.0e05 Reads ≤ 1.0e06 Reads > 1.0e06 Reads

1 : Most likely due to index hopping 2 : Probable contaminations

✔ : Positive by r(RT-)PCR

# : Undetected by r(RT-)PCR

⌘ : Insufficient volume to be tested by r(RT-)PCR Groups :

Encephalitis, Meningoencephalitis Meningitis

Meningomyelitis, Myelitis

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. ..1 ..2 ..3 ..4 ..5 ..6 ..7 ..8 ..9 ..10 P35 P04 P19 P37 P33 P26 P31 P29 P23 P02

Human pegivirus 1 Human papillomavirus Merkel cell polyomavirus Herpesvirus 7

Gemycircularvirus Anelloviridae

✔

2 2 2 2 2 2 2

2

# ✔ 2

# 2

#

#

⌘ = 0 Read ≤ 10 Reads ≤ 1.0e02 Reads ≤ 1.0e03 Reads ≤ 1.0e04 Reads ≤ 1.0e05 Reads ≤ 1.0e06 Reads > 1.0e06 Reads

2 : Probable contaminations

✔ : Positive by r(RT-)PCR

# : Undetected by r(RT-)PCR

⌘ : Insufficient volume to be tested by r(RT-)PCR

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