








key: cord-291553-j9nn5g70
authors: Fridholm, Helena; Østergaard Sørensen, Line; Rosenstierne, Maiken W.; Nielsen, Henrik; Sellebjerg, Finn; Bengård Andersen, Åse; Fomsgaard, Anders
title: Human pegivirus detected in a patient with severe encephalitis using a metagenomic pan-virus array
date: 2016-01-29
journal: J Clin Virol
DOI: 10.1016/j.jcv.2016.01.013
sha: 
doc_id: 291553
cord_uid: j9nn5g70

We have used a metagenomic microarray to detect genomic RNA from human pegivirus in serum and cerebrospinal fluid from a patient suffering from severe encephalitis. No other pathogen was detected. HPgV in cerebrospinal fluid during encephalitis has never been reported before and its prevalence in cerebrospinal fluid needs further investigation.

Metagenomic methodologies are excellent complement in cases where a diagnosis is difficult to establish with conventional laboratory tests and its usage is ever increasing. Metagenomic approaches reveal presence of both pathogenic and commensals in patient samples where the focus is on identifying an underlying ethological agent for a specific disease condition. We report a case of severe encephalitis where the only microbe detected in the CNS was human pegivirus (HPgV), hitherto only known to cause asymptomatic infections in humans. One previous report describes the detection of HPgV in brain tissue and CSF [1] . In both cases it is uncertain if HPgV is pathogenic but it is noteworthy to detect a virus at a high viral load in the CNS. In other cases, HPgV infections have been associated with beneficial outcomes in patients dually infected with HPgV and HIV or Ebola [2] [3] [4] [5] .

A 25-year old Danish female was admitted to the hospital for abdominal pain, vomiting, dizziness and lower extremity pain.

She was working as a bartender on a cruise ship, was sexually active but had no travel history outside Scandinavia or exposure to blood transfusions, intravenous drugs or close contact to animals albeit recently received a tattoo. Her past medical history included radiofrequency catheter ablation for Wolf-Parkinson-White syndrome and she awaited elective cholecystectomy due to prior gallstone attacks. Upon admission she was alert and circulatory stable with a fever of 38.5 • C, Glasgow coma score of 15 and a BMI of 20. Routine blood test showed haemoglobin of 8.0 mmol/L, white blood cell count of 2.5 × 10 9 /L with lymphocytopenia, normal platelet count, c-reactive protein (CRP) of 33 mg/L, alanine aminotransferase 270 U/L, lactate dehydrogenase 281 U/L, alkaline phosphatase 110 U/L and bilirubin of 8 mol/L. An acute laparoscopic cholecystectomy was performed but no pathology of the gall bladder was found. Immediately following the surgery she developed increasing abdominal pain and fever and she underwent an explorative laparoscopy, again with normal findings. Post-operatively the patient complained of headache and diplopia, which both disappeared within 24 h and she was discharged from the hospital. However, she was readmitted the following day with fever, headache, sudden behavioral change, photosensitivity and ataxia. She presented with somnolence and neck stiffness on physical examination (Glasgow coma score 14). Vital signs were within normal limits but she had a fever of 38.0 • C. Peripheral blood showed CRP of 17 mg/L, white blood count 3. pleocytosis of 150 cells/mm 3 (99% lymphocytes) and increased protein concentration of 3.5 g/L, indicative of a viral infection. An MRI of the brain revealed leptomeningeal enhancement over the right hemisphere together with parenchymal changes, consistent with meningoencephalitis. She was treated with aciclovir for suspected viral encephalitis and with meropenem for possible bacterial infection.

Over the following days the patient worsened with mental status deterioration and progressed into coma and was transferred to an intensive care unit for mechanical ventilation. Repeated lumbar puncture on day 9 disclosed an increase of mononuclear cells to 333 leukocytes/mm 3 (99% lymphocytes) with a protein concentration of 2.8 g/L. She received dexamethasone, methylprednisolone and later prednisolone. Serum and CSF were tested for relevant pathogens, all returned negative ( Table 1) . Because of the lack of a specific diagnosis serum and CSF were sent to Statens Serum Institut, Copenhagen, where the specimens were run on a Lawrence Livermore pan-microbial array. This array contains 360000 probes against all sequenced bacteria and viruses present in the NCBI database as of 2010 [6, 7] . The only positive signal was for human pegivirus (HPgV) (Acc. nr. GSE67021), and two separate diagnostic laboratories subsequently confirmed HPgV RNA in both serum and CSF [8, 9] . The Ct value for HPgV during the acute phase in serum and spinal fluid was 23.4 and 32.1, respectively. An RNAseq library was prepared from serum and sequenced on an Illumina platform to obtain type information. The reads mapped to the entire reference genome (Acc. nr. NC 001710) with a mean sequence depth of 48, a pariwise identity of 89.7% (nt) and 98,3% (aa), respectively. The assembled sequence (Acc. nr. KP259281) clustered within genotype 2 [10] . After eight days with severe neurological symptoms the patient gradually recovered and was discharged from the hospital four weeks later for rehabilitation. Five weeks after discharge she was still viremic for HPgV in serum but viral load had decreased 21 times (Ct 27.8).

HPgV is a flavivirus, characterized by enveloped virions with a single-stranded, positive-sense RNA genome and is closely related to Hepatitis C virus [11] . Upon discovery HPgV was initially thought to cause acute hepatitis but this has later been disproven and an HPgV infection has not been linked to any clinical disease in humans [12, 13] . On the contrary, an infection has been associated with a beneficial outcome in HIV patients and also recently in ebola infected individuals [2, 4] .

In 1998 Radkowski et al. screened 17CSF samples from patients suffering from aseptic meningitis or encephalitis by RT-PCR but could not detect HPgV in any of the samples [14] . More recently, both positive and negative RNA-strands of HPgV was detected in post mortem brain tissue from a multiple sclerosis (MS) patient, implying that the virus was replicating in the brain tissue [1] . The authors also detected HPgV in the CSF of the same patient, but screening of CSF from an additional 27 MS patients were all negative.

In developed countries 1-4% of healthy blood donors are viremic for HPgV [15] [16] [17] [18] [19] [20] (2.2% in Denmark) [21] whereas in developing countries and for people with blood-borne or sexually transmitted diseases the prevalence reaches 20-40% [11] . Clearance of HPgV coincides with the appearance of HPgV E2 antibodies [22] [23] [24] and healthy individuals normally clears the infection within two years. Between 5-20% of the population are seropositive against the glycoprotein E2 reflecting a previous infection [11, 21] .

HPgV is not routinely tested for during illnesses of the CNS and it was surprising to detect it in CSF of our patient as the only positive finding. We analyzed an additional 40 CSF samples from encephalitis patients and 13 from patients with a relapse of MS for HPgV by qPCR. For 20 of the encephalitis samples we tested serum collected concurrently with the CSF sample. All CSF samples where negative for HPgV but one encephalitis patient was positive in serum (Ct 27.2). Interestingly, this patient also had CSF pleocytosis, as our case patient. As HPgV is lymphotropic [25] the presence of HPgV in the CSF could be attributed to passive transport of the virus as the cells are recruited to CSF. It is uncertain if the HPgV infection and its presence in CSF in any way influenced the clinical presentation but this possibility cannot be excluded. A few weeks after discharge she was still viremic for HPgV but the viral load in serum had decreased from Ct 23.4 to 27.2.

It seems that on rare occasions HPgV can enter the CNS in high viral titers but it is unknown for how long it persists. Clearance of virus from the CSF is not a well understood process. Herpesvirus (CMV, EBV, VZV and HSV 1/2) and flavivirus (TBEV) can persist in individuals after encephalitis and may cause recurrent encephalitis. We have also excluded the possibility that the encephalitis is a result of a reactivation event due to the surgical procedure as the patient was negative for HSV and the symptoms coincided with the time of surgery.

Several viruses, bacteria and antibodies are known to be able to induce encephalitis but in many clinical cases the etiology remains unknown [26, 27] . Metagenomic methodologies target a multitude of microbes simultaneously thereby reducing the number of tests and the total cost. Due to their unbiased presentation of the nucleic acids present in a sample they have an unprecedented potential as a diagnostic tool for differential diagnosis. NGS has been used to resolve the presence of Leptospira santarosai [28] and an astrovirus [29] in CSF in cases of encephalitis, where comprehensive microbiological testing was inconclusive. More precise interpretations need to be developed as both pathogenic and non-pathogenic microbes will yield a signal and perhaps disclose known virus with unexpected pathogenetic potential. Whether presence of HPgV in CSF affects disease progression remains to be established but these findings needs to be reported as they add to our knowledge of the microbial flora and aids in pinpointing which microbes warrants further attention and study.

Deep sequencing for the detection of virus-like sequences in the brains of patients with multiple sclerosis: detection of GBV-C in human brain

GB virus C: the good boy virus?

Diagnostic value of anti-GBV-C antibodies in HIV-infected patients

GB virus C coinfections in West African Ebola patients

Acquisition of GB virus type C and lower mortality in patients with advanced HIV disease

The microbial detection array for detection of emerging viruses in clinical samples-a useful panmicrobial diagnostic tool

A microbial detection array (MDA) for viral and bacterial detection

Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions

Hepatitis C virus and GB virus C/hepatitis G virus viremia in Swedish blood donors with different alanine aminotransferase levels

Evidence for extensive genotypic diversity and recombination of GB virus C (GBV-C) in Germany

The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae

G-pers creepers, where'd you get those papers? A reassessment of the literature on the hepatitis G virus

GB virus-C-a virus without a disease: we cannot give it chronic fatigue syndrome

Lack of GB virus C/hepatitis G virus sequences in cerebrospinal fluid in patients with central nervous system infections

A case-control study of transmission routes for GB virus C/hepatitis G virus in Swedish blood donors lacking markers for hepatitis C virus infection

GB virus C/hepatitis G virus infection in patients investigated for chronic liver disease and in the general population in southern Sweden

Prevalence of GB virus C (also called hepatitis G virus) markers in Norwegian blood donors

Seroprevalence of GB virus C and persistence of RNA and antibody

A novel T cell evasion mechanism in persistent RNA virus infection

Humoral immune response to the E2 protein of hepatitis G virus is associated with long-term recovery from infection and reveals a high frequency of hepatitis G virus exposure among healthy blood donors

GB Virus C epidemiology in denmark: different routes of transmission in children and low-and high-risk adults

Detection of antibodies to a putative hepatitis G virus envelope protein

Antibody to GBV-C second envelope glycoprotein (anti-GBV-C E2): is it a marker for immunity?

Prevalence of GBV-C/hepatitis G virus RNA and E2 antibody among subjects infected with human immunodeficiency virus type 1 after parenteral or sexual exposure

Evidence that the GBV-C/Hepatitis G virus is primarily a lymphotropic virus

Causes of encephalitis and differences in their clinical presentations in England: a multicentre, population-based prospective study

Etiology of encephalitis in Australia

Actionable diagnosis of neuroleptospirosis by next-generation sequencing

Diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing

The authors declare no conflicts of interests.

This study was partially funded by Familiefonden Egetrae.

