Whole-genome sequencing of human Pegivirus variant from an Egyptian patient co-infected with hepatitis C virus: a case report
Human pegivirus (HPgV) is structurally similar to hepatitis C virus (HCV) and was discovered 20 years ago. Its distribution, natural history and exact rule of this viral group in human hosts remain unclear. Our aim was to determine, by deep next-generation sequencing (NGS), the entire genome sequence of HPgV that was discovered in an Egyptian patient while analyzing HCV sequence from the same patient. We also inspected whether the co-infection of HCV and HPgV will affect the patient response to HCV viral treatment. To the best of our knowledge, this is the first report for a newly isolated HPgV in an Egyptian patient who is co-infected with HCV.
The deep Next Generation Sequencing (NGS) technique was used to detect HCV sequence in hepatitis C patient’s plasma. The results revealed the presence of HPgV with HCV. This co-infection was confirmed using conventional PCR of the HPgV 5′ untranslated region. The patient was then subjected to direct-acting-antiviral treatment (DAA). At the end of the treatment, the patient showed a good response to the HCV treatment (i.e., no HCV-RNA was detected in the plasma), while the HPgV-RNA was still detected. Sequence alignment and phylogenetic analyses demonstrated that the detected HPgV was a novel isolate and was not previously published.
We report a new variant of HPgV in a patient suffering from hepatitis C viral infection.
KeywordsHuman pegivirus HCV Deep NGS Egyptian patient
Basic Local Alignment Search Tool
Ethylenediamine tetraacetic acid
GB virus C
Hepatitis C virus
Next generation sequencing
Non-structural protein 5A
Non-structural protein 5B
Polymerase chain reaction
quantitative reverse-transcription PCR
Single nucleotide polymorphisms
There are several techniques that can be used in the detection of the novel virus . In the last decade, the next-generation sequencing (NGS) is a technology perform rapid sequencing  and offer a significant way for detecting known viruses, and for discovering novel viruses with several applications in clinical diagnosis . The deep-sequencing is a recently applied technique that yields millions of short sequences and enables investigation of viral diversity and quasi-species. In addition, it enables the simultaneous characterization of different pathogens . Hepatitis C virus (HCV) is an RNA virus that replicates in hepatocytes and liver tissues  causing acute, chronic hepatitis, and cirrhosis, which finally may lead to hepatocellular carcinoma . It has been showed that 10–20% of patients infected with chronic HCV are co-infected with human pegivirus (HPgV) in which both HCV and HPgV can be transmitted by parenteral route .
The HPgV, which is also known as GB virus C (GBV-C), is a member of the family Flaviviridae and the genus Pegivirus . It has a positive-sense RNA genome of ~ 9.3 kb that is translated to produce a single polyprotein . The polyprotein is cleaved by viral protease into smaller viral proteins including two putative envelope proteins (E1 and E2) and several nonstructural proteins (NS2–NS5). The coding region is flanked by long 5′ and 3′ untranslated regions (UTRs) . On the basis of phylogenetic comparisons six genotypes of HPgV have been identified, genotype-1 in West Africa, genotype-2 in North America and Europe, genotype-3 in Asia, genotype-4 in Southeast Asia, genotype-5 in South Africa, and genotype-6 in Indonesia [11, 12]. HPgV is a lymphotropic, non-pathogenic virus which is not associated with any known disease ; however its clinical significance still uncertain. It replicates in bone marrow, lymphoid tissue, and peripheral blood mononuclear cells, but is not thought to be hepatotropic  while others suggested its hepatotropicity and pathogenicity . Worldwide, the prevalence of HPgV varied from 0.5 to 4% in healthy adults, with much higher levels in particular risk groups, including HIV patients . In Egypt, the high prevalence of HPgV (61%) has been reported in multiple transfused children, and 15% in healthy controls . HPgV viremia can be cleared within the first year of infection followed by protection against reinfection, but it may persist for longer periods . In this case report, we find a new variant for HPgV in a patient suffering from HCV infection by using NGS.
Clinical features of plasma samples taken throughout the study
AST level (IU/ml)
R (up to 41)
ALT level (IU/ml)
R (up to 41)
10th April 2018
13th May 2018
13th June 2018
13th July 2018
Before the treatment, the blood sample was collected on EDTA containing tube. RNA sample library was prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA). RNA fragmentation, cDNA synthesis/indexing, PCR amplification/clean-up, and library normalization/pooling steps were conducted according to the manufacturer’s instructions. Sequencing was performed on a MiSeq sequencer with the MiSeq reagent kit v2 (300 cycles; Illumina), as described previously . Paired-end reads (2 × 150 nucleotide) were analyzed to identify the virus. An in-house workflow was used, as previously described . The identification on the HPgV isolate was done as followed: the first step in the pipeline included removal of the adaptor sequence from the reads and trimming low-quality bases using the program Fastx toolkit [http://hannonlab.cshl.edu/fastx_toolkit]. The next step was to map the reads to a database of viral sequences using the specific programs SNAP DB [https://www.freewarefiles.com/Snap-DB_program_86429.html] and BWA [http://bio-bwa.sourceforge.net]. The reads mapped to the same taxa were grouped together and assembled with SPAdes program, to yield a set of contigs. The Un-mapped reads (which could represent novel sub-sequences) were collected and assembled with the contigs to close any potential gaps and to improve the assembly. Then final contigs were aligned to the reference genome of each group using pairwise clustalw2. Furthermore, the whole viral genome sequences for the most significant group were collected from the GenBank database and the contig was aligned to each of them in order to check if the target sequence has better similarity to a sequence rather than those in the RefSeq database. The assembly yielded a single contig of 9370 bases for the HPgV sequence and another contig of 9291 bases for the HCV sequence, which was submitted to GenBank database and assigned with accession numbers MK234885 and MK799639, respectively. The contig was identified as HPgV by matching the sequences to the set of whole-genome pegivirus sequences in the GenBank database. The most significant hits were 75 sequences with a minimum average depth 100× and minimum 90% genome coverage (Additional file 1). The supplementary file also included the statistics for our sequence alignment and each pegivirus sequence in the hit list. These results indicate a high level of confidence in viral identification and give a strong hint regarding its genotype.
Discussion and conclusions
In spite of the discovery of the first pegiviruses for 20 years, the natural history of this viral group and its pathogenicity remains unclear. The pathogenicity of HPgV has caused many controversies. While some studies documented that HPgV is a lymphotropic virus and its infection considered nonpathogenic [14, 21, 22], others showed that it is a hepatotropic virus and pathogenic to humans and animals . This contradiction may be due to the occurrence of mixed infection between HPgV and other hepatitis viruses in which patients with single infection are rare . The advanced molecular studies (the sequence-specific, sequence-independent PCR-based approaches, and massive deep sequencing approaches) improved the understanding of genetic diversity of viruses. By using these approaches, we were able to sequence the new variant of HPgV that was present in the Egyptian HCV-infected patient. According to the bioinformatics analysis, we confirmed the presence of a new variant of HPgV genotype-2. The HPgV genotype-2 had been identified in North and South America, Europe, East Africa, and the Indian subcontinent .
At the end of the treatment, the patient responded to the DAA treatment in spite of being infected with HPgV. In the present case, the co-infection of HPgV with HCV didn’t result in a significant change in the clinical presentation, biochemical changes, viral load or response to treatment . Daclatasvir and sofosbuvir are being used as inhibitors of the NS5A and NS5B proteins, of HCV respectively , since both of them have key roles in the replication of HCV RNA . The differences in treatment response between HPgV and HCV infected patient may be due to the different structures of NS5A and NS5B in the two viruses. To the best of our knowledge, this is the first study from Egypt and the Middle East to analyze the whole genome sequence of HPgV using deep sequencing technique.
In conclusion, this is the first study from Egypt and the Middle East reporting the presence of a new variant of HPgV genotype-2 in Egyptian patient infected with HCV. The whole-genome sequencing identified this isolate as a novel variant of HPgV, and provided evidence that co-infection by HPgV and HCV has no influence on patients’ response to HCV treatment.
This study was conducted using MIseq device, Illumina, USA at NGS laboratory, the Egyptian Pediatric Cardiology Center of Excellence (STDF), Cairo University Children’s Hospital. The authors would like to thank Dr. Amira Salah El-Din Youssef from the Cancer Biology Department, National Cancer Institute, Cairo University for constructing the phylogenetic tree. All authors revised the final version of the manuscript and declare that they have no conflict of interest.
Hany K. Soliman, Mohammed M. Hafez, and Zeinab K. Hassan carried out the practical work, data analysis wrote and edited the manuscript. Mahmoud N. El Rouby and Yehia A. Osman participated in coordination and supervised the work. Dina A. Mehaney, Rania K. Darwish and Ola S. Ahmed participated in practical work, Gamal E carried out the treatment and the follow-up. Manojkumar Selvaraju retrieved sequences from the NCBI website. Mohamed A. Hoda participated in data analysis. Abdel-Rahman N. Zekri participated in the study design and coordination and supervised all the work. All authors read and approved the final manuscript.
This work was funded by Cairo University and a grant from the Science & Technology Development Fund in Egypt to Abdel-Rahman N Zekri (Grant number 5193).
Ethics approval and consent to participate
The study was ethically approved by the Institutional Review Boards (IRB) of the National Cancer Institute, Cairo University. Organization No·IORG0003381 (IRB NO·IRB00004025).
Consent to publication
The patient provided written informed consent for the publication of this case report.
The authors declare that they have no competing interests.
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