First detection of human hepegivirus-1 (HHpgV-1) in Iranian patients with hemophilia

A novel blood-borne virus called the human hepegivirus 1 (HHpgV-1) was recently discovered in hemophilia patients. The present study aimed to investigate the presence of HHpgV-1 in hemophilia patients. A total of 436 serum samples were investigated for the presence of hepatitis C virus (HCV), human pegivirus-1 (HPgV-1), torque teno virus (TTV), and HHpgV-1. Out of the 436 patients, 163 (37.4%), 19 (4.4%), 76 (17.4%), and four (0.9%) patients were positive for HCV, HPgV-1, TTV, and HHpgV-1, respectively. HHpgV-1 patients had a mean viral load of 4.9 ± 0.3 log RNA copies/mL and were co-infected with HCV-1a, HPgV-1, and TTV. Moreover, three HHpgV-1-positive patients exhibited stage F0 liver fibrosis. HCV viral load in HHpgV-1-positive patients was lower than those of HHpgV-1-negative patients. Results also revealed that co-infection of HHpgV-1 with HPgV-1 and HCV may play a protective role in patients with chronic HCV. In conclusion, we detected a low frequency of HHpgV-1 infection in hemophilia patients, and results suggested that HHpgV-1 infection was correlated with the presence of other blood-borne viruses and is likely to also correlate with low HCV viral load and reduced severity of liver disease. Additional studies are required to further investigate the clinical importance of HHpgV-1.


Discussion
This is a preliminary study to provide first evidence of the frequency of HHpgV-1 as new blood-borne virus in Iranian patients with hemophilia. Subsequent specific semi-nested PCR screening of hemophilia samples from 432 subjects failed to detect any HHpgV-1-positive samples. HHpgV-1 was also infrequently identified in four hemophilia patients, and subsequently, they were confirmed using Illumina sequencing. This result was in keeping with that of three previous studies, which detected low frequency of HHpgV-1 in hemophilia patients 5,14,15 .
In the current study, four positive samples had co-infection of HHpgV-1 with HCV, HPgV-1, and TTV. This result is completely consistent with data from four published reports, in which HHpgV-1 was mainly or only identified in serum containing HCV, HPgV-1 RNA, and other blood-borne virus. These results indicate the conclusion that HHpgV-1 circulates less frequently in humans but especially in patients with HCV infection is relatively frequent 5,[13][14][15] .
Compared to HCV and HPgV-1, the low frequency of detection of HHpgV-1 in hemophilia patients as a high-risk group may be described by the fact that (I) HHpgV-1 is an uncommon viral infection in humans, (II) HHpgV-1 is less persistent, (III) due to the limited sample numbers and areas tested, or (IV) the PCR-based screening method was incapable to amplify sequences of more genetically divergent variants of HHpgV-1 5 . Whole-genome sequencing has been demonstrated to have high accuracy and sensitivity for the detection of new viral RNA, such as HHpgV-1 RNA, in human blood, which can be highly divergent and indiscernible using  conventional PCR techniques 14,15 . One limitation of the present study is that not all samples were subjected to whole-genome sequencing because of limited funding. After discovering HCV in 1989, HPgV-1 in 1995, TTV in 1997, TTV-like minivirus (TLMV), and SEN virus (SENV) in 2000, no more transfusion-associated virus infections have been reported 16 . In addition, many cases of post-transfusion hepatitis with unknown etiology still occur among blood-transfusion recipients. However, chronic HCV infection is a major health problem and leads to liver cirrhosis, hepatocellular cancer, liver transplantation, and death [17][18][19][20][21] . In return, it was reported that most HPgV-1 infections were usually asymptomatic, transient, and self-limiting 22 .
Interestingly, several studies have also shown that co-infection with HIV and HPgV-1 can delay progression to acquired immune deficiency syndrome (AIDS), which can be potentially explained by reduced viral replication and/or impairment in the host immune system [23][24][25] . Moreover, previous studies have revealed that co-infection with Ebola and HPgV-1 viruses was correlated with improved patient survival 26 .
In the current study, the mean HCV viral load in HHpgV-1-positive patients was lower than that in HHpgV-1-negative patients and three patients were in the F0 liver disease stage. Results suggested that co-infection of HCV with HHpgV-1 and HPgV-1 caused a reduction in HCV viral load and also decreased the severity of liver damage in chronic HCV. Berzsenyi et al. showed that HPgV-1 infection in chronic HCV was correlated with reduced liver disease severity 27 . Moreover, Kandathil et al. showed similar results that is, no evidence that HHpgV-1 leads to liver disease 15 . HHpgV-1, like HPgV-1, is presumably not a significant cofactor in the development of HCV-related liver disease. These results give us with clues about the medical consequences of HHpgV-1 infection, raising the eventuality that HHPgV-1 infection may lead in limited or absence of pathogenicity 13 . However, further studies are required to verify the mechanisms underlying the interactions of HCV with HHpgV-1 and HPgV-1.
In a very recent study, Wang et al. reported the tight association HHpgV-1 with HCV. Also, in line with our study, HHpgV-1 seems not to worsen HCV-related liver fibrosis. As a result, they showed that HHpgV-1 could effect on the replication and pathogenesis of HCV 13 .
Whether HHpgV-1 plays an important role in the development of human hepatitis remains unclear. HHpgV-1 has the characteristics of both hepaciviruses and pegiviruses, thereby increasing the risk of chronic infections like HCV and HPgV-1 infection. Given that the effects of HHpgV-1 on human health have not been well characterized, a more precise evaluation of HHpgV-1 frequency in human populations is warranted 28 .
HHpgV-1 is likely to be infectious and can potentially be added to the list of viral infections that are investigated at Hemotherapy Services; further experiments can be conducted to prove this issue 16 . Historically, the prevalence of HCV infection was underestimated and was resolved only when the full range of the genetic variation of HCV was characterized. However, continued efforts to recognize more cases of HHpgV-1 infection are ongoing, for better understanding of the epidemiology, pathogenesis, and clinical relevance of HHpgV-1 11 .
In conclusion, our results demonstrated that the low prevalence of HHpgV-1 infection in hemophilia patients may be correlated with ongoing HCV, HPgV-1, and TTV infection and may be associated with lower HCV viral load and reduced liver disease severity. However, additional studies are required to further characterize the clinical importance of HHpgV-1.

Patients and Methods
Study population. The current study was performed at the Pasteur Institute of Iran (PII) from October 2016 to August 2017 and conducted in accordance with the 1975 Declaration of Helsinki and relevant local regulations. The study was approved by the Ethical Committee of PII, and written informed consent was directly obtained from all participants. Serum samples from a total of 436 hemophilia patients were collected at the Iranian Comprehensive Hemophilia Care Center (ICHCC) and PII. Severity and staging of liver disease patients were extracted from the patient records. Patients were classified into the following five groups based on the severity of fibrosis: no fibrosis (F0), portal fibrosis alone (F1), portal fibrosis with rare septae (F2), portal fibrosis with many septae (F3), and cirrhosis (F4) 6 . Aspartate Aminotransferase (AST), alkaline phosphatase (ALK), and Alanine Aminotransferase (ALT) were detected according to laboratory protocols. All patients were negative for both hepatitis B surface antigen (Radim SPA, Pomezia-Rome, Italy) and HIV antibodies (Radim SPA, Pomezia-Rome, Italy). DNA and RNA extraction. Total RNA was extracted using the High Pure Viral RNA Kit (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) and cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics Deutschland GmbH, Mannheim, Germany), according to the manufacturer's instructions. TTV DNA was extracted using High Pure PCR Template Preparation Kit (Roche Diagnostics Deutschland GmbH, Mannheim, Germany), according to the manufacturer's instructions.
HCV detection and genotyping. A nested PCR assay was prepared for detection of HCV using the primer pair HCV-251F (5′-AGCGTCTAGCCATGGCGT-3′) and HCV-22R (5′-GCACGGTCTACGAGACCT-3′) during in the first round of PCR (264 bp) and HCV-FN-F2 (5′-GTGGTCTGCGGAACCGG-3′) and HCV-RN-R2 (5′-GGGCACTCGCAAGCACCC-3′) during the second round of PCR (174 bp). PCR conditions for the first round were 95 °C for 5 min, followed by 30 cycles of 94 °C for 45 s, 59 °C for 45 s, and 72 °C for 45 s, and 72 °C for 5 min as a final extension. PCR conditions for the second round were to the same as the first, but annealing temperature (62 °C) was different. PCR-restriction fragment length polymorphism assay (PCR-RFLP) was used to detect HCV genotypes. HCV-genotyping by RFLP assay has also been confirmed by sequencing of the 5′ non-coding region fragments 7 .