Human pegivirus (HPgV, GBV-C) RNA prevalence, genome characterization and association with HIV coinfection among volunteer blood donors from a public hemotherapy service in Northern Brazil.

Background Human pegivirus (HPgV) - formerly known as GBV-C - is a member of the Flaviviridae family and belongs to the species Pegivirus C. It is a non-pathogenic virus and is transmitted among humans mainly through the exposure to contaminated blood and is often associated with human immunodeficiency virus (HIV) infection. This study aimed to determine the prevalence of HPgV viremia, its association with HIV and clinical epidemiological factors, as well as the full-length sequencing and genome characterization of HPgV recovered from blood donors of the HEMOPA Foundation in Belém-PA-Brazil Methods Plasma samples were obtained from 459 donors, tested for the presence of HPgV RNA by the RT-qPCR. From these, a total of 26 RT-qPCR positive samples were submitted to the NGS sequencing approach in order to obtain the full genome + . Genome characterization and phylogenetic analysis were conducted. its circulating strains among the Brazilian population, particularly blood donors. This study aimed to determine the prevalence of HPgV viremia, its association with HIV and clinical epidemiological factors, as well as the complete genome characterization of HPgV present in blood donors of the HEMOPA Foundation in Belém-PA-Brazil.

genome is similar to the genome of the hepatitis C virus and contains a single open reading frame (ORF) located between the untranslated regions (UTRs) at the 5¢ and 3¢ ends of the viral genome.
The 5¢-NTR region is highly conserved with an internal ribosome entry site (IRES) and is responsible for the initiation of the translation of the viral RNA, resulting in the synthesis of a polyprotein of approximately 3,000 amino acid residues. Through the action of cellular peptidases and viral proteases, the polyprotein is cleaved to produce eight mature yet incompletely characterized proteins, including the two structural (E1 and E2) and seven non-structural (NS) proteins [2][3][4].
HPgV is transmitted among humans mainly through exposure to contaminated blood. This transmission profile deems HPgV as a common coinfection with other viruses such as HIV-1, hepatitis C virus (HCV), and Ebola virus [5][6][7]. Up to 40% of the individuals infected with HIV and/or HCV are positive for HPgV infection [8,9] People HIV-1 co-infected with HPgV experience slower disease progression that may be influenced by the interference of HPgV on the pathogenicity of HIV-1, however, the mechanism by which HPgV mediates this protective effect still remains unknown [10,11].
Several studies carried out in different populations in the last decades in Brazil have shown varying prevalence rates [12,13]. In studies among healthy blood donors conducted in Brazil, prevalence rates of 19.5% and 9.7% were observed among individuals with prior exposure and active infection, respectively [14]. However, the most significant prevalence has been reported among patients with HIV, with a value reaching up to 34% [15].
The prevalence of the virus is lower in the developed countries (1-5%) than in the developing countries -(approximately 20%), with South America exhibiting a prevalence rate of up to 14.6% among blood donors [4]. Seroprevalence studies in Brazil reveal the presence of anti-E2 antibodies in 19.5% of healthy blood donors [16], however, little is known about the prevalence of HPgV viremia and its circulating strains among the Brazilian population, particularly blood donors.
This study aimed to determine the prevalence of HPgV viremia, its association with HIV and clinical epidemiological factors, as well as the complete genome characterization of HPgV present in blood donors of the HEMOPA Foundation in Belém-PA-Brazil.

Methods
Blood donors and the collection of serum samples A cross-sectional study was performed in order to determine the prevalence of HPgV infection among blood donors from the HEMOPA Foundation between March 2017 and April 2018. Epidemiological data were obtained through access to the HEMOPA Foundation donor registry. The sample size was calculated using EpiInfo™ software [17] based on the presumed prevalence of 5-10% of HPgV in Brazil [15,18]. For this calculation, the number of blood donors registered in 2016 at the HEMOPA Foundation (63,501), 95% confidence level, and 20% margin adjustment was used to obtain a total of 366 individuals. A total of 459 serum samples (400 µL) from the blood donors from the HEMOPA

Sequencing
The RNA, obtained in the nucleic acid extraction step, was quantified in Qubit 2.0 fluorometer (Thermo Fisher Scientific), using the QubitTM RNA HS Assay Kit 500 assays (Invitrogen by Thermo Fisher Scientific). Then, cDNA was synthesized using the cDNA Synthesis System Roche® kit (Roche Applied Science), as described by the manufacturer. The subsequent step was the quantification of cDNA using the qubit 2.0 fluorometer (Thermo Fisher Scientific), using the QubitTM dsDNA HS Assay Kit (Invitrogen by Thermo Fisher Scientific) and analysis of cDNA integrity in the equipment 2100 Bioanalyzer (Agilent Technologies) using the high sensitivity DNA reagents kit (Agilent Technologies).

Genome characterization and phylogenetic inference
The HPgV genomes, identified by the Blastx algorithm, were used for predicting the coding region (Open Reading Frame; ORF), as well as the 5'-UTR and 3'-UTR regions using the Geneious v9 tool.
Viral genomes were aligned with other HPgV complete genomes available in GenBank database using the MAFFT v7 software [27]. For phylogenetic analyses, ORFs from aligned genomes were used to perform the phylogenetic reconstructions using the maximum-likelihood method, generated by RAxML v.8.2.12 [28], applying 1000 bootstrap replicates [29] and the best nucleotide replacement model calculated by JModelTest [30]. Complete genome sequences of HPgV obtained in this study have been submitted to GenBank (accession numbers MN215894-MN21591).

Statistical analysis
The differences between the groups were analyzed with the chi-square test, G test of independence, Student's t-test, and odds ratio. The level of significance of α = 0.05 was adopted for the rejection of the null hypothesis. Statistical analyses were performed using the BioEstat program version 5.  Table 1). with HPgV genotype 2 sequences (Fig. 1).

Discussion
The prevalence of HPgV-1 among blood donors was 12,4%, which is consistent and not significantly different from the expected prevalence in developing countries (up to 20%) [4,31]. The prevalence calculated in this study was 2,8% higher than reported Slavov et al. [32] [39]. Another issue to consider is that sexual activity is more evident among young people so this population has shown a greater risk to be infected with HPgV, as suggested Da Mota et al. [15] and Miao et al [40].
The prevalence of HPgV among the individuals diagnosed with HIV-1 in this study was 9,7% higher than that reported by Miranda et al. [38]. The high prevalence of HPgV among HIV-1 individuals has been reported in several studies in Brazil and the world [40][41][42]. The association between the presence of HPgV and HIV is owing to the fact that HPgV likely acts as a protective factor for the development of HIV [40,43,44].
HIV-1 infected people have reduced mortality when co-infected with HPgV, nonetheless the mechanism by which HPgV mediates this protective effect remains unknown [45,46]. Nevertheless, the present study showed no evidence of viral load value that corroborated with the protective effect of HPgV in the evolution of HIV, instead, HIV-1 viral load in the coinfected group (HIV-1 + HPgV positive) was 0.72 Log 10 (p = 0,002) higher than in a monoinfected group (HIV-1 positive). One interpretation of this finding is that the increase in T cells during the expansion phase of viral infection leads to an increase in both viral loads [47,48]. Another consideration is that all individuals in our sample were newly diagnosed with HIV during the acute phase, suggesting that HPgV does not exert a protective effect on the pathogenesis of HIV during the acute phase of HIV infection as suggested Bailey et al. [49]. We hypothesize that HIV-1 would have an advantage in lymphocyte infection since HPgV may infect the same cells as HIV-1 [50].
The phylogenetic analysis revealed the presence of genotype 2 and the subtypes 2a and 2b in the studied population. These findings corroborate previous studies that identified these same genotypes in other regions of Brazil [18,33,41] and in Brazilian Amazon [32].
HPgV is known as a non-pathogenic virus and is not part of the routine diagnosis in the HEMOPA

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Competing interests
The authors declare that they have no competing interests

Authors' contributions
AS contributed to the conception of the work; acquisition, analysis and interpretation of data for the work; revising the work critically for important intellectual content. CS worked on the acquisition, analysis and interpretation of data RB contribuited to the acquisition, analysis and interpretation of the data. PS and PM contributed on the acquisition of data. LL and RB contributed to the conception of the work; acquisition, analysis and interpretation of data for the work; revising it critically for important intellectual content. MN and PM contributed to the conception of the work; acquisition, analysis and interpretation of data for the work; revising it critically for important intellectual content.
All authors read and approved the final manuscript. Figure 1 Phylogenetic tree of Human pegivirus (HPgV) generated using RAxML with the GTR+I+G+F nucleotide substitution model using 1000 bootstrap replicas displaying only values greater than 50.