A panel of 30 top gastroenterologists of the country met in 2004 at a conference and reported that 75%-90% of HCV Pakistani patients were harboring genotype 3a, followed by genotype 1, also confirmed in current and in other different Pakistani population.

The prevalence was higher among the males. High prevalence in male as compared to female could be due to their exposure to various HCV risk factors particularly barber community and multiple sexual exposures. In current study 3 was more prevalent in females and 1 in male, as stated in a study that genotype 1 was more common in male as compared to females. For HCV prevalence and genotype distribution statistically no significant difference was found between male (57.14%) and female patients, verified by other study. Old age groups were found more infected with HCV. It has been suspected that fragile health structure, unsterilized instruments and use of contaminated razor by barbers may be contributing to the spread of HCV.

Blood transfusion(s) constitute a part of treatment in many HD patients and thus exposed greatly to HCV [2,14,29]. The risk of hepatitis transmission through blood transfusion is considered to be high in Pakistani population due to a lack of appropriate screening of blood in past. Several studies confirmed the prevalence of HCV among people with a history of blood transfusion before the advent of blood screening procedures in Pakistan. Percentage prevalence of HCV was 4.95% ± 0.53% in the general adult population and 7.94 ± 1.49% in multi transfused population. The prevalence found in this study (75%) was greater than that estimated for general adult population in the country. The multitransfused patients in this study was more prone to HCV, also supported by various studies but could not recognizes as independent risk factor in other studies.

Needle sharing and household contacts were not significant risk factors in this study, similar to other literature. There is an indication that environment of dialysis treatment itself function as a vehicle in dissemination of HCV among HD patients. A high prevalence of patients with HCV infection in HD facilities has been considered a risk factor for transmission of the infection. Several reports have linked a high incidence of HCV infection in dialysis patients who shared dialysis machines in dialysis unit.

Study Sample and Data Collection

384 HD patients were randomly selected collected from three hospitals of Peshawar, Khyber Pakhtunkhwa: Khyber Teaching Hospital, National Diagnostic Dialysis Center and Dialysis Ward Hayatabad Medical Complex. All patients were briefed about the study and proper willing consent was signed.

All patients were interviewed for demographic data and risk factors to HCV infections including history of number of blood transfusion, intravenous drug use (IDU), surgical interventions, and dental treatment, multiple sexual partners, barber shop, piercing instruments and exposure to known HCV-positive persons, number of years on dialysis and change of the center. Blood (5CC in sterile syringes) were collected from HD patients; sera were separated in two aliquots and frozen at -70°C for HCV RNA detection and genotyping.
Antibodies Screening

All the subjects were screened for anti HCV antibodies third generation test according to the manufacture instructions (Accurate Diagnostics USA).
RNA Isolation, PCR amplification and Detection

RNA was isolated from 100 μL serum, using RNAzole RNA purification kit (Trizole Inc. USA) as per manufacturer protocols. HCV RNA was Reverse transcribed with 200 U of Maloney murine leukemia virus reverse transcriptase (Fermentas USA) and amplified with nested primers specific for 5′ untranslated region of HCV genome. Amplified product was subjected in 2% agarose gel for electrophoresis.

HCV genotyping

HCV RNA positive samples were genotyped with primers specific for core region of different HCV types by type specific PCR as mentioned elsewhere.

For contamination control RNA extraction, cDNA amplification and electrophoresis were carried in separate areas. Both negative and positive controls were run parallel to the patient samples in each batch.

Methods

Polymerase chain reaction was performed for HCV RNA detection and genotyping in 384 HD patients. The data obtained was compared with available past studies from Pakistan.

Results

Anti HCV antibodies were observed in 112 (29.2%), of whom 90 (80.4%) were HCV RNA positive. In rest of the anti HCV negative patients, HCV RNA was detected in 16 (5.9%) patients. The dominant HCV genotypes in HCV infected HD patients were found to be 3a (n = 36), 3b (n = 20), 1a (n = 16), 2a (n = 10), 2b (n = 2), 1b (n = 4), 4a (n = 2), untypeable (n = 10) and mixed (n = 12) genotype.

Conclusion

This study suggesting that i) the prevalence of HCV does not differentiate between past and present infection and continued to be elevated ii) HD patients may be a risk for HCV due to the involvement of multiple routes of infections especially poor blood screening of transfused blood and low standard of dialysis procedures in Pakistan and iii) need to apply infection control practice.

Introduction

Hepatitis C virus (HCV) infection is a major public health problem, with an estimated global prevalence of 3% occurring in about 180 million carriers and approximately 4 million people have been newly infected annually. The prevalence of HCV infection among dialysis patients is generally much higher than healthy blood donors and general population. Studies held in dialysis centers from different countries revealed that prevalence ranges form 1-84.6% and there is a particular concern because HCV chronic infection causes significant morbidity and mortality among patients undergoing hemodialysis (HD).

In Pakistan currently, approximately 10 million people are suffering from this tremendous disease which cover 6% of the overall population. A high prevalence of HCV Ab (38% weighted average) was described in the studies of patients undergoing chronic dialysis in Pakistan. The spread of HCV in Pakistan is fuelled due to lack of education and awareness of disease, shortage of medically qualified and scientifically trained health care workers especially dentists, lack of health infrastructure such as use unsterilized instruments, use of high numbers of therapeutic injections and practice of daily face and armpit shaving in community barber shops. New HCV infection was evidently more frequent at dialysis centers with higher anti-HCV prevalence and failure in infection control measures. In some countries, both prevalence and incidence remain very high, indicating major ongoing nosocomial transmission, probably due to the limited resources available to treat a rapidly growing HD population.

Risk assessment for HCV infection

The frequency distribution of genotypes in relation to age revealed that genotype 3a was most common in nearly all age groups while 3b and 2a were comparatively less frequent. Age group with > 46 years were more affected with mixed genotype while untypable genotype were more common in age group < 45 years. Genotype 3 (a and b) were common in female as compare to male while genotype 1a, untypable and mixed were more common in male as compared to female. Genotype 1b and 4a were found only in male. Prevalence of genotype subtypes within age groups (χ2 = 22.076, p = 0.004) were found significant while gender (χ2 = 42.48, p = 0.0113) and marital status (χ2 = 62.803, p = 0.247) of the patients were not statistically significant.

Mixed genotype was more prevalent in HD patients with history of blood transfusion and barber shops. Mixed and untypable genotype was found significantly more often in HD patients with history of mean number of 2.28 blood transfusions and genotype 3a, 3b and mixed genotype were found more commonly in patients with mean number of 4.61 blood transfusions. IDUs were found with untypable and mixed genotypes only. In patients infected through barber shops, genotype 3a and 2a were more prevalent, followed by 3b and 1a genotypes. The most prevalent risk factor for untypable genotype was found to be dental treatment and barber shop.

Regarding the duration on HD, 42% patients were being dialyzed during 1997-2009 and 58% from 2004-2009 with most prevalent genotypes 3a, 3b and 2a, and untypable and mixed genotypes, respectively.

Discussion

The HCV infection continues to be a major disease burden on the world. For example, the prevalence of HCV antibodies among dialysis patients has been reported to range from: 8 to 36% in North America, 39% in South America, 1 to 54% in Europe, 17 to 51% in Asia1.2 to 10% in New Zealand and Australia. The first description of HCV in Pakistan was recorded in 1992 and about 6%, i.e. at least ten million persons are carriers among a population of 140 million, showed there is no proper review of HCV and it is becoming a Herculean challenge. With the current disease burden, Pakistan has left behind the surrounding countries like India, Nepal, Myanmar, Iran and Afghanistan. HCV gained importance particularly as major complication in multiple transfused patients during the last decades especially in the countries where HCV is more prevalent in general population and amongst the blood donors. This was the first study conducted in patients treated with HD in Pakistan to determine the distribution of HCV genotypes and their interrelation with risk factors. Even though considerable progress has been attained during the last years, HCV prevalence rate among HD patients does not seem to have changed considerably.

Plasmid construction
DNA preparations were performed using standard methods as described in manufacturers’ instructions. The reporter plasmids pGEM-CAT/EMC/LUC, containing the EMCV IRES cDNA, and pGEM-CAT/LUC were kindly offered by Ian Goodfellow (Imperial College, London, UK). These plasmids allow T7 promoter-directed expression of dicistronic mRNAs encoding chloramphenicol acetyl transferase (CAT) and firefly luciferase (LUC). For the construction of the DHV-1 5′-UTR-containing plasmid, a pair of primers (DHV-1 UTRF: 5′-GAGGATCCTTTGAAAGCGGGTGCATG-3′; DHV-1 UTRR: 5′-CGGGATTCTGCATGAAAG TCTACTGGT-3′) were used to amplify the DHV-1 5′-UTR from wild DHV-1 strain (ZJ-V, GenBank No. EF382778). The purified PCR fragments were digested with BamHI, and then inserted into similarly digested and phosphatased pGEM-CAT/LUC between the two open reading frames (ORFs). The plasmid, containing the DHV-1 5′-UTR cDNA, was designated pGEM-CAT/DHV-1/LUC (Figure 1A). The further constructs containing the cDNA corresponding to the DHV-1 5′-UTR plus 10, 40, 60 nt of coding sequence were generated similarly using three pairs of specific primers and the resulting plasmids were termed pGEM-DHVUTR+10nt, pGEM-DHVUTR+40nt, pGEM-DHVUTR+60nt, respectively. Initiating AUG codon of the DHV-1 IRES was mutated to allow the expression of the luciferase from its own AUG.

Mutagenesis of the DHV-1 cDNA
In order to introduce mutations in the predicted IIIe region, Quick-Change site-direct mutagenesis kit (Stratagene) was used in order to change the sequence in the loop region 570-573 from GAUA to AAAA. The plasmid pGEM-CAT/DHV-1UTR+60/LUC was used as the template with two specific primers: P1 (sense primer): 5′-CCTACACTGCCTAAAAGGGTCGCGGCTGGT-3′; P2 (antisense primer): 5′- CAGCCGCGACCCTTTTAGGCAGTGTAGGTT-3′. The resultant plasmid was named pGEMDHV IIIe mut. Similar strategies were used to introduce mutations within the stem sequences of the predicted pseudo-knots (termed Stem1-mut and Stem2-mut). For the generation of the Stem1-mutant, the nts 447-449 (TGT) were changed to CCC. The mutagenic primers were as follows: P3 (sense primer): 5′-TGTAGGTGAGTGCCCGGTCTAGAGTAGGC-3′; P4 (anti-sense primer): 5′-CTACTCTAGACCGGGCACTCACCTACAAC-3′. For the generation of the Stem2 mutant, the nts 604-605 (CC) were changed to GG. The mutagenic primers were as follows: P5 (sense primer): 5′-TGATAGGGTCGCCCCTGGTCGAGTCCCA-3′; P6 (antisense primer): 5′-GGACTCGACC AGGGGCGACCCTATCAGG-3′. The presence of all the expected mutations in the plasmids (pGEMDHV IIIe mut, pGEMDHV S1 mut and pGEMDHV S2 mut) was confirmed by DNA sequencing. Similar strategies were used to introduce the compensatory mutations in Stem1 mut’ and Stem2 mut’ constructs.

In vitro translation reactions
For in vitro translation reactions, transcription of capped dicistronic mRNA was performed from the above plasmids, linearized with XhoI, using the Megascript transcription system (Ambion). Addition of the 7-mGTP cap 0 structure was performed using ScriptCap™m7G Capping System (Epicentre Biotechnologies) and mRNA was poly-adenylated using poly-A polymerase (PAP) following the suppliers’ recommendations. In vitro transcribed mRNAs were added to the Flexi rabbit reticulocyte lysate (RRL) system (Promega) to a final concentration of 10 μg/ml. This concentration of RNA was previously determined to give a linear yield of the translated product over the time course of the experiment (90 min). In reactions that required the addition of either recombinant 4E-BP1, L-protease, the dominant negative mutant forms of eIF4A or hippuristanol, the RRL were pre-incubated with the recombinant proteins or with the eIF4A inhibitor at 30°C for 15 min prior to the addition of the different plasmids. After 90 min, the reactions were terminated by the addition of SDS-PAGE sample buffer and subsequently resolved on 12.5% polyacrylamide gels.

Finally, we have characterized the DHV-1 IRES requirement for the eIF4F complex components and, as well as the EMCV and HCV-like IRESes, is insensitive to the FMDV L protease and the 4E-BP1 activities. These results indicate that the translation initiation takes places in the absence of fully functional eIF4E and eIF4G proteins. However, while the DHV-1 and HCV-like IRESes are not inhibited by hippuristanol or dominant negative forms of eIF4A, the EMCV IRES is strictly dependent on the eIF4A function. Although the EMCV and DHV-1 IRESes share some common structural organization, they initiate translation by different mechanisms involving different cellular translation initiation factors. Moreover, as the DHV-1 and HCV share common structural motifs, they have similar requirement for the eIF4F complex components. Thus, it is likely that the translation initiation of DHV-1 could proceed similarly to PTV-1 and to other HCV-like IRES containing picornaviruses, by the direct recruitment of the 40S ribosomal subunit to the viral mRNA.

All together, these results demonstrate, for the first time, the existence of an IRES element within the highly structured 5′-UTR region of the DHV-1 genome. The DHV-1 IRES could be classified as type IV IRES attending to its secondary structure and biological functions and, consequently, it exhibits functional similarities with the HCV IRES. These new advances in the understanding of the DHV-1 IRES structure, function and interaction with translation-initiation factors provides a foundation for developing therapeutics to prevent the viral protein synthesis.

To further analyze the eIF4A requirement and confirm these results, in vitro experiments were carried out using increasing amounts of a small molecule inhibitor of eIF4A. The hippuristanol is a sterol isolated from the coral Isis hippuris and identified via a high throughput screening for general translation inhibitors. It has been shown to block the eIF4A-dependent translation by inhibiting its RNA binding, ATPase, and helicase activities by interaction with the C-terminal domain of the eIF4A. The in vitro translation efficiency of the different dicistronic RNAs was evaluated in the RRL in the presence and in the absence of hippuristanol. In this experiment, PTV IRES, a class IV IRES which direct translation initiation independently of eIF4A, was used as positive control. Cap-dependent translation was efficiently inhibited by the eIF4A inhibitor, while PTV and DHV-1 IRES-directed translation were unaffected in these conditions. These results indicate that, DHV-1 5′-UTR, as well as the class IV HCV-like IRESes, do not require functional eIF4A to initiate translation.

Discussion

Picornavirus mRNAs are translated by an internal initiation mechanism, in which the ribosome enters directly at an internal site within the mRNA rather than scanning from the physical 5′ end. So far, internal ribosome entry sites (IRESs) have been identified for all the picornaviruses as well as an increasing number of cellular mRNAs. The IRES elements in picornaviruses have been located within the 5′ non-coding region, where the sequence and the RNA secondary structure are well conserved among viral serotypes in each genera. Based on sequence alignments, the genome of DHV-1 showed a typical picornavirus genetic organization and a putative class IV IRES element within DHV-1 5′-UTR. Therefore, DHV-1 should use similar picornavirus-like strategies for the initiation of translation. In this paper, we demonstrate, for the first time, that the DHV-1 5′-UTR has typical IRES activity, and that it can drive the translation of a downstream reporter gene both in vitro and in vivo. According to the prediction of the IRES structure, the DHV-1 IRES belongs to the type IV HCV-like IRESes, but it shares some common features with the IRES of viruses included in different groups.

To further characterize the presence of accessory regulatory sequences downstream of the 5′-UTR of the viral genome, 4 additional dicistronic reporter plasmids were constructed, containing DHV-1 5′-UTR and 10, 40 or 60 nucleotides of the coding region adjacent to the viral 5′-UTR (DHV-1 UTR+10nt, UTR+40nt, UTR+60nt, respectively). Initiating AUG codon of the DHV-1 IRES was mutated to allow the expression of the luciferase from its own AUG. The dicistronic plasmids were in vitro transcribed and individually added to Flexi rabbit reticulocyte lysate (RRL) system (Promega). The translation of the reporter proteins was evaluated measuring 35S-methionine incorporation. Cap-dependent translation from the first ORF was determined by the level of CAT expression and internal initiation activity of the different sequences included between the two reporter genes was estimated by the accumulation of fLUC. All the constructs expressed CAT efficiently as expected but the efficiency of the DHV-1 UTR+10nt, UTR+40nt and UTR+60nt sequences was stronger than that of the DHV-1 UTR (Figure 5A). Moreover, the dicistronic plasmids were transfected into vFT7-infected BHK-21 cells and, in order to evaluate the in vivo internal initiation activity, the accumulation of fLUC protein expressed from the different constructs was determined and corrected by CAT expression levels. The experiments were performed by triplicate. As observed in the in vitro experiments, the translation initiation efficiency of DHV-1 UTR+10nt, UTR+40nt and UTR+60nt sequences was stronger than that of the DHV-1 UTR. These results indicate the presence of accessory regulatory sequences downstream the DHV-1 putative IRES, which are not required for translation initiation but positively modulate the IRES activity.

Characterization of the putative DHV-1 IRES requirement for eIF4F complex components
Translation initiation of DHV-1 IRES is not inhibited by 4E-BP1

Picornavirus IRES-mediated translation is independent of the cellular cap-binding protein eIF4E. In order to evaluate the putative DHV-1 IRES requirement for the eIF4E, the in vitro translation efficiency of the dicistronic constructs was evaluated in RRL as described upon addition of increasing amounts of purified recombinant 4E-BP1 protein. The eIF4E-binding proteins (4E-BPs) are a family of three small polypeptides that inhibit cap-dependent translation by binding to the eIF4E, obstructing its interaction with eIF4G. However, the internal translation initiation on IRES elements is not affected in these conditions. As shown in Figure 6A, whereas the recombinant 4E-BP1 efficiently inhibited the cap-dependent CAT expression in RRL, the EMCV IRES-directed and DHV-1 5′-UTR-directed translation were unaffected.

The DHV-1 IRES does not require intact eIF4G

According to the prediction of the 5′-UTR secondary structure, DHV-1 could be included among the type IV picornaviruses. Previous studies have shown that the type IV IRES elements (HCV-like IRESes) do not require the eIF4G activity to direct translation initiation and, consequently, that are insensitive to the eIF4G cleavage induced by different viral proteases, such as the foot-and-mouth disease virus (FMDV) L protease. The FMDV L protease efficiently cleaves the eukaryotic eIF4G resulting in a partial inhibition of the cap-dependent translation while the IRES-directed translation is not inhibited in these conditions and it can even be enhanced. To confirm that the translation of DHV-1 5′-UTR is cap-independent and to examine its eIF4G dependence, the effect of foot-and-mouth disease virus (FMDV) L protease addition in the RRL system was evaluated. The dicistronic RNA constructs indicated in Figure 7A were individually added to FMDV-L treated or control Flexi RRL and the translation of the reporter proteins was evaluated. Whereas the cap-dependent CAT expression was efficiently inhibited, the EMCV-directed and the DHV-1 5′-UTR-directed translation were insensitive to FMDV L protease addition. Western blot analysis was performed to control the efficiency of the eIF4G cleavage by FMDV L protease. These results indicate that the DHV IRES directs cap-independent internal initiation of translation and not requires an intact full-length eIF4G.

To confirm and extend the results from these in vitro assays, the same dicistronic plasmids were tested in vivo by transient-expression experiments in mammalian cells. The dicistronic plasmids were transfected into vFT7-infected BHK-21 cells, and 20 h post-transfection, cell extracts were prepared and protein synthesis was analyzed by SDS-PAGE and immunoblotting to detect CAT and LUC expression. As expected, all plasmids expressed CAT efficiently. Moreover, the DHV-1 5′-UTR and the EMCV IRES containing plasmids also produced efficient fLUC accumulation. These results indicated that the putative DHV-1 IRES element is able to efficiently initiate protein synthesis in vivo.

Stem1, Stem2 and domain IIIe are essential to the function of DHV-1 IRES

Although there is only limited primary sequence relatedness among the 5′-UTRs of DHV-1, AEV and HCV, the structure of domain IIIe appears remarkably conserved among these viral IRESes [2,35,11]. It has been previously reported that domain IIIe is critical for the HCV, PTV-1 or AEV IRES-mediated initiation of translation. To evaluate the relevance of the putative IIIe region in DHV-1 IRES activity, the most important loop sequence within the IIIe region (GAUA) was mutated to AAAA (DHV IIIe mut) (Figure 3A) and the effect of the mutation on the translation initiation was evaluated. Mutant constructs were analyzed both in Flexi RRL and in DF-1 cells. The results reveal that these mutations partially inhibited translation initiation both in vitro and in vivo, confirming the relevance of domain IIIe for DHV-1 IRES activity. Surprisingly, the reduction in the translational activity of the DHV-1 IRES upon disruption of the domain IIIe (50%) is moderate when compared with similar mutations in other type IV IRESes (90-95%).

Different experiments carried out with other picornaviruses, such as the HCV, PTV or AEV, showed that stem 1 and stem 2 within the IRES structure are crucial for translation initiation. The disruption of the base pair interactions in these regions seriously damages the ability to initiate translation. To analyze the role of the putative stems loops of the DHV-1 IRES, different mutations were introduced to disrupt the predicted base pairing of stem 1 (DHV stem1 mut) and stem 2 (DHV stem2 mut). The mutant constructs were analyzed by triplicate both in Flexi RRL and in DF-1 cells. The results showed that both mutations resulted in a slight reduction in translation initiation ability of DHV-1 IRES both in vitro and in vivo. Moreover, the function of the IRES was partially recovered with the corresponding mutations that restore the structure (DHV stem1 mut’ and DHV stem2 mut’ respectively), confirming that the predicted pseudo-knot structures are formed in vivo and that they play a role in viral translation regulation. Once more, the reduction in the translational activity of the DHV-1 IRES upon disruption of these structures (50%) is moderate when compared with similar mutations in other type IV IRESes (90-95%).

Results
Conserved secondary structure elements in DHV-1 and HCV-like IRES RNAs

Sequence analysis of the DHV-1 5′-UTR display a secondary structure with two major domains, II and III, which contain all the structural elements that have been described as crucial for internal translation initiation. The larger domain III consists on several branching high conserved hairpin stem-loops (III abcdef) (Figure 1A), which were also found in several members of the Picornaviridae family, such as the porcine teschovirus (PTV) and the avian encephalitis virus (AEV), and in some viruses from the Flaviviridae family, such as the classical swine fever virus (CSFV) and the hepatitis C virus (HCV). DHV-1 5′-UTR is able to initiate cap-independent translation

In order to evaluate the IRES activity of the 5′-UTR region of the DHV-1 genome, a dicistronic reporter plasmid was constructed by the insertion of a cDNA corresponding to the DHV-1 5′-UTR (nts 1 to 626) between two reporter gene sequences, the first encoding CAT protein and the second encoding fLUC (DHV UTR). Dicistronic plasmid pGEM-CAT/EMCV/LUC (EMCV-IRES), which contains the EMCV IRES, was used as a positive control for internal translation initiation activity, and the plasmid pGEM-CAT/LUC (CAT/LUC), which lacks any IRES sequence, was used as a negative control. The dicistronic plasmids were in vitro transcribed as indicated in Materials and Methods and the resultant RNAs were individually added to Flexi rabbit reticulocyte lysate (RRL) system (Promega).