Seroprevalence and Phylogenetic Characterization of Hepatitis E Virus ( Paslahepevirus balayani ) in Guinean Pig Population

Abstract

Background:

Hepatitis E virus (HEV) is transmitted by the fecal route, usually through contaminated water in humans and/or infected animals, especially pigs. The objective of this study was to evaluate the level of anti-HEV antibodies in a panel of pig sera and to identify HEV in pig feces in farms.

Methodology:

The presence of HEV antibodies was tested by an in-house ELISA and a commercial ELISA IDvet. HEV genome was assessed by nested RT-PCR, and then, genotype was identified by sequencing (MinION Nanopore technology).

Results:

In 2017–2019, the 43% seroprevalence found in Forest Guinea was significantly higher than the 7% found in the Lower region (p < 0.01). Presence of HEV genotype 3c was demonstrated during a secondary study in the Lower region (Conakry) in 2022.

Conclusion:

The presence of HEV-3c in pigs calls for an evaluation of seroprevalence in human populations and for a HEV genotype human circulation check.

Contribution Heading:

This study is the first report, to our knowledge, of seroprevalence and characterization of HEV infection in pigs in Guinea.

Introduction

Hepatitis E virus (HEV) is one of the five known human hepatitis viruses. HEV infection is often mild but can lead to severe, acute liver disease in humans and sometimes progress to fulminant hepatic failure. In pregnant women, it may also lead to severe complications resulting in fetal and/or maternal mortality. Out of more than 20 million yearly global infections, 44,000 result in death (https://www.who.int/news-room/fact-sheets/detail/hepatitis-e), mostly in low-income countries in Asia, Africa, and Latin America (Aubry 2013; Motoya et al. 2019). HEV was originally described to be transmitted primarily by the fecal–oral route but for the past 20 years, HEV has also been considered as a zoonotic pathogen that can be transmitted by domestic or wild animals mainly from swine lineages (Khuroo et al. 2016).

HEV is a quasi-enveloped, positive-sense single-stranded RNA virus within the Hepeviridae family, Paslahepevirus genus, Paslahepevirus balayani species that comprises eight genotypes: genotypes 1–4 are involved in human infection, whereas genotypes 5 and 6 are restricted to wild boars (de paula et al. 2013; Smith et al. 2020), and genotypes 7 and 8 have been described to be able to infect camels, with rare cases in humans (Takahashi et al. 2020). Despite the existence of many genotypes, there is only one serotype (Kamar et al. 2012). Over the past 10 years, new members of the Hepeviridae family have been found in rodents, bats, birds, and fishes, sometimes referred to as emerging infectious agents (Purdy et al. 2022).

The recognition of the zoonotic status of HEV genotypes 3 and 4 led to a reassessment of the HEV prevalence worldwide to evaluate the related health burden in humans. Until now, several studies in African countries such as Burkina Faso, Ghana, and Cameroon have shown a high HEV seroprevalence and HEV virus detection in pigs (Cooper et al. 2005; Traoré et al. 2015; El-Duah et al. 2020). A recent epidemic was also described in southeastern Senegal close to the northern Guinea border (Sadio et al. 2022). The assessment of HEV seroprevalence among pigs, known as a reservoir of HEV virus, informs the need for a HEV surveillance system to prevent human cases. Following our first-step survey performed on animals sampled in 2018, an evaluation of hepatitis viruses was recently done in a group of 74 health care workers in Guinea after the COVID epidemic peak using HEV IgM commercial ELISA (Ostankova et al. 2023). In this study, three cases of HEV infection were detected, demonstrating the circulation of HEV in the country, but no identification of the involved genotype was provided. The prevention strategies to avoid HEV infection will depend on the involved virus: from human to human in genotypes 1 and 2, whereas genotypes 3 and 4 may be maintained by an animal reservoir. Thus, the identification of the involved genotype and the animal reservoir should be a very first step to any human health intervention.

The aim of this study was to determine the circulation of HEV among pigs in Guinea as a surveillance tool to prevent human cases. HEV infection was determined indirectly by testing for specific antibodies using ELISA or directly by PCR to detect the virus and characterize the genotype by sequencing.

Materials and Methods

Study population

There was no sample size calculation, as our study was derived from a program in which the main objective was the research of an Ebola reservoir. Because of the relative population sensitivity, we chose to sample the larger number of animals in the farms that accepted the study.

The studied population were pigs from Guinean farms in the two main regions developing pig farming in Guinea, mainly Lower and Forest Guinea. Currently, Guinea Agricultural Ministry services estimated a total of 147,000 pigs in Guinea, with 98% in the two main regions. In total, 20 piggeries in 11 prefectures were surveyed. We collected 886 samples from pigs, all of which were reared and sampled in pens. Sample were collected between 2017 and 2019 in the two main regions (Lower and Forest Guinea) and then in 2022 in Conakry (105 feces samples). The pig farming system is described in Supplementary Table S1. Age, sex, place, city, and day of collection were recorded for each sample.

Sampling blood and feces

Whole blood was collected from the jugular vein in a dry tube of 5 mL. Tubes were centrifuged on site (3000 × g, 10 min) to collect serum and aliquoted per 0.5 mL. Sera were stored at −20°C on the field and at −80°C back in the laboratory until use in the biobank of Institut Pasteur de Guinée. During the 2022 study, feces were sampled from the rectum from each individual animal. Pigs sampled were at least 1 month of age and not gestating sows. The feces samples were taken by rectal examination, with a finger inserted into the animal’s rectum to remove a small portion with sterile gloves (around 1 g). Each animal was gently restrained by three people to collect the feces (at the same time for blood collection), which was placed in a tube containing 1× PBS (2 µL), sent to the laboratory in a −20°C cooler, and then stored in the laboratory at −80°C before handling.

Serological assay

Because the exposure of pigs to pathogens (parasites and others) and the weather conditions in Africa are largely different from those in Europe or the United States (where most of the ELISA kits were developed), the current procedure is to test animals’ sera with different kits.

Two immunoserological ELISA techniques were used. In a first approach, an in-house ELISA set up at Friedrich Loeffler Institute (FLI) for the detection of HEV antibodies in swine serum was used. The HEV-specific bait was a recombinant, bacterially expressed p239 from the open reading frame 2 (ORF2) of HEV genotype 3 comprising amino acid sequence 368–606 of the ORF2 capsid protein. Briefly, in Conakry, we sensitized the ELISA plates (Nunc MaxiSorp 96) with the recombinant protein (1 µg/mL in 0.05 M carbonate buffer, 4°C overnight) and blocked with milk (Difco skimmed milk in 1× PBS, 1 h, 37°C) to limit nonspecific protein interactions and then washed three times with PBS/0.1% Tween 20. We placed test samples (diluted sera 1:25 with PBS/2% skimmed milk) and controls in wells after incubation, and after three washing steps, we added the antibody conjugate (Protein G horseradish peroxidase): this resulted in the formation of the antigen–antibody-conjugate–peroxidase complex.

In a second approach, sera were tested with the indirect commercial ELISA kit ID Screen Hepatitis E Indirect Multispecies (IDvet, Paris, France) with a sensitivity and specificity of 99.5% and 100%, respectively, that also used a recombinant genotype 3 HEV capsid.

In Germany, the in-house ELISA was validated against European pig sera that were pretested with two commercial ELISA kits—PrioCHECK™ HEV Antibody ELISA (ThermoFisher, Dreieich, Germany) and ID Screen® Hepatitis E Indirect Multispecies ELISA (IDvet, Montpellier, France).

Molecular detection

Total RNA was extracted from the fecal samples from pigs collected in Conakry using the QIAamp viral RNA extraction mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. A nested RT-PCR was performed to check for the HEV sequence: the first round of RT-PCR (GoTaq® 1-Step RT-qPCR System [Promega, New York, USA]) generated a 748-base-pair (bp) segment in the ORF2 with primers 3156N (position 5711–5732 along the HEV genome) and 3157N (position 6419–6441), whereas the second round of PCR generated a 325-bp segment at the 5′ end of the ORF2 amplified fragment using primers 3158N (position 5996–6017) and 3159N (position 6322–6343) (Cooper et al. 2005). All steps were done with the GoTaq® Probe 1-Step RT-qPCR System (Promega, New York, USA). The amplification products were separated by electrophoresis on a 1% agarose gel for 45 min at 100 V stained with SYBR Safe Dye (Invitrogen, Carlsbad, USA).

PCR and sequencing

For sequencing, new DNA fragments were obtained from extracted RNA by optimization using Superscript IV reverse transcriptase (Invitrogen, Vilnius, Lithuania) and diluted 1:10 in nuclease-free water and then PCR was run with Q5 enzyme (NEB, Ipswich, MA, USA) to obtain a 748 partial segment of the capsid gene and a 338 base segment of the polymerase gene using two sets of primers: first HEV-F4228 (position 4279–4307)–HEV-R4598 (position 4627–4649) and the second HEV-F4228–HEV-R4565 (position 4591–4616) (Cooper et al. 2005; Drexler et al. 2012). Hepatitis E Virus Genotyping Tool version 1.0 was used for initial genotyping (Vennema et al. in press).

Phylogenetic analysis

Phylogenetic tree was constructed with Mega 7 software with the maximum likelihood method using 16 HEV references sequences retrieved from GenBank. Sequences were submitted to EMBL GenBank (AC number OR283252–OR283253).

Results

Comparison of the FLI in-house kit with the IDvet

ROC analysis performed in Germany with two different commercial kits as references gave a sensitivity of 96.1% and a specificity of 96.8% for German pigs. In our laboratory assay, we performed a complementary ROC analysis of the in-house assay using the Guinean pigs’ pretested sera with the single commercial ID Vet kit and found a sensitivity of 90.1% and specificity of 82.1% for the in-house test (XLSTAT statistical and data analysis solution: www.xlstat.com). Using the in-house ELISA, developed by the FLI team, an overall seroprevalence of 22% (IC95%: 19.0–25%) (193/886) was found to signify circulation of HEV in pigs. Among the 510 samples double tested with the two kits, we obtained 176 positives with the in-house kit. Thus, 80.11% of the positive sera from in-house ELISA (141/176) were found to be positive according to the commercial kit “IDvet” in Supplementary Table S2. However, a Kappa comparison test of the two systems using the data of the 510 double-tested sera indicated an almost perfect accordance between the two kits (kappa = 0.84; Table 1; https://www.graphpad.com/quickcalcs/kappa1/?K=3).

Table 1.

Comparison of the Two ELISA Kits (In-House FLI versus Commercial IDvet) on 510 Samples Selected by Chance within the 886 Pig Samples

Elisa IDvet
Elisa FLI	Positive	Doubtful	Negative	Total
Positive	141	8	27	176
Doubtful	0	0	0	0
Negative	0	0	334	334
Total	141	8	361	510

Analysis of the HEV seroprevalence

The pig breeder sites are found mainly in two regions: the Lower region and Forest Guinea (Fig. 1). Farms in Forest Guinea are home to 89% of the registered Guinean pig population, and most of them keep the animals in enclosures. In Lower Guinea (8.4% of the pig population), on some farms, the pigs also feed freely in the bushes around the houses. We thus performed separate analysis of the two regions.

FIG. 1.

Spatial distribution of positives samples by ELISA using the FLI kit. The 886 pig sera that were collected in 11 prefectures are indicated in light gray. In each prefecture, the number of seropositives out of the number of animals tested is noted (xx/xx). Conakry (11/53) and Coyah (15/169) are so small, so a zoomed image (left corner) was created to identify these two prefectures in the black square indicated on the map.

Presence of anti-HEV IgG among pigs

Figure 1 and Table 2 show the distribution of positive samples obtained using the in-house ELISA in the two regions and the various prefectures of interest. After a multivariate logistic regression with ELISA in-house results as the outcome, results in-house are significantly related to region where the samples were collected. The seroprevalence in Forest Guinea prefectures (43% [IC95% 38–48%]) was significantly higher than the one in Lower Guinea (7% [IC95% 5–9%]) (Table 2).

Table 2.

Results of the ELISA FLI Kit by Prefectures and Prevalence with Confidence Interval

Population	Testedpopulation	Casenumber	Prevalence	Confidenceinterval95%
Total	886	193	22%	19%	25%
Boffa	47	6	13%	3%	22%
Boke	29	0	—	—	—
Conakry	53	11	21%	10%	32%
Coyah	169	9	5%	2%	9%
Dalaba	15	0	—	—	—
Dubreka	86	9	10%	4%	17%
Forecariah	40	0	—	—	—
Kindia	84	1	1%	−1%	4%
Total Coast Guinea	523	36	7%	5%	9%
Kissidougou	94	34	36%	26%	46%
Nzerekoré	195	97	50%	43%	57%
Guékédou	74	26	35%	24%	46%
Total Forest Guinea	363	157	43%	38%	48%

With a 5% risk, adjusting by region, sex, and HEV in-house result, we have not been able to show any statistically significant relationship between results in-house and age (month). All these statistics tests are reported in Supplementary Data S1.

Genotype 3c HEV was found in pigs

Following the results from the serological survey on pig farms, we decided to assess the presence of HEV in pig feces where the virus is naturally excreted. This has been done on 105 feces samples from free-range pigs in the Conakry suburbs, different from those tested serologically, all of them between 6 and 12 months of age. Two of these 105 porcine feces samples were positive for HEV-RNA (two animals around 6 months of age) using nested PCR targeting ORF2 (Cooper et al. 2005).

Sequencing and phylogenetic analysis

Two PCR products targeting the capsid and RNA polymerase coding region were sequenced using the Nanopore MinION technology. Only one of the two positive samples (sample 76) provided a sufficient sequencing depth (>30×) to obtain the consensus for capsid and polymerase partial sequences. A phylogenetic analysis of the two combined sequences was compared with reference sequence strains from GenBank (Smith et al. 2016). This analysis identified HEV from genotype 3c (Fig. 2).

FIG. 2.

Phylogenetic analysis of the sequences obtained from the feces samples (polymerase and capsid combined). Sample 76 sequences as indicated by a square were aligned with 16 reference sequences retrieved from GeneBank using MEGA7 software. The evolutionary history was inferred by using the maximum likelihood method based on the general time reversible model. The tree with the highest log likelihood (−5757, 6931) is shown. The robustness of the branching order was assessed by the bootstrap method (1000 replicates), as shown next to the branches (values below 50% are not shown). Initial tree(s) for the heuristic search were obtained automatically by applying the neighbor-join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with superior log likelihood value. A discrete gamma distribution (+G) and portion of invariable sites (+I) to model evolutionary rate differences among sites was used (five categories; +G = 1, 1356; +I = 53, 3484% sites). All positions containing gaps and missing data were eliminated, leaving 762 positions in the final dataset.

Discussion

In Guinea, pork meat is consumed mainly in the Lower and Forest regions and not in the two other regions (Middle and Upper regions). We thus checked for the presence of HEV in the two larger pig production regions with ELISA and found 43% (IC95% [38–48%]) positive in the Forest region and 7% (IC95% [5–9%]) in the Lower region sera as a proof of HEV circulation. A rough comparison of the HEV seroprevalence with surrounding countries indicated higher seroprevalence in Ghana, where it reached 62.4% in 2011 (Bagulo et al. 2022) but close to the one found in Cameroon (43.2% in 2017–2018) (Modiyinji et al 2020).

Interestingly, the seroprevalence in Lower Guinea was substantially lower (7%) than in Forest Guinea (43%). Many Ebola, Lassa, and other human cases have recently been diagnosed in the Forest region, indicating that the climate, wildlife, and environmental conditions are favorable for zoonotic transmission. Animal husbandry systems include intensive and semi-intensive forms, where the spatial proximity of the animals promotes virus transmission (Forest Guinea), and extensive or free-range farming (Lower Guinea), where pigs are allowed to roam freely for food. However, although in 2022, in Conakry, we found mainly semi- or free-range farming, during 2017–2019, most of the farms visited in Lower Guinea were totally enclosed. Interestingly, the seroprevalence in pigs sampled in Conakry was higher than in the surrounding prefecture.

Therefore, in Conakry, the virus is present in pig farms, as shown by the two positive samples detected by RT-PCR. Upon sequencing of the gene fragments and phylogenetic analysis, the circulating HEV virus belonged to the genotype 3c. This first result in the West African coast reveals the very same genotype previously identified in Cameroonian and Ghanaian pig sera as well as in pig’s feces from Madagascar (El-Duah et al. 2020; Modiyinji et al. 2020). No data are available from nearby countries such as Senegal and Mali, where pig breeding is very low. In the Ivory Coast, consumption of pork meat is more common, but there are no data about the animal population or the human one. Recently in Guinea, a study described some human HEV-positive cases but did not provide any information about the sampling date or genotype and only sporadic information about the detailed risk factor. However, this highlights the interest of the HEV studies in Guinea (Ostankova et al. 2023). In the absence of a recognized gold standard for the HEV ELISA specific to pigs, the use of two diagnostic kits (in-house validated in Germany and a commercial IDvet kit) that were not very different in terms of sensitivity and specificity (ROC curve and the Kappa test) strongly supports our conclusion and the results we obtained However, such tests are never perfect, and this also calls for new studies to clearly identify the involved viruses either in the animals or in the environment within the two regions of pig breeding.

Some limitations were noted in this study, as the limited number of sampling sites may not be representative of the entire pig population. However, it provided a first snapshot of the situation. Hence, it was previously shown that the HEV seroprevalence may vary largely from site to site, but in our case, this is only significant when we compare the two regions. Within the Forest region, the sites where sampling was done by chance always reported a relatively high seroprevalence, whereas in the Lower region, we only found a few positive pigs or none in some sites. All these results remained within the confidence interval of the computed percentage.

Conclusion

Widespread HEV infections were demonstrated in pigs in Guinea by the seroprevalence study. The molecular study indicates that the HEV genotype 3c is circulating in the Guinean pig population. Further studies will address the reasons for the discrepancy in seropositivity between the Lower and Forest regions. Additional sequencing of HEV will need to be carried out to determine the HEV diversity in the pig population. It is also important to conduct a similar study in humans to identify the risk factors for HEV infection.

Footnotes

Acknowledgments

The authors thank the Guinean authorities and are grateful to the Guinean breeders and veterinarians for their expert collaborations during the sampling process, particularly Dr. Traore.

Ethical Consideration

The study was validated by the National Ethics Committee for Health Research (CNERS) from Guinea (ref 040/CNERS/17, June 1, 2017). All procedures performed in studies involving animals followed all international, national, and/or institutional guidelines for the care and use of animals.

Authors’ Contributions

P.R., N.T., and C.T.: Conceptualization of study; B.D., S.G., and P.R.: Data curation; B.D. and P.R.: Formal analysis; M.E. and M.H.G.: Resources; B.D., C.T., N.T., and Y.L.P.: Investigation; B.D., P.R., and N.T.: Writing. All authors read and approved the final version.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

The following programs contributed to fund the current research: EBOLA FORESIGHT project financed by the German Federal Ministry of Food and Agriculture (File Ref. 323-06.01-03-2815FSEBOL) and the European Union grant EBOSURSY N° FOOD/2016/379-660. Sequencing was supported by Agence Française de Développement through the AFROSCREEN project (grant agreement CZZ3209), coordinated by ANRS | Maladies infectieuses émergentes in partnership with Institut Pasteur and IRD. The authors would additionally like to thank members from the AFROSCREEN Consortium () for their work and support on genomic surveillance in Africa. Formerly receiving stipend from IPGui, B.D. is currently funded by a PhD grant from CADEIRA Project DAAD fund.

Data Availability

Data supporting the findings of this study are available from the corresponding author, B.D., on request.

Disclaimer

The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.

Supplementary Material

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