Abstract

Hepatitis E is an emerging zoonotic disease caused by the hepatitis E virus (HEV). HEV is a member of the genus Orthohepevirus A and can be divided into eight major genotypes¹1. Purdy MA, Harrison TJ, Jameel S, Meng XJ, Okamoto H, Van der Poel WHM, et al. ICTV virus taxonomy profile: hepeviridae. J Gen Virol. 2017; 98(11): 2645-6. and 43 subtypes (assigned and unassigned).²2. Smith DB, Simmonds P, Izopet J, Oliveira-Filho EF, Ulrich RG, Johne R, et al. Proposed reference sequences for hepatitis E virus subtypes. J Gen Virol. 2016; 97(3): 537-42. Among them, only HEV-3 and HEV-4 have been reported in humans and animals worldwide, and they are known to cause cross-species infection, posing a threat of zoonotic transmission.³3. Nan Y, Wu C, Zhao Q, Zhou EM. Zoonotic hepatitis E virus: an ignored risk for public health. Front Microbiol. 2017; 8: 2396. In Brazil, HEV-3 has been detected in humans and in swine in the South, Southeast, and Central-West regions.⁴4. dos Santos DL, de Oliveira-Filho EF, Pinto MA. Hepatitis E worldwide and in Brazil: review. J Vet Med An Sci. 2013; 20(3): 416-33.^,55. dos Santos DR, Vitral CL, de Paula VS, Marchevsky RS, Lopes JF, Gaspar AM, et al. Serological and molecular evidence of hepatitis E virus in swine in Brazil. Vet J. 2009; 182(3): 474-80.^,66. dos Santos DR, de Paula VS, de Oliveira JM, Marchevsky RS, Pinto MA. Hepatitis E virus in swine and effluent samples from slaughterhouses in Brazil. Vet Microbiol. 2011; 149(1-2): 236-41.^,77. Lopes Dos Santos DR, Lewis-Ximenez LL, da Silva MF, de Sousa PS, Gaspar AM, Pinto MA. First report of a human autochthonous hepatitis E virus infection in Brazil. J Clin Virol. 2010; 47(3): 276-9.^,88. da Costa Lana MV, Gardinali NR, da Cruz RA, Lopes LL, Silva GS, Caramori Jr JG, et al. Evaluation of hepatitis E virus infection between different production systems of pigs in Brazil. Trop Anim Health Prod. 2014; 46(2): 399-404. To date, HEV isolates have not been detected in Northeast Brazil. Swine production in this area is characterised by a low technical level and by poor investments in mechanisation, technology, and biosecurity practices. In this study, we screened for and performed phylogenetic analyses based on different genome segments of HEV strains circulating among domestic swine in Northeast Brazil.

A total of 119 faecal samples were individually collected in 2017 from two to six-month-old animals from eight farms using both intensive and extensive production systems located within a high HEV seroprevalence area.⁹9. de Oliveira-Filho EF, Lopes KG, Cunha DS, Silva VS, Barbosa CN, Brandespim DF, et al. Risk analysis and occurrence of hepatitis E virus (HEV) in domestic swine in Northeast Brazil. Food Environ Virol. 2017; 9(3): 256-9. Viral RNA was extracted from a 200 µL faecal suspension using the ReliaPrep™ Viral Total Nucleic Acid Purification Kit (Promega, Brazil) and screened by a previously described real-time polymerase chain reaction (RT-PCR) method¹⁰10. Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR. A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Methods. 2006; 131(1): 65-71. using the NextGeneration ECO One-Step HotStart RT-qPCR Kit (DNA Express Biotecnologia, Brazil). Conventional HEV RT-PCR, using a set of previously designed primers to amplify the ORF2 region,¹¹11. Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, et al. A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci USA. 1997; 94(18): 9860-5.^,1212. Huang FF, Haqshenas G, Guenette DK, Halbur PG, Schommer SK, Pierson FW, et al. Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. J Clin Microbiol. 2002; 40(4): 1326-32. was used to confirm the positive samples. Primers for sequencing the complete capsid region were designed based on the pertinent literature¹³13. Oliveira-Filho EF, Bank-Wolf BR, Thiel HJ, König M. Phylogenetic analysis of hepatitis E virus in domestic swine and wild boar in Germany. Vet Microbiol. 2014; 174(1-2): 233-8.^,1414. Müller B, Klemm U, Mas Marques A, Schreier E. Genetic diversity and recombination of murine noroviruses in immunocompromised mice. Arch Virol. 2007; 152(9): 1709-19. and on an alignment using 156 partial genomic Brazilian and HEV-3 subtype reference sequences (Tables I-II). RT-PCR was performed using the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Invitrogen, USA) according to manufacturer’s instructions. Nested PCR was performed using PCR Master Mix 2X (Promega, Brazil), and the obtained PCR products were purified with the QIAquick Gel Extraction Kit^TM (Qiagen) before being sent for sequencing.

Initially, sequences were aligned using MUSCLE v3.6 software.¹⁵15. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004; 5: 113-29. Markov models of nucleotide substitution were chosen using jMoldelTest v.2 software.¹⁶16. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9(8): 772-5. Sequences were retrieved from GenBank, and the accession numbers are displayed on the phylogenetic trees. Evolutionary studies were conducted using the maximum likelihood (ML) and maximum parsimony (MP)¹⁷17. Moratorio G, Vignuzzi M. Monitoring and redirecting virus evolution. PLoS Pathog. 2018; 14(6): e1006979. inference methods implemented in FastTree v.2.1.7¹⁸18. Price MN, Dehal PS, Arkin AP. FastTree 2 ? approximately maximum-likelihood trees for large alignments. PLoS One. 2010; 5(3): e9490. and Mesquite v.3.5.1¹⁹19. Maddison WP, Maddison DR. Mesquite: a modular system for evolutionary analysis. Version 3.51 2018 [cited 2018 30.09.2018]. Available from: http://www.mesquiteproject.org.
http://www.mesquiteproject.org... software. ML analysis was conducted with the standard default GTR + CAT with 20 gamma distribution parameters and a mix of the nearest-neighbour interchanges (NNI) and sub-tree-prune-regraft (SPR). The MP method, in which the best phylogenetic tree is determined based on favouring the fewest evolutionary changes, was performed using the parsimony ancestral states algorithm with the SPR rearranger to trace the history of character evolution on 1.000 trees.¹⁷17. Moratorio G, Vignuzzi M. Monitoring and redirecting virus evolution. PLoS Pathog. 2018; 14(6): e1006979. The reliability of the tree nodes was obtained from Shimodaira-Hasegawa (SH-like) test support values with 1000 replications.²⁰20. Anisimova M, Gascuel O. Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Syst Biol. 2006; 55(4): 539-52. Phylogenetic signals were assessed via likelihood mapping analysis using Tree-Puzzle v.5.2 software.²¹21. Schmidt HA, Strimmer K, Vingron M, von Haeseler A. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics. 2002; 18(3): 502-04.^,2222. Strimmer K, von Haeseler A. Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment. Proc Natl Acad Sci USA. 1997; 94(13): 6815-9. The pairwise homoplasy index (PHI) for the Recombination search was implemented in SplitsTree v.4.14.6 following the default settings.²³23. Huson DH, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006; 23(2): 254-67. Lastly, pairwise and patristic distances were inferred using MEGA v.7²⁴24. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33(7): 1870-4. and PATRISTIC²⁵25. Fourment M, Gibbs MJ. PATRISTIC: a program for calculating patristic distances and graphically comparing the components of genetic change. BMC Evol Biol. 2006; 6: 1. software, respectively.

From a total of 119 samples, two samples (1.68%) originating from three- (S67) and five-month-old (S26) clinically healthy piglets yielded 304-nucleotide (nt)-long DNA fragments corresponding to the HEV capsid region (Accession numbers: MH664123 and MH664124). The positive animals were from two distinct farms [Supplementary data (Fig. 2)]. Evolutionary analysis showed that both the S26 and S67 HEV sequences clustered within genotype HEV-3 together with the subtype 3f derived from a patient with acute hepatitis E in Japan who is known to have travelled to Bangladesh (see AB369387) (Fig. 1A). Additionally, we performed further analyses on a 1503-nt-long capsid sequence fragment isolated from sample S26. Analysis of this larger fragment along with 96 HEV-3 representative reference sequences retrieved using Boolean terms [Hepatitis E virus(Organism)] AND ORF2(Gene name) from GenBank, 43 of which had been previously assigned to clade 3e-3f-3g, thus confirming S26 to be phylogenetically related to clade 3f (see Fig. 1C clade two highlighted by the dotted lines, SH-like support ≥ 80%). These data were extrapolated from the consistent monophyletic origin and asymmetric phylogenetic tree backbone support [Fig. 1B, Supplementary data (Fig. 1)]. Intriguingly, although the sequence exhibited a high divergence, as demonstrated by the length of the tree branch (red line), we observed a proximity to the data obtained from the pairwise and patristic distances of HEV isolated from humans (KT591533.1 and KT591532.1) (Fig. 1B, Table II). Further gene flow analysis of S26 showed an ancestry degree and viral gene stream among HEV-3 virus populations typically found in human and animal hosts, suggesting both a switch of host direction and that this HEV-3 subtype 3f strain is probably of zoonotic origin (Fig. 1C). Similar patterns were also observed among strains of the HEV-e-f-g monophyletic clade, revealing a continuous flow of viral gene interchange and spread among sequences from human and animal hosts.

Phylogenetic analyses based on a 304 nt fragment (A) and 1503 nt (B and C) fragment of the hepatitis E virus 3 (HEV-3) capsid gene. Maximum likelihood (ML) phylogenetic tree showing the HEV samples in this study (blue gradient background: S26 and S69) clustering with subtype 3f isolated from humans (A). In (B), sequence S26 is in a rectangular dashed box. Subtype reference sequences⁽²⁾ are in bold. The red line under a tree branch highlights the sequence divergence according to the tree bar scale. The branches in orange colour highlight the HEV 3f and 3e clades. The asterisks along tree branches represent SH-like support values of ≥ 0.75 (B). Parsimony ancestral states analysis inferring the character history of the sequences extracted from the second large clade from (B). Each colour represents HEV-3 isolated from different hosts, countries, and different sampling years (see Table II). The dotted blue and green arrows show our hypothesis about the continuous flow showing viral gene interchange and spread among HEV isolated from different hosts (C).

Although a human virus was recently suggested to be the most likely ancestor of Orthohepevirus A,²⁶26. Forni D, Cagliani R, Clerici M, Sironi M. Origin and dispersal of hepatitis E virus. Emerg Microbes Infect. 2018; 7(1): 11. the human or animal origin of HEV remains under debate. In addition, with regard to its progressive host range expansion, it appears that host switching has played an important role during the evolutionary history of HEV and that genotype differences might have arisen via cyclical adaptation to different hosts.²⁷27. Brayne AB, Dearlove BL, Lester JS, Pond SLK, Frost SD. Genotype-specific evolution of hepatitis E virus. J Virol. 2017; 91(9): pii: e02241-16. Although there is a clear host range distinction among HEV genotypes, HEV-3 and HEV-4 viruses appear to be circulating between animal and human hosts.

Our results are the first confirmation of HEV circulating among domestic swine farms in Northeast Brazil. The HEV RNA prevalence rate found here is markedly low considering the seroprevalence rates of up to 95% found in the same region.⁹9. de Oliveira-Filho EF, Lopes KG, Cunha DS, Silva VS, Barbosa CN, Brandespim DF, et al. Risk analysis and occurrence of hepatitis E virus (HEV) in domestic swine in Northeast Brazil. Food Environ Virol. 2017; 9(3): 256-9. Nevertheless, our findings are similar to those obtained in other countries such as Germany, China, Japan, and India.¹³13. Oliveira-Filho EF, Bank-Wolf BR, Thiel HJ, König M. Phylogenetic analysis of hepatitis E virus in domestic swine and wild boar in Germany. Vet Microbiol. 2014; 174(1-2): 233-8.^,2828. Geng Y, Wang C, Zhao C, Yu X, Harrison TJ, Tian K, et al. Serological prevalence of hepatitis E virus in domestic animals and diversity of genotype 4 hepatitis E virus in China. Vector Borne Zoonotic Dis. 2010; 10(8): 765-70.^,2929. Sakano C, Morita Y, Shiono M, Yokota Y, Mokudai T, Sato-Motoi Y, et al. Prevalence of hepatitis E virus (HEV) infection in wild boars (Sus scrofa leucomystax) and pigs in Gunma Prefecture, Japan. J Vet Med Sci. 2009; 71(1): 21-5.^,3030. Vivek R, Kang G. Hepatitis E virus infections in swine and swine handlers in Vellore, Southern India. Am J Trop Med Hyg. 2011; 84(4): 647-9. Differences between serological and molecular prevalence rates are expected and likely to be related to animal age because HEV RNA is more easily detected in piglets that are two to six months old.⁵5. dos Santos DR, Vitral CL, de Paula VS, Marchevsky RS, Lopes JF, Gaspar AM, et al. Serological and molecular evidence of hepatitis E virus in swine in Brazil. Vet J. 2009; 182(3): 474-80.

Following the ICTV recommendation,²2. Smith DB, Simmonds P, Izopet J, Oliveira-Filho EF, Ulrich RG, Johne R, et al. Proposed reference sequences for hepatitis E virus subtypes. J Gen Virol. 2016; 97(3): 537-42. S26 was assigned to subtype 3f, which has already been detected in Brazil.⁷7. Lopes Dos Santos DR, Lewis-Ximenez LL, da Silva MF, de Sousa PS, Gaspar AM, Pinto MA. First report of a human autochthonous hepatitis E virus infection in Brazil. J Clin Virol. 2010; 47(3): 276-9. Although we were able to assign it successfully, a clear subtype separation based on pairwise or patristic distances was not possible (Table II). This divergence is likely related to differences in HEV-3 evolutionary rates among different subtypes, which seem to be higher among human-associated lineages.²⁷27. Brayne AB, Dearlove BL, Lester JS, Pond SLK, Frost SD. Genotype-specific evolution of hepatitis E virus. J Virol. 2017; 91(9): pii: e02241-16. In addition, the marked heterogeneity observed within the subtype 3f and 3e-3f-3g clade might be an evolutionary hallmark, for example resulting from the accumulation of mutations positively selected through successful infection in different hosts.²⁷27. Brayne AB, Dearlove BL, Lester JS, Pond SLK, Frost SD. Genotype-specific evolution of hepatitis E virus. J Virol. 2017; 91(9): pii: e02241-16. Thus, these results illustrate that subtyping inside this clade can be challenging and might indicate the possibility of the future emergence of new subtypes if additional new heterogenic sequences appear.

The close relationship between HEV strains isolated from different host species points towards interspecies transmission. In addition, the gene flow study corroborated a possible evolutionary host switch origin and zoonotic potential. Thus, the molecular evidence indicates that the HEV strains identified here are potentially zoonotic and pose a threat regarding the infection of animal handlers, veterinarians, and consumers of non-cooked swine meat and pork products. Future studies should address HEV molecular epidemiology and explore the genetic variability and potential transmission among human and animal populations in Brazil.

ACKNOWLEDGEMENTS

To Louisa Ludwig-Begall for the careful English proofreading of this manuscript.

REFERENCES

Publication Dates

History