Molecular Detection of Hepatitis E Virus in Layer Chickens: A Possible Public Health Risk in Pakistan

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus which causes self-limiting infection. However, HEV infection in individuals with weakened immune system is linked with developing chronic hepatitis having higher death rates (Kmush et al., 2015). The HEV clinical symptoms include nausea, fever and followed by anorexia, vomiting, abdominal pain, jaundice and hepatitis (Mansuy et al., 2009). The HEV mortality rate ranges from 0.5 % - 4.0 % in overall population but it could reach up to 30 % in pregnant women particularly in third trimester (Navaneethan et al., 2008). The water-borne transmission of HEV causing repeated outbreaks of HEV genotype (gt) 1 and 2 is considered as emerging public health risk, especially in Asia and Africa (Hakim et al., 2017). The estimated HEV infections caused by gt-1 and 2 in 2005 were almost 20.1 million, out of which 3.4 million were found symptomatic with 70,000 fatalities while 3000 still births were recorded (Rein et al., 2012). The outbreaks burden of HEV is relatively higher in Asian countries including Pakistan, as compare to rest of the world. In Pakistan, so far, four major outbreaks were reported where prevalence rate ranged from 10.4 % to 20 % (Iqbal et al., 1989; Rab et al., 1997). Some small outbreaks were also reported times to times (Iqbal et al., 2011).

The HEV belongs to Hepeviridae family and members of which, on the basis of host range and phylogeny, are classified into two genotypes; Orthohepevirus and Piscihepevirus (Purdy et al., 2017). Orthohepevirus reported to contain four species (A-D) while Piscihepevirus consists of single species only (A). The HEV infecting human, deer, wild boar, pig and rabbit were placed in Orthohepevirus A and those isolated from bats were placed in Orthohepevirus D. Orthohepevirus B contains HEV isolated from chicken (layer and broiler breeders) while Orthohepevirus C consists of HEV identified in ferret and rats (Purdy et al., 2017).

The human HEV genome size is approximately 7.2 kb which encode three open reading frames (ORFs). The ORF1 encodes non-structural polyprotein having different functional domains like, methyl transferase, Y, protease, hypervariable region, X, helicase and RNA polymerase (RNA dependent). Whereas, ORF2 and ORF3 code for capsid protein and a multifunctional protein, respectively. In most of the strains, ORF2 and ORF3 are overlapped with each other but separated from ORF1 through intergenic junction region (Huang et al., 2004). On the other hand, the aHEV genome is bit short (6.6 kb) but contains components and protein products same as other HEV strains mentioned above. Almost 600 bp putative short region, as compare to other HEVs, is located between methyl transferase and helicase domain in ORF1 (Reuter et al., 2016).

Identification of many animals HEV strains suggests cross species infection which poses a big public health challenge in term of zoonosis and food borne transmission. So far, only genotype 3 and 4 are reported to be zoonotic which putatively caused infection in human through food borne transmission (Meng, 2013; Doceul et al., 2016). The cross-species infection of aHEV is reported within birds but no attempt has been successful in describing infection in non-human primates or other mammals (Sun et al., 2004; Huang et al., 2004). So, the trend of cross-species infection of aHEV within birds may be an indication of zoonosis in future. Moreover, the risk of food borne transmission cannot be underestimated as chicken based food stuff, especially the semi-cooked or under-cooked, may play important role in this regard. To the best of our knowledge no data about the circulation of aHEV is reported from Pakistan till date, so the present study was designed to isolate and characterize aHEV strain circulating in layer chickens from Pakistan.

The bile fluid, liver and spleen tissue samples were collected from overnight dead layer chickens (n = 8) through necropsy from Pattoki region of Punjab province, Pakistan. The chicken specimens were designated as PT7, PT9, PT10, PT11, PT12, PT13, PT14 and PT16. Similarly, bile samples obtained were designated as PT7B, PT9B, PT10B, PT11B, PT12B, PT13B, PT14B and PT16B. Notably, no outbreak was reported in the area during sampling. The tissues (liver and spleen) were fixed in 10 % formalin for histological studies while bile fluid samples collected in sterilized 1.5 ml tubes were stored at -80°C till future analyses.

For histological examination, formalin fixed paraffin embedded block of liver and spleen tissues were made. Briefly, formalin-fixed tissues were cut into cubical shape and dipped into different percentages of ethanol for 16 hours followed by xylene treatment. The tissues were then embedded in paraffin wax at 58-60°C and placed into embedding cassettes. The paraffin embedded tissues were sectioned through microtome and proceeded for hematoxylin and eosin (H&E) staining according to the described procedure (Cardiff et al., 2014).

Total RNA was isolated from bile samples by using Trizol reagent (Invitrogen). The RNA was converted into cDNA through Reverse Transcription PCR (RT-PCR) using VILO 2X master mix kit (Invitrogen), according to the protocol described previously (Iqbal et al., 2018). The Kwon et al., 2012 described method was used for viral genome detection in bile samples through amplification of partial helicase (ORF1) 186 bps and partial capsid protein (ORF2) 280 bps fragments using PerfeCta SYBR Green Fast Mix 2X kit (Quanta Biosciences). The PCR strategy was modified to two steps-PCR and amplification was carried at; initial denaturation 95 °C for 2 min after which 40 cycles of 95 °C for 30 secs, 50 °C for 30 secs, 72 °C for 1 min and final extension 72 °C for 10 min.

The PCR products were purified and sequenced through Sanger method using dideoxynucleotides (Big dye Invitrogen) while obtained sequences were processed and compiled through DNASTAR (Lasergene). Phylogenetic analysis was performed through Molecular Evolutionary Genetics Analysis version 6 (MEGA6) (Tamura et al., 2013) using Neighbor-Joining method with 1000 bootstrapping replicates (Tamura et al., 2004). The obtained sequences were submitted in Gen Bank. The accession numbers of sequences used in this study are given in Fig. 3 and 4.

The bile samples of total eight specimens were screened for aHEV RNA and two samples (PT12B and PT16B) were found positive for aHEV RNA. The 186 bps (2769-2954 nt, reference AM943647) from helicase domain (ORF1) and 280 bps (5461-5741 nt) from capsid protein (ORF2) were amplified from bile fluid of infected layer chickens (PT12 and PT16) (Fig. 1) while complete ORF3 of these two aHEV isolates was amplified in our earlier study (Iqbal et al., 2018).

Histological analyses of infected/viral genome (RNA) positive layer chickens (PT12, PT16) revealed lymphocytic infiltration causing portal phlebitis and periphlebitis in liver tissue (Fig. 2A). Additionally, granulocytes and multifocal necrosis was also observed in infected liver tissue (Fig. 2B). Similarly, spleen tissues demonstrated infiltration of monocytes, granulocytes, basophils and other large cells (Fig. 2C and 2D).

The obtained PCR products (ORF1=186 bps and ORF2=280 bps) were sequenced and sequences were submitted to Gen Bank database with following accession numbers; helicase domain (ORF1) MH243320, MH243321 and capsid protein (ORF2) MG692744, MH094852. These sequences were further subjected to the phylogenetic analysis. Our results revealed that on the basis of partial nucleotides sequence of both helicase and capsid proteins, Pak aHEV strains made monophyletic clade with Orthohepevirus B species of Orthohepevirus genus upon comparison with Orthohepevirus A species (HEV members from mammals including human), Orthohepevirus B species (HEV strains from chickens), Orthohepevirus C species (HEV only from Rats), Orthohepevirus D species (HEV specifically from Bats) and Piscihepevirus (HEV from cutthroat trout) (Fig. 3 and 4). On other hand, the results of this study manifested that Pak aHEV strains showed distant clustering within Orthohepevirus B species as compared to other member strains belonging to gt1, gt2, gt3 and gt4 which is evident from low bootstrap values on the branch point within monophyletic clade of Orthohepevirus B species showing that Pak aHEV strains may represent a novel genotype. Interestingly, phylogenetic analysis of Pak HEV strains with each other showed 100% bootsrap value for helicase domain and 99% for capsid protein gene showing that same HEV is circulating in layer chickens’ population in the area.

The infectivity of HEV in human and other animal species poses a concern of cross species infection which further extends to zoonosis. There are many factors associated to virus itself, potential host and environment which may promote its transmission and infection (Sooranarain and Meng, 2019). Human HEV gt1 and 2, infecting only human, are endemic to developing countries and their transmission is associated with sanitary contaminated drinking water through oral-fecal route (Khuroo, 2011). On the other hand, gt3 and 4 are zoonotic and have been reported from industrialized and developing countries as well and commonly follow foodborne transmission route The main foodborne transmission of gt3 and 4 is associated with consumption of contaminated animal meats like pig and deer (Kmush et al., 2015; Clemente-Casares et al., 2016). Pig has been reported as a reservoir of HEV multi-genotypes including gt3, 4, 5, and 6 while gt7, gt8 primarily infect camel and these genotypes possibly cause infection in human too (Meng, 2016; Sooryanarain and Meng, 2019). Recently cow has been identified as another potential reservoir for HEV (gt4) in China (Huang et al., 2016). Similarly, rabbit HEV is also known to cause infection in human (Abravanel et al., 2017; Kaiser et al., 2018). In this scenario the handler and people in close contact with these animals are at high risk of HEV infection which is evident by high rate of anti-HEV seropositivity among these peoples from different countries (Sooryanarain and Meng, 2019). In this context the characterization and monitoring of HEV animal strains is needed to understand HEV epidemiology.

Little is known about the pathogenesis and replication of HEV due to the lack of a cell culture system and a practical animal model for the propagation of this virus. In the present study, we selected layer’s flock with a history of signs and symptoms associated with aHEV that mainly include reduced egg production and HSS (Haqshenas et al., 2001; Zhao et al., 2017). We cannot say with surety that selected birds died because of aHEV infection. The reason of death in selected birds could be the HEV or any other infection sharing the signs of hepatitis, peritonitis and airsacculitis, for instance, Fasciola hepatica or Escherichia coli. Presence of virus in few (2 out of 8) birds itself is an indication that HEV was not the sole reason behind death of birds. In fact, HEV infection is an essential but not only the one factor for the development of HS syndrome in poultry (Billam et al., 2005).

Molecular identification through phylogenetic analysis demonstrated Pakistani strains as member of Orthohepevirus B species but distantly divergent as novel aHEV strains as compared to other aHEV genotypes. The molecular identification of aHEV in layer chickens in this research work seemed to be sporadic infection as no outbreak was reported in the area. So far, global distribution of aHEV is reported from United States of America (USA), Australia, China, Hungary, South Korea and Taiwan (Park et al., 2016) and this data may be the first report from Pakistan. The association of aHEV infection with histopathalogical lesions in liver and spleen tissue causing hepatitis-splenomegaly syndrome (HSS) in layer chickens and broiler breeders has been reported in different parts of the world (Crespo et al., 2015; Moon et al., 2016) which are evident in the present study and same have been reported in many animal species as well (Meng et al., 1999).

Phylogenetic analysis has revealed that avian HEV is genetically related to, but distinct from, other known HEV strains (Haqshenas et al., 2001). It has similar genomic organization and share 60% nucleotide sequence with human HEV viruses (Haqshenas et al., 2002). Likewise, the target organ (liver) and receptors (Heparan Sulfate) used by aHEV and human HEV are same (Kalia et al., 2009). Our data suggests that Pakistani aHEV described in this study is a novel strain because of its clustering in a separate clade upon comparison with previously known genotypes. Further studies will be required in order to describe pathogenic and zoonotic potential of this newly described strain.

Zoonosis of HEV is a big public health risk not only in developing countries but also in developed world. Foodborne transmission of zoonotic HEV gt3 and gt4 in developed countries is well established. In this regard under-cooked or un-cooked pork liver sausages, wild boar and shellfish have been recognized as potential risk factor (Capai et al., 2018). Poultry industry is well established all over the world and full fills a large portion of human food needs. The poor hygiene of poultry farms and substandard poultry wastes disposal play very important role in aHEV transmission among chicken flocks (Liu et al., 2017). However, aHEV has not been yet reported as zoonotic HEV but its cross-species infection within birds and sharing of conserved antigenic epitopes in capsid protein gene with swine HEV may be an indication of zoonosis in future as virus evolve (Haqshenas et al., 2002; Sun et al., 2004). Having this assumption, the foodborne transmission of aHEV through under-cooked or un-cooked chicken meat and other derived products may emerge as a potential public health risk in future. Moreover, Pakistani aHEV strains showed diverged clustering as compared to other aHEV genotypes and they may have different cross-species infection behavior. So, further investigations are needed on zoonosis and cross-species infection in this regard. Keeping in view the above scenario, detection of aHEV in layer chickens in Pakistan may pose a potential public health challenge.

The data presented in this study suggested that aHEV strains reported in this study are novel strains. Furthermore, comparison with each other showed close sequence homology which further suggests that single aHEV is circulating within the layer chickens’ population in Pattoki region of Punjab Province, Pakistan.

This research work was partially funded by Higher Education Commission (HEC) Pakistan under International Research Support Initiative Program (IRSIP). The positive aHEV control samples were kindly provided by X. J. Meng, Center for Molecular Medicine and Infectious Diseases, Virginia Tech VA, USA.

All authors declared that no conflict of interest.