Current knowledge on Hepatitis E virus infection

The knowledge about epidemiology and genomic characteristics of Hepatitis E virus (HEV) have developed and changed over the past 30 years. HEVgt1 and HEVgt2 are basically met in South-East Asia and Africa and their transmission is by waterway. HEVgt3 and HEVgt4 are mainly established in Europe and North America, they have native character and transmitted by consummation of contaminated food. The current article presents systematic analysis of the epidemiology, etiology, clinical signs, diagnosis, therapy and prevention of HEV infection.


Introduction
Hepatitis E is an emerging viral disease affecting both humans and different kinds of domestic and wild animals.In developing countries, human HEV has trend for epidemic spread with benign outcome, except pregnant women 1 .The death rate due to HEV exceeds 25% in the third trimester 1 .In developed countries, the autochthonous cases of human HEV infection are associated with a consummation of poorly heat-treated meat and meat products (mostly domestic and wild swine) 1 .
The current article presents systematic analysis of the epidemiology, etiology, clinical signs, diagnosis, therapy and prevention of HEV infection.

History
Hepatitis E is "recognized" in 1980 during the epidemic in the valley of Kashmir (India) 2 .The affected people were between 11-40 years old, native citizen of the valley with common source of water 2 .The infected area was characterized with high level of viral distribution and mortality among pregnant women 2 .The epidemic spread, the incubation period, clinical signs and biochemical results of the examined patients were similar to manifestation of Hepatitis A virus infection 2 .A few months later Wong et al, published results from retrospective serological study of stored samples from a large hepatitis epidemic in Delhi, India (1955-1956) and two smaller infected areas in Ahmedabad, India (1975-1976) and Pune, India (1978-1979)  3 .The results from that study established a few cases of acute hepatitis B and none acute hepatitis A 3 .Owing that fact was given the idea for existence of "non-A, non-B hepatitis agent" 3 .The next serious breakthrough was in USA (1997), when was found a swine virus, named "swine hepatitis virus" 4 .At the same time has been described the first case of human HEV, the isolated virus had similar genomic characteristics to the swine HEV 5,6 .That disclosure determines the zoonotic character of the virus 1 .

Etiology
Hepatitis E virus belongs to family Hepeviridae, genus Hepevirus 7 .According to the current classification, family Hepeviridae is divided into two genera: Orthohepevirus and Piscihepevirus.Orthohepevirus includes four species 8 :  Orthohepevirus A: isolated from human, swine, deer, mongoose, rabbit, camels;  Orthohepevirus B: isolated from birds;  Orthohepevirus C: isolated from rats, big Indian rat, Asian kind of mole, ferret and mink;  Orthohepevirus D: isolated from bats.
Until now, there are four main genotypes, with more than 24 subtypes and only one serotype [9][10][11] .Genotypes 1 and 2 (HEVgt1 and HEVgt2) are linked with large human epidemics in countries with poor hygiene 12 .Genotypes 3 and 4 (HEVgt4 and HEVgt3) infect humans and other mammals, which cause sporadic cases of Hepatitis E in industrialized countries 12 .
HEV is a small virus with a diameter approximately 27 to 32 nm, icosahedral symmetry, spherical shape and simple structure 12 .The virion contains single positivestranded RNA with size 7.2-7.5 kb 13 .HEV genome includes 5′ untranslated region (UTR), three opened-reading frames (ORF1, ORF2 and ORF3) and 3' UTR, followed by poly-A tail 13 .Each one reading frame has different functions 12,13 :  ORF1: is situated next do 5′ and encodes non-structured proteins with enzyme function (Methyltransferase, Papain-Like Cysteine Protease, Macrodomain, Helicase and RNA-dependent RNA Polymerase);  ORF2: is situated next do 3′ and encodes viral capsid protein, build from 660 amino acids, which is responsible for the viral cutting, interaction with target cells and immunogenicity properties;  ORF3: encodes small protein, build from 113-114 amino acids, which is responsible for replication and building cytoskeleton, also decreases inflammatory response and protects viral-infected cells.

Epidemiology and prevalence
Hepatitis E is an endemic disease for Central and Southeast Asia, for tropic and subtropical countries in Africa and Central America 1 .In the endemic area large waterborne epidemics were described.Hepatitis E is sporadically reported in USA and Europe 1 .In the developed world, it has been thought that the infection was associated with traveling to the endemic regions 14 .But in nowadays it is known that it is a local, autochthonous transmission.
HEVgt1 and HEVgt2 are responsible for enterically-transmitted epidemics in tropical and some subtropical areas 15 .They are associated with contamination of water (water supplies) and poor sanitation conditions 15 .Both genotypes cause acute hepatitis in humans, the virus is found in feces, an environment contaminated with human's feces 15 .A study in Uganda showed that environmental factors could be much more important for transmission, than it was thought until now 16 .
In non-endemic regions the mechanisms of transmission are much less known, in contrast in endemic areas the contaminated water is a documented source of infection 17 .
HEV is the only one among other hepatotropic viruses with zoonotic character and animal reservoir 17 .The literature search presents that most of the autochthonous human cases are associated with the consumption of raw and undercooked meat infected with HEV [18][19][20] .
Pigs are considered to be the main reservoirs for HEVgt3 and HEVgt4, and the two genotypes are found in pigs all over the world 21 .Antibodies against HEV are found in chickens, dogs, rodents, cows, sheep, goats, monkeys and other animals 22 .HEVgt3 is responsible for most of human HEV infections in Europe, North America and East Asia 23 .
HEVgt3 is dominated in swine samples in Europe and America.The virus was detected in pork products 19,24 .Strains of HEVgt3 were recently found in pigs in Africa 25 .In 1998 HEVgt4 was responsible for sporadic human HEV cases in Taiwan, and after that the virus was found in pigs at the same geographical area 26,27 .In China, HEVgt4 is the most common virus in humans and swine 28,29 .Also it is endemic in Japan 30 .In Europe, Japan and USA, specific antibodies against HEV are often detected in domestic pigs, which prove their role as a source of HEV infection 31,32 .Studies done in Japan and France presented the transmission of the virus through a consumption of meat and sausages, made of domestic pigs, wild boars and deer 19,33 .Acute hepatitis E was described after eating pork meat infected with HEVgt4 in Japan 34 .In Japan was reported severe human case after eating raw liver from a wild boar, whereas in Europe the severe human infections were related to a consumption of pork meat 19,35 .A phylogenetical analysis of HEV samples from Japan indicated a previous transmission of the virus from domestic pigs to wild boars 36 .Urine was identified as a possible source for swine HEV infection 1 .It has been established that swine HEV could pass colostrum, while transplacental transmission is arguable 1 .Another possible way of HEV transmission is the direct contact between people and swine 1 .
Serological studies in USA reported that veterinaries and people, who are working in slaughterhouses had high positive results for anti-HEV IgG compared to population with lower risk for direct contact with pigs and pork products 37 .A higher rate of seroprevalence among foresters was found in comparison with the seroprevalence among blood donors 1,21 .
HEV infection could be transmitted by transfusing blood and blood products 38 .Swine products, such as swine heparin and others, used in human medicine, could be a risk factor for HEV spread 39 .Other possible risk for HEV source could be feces or manure 40 .A study reported the presence of HEV in manure storage facilities 40 .
Nowadays, wild boars are thought to be an important natural reservoir for HEVgt3 and HEVgt4 1 .Recent study done across Asia and Europe showed a high rate of HEV seroprevalence likewise a molecular evidence of HEV infection in wild boars 1,12,21 .
Takahashi et al found HEV RNA in 1.1-13.3% of examined wild boars and seropositivity varied between 4.5% to 34.4% 41 .In Germany, wild boars are considered as one of the main sources for HEV transmission 42 .HEV RNA could be found in the serum, gall and liver from wild boars 43 .HEV samples collected from the wild boars showed great genetic variability 9,11 .
In many European countries different serological studies for human HEV seroprevalence were conducted over the past years.We present the results of HEV seropositivity in blood donors from 24 studies (Table 1)   In worldwide, the main animal reservoir for HEVgt3 and HEVgt4 are domestic pigs and wild boars 1,68 . Data or swine HEV seroprevalence in European countries are summarized in Table 2 32,67,[69][70][71][72][73][74][75][76][77][78] .There is a broad spectrum of variety in seropositivity among different countries. The valuated Mean±SD swine HEV seroprevalence is 47.93±19.75(95%CI = 9.23-86.64). The presentd average percentage for seropositivity illustrates the existence and persistence of the virus among pigs and their potential animal reservoir.

Clinical manifestation
The most common clinical manifestation of HEV among people in endemic areas is acute icteric hepatitis with typical clinical and laboratory signs 14 .Sometime prolonged cholestasis could be developed or asymptomatic infection may occurred 14 .High rate of fulminant hepatic failure and death were mentioned among pregnant women in hyperendemic areas 79,80 .In non-endemic areas the virus affected mainly elderly men, presence of accompanying liver diseases and alcohol abuse [81][82][83] .The autochthonous cases could be manifested as an acute hepatitis, asymptomatic infection, and nonspecific symptoms with anicteric diseases 48 .In contrast, severe illness does not show during pregnancy in non-endemic regions 81,82 .Chronic HEV infection has been described in solid-organ transplant recipients, patients with hematological diseases, HIV patients, people under immunosuppressive conditions and anticancer chemotherapy 81,82,[84][85][86] .In such case of patients the liver biopsy illustrated liver fibrosis, which predicts the progress to cirrhosis 87 .
Swine HEV infection does not present with typical clinical symptoms and signs.
Usually animals' diseases are characterized with fluctuations in body temperature or body weight 1 .After a subclinical HEV infection mild microscopic lesions in the liver could be developed 11 .Pathological findings include viral antigen in the hepatocytes, positive immunohistochemical changes in the small and large intestines, lymph nodes, tonsils, spleen and kidneys 12 .Spanish study reported no correlation between HEV RNA and the histological changes in the liver 88 .So it's arguable whether or not a natural HEV infection causes any histological changes in the liver.

Laboratory diagnostics
The most common method for routine diagnosis of HEV infection is serological examination.Laboratory diagnostics use serum samples for detection of HEV antibodies by enzyme-linked immunosorbent assays (ELISA) and western blot assays 1 .The tests estimate the presence of antibodies of class IgM and IgG (rarely IgA) against HEV.In the first stage of the infection antibodies of class IgM appear and mark acute present infection 1 .After that antibodies of class IgG follow up and show a recent or past infection.Serum samples are collected for serological tests in humans, for swine examination it could be collected sera or meat juice 89 .In humans, anti-HEV IgM levels peak around the time of the ALT peak and may persist up to five months after the onset of the illness (Figure 1) 14 .A little later anti-HEV IgG begin to produce, they remain during the acute phase, the recovalescent period and also maintain high levels at least one year after the recovering (Figure 1).Commercially available immunoassays differ substantially in their sensitivity and specificity, and the false-positive results varying from 0.3% to 2.5% 90,91 .
The majority of pigs are naturally infected with HEV at the age of two to four months 4 .Eighty six percentage of pigs are naturally infected with the virus until their eighteenth week 92 .The maternal antibodies decline at the age of 8 to 10 weeks 17 .After the reduction of them, the piglets could be attacked by the virus around the second weeks after birth 11 .Swine HEV infection is accompanied with a transient viremia lasting one to two weeks and a fecal-oral emission of the pathogen continuing three to seven weeks 9 .The number of viremic pigs increase from nine weeks with peaking around 15 weeks, following decline to slaughter age 93 .Seroconversion of anti-HEV IgM, which is related to the peak of the virus excretion through feces, is followed by seroconversion of anti-HEV IgG with the highest concentrations at the age of four months (Figure 2) 17 .Interestingly enough, the presence of antibodies does not always assure the absence the virus because HEV RNA and the anti-HEV antibodies are found in pigs together.This leads to the conclusion that these animals are HEV-reservoirs 21 .
Nowadays, the detection of HEV RNA using molecular-genetic methods is considered as the "golden standard" in the laboratory diagnosis 1 .The detection of RNA is performed by different RT-PCR methods, amplifying genomic fragments in one of the three ORFs 11,21 .HEV RNA could be found in patients' blood and/or feces in the prodromal period, after that the virus could be detected in feces for another two weeks 12 .
The viremic period is very short, wherefore HEV RNA not always could be found in sera.
The presence of HEV RNA is a definitive marker for a current infection.
Recently, the establishment of HEV antigen is introduced as an early diagnostic method 94,95 .However, the test has a low sensibility compared to methods that use the amplification of nucleic acid 96 .The presence of HEV antigen in different swine tissues using immunohistochemistry was recently demonstrated 97 .The detection of HEV antigen in liver tissue representing a valuable tool for the viral establishment in biopsy, autopsy and explant liver tissues 98 .

Therapy and prophylaxis
There is no specific therapy for acute HEV infection in humans, because in most cases the illness is self-limiting.The management of acute illness in immunocompetent patients include a strict diet, administration of fluids and hepatoprotective medications.In case of acute liver failure intensive care treatment is required and sometime liver transplantation needs 99 .In chronic HEV infection the administration of Pegylated interferon alpha-2a/alpha-2b or Ribavirin for 3-12 months were applied as specific antiviral therapy 100,101 .A recombinant vaccine showed 94-100% efficacy in a phase III study of >100 000 Chinese adults 102 .The vaccine protected from HEVgt1 and HEVgt2 102 .
There are no specific therapeutic medications for animals.Swine HEV vaccine has not been developed yet.
The prevention measures are guided to improving sanitation and hygiene in developing countries 17 .In developed countries, population with high risk could be informed for the virus and his zoonotic characteristic, consequently should be asked to reduce and/or avoid consummation of raw or undercooked meat and meat products from pigs, wild boars, deer and direct contact with infected animals.

Conclusion
Swine are defined as the main reservoirs for the zoonotic HEVgt3 and HEVgt4.The infection is widely spread in pigs all around the world.Just like in humans, the fecal-oral mechanism of the transmission is thought to be the main one in animals as well.The nature . The calculated Mean±SD human HEV seroprevalence is 15.21±14.20 (95%CI = 12.61-43.04).The great variety of positive results are affected by geographic location, national traditions and customs, design of the study, year of projects conducted and type of diagnostic tests.Nevertheless of the published diversity, these data confirmed the seroprevalence of HEV among blood donors in different European countries.

Table 1 Seroprevalence of hepatitis E virus in blood donors in European countries
Note: BD -Blood donors;