Hepatitis E: Current Status in India and Other Asian Countries

Six types of hepatitis infections are accounted for the inflammation of liver in human throughout the world and assigned the letter A, B, C, D, E and G. Out of these, Hepatitis E, caused by Hepatitis E virus (HEV), currently categorized as a member of the genus Hepevirus in the family Hepeviridae. Hepatitis E is considered highly significant, on account of its predominance in both developed and developing nations, due to poor sanitation and cleanliness condition related to drinking water. This review focuses on general epidemiological study update of hepatitis E virus in India and rest of Asia for the selected period and also throws light on some diagnostic approaches available for the same. An extensive literature search was performed with published work and search in PubMed using term “HEV” combined with “country name” restricting the search publication between year 2008 to 2018 for India and 2013 to 2018 for the rest of Asia. The data was analyzed in detail to meet the requirements of the objectives. Study showed that in India the major causes of Hepatitis E was water contaminated with fecal matter. Reports from East and Southeast Asia signified the role of animal reservoir especially the pigs for HEV. Whereas in Indian neighboring countries like Nepal, Pakistan and Western Asia the major cases of Hepatitis E were associated with blood transfusion and pregnancy related cases. Majority of the diagnostic approaches used in India at medical facilities are based on anti-HEV IgM detection. However, the molecular based approaches played very crucial role in HEV detection. It is observed that the incidence of HEV is gradually expanding and its objective range is not only just developing countries, but also showing an enhancement. In overall, there is need for improvements in detection of HEV disease and methodologies used for the development of HEV vaccines.


INtRODUCtION
Hepatitis is an augmentation of the liver; a condition that can act naturally restrictant or can lead to cirrhosis, fibrosis (scarring), or liver cancer 1 . Researchers have recognized 6 types of hepatitis infections, distinguished by the letters A, B, C, D, E and G 2 . Hepatitis A virus (HAV) was first perceived in 1947 in worldwide yet is exceptionally common in the developing nations and Greenland and further, its worldwide occurrence is diminishing as a direct result of enhanced clean and living conditions 2 . Hepatitis B virus (HBV) is usually transmitted via exposure of contaminated semen, blood, and other body fluids. Hepatitis C virus (HCV) is mostly transmitted due to exposure to infective blood, transfusions of HCV-tainted blood and blood items 1 . Hepatitis D virus or delta virus, is a defective RNA virus that can replicate itself, still it requires simultaneous HBV for assembly and discharge; accordingly, patients with HDV in every case are dually infected with HBV 3 . The hepatitis G virus (HGV) is again a blood-borne virus spread by tainted blood and blood items 2 . The Hepatitis E virus (HEV), similar to HAV, is transmitted through utilization of debased food and water, and its pervasiveness is regular in both developing and developed countries.
An enteric transmitted non-A, non-B hepatitis virus was first reported in view of epidemiological examinations during a flare-up of viral hepatitis in [1978][1979] in Kashmir locale 4 . Soon after the fact, serological testing of sera was done after an extensive episode of hepatitis in New Delhi amid 1955-1956 and two reports in Ahmadabad (1975Ahmadabad ( -1976 and, Pune (1978)(1979) in India 5 . Samples from none of the three episodes demonstrated confirmation of acute hepatitis A and just few reports showed markers of acute hepatitis B, thus providing important help in confirmation of an enteric non-A, and non-B hepatitis. The non-A, and non-B hepatitis virus was also found after an episode of mysterious hepatitis in Soviet military in Afghanistan 6 . A member of research team ingested a pooled fecal extract from affected military personnel, and finally suffered with acute hepatitis and small viral particles were identified in his stool through immune electron microscopy 6 . The term hepatitis E virus (HEV) was coined, in view of its enteric transmission and relationship with hepatitis scourges 7 . Currently HEV is classified as a member of genus Hepevirus in the family Hepeviridae 8 . This review focuses on general epidemiological study update of HEV in India and rest of Asia for the selected period and also throws light on diagnostic approaches available for the same. For this study, keyword literature search was performed in PubMed using term "HEV" combined with "country name" restricting the search publication between year 2008 to 2018 for India and 2013 to 2018 for the rest of Asia. Any article that has been published between 2008 and 2018, but, in which sampling data was taken before 2008, has not been included. Similarly for rest Asia, in which sampling data was taken before 2013, has not been included.

Molecular Biology and Classification of HEV
HEV is a non-enveloped virus, with an icosahedral capsid of 27-34 nm size. The virus possesses a single-stranded, positive-sense, 7.2-kb RNA genome showing capping and polyadenylation at the 5' and 3' ends, individually 7,9 . The HEV genome has three open reading frames (ORFs) as shown in figure 1. ORF1 encodes a protein of 1,693 amino acids comprising functional domains itself present in the nonstructural proteins of other positive-strand RNA viruses 10 . These functional domains contain cysteine protease, methyltransferase, RNA helicase, and RNA-dependent RNA polymerase areas. ORF2 encodes 660 amino acids, a viral capsid protein responsible for virion assembly, immunogenicity, and interaction with target cells [11][12][13][14] . The ORF2 protein having three linear domains: the shell domain (S) (amino acids 129 to 319), the center domain (M) (amino acids 320 to 455), and the protruding domain (P) (amino acids 456 to 606) harboring the neutralizing epitope(s) [15][16][17]8,18 . ORF3, which overlaps ORF2, encodes a small protein of 113 or 114 amino acids, is associated with virion morphogenesis and discharge 19-21 . The HEV genomes of a few topographically distinctive isolates demonstrate a high level of sequence conservation 22 . Four phylogenetically unique genotypes have been characterized, which disseminate by geographic regions. In Asian and African regions HEV strains belonging to genotype 1 was more prevalent, genotype 2 incorporates the single Mexican HEV strain and few of variants identified as endemic in African nations. In industrialized nations genotype 3 with human and swine HEV strains are common, whereas in East Asia especially China, Taiwan and Japan, genotype 4 incorporates swine HEV and human strains 23 . The avian HEV was proposed to exhibit a place with another genotype 5 , but this has not yet been affirmed 24, 25 .

HEV in India from 2008-2018
At first the epidemics of acute hepatitis E, transmitted by means of polluted water, were reported in developing nations including India. Now the examples have changed with pace of time; and water-borne fecal-oral transmission is unable to justify the locally-acquired cases of hepatitis E in developed countries, as sterile and clean conditions have improved a lot. In such nations, now the zoonotic transmission via ingestion of uncooked meat has been considered to be the principle cause of HEV infection 26, 27 . Defiled water is still considered the prime suspect for spread of hepatitis E (Table 1) although there are also some reports based on transfusion-mediated HEV transmissions 28 . In, 2011, a flare-up of water linked hepatitis in Bathinda city, Punjab, showed the presence of 64.19% cases of HEV in the blood tests of the patients 29 . Study showed that the poverty and lack sanitation is firmly related with HEV infection. Blood tests reports gathered from the patients, in Nanital, Uttrakhand revealed the presence of high rate of HEV in drinking water due to sewage tainting of drinking water supply 30 . Two suburban (Nawanshahar and Palsora) of Punjab were suspected with the viral hepatitis episode related with sewage defilement of drinking water 31 . Another research in similar zones, compared the acute viral hepatitis patients (AVH) along with healthy and subclinical subjects (apparently healthy) and observed that both the strains were having a common source of infection, but with low viral load 32 . Another research also reported the flare-up of HEV in Northwest region of Punjab with fecal contamination of drinking water among 21-30 years of age group 33 . Vertical transmission of HEV contaminations at the rate of 78.9% (based on IgM, IgG anti-HEV and HEV RNA positive) was confirmed in newly conceived babies 34 . The biochemical, clinical, serological and virological profile of survived children showed that HEV contamination (mother-to-foetus transmission) in neonates is a self-restricting disease and does not cause unending viremia or delayed clinical course.
Duel infection of hepatitis E and B was accounted in Delhi for first time, and 26 patients with double infections showed that hepatitis B virus (HBV) infected chronic carriers patients were secondarily infected with HEV and resulted in acute hepatitis 35 . A report from Ahmadabad, Gujarat showed 4.78% HEV cases among the blood donors 36 . The age of the study group ranged from 18 to 60 years and age of 41-60 years tended to have higher HEV seroprevalence rates of 10.34% (positive IgM anti-HEV). In general less seroprevalence among blood donors is connected with proper sanitation facilities in their respective areas and to some extent the social status. Anti-HEV IgG rate was 60.5% among the blood donors in the range of 18 and 63 years in Lucknow and showed comparable anti-HEV rates among male and female 28 . A hospital-based investigation in West Bengal, revealed the detection of anti-HEV IgG in 41.8% AVH patients and 23% in healthy people. The high prevalence of anti-HEV IgG in healthy control shows the presence of subclinical infection 37 . Another hospital-based investigation showed that low HEV prevalence in kids (9.79%) may be related with improvement in living standards of the population 38 . HEV antigen measure can be used as an extra analytic marker to show the viral replication in HEV infection 39 . HEV strains isolated from sewage samples collected from Vellore belonged to genotype 1 and have shown 94-100% sequence similarity with HEV strains have isolated earlier from acute hepatitis E cases at Vellore hospital 40 . An examination report from Punjab has shown pigs as a reservoir of HEV with 65.0% rate and high seropositivity (18 . Another investigation on goat, exhibited 2 HEV strains belonging to genotype 4 and showing sequence similarity with cows, humans and pigs 51 . Blood sera gathered from sheep and sheep butchers in China showed 35.2% anti-HEV antibody in sheep blood, 57.7% in butcher's blood, and among sheep the most elevated cases were among adult in contrast with young ones and all the isolates belonged to genotype 4 52 . Study was done in HEV patients and different food items in Hong Kong and all HEV patients had 100% IgM anti-HEV and 82% of HEV RNA from pig liver, intestine and oysters 53 . Experts from Japan have shown the presence of HEV in wild boars in few territories and phylogenetic tree examination showed that the majority of the HEV strains resembled with genotype 3 54 . Another report from Japan found the highest positivity of HEV RNA among 3-months old pigs 55 . HEV was also additionally found in Japanese bats and BLAST study showed the highest sequence similarity with German strain. Although, the research only commented on the geographical distribution of BatHEV among different bat species; yet it failed to connect the presence of same HEV strains in Japanese and German bats 56 . In South Korea, fecal sample of the pigs were gathered from 14 farms in which 5 farms contained HEV-3, while other 2 had HEV-4 only 57 . HEV RNA detected from rabbit's feces in Korea is arranged into rabbit clad within HEV-3 and one of the rabbit HEV isolate was named as KOR-RB-1. The full length genome of such isolate showed 86    Healthy Camels NS (Not Specified); ND (Not Determined) * (Swine, Rabbits, Foxes, Sheep, Sika Deer, Wild Boars, Yaks, Camels, Asiatic Black Bears, African Lion, Red Pandas, Civets, Wolves, Jackals,); ** Birds(Species not defined); *** Pig stool RNA; **** Rabbit stool RNA; ***** Marek's disease; ****** Liver, Lung and Intestine; ******* Collective RNA (Liver and Fecal); ******** Specific-pathogens free; ********* Different sampling site report from Taiwan, showed the presence of Avian HEV (aHEV) in chickens, and its full genome ( 63 .

Cases Related to Blood Donors
Transfusion safety is having utmost significance in transfusion services and with time the transfusion safety is enhancing with screening of blood and blood items with regular introduction of better tests. The presence of anti-HEV IgG antibody has generally been taken as proof of prior exposure to HEV 64 . Report from Nepal showed the presence of hepatitis E viremia among the healthy blood donors with a rate of 1.54% and phylogenetic examination of these samples demonstrates the infection with homology of 95% with strain from India and Nepal outbreak in 2014 65 . An investigation conducted to check HEV seroprevalence among the blood donors in Nepal after devastating earthquakes in 2015 showed 3.0% donors positive for anti-HEV IgM, and 2.7% were found with both HEV IgM and IgG antibodies. HEV IgM predominance was related with donors having history of jaundice and pork consumption was found to be a possible risk factor for such infection. Anti-HEV IgG positivity rate was 41.9% and the predominance HEV IgG increased with increasing age and was higher in repeat blood donors, with a history of jaundice and pork consumption 66 . Among the blood donors in Cambodia 28.2% of Anti-HEV IgG were found 67 . One of the isolate belonged to genotype 3 and researcher found a strong genetic link between human transfusion-related HEV-3 strain and one aquatic HEV-3 strain from the different geographical regions in Cambodia. The study suggested that river water, contaminated with HEV-3 by human feces, may be additional source of HEV-3 infection in Cambodia. In another report the blood samples collected from the donors in one of the Iranian city showed 46.1% anti-HEV IgG antibody with noteworthy rate of anti-HEV seroprevalence in subjects in the age group of 61-70 years (90.9%). On the other hand, the amount of anti-HEV IgM was 1.4% and the rate was high among the subjects with 18-30 years of age 68 . A study in the healthy blood donors in Bushehr city, Iran showed that the anti-HEV IgG was 39.1% in the age group 40-60 years and 71.4% in the group older than 60 years: thus indicating that anti-HEV prevalence increased with increase of age 69 . In Tehran, Iran; the HEV seroprevalence was essentially extraordinary with regards to level of education among the healthy blood donors and showed low HEV predominance in university degree holders 70 . A study on the blood donors in Southwest of Iran showed 7.1% of HEV seroprevalence (on the basis of HEV IgG and IgM antibodies), however the HEV RNA from all the isolates reveled 88% to 99% nucleotide sequence similarity with HEV genotype 1 71 . Similarly, 20.7% anti-HEV seroprevalence was found among the blood donors in Qatar amid 2013 and 2016 72 . The high seroprevalence in Qatar may be due to blended population where a substantial extent is originating from hyperendemic nations such as, Egypt and Indian subcontinent.

Cases Related to General Population
A large number of HEV infection cases were also reported from general population. Serum anti-HEV positive rate was 13.92% in Yunnan region, China 73 . HEV infection rate was 16.13% in male in contrast to females 10.97%, however the highest cases were found among the age group of 20-30 years in both men and women. Farmers exhibited high HEV infection rate (20.35%) and showed that poor health and living condition and regular contact with animals might have caused above phenomenon. In another study a total of 1500 healthy children were screened for the presence of anti-HEV antibodies and out of these 14.93% were found positive for HEV seroprevalence 74 . Blood tests of the workers, in seafood processing plants also revealed the presence of anti-HEV IgG antibody and persons handling the crude seafood had higher anti-HEV IgG (32.54%) than those handling semi-processed items (24.74%) 75 . A study conducted in 4 ethnic (Naxi, Bulang, Wa and Hani) population in China detailed the predominance of IgG in Hani, Naxi and Bulang than in Wa ethnic individuals. Univariable analysis showed that HEV IgG pervasiveness was related with origin, gender, education level, smoking habit, body fat ratio and visceral fat index. Study additionally stressed that there might be tendency for eating half-cooked or crude meat and drinking from dairy animals and goat's crude milk as one of the reason of high HEV IgG in these ethnic people. On the other hand, HEV IgG pervasiveness was most elevated in Hani ethnic group (82.3%) Journal of Pure and Applied Microbiology as they have the habit of eating raw pork liver and fresh blood from pigs, goats and dogs 76 . An investigation in Dhaka city of Bangladesh had shown 30% cases of HEV among the healthy urban individuals, and has shown relentless increase among persons over 60 years of age. In 18-40 years of age group the prevalence of anti-HEV IgG is nearly double in females than that in males; however, the study did not define the actual causes of HEV in report 77 . Study in Laos villages figured out the higher anti-HEV IgG seropositivity rates among the people who were in close contact with cattle and had 3 times higher probability to be seropositive than members without contact. Consumption of crude animal organs, blood, or risky water was not related with high anti-HEV IgG seropositivity and overall 7.0% of the ruminants had antibodies against HEV, yet this study did not report direct or indirect zoonotic transmission from cattle 78 . In Lop Buri and Narathiwat provinces of Thailand, 37.3% and 8.9% of anti-HEV IgG were found in healthy persons. Majority of residents such as abattoir worker, animal transporters, swine farmers, pork handlers or consumers in Lop Buri who follows Buddhism have no restrictions on consuming pork products and these individuals had the chances to get exposed to HEV. These factors may therefore increases the exposure of HEV in Lop Buri populations, whereas, most Narathiwat residents stick to Islam and were not engaged in any swine related activity and showed less HEV among them 79 . During 2015 and 2016, 30.9% seroprevalence cases of HEV were noticed among the healthy people of Jordan 80 . The study showed that the individuals with formal education had significantly lower HEV seroprevalence as compared to those who had no education.
Interestingly, eating undercooked meat was highly associated with HEV seropositivity. In Denizli province of Turkey, 12.4% Anti-HEV seroprevalence was found among the healthy primary school kids in 2013 81 . Anti-HEV antibodies were found positive in 17 (18.1%) kids of the age group seven-year, and 6 (6.6%) kids of the age group fourteen-year among 185 grade school youngsters. There was no association between the anti-HEV antibody and gender, parental educational level, and socioeconomic level, and concluded that insufficient sanitation could be the reason for such prevalence.

Cases Related to Acute and Chronic Viral Hepatitis Patients
Acute viral hepatitis is exhibited by symptoms, such as darkened urine, uncolored stool, vomiting, myalgia, weakness and jaundice. Serological markers include high levels of liver transaminases, bilirubin, alkaline phosphatases and β-glutamyltransferase, and detection of anti-HEV IgG/IgM antibodies and HEV-RNA in serum and stool 82 . HEV-4 genotype were found among the acute viral hepatitis (AVH) patients in Shandong province of China and showed that direct contact with pigs, pig farmers; workers in meat handling plant; ingestion of pork, pork items and crude vegetables are essential epidemiological elements that related with HEV infection 83 . An outbreak of HEV in old age nursing home people in Japan showed that some uncooked food items were responsible for such infection 84 . In Mongolia, 20.8% human HEV cases were reported in AVH patients ranging between the years 2014-2015 and 99.8% isolates showed their nucleotide sequence closeness with Nepalese genotype 1. It was presumed that in Mongolia, people with HEV infection or contaminated food came from Nepal and other neighboring countries and brought genotype 1 HEV strain 85 . An episode of acute hepatitis E in a hospital of Bangladesh showed 13 patients with history of jaundice although their etiological factors were unknown, but most of the patients shared common drinking water, common cooking and dining facilities and even two persons showed acute hepatitis 86 . Epidemic of HEV occurred in one of the province (Biratnagar) in Nepal in 2014 and >7,000 patients were affected during this epidemic and the epidemic was presumed to be caused by the drinking water. In 2014, water and sewerage pipelines were harmed in various territories of Biratnagar amid development, and, repair of streets and serum tests from 48 AVH patients demonstrated the presence of IgG, IgM and IgA against HEV and all the isolates belonged to genotype 1a 87 . Patient with chronic HEV infection also secreted HEV-RNA and HEV-Ag persistently in urine 88 .

Cases Related to High Risk Patients
Hepatitis E virus (HEV) infection was earlier thought to be a self-limiting disease having no risk of chronicity. But recently several chronic cases of hepatitis E have been reported among immunosuppressed patients 89 . Over all rate of HEV among the HIV patients in China were 44.68% and the anti-HEV prevalence expanded by 4.1% every year with patient's age. Beside age the other factors such as gender, CD4 cell count, WHO stage, marital status and total cholesterol levels are other risk factors that are associated with HEV co-infection in HIV-infected population 90 . Another investigation from China additionally detailed the HEV among disease patients and the rate was 26.0% and the highest HEV seropervalence occurred in patients with leukemia followed by liver and gastric cancer. This study showed that the HEV seroprevalence was higher in cancer patients than in controls (Healthy people) (26.03% versus 13.0%). The diseased patients are normally immuno-suppressed or immunocompromised, and received blood transfusions, immunosuppressive medications or cancer chemotherapy which prompts their higher vulnerability to HEV. In rural cancer patients the seroprevalence of HEV was 27.8% marginally higher and pigs act as main reservoir of HEV in these areas as compared to urban cancer patients (24.2%) 91 . Prevalence of HEV was also examined in the Nepalese HIV An examination of kids with neurological issues, immune deficient kids and healthy kid's sera tests in Russia revealed the higher presence of anti-HEV IgG among immune deficient in contrast with healthy kids. Besides this anti-HEV IgM rate was higher (1.1-1.6% versus 0.25%) in immune deficient in contrast with healthy children. This study stressed that immune suppression is a factor of increased risk of infection in children with HEV 93 . Study from Iranian hospital showed the presence of anti-HEV antibody among the patients with end stage renal disease (10.6%), but failed to report any association between HEV, age, gender, duration of hemodialysis (HD), and HCV antibody among the patients 94 . Another study conducted on high risk patients showed 10.0% and 1.8% of anti-HEV IgG and IgM antibodies respectively among the thalassemic patients and the seroprevalence of IgG increased with age, raising from 0% in patients below 10 years to 10% above 10 years of age group 95 . Study emphasized the importance of HEV infection concerning blood multi-transfusion and possibility that the virus could be parenteral exposed or there is another possibility of shared route of transmission.

Cases Related to Pregnancy
In developing countries, the incidence of HEV infection in pregnancy is high and a significant proportion of pregnant women shows mortality rate varying from 30-100% 96 . However the severe liver injury in pregnant women with Hepatitis E remains unknown. In Pakistan, 15.55% of HEV cases were reported among the pregnant women admitted in various hospitals and none of ladies had jaundice of pregnancy or hepatocellular carcinoma at the time of hospital admission 97 . The study showed that HEV infection increased with repeated exposure of virus, and during pregnancy the severity increases with every trimester and it was more predominant in multigravida ladies and in third trimester, all the babies that were born from infected mothers survived. In Urmia an Iranian city, researchers have reported presence of anti-HEV IgG antibody (3.6%) among the healthy pregnant ladies and none of the subject had history of blood transfusion and tattooing or Hijma. The study did not show any significance between age, pregnancy time, gestational age and income level and concluded that low level of HEV among pregnant ladies may be related with better sanitation, efficient health system and arrangement for safe water than other locales of Iran 98 . Another examination on HEV among the pregnant ladies was conducted in Bushehr the biggest province of Iran and the rate was 6.3% 99 . The Bushehr city is having seaport with a high migration flow, and this may be the solid reason of high seroprevalence of HEV in this city.

Cases Related to Sewage
HEV, which is shed in the feces of infected individuals and animals, has been detected in sewage samples, suggesting that HEV contamination of aquatic environments may also be present 100 . In China 1.32% of HEV was found in sewage plants and BLAST analysis showed 95%-99% nucleotide sequence similarity with swine isolates and belonged to genotype 4 101 . A report from Israel found 8.8% HEV RNA in the sewage water and 6.1% among the hepatitis patients respectively 102 . All of these patients' and sewage samples sequences belonged to genotype 1 and genotype 3, respectively.

Diagnosis and Vaccine
Various approaches including demonstrative and diagnostic procedures were applied for confirming and collecting information for HEV. Elevated values of liver parameters at routine intervals, including alanine amino-transferase (ALT), aspartate aminotransferase (AST), bilirubin, gamma-glutamyl transpeptidase, and soluble phosphate are delicate yet nonspecific, marker of liver damage 103 . Immune electron microscopy (IEM) identifies infection like particles in fecal samples and, in this method HEV particles were precipitated with native antibody to recover HEV from sera 104 . HEV antigen can be detected in liver tissue utilizing immune fluorescence microscopy in which liver biopsy tissue having HEV particles are absorbed with fluorescent labeled anti-HEV IgG antibody and complex are watched with microscope equipped with epifluorescent gadget 105 . The various approaches used for identification of HEV are shown in figure 2.
Patients' initial introduction, timely collection of feces or serum samples and suitable transport and processing determined the sensitivity of the molecular test employed for the detection of HEV RNA. In this way, untraceable viral RNA does not get ruled out from HEV infection 106 . The HEV RNA can be recognized in the beginning of illness and up to 6 months in stool and serum thus, reduces the catch window for HEV 107 . The detection of viral RNA in biological example is the gold standard for identification of acute HEV hepatitis as nucleic acid amplification techniques (NATs) can precisely recognize active infection and help in affirming serological findings 108 . On the other hand NAT-based identification is a costly methodology that is generally not available at diagnostic research centers, and also it requires exceptionally specialized techniques and trained work force 109 . Over the couple of years, a few NAT tests based on reverse transcription followed by PCR, real-time PCR, and reverse transcription loop-mediated isothermal amplification have been reported for identification and detection of HEV RNA in serum and stool samples. For the detection of four human HEV genotypes, NATs assays have been designed and optimized broadly [110][111][112] .
Extraordinary fluctuation has been seen in the execution of in-house tests because of the non-standardized nature of the HEV nucleic acid detection methods 113 . For the detection of hepatitis A, B, C and E nucleic acids in sera of infected patients, a single step-multiplex RT-PCR was created in which the conserved regions of all the viral genomes were used as target sequence for amplification and the method was found to be rapid, sensitive, precise and reproducible in nature. The detection limit was found to be 280 copies/ml for hepatitis A virus; for hepatitis B 290copies/ml; for hepatitis C 30copies/ml and 300 copies/ml for hepatitis E in a single tube assay system 114 .
A very little knowledge regarding immunological parts of the HEV infection was available until a pattern of antibody response to HEV was recognized and studied. Anti-HEV IgM appears in the acute phase of the HEV infection and is detectable 4 days after the beginning of jaundice and holds on for 5 months 115 . Generally, 90% of patients suffering with acute hepatitis E infection show presence of detectable anti-HEV IgM within 14 days of infection, while anti-HEV IgG antibodies are noticeable soon after the presence of anti-HEV IgM 115 . The way these two classes of antibodies develop at the same time in acute infection makes it hard to build up a precise serological conclusion of acute HEV infection. Therefore, conventional immunoassays for IgM are only effective from 90% to 97%, with false positive aftereffects of up to 2.5% and a few of these results also create false negative outcomes in patients infected with genotype 1 strains [116][117][118] . ORF2 and ORF3 antigens and immunodominant peptides are broadly utilized for recognizing IgM, IgG, and IgA antibodies against HEV in commercial HEV serological tests 119,120 . The cross-serological reactivity of HEV with other viruses acknowledged to causes hepatitis was also additionally researched 121 . Most of the research is engaged to the analysis of acute HEV infection in immunosuppressed patients, for example, AIDS, leukemia and lymphoma and also solid organ transplant beneficiaries. During serological testing those conditions of patients must be considered, where seroconversion to anti-HEV antibodies during infection is delayed or may not occur at all. A quantiferon assay has been developed for the fast result of HEV infection 122 . In this assay, a genotype 3a peptide library (616 overlapping peptides spanning open reading frames [ORFs] 1-3) was used in interferon-gamma (IFN-γ) T-cell ELISpot assay.
Two hepatitis E immunizations have been assessed in clinical preliminaries and the first of these, rHEV5, was produced by GlaxoSmithKline (GSK, Rixensart, Belgium) 123 . The other HEV 239 (exchange name Hecolin®), was created by Innovax (Xiamen, China) [124][125][126] . HEV 239 was authorized in China in 2012 and at present, Hecolin® is accessible in Chinese market and has been affirmed for use in individuals aged more than 16 years. It is used more frequently for inoculating people at high danger of HEV disease, for example, those engaged with creature cultivation, understudies, individuals from the military, sustenance handlers, ladies of childbearing age, and explorers to endemic locales. The Chinese Center for Disease Control and Prevention (CDC) is using an internet framework to gather post-showcasing unfriendly responses/ occasions, and has not recognized any worries with the immunization. In spite of the fact that the current 4.5 years of security and adequacy the information for Hecolin® are reassuring, and the information has not yet been gathered in Journal of Pure and Applied Microbiology some areas, including pediatric subjects (< 16 years old), the elderly (> 65 years old), pregnant ladies, people with hidden liver sicknesses or the immunosuppressed individuals. These groups confront a more prominent danger of HEV contamination and are consequently in high need of this vaccine 123 .

CONCLUSION
It is being observed that the incidence of HEV is gradually expanding and its objective range is not only just developing, but also showing enhancement as well. So, in overall, there is need for improvements in detection of HEV disease and methodologies used for the development of HEV vaccines. Advancement of methods using biosensors for the brisk location of HEV in clinical, sustenance and ecological examples is the need of the hour. The research has been done to develop the amperometric sensor for quickly recognizing Streptococcus pyogenes causing rheumatic heart disease; and ultrasensitive transglutaminase based nano-sensor for early detection of celiac disease in human 127,128 . Additionally, consideration is required for more proficiency in planning of HEV vaccines development with new innovations to meet the future requirement.