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Hepatitis E virus infection and blood transfusion in Japan

2011; Wiley; Volume: 6; Issue: 2 Linguagem: Inglês

10.1111/j.1751-2824.2011.01512.x

1751-2824

Keiji Matsubayashi, Hidekatsu Sakata, Hisami Ikeda,

Viral gastroenteritis research and epidemiology

Nearly 30 years ago, a volunteer immune to hepatitis A virus (HAV) developed acute hepatitis after experimental ingestion of faecal suspension collected from nine acute phase patients from a water-borne outbreak of non-A, non-B hepatitis in Afghanistan. The virus-like particles (VLPs) different from hepatitis A and B viruses were visualized in his stool [1, 2]. Despite serological and morphological evidences for the novel enterically transmitted non-A, non-B hepatitis virus, it was very difficult to identify the sequence of this virus because the viral load in stool samples was extremely low and insufficient for cloning and sequencing [2]. It needs to be noted that PCR was not available at that time. Using the bile sample of the experimentally infected monkey, cDNA of this virus was finally cloned and sequenced. The nucleotides sequence was not homologous to any in the GenBank database at that time [3] and named hepatitis E virus (HEV) after 'enterically' transmitted hepatitis virus. A few years later, full length HEV genome was sequenced and determined (GenBank accession no. M73218) [4]. The epidemic of enterically transmitted non-A, non-B hepatitis in New Delhi, India in 1955 through 1956 was described first as an epidemic of hepatitis E [5]. Large epidemics of HEV have been reported from many countries in Central, East and South East Asia, Central America and North and West Africa. HEV is the major cause of acute hepatitis in developing countries with insufficient sanitary conditions and is transmitted mainly by water-borne route in association with contaminated drinking water and food [6]. In general, hepatitis E is a self-limited disease and asymptomatic HEV infection is common. Typical clinical manifestation of hepatitis E is similar to that of acute viral hepatitis; anorexia, dark coloured urine, vomiting, fever, epigastric pain and jaundice. Severe hepatic failure and high mortality rate of nearly 20% among pregnant women in hepatitis E are observed only in endemic area, which is a distinctive feature of this disease and most important issue to be solved in endemic area. Recently, persistent HEV infection and chronic hepatitis E along with progression of liver inflammation and fibrosis with HEV-related cirrhosis have been described in immunocompromised patients with lymphoma, recipients of organ transplant and patients with HIV infection [7-10]. HEV is a small spherical (27–34 nm in diameter), non-enveloped virus with a single-stranded, positive-sense RNA genome of 7·2 kb which encodes three open reading frames (ORFs: ORF1, ORF2 and ORF3) between cap structure at 5′-end and poly-(A) tail at 3′-end. The three ORFs encode for non-structural polyprotein, capsid protein and phosphorylated protein, respectively [11]. Although HEV has a single serotype, high genetic diversity of HEV genome is observed. HEV is classified into four major genotypes; genotype 1, 2, 3 and 4 [11]. Their features in epidemiology and virulence are different from each other (Table 1). Genotypes 1 and 2 are distributed in hyper endemic area in Asia, Central America and Africa and spread by water-borne route. In contrast, genotype 3 is found worldwide including Asia, North and South America, Europe and Oceania. Genotype 4 is restricted in East, South East Asia and rarely in Europe and seems to cause more severe hepatitis than genotype 3. Genotypes 3 and 4 are also isolated from sera, stool, liver and meat products of several animal species such as farmed pigs, wild boars and deer. Very recently, in addition to the four genotypes, other new genetic groups of HEV strain were isolated in wild boars in Japan [12, 13]. Hepatitis E is diagnosed by detection of IgM, IgA, or IgG anti-HEV or HEV RNA. N-terminally truncated HEV capsid proteins can be efficiently expressed and self-assembled as empty VLPs in insect cells infected with a recombinant baculovirus. The VLPs are used as immobilized antigens for the sensitive ELISA for HEV-specific antibodies [14]. Nested RT-PCR and real-time RT-PCR targeting ORF1 region or ORF2/3 overlapping region are used for detection of HEV RNA [15-19]. Hepatitis E had been long considered as a disease of developing countries with poor sanitary conditions and recognized as an imported disease of travellers returned from the endemic area. However, recent evidences have indicated that autochthonous HEV infection occurs in developed countries including Japan, and appears to be more prevalent than previously thought. Hepatitis E patients without history of recent foreign travel were found in the USA and European countries and novel HEV strains of genotype 3, different from genotypes 1 and 2 in endemic area, were identified [20-22]. At the same time, a novel swine HEV of genotype 3 was isolated from a pig in the USA which was closely related to human HEV of genotype 3 strains [23]. High prevalence of IgG anti-HEV was also observed in pig handlers in the USA [24]. These findings suggested a possibility of autochthonous HEV infection as zoonosis in developed countries but it had been controversial whether HEV was a zoonotic agent or not. In Japan, a few years later, novel HEV strains of genotype 3 indigenous to Japan were isolated from a hepatitis patient with unknown aetiology and a farmed pig, respectively [15, 16]. Following the discovery, many sporadic cases and several small outbreaks of HEV infection have been recognized in Japan and many Japan-indigenous HEV strains of genotype 3 and 4 have been identified in human as well as pigs, wild boars, deer, mongoose and bivalves Yamato-shijimi. Thus, HEV has a broad host range and pigs are recognized as an important reservoir of HEV. Furthermore, important studies on the route of transmission of HEV were reported from Japan. Members of two families developed hepatitis E after consumption of raw meat from Japanese Sika deer. HEV RNA sequences of genotype 3 isolated in their blood were identical to that in the leftover meat refrigerated in a freezer [25]. Although there had been several reports of the indirect evidences of HEV infection, this was the first direct evidence of zoonotic infection of HEV from animal to human. Soon after the report by Tei S, et al. [25], Li TC et al. described another case report in which patients infected with HEV through consumption of wild boar meat contaminated with HEV of genotype 3 [26]. These findings settled the debate over whether HEV is a zoonotic agent or not. Genotypes 3 and 4 of HEV are zoonotic agents. As many as 253 of incidents of HEV infection were extensively collected in Japan and analysed by a group led by Mishiro [27]. The status of HEV infection in Japan can be summarized briefly as follows: (1) HEV infection spreads widely across Japan; (2) hepatitis E is a disease of middle-aged people with a predominance of male over female; (3) HEV strains of genotype 3 and 4 are autochthonous in Japan; (4) the older the age the severer the disease; (5) genotype 4 is associated with more obvious and severer clinical manifestations of hepatitis than genotype 3; (6) no seasonality in its incidence is observed; and (7) transmission routes remain unknown in approx. 60% cases, although about 30%, 8%, and 2% are ascribable to zoonotic food-borne transmission, imported infection, and blood-borne, respectively [27]. Thus, the status of HEV infection in Japan seems to be different from that in hyper endemic countries. Blood-borne transmission of HEV will be referred in more detail, next. As mentioned above, in hyper endemic area the route of transmission of HEV is extensively water-borne but a possibility of vertical transmission as well as blood-borne transmission has been suggested. In India, two cases of transfusion-transmitted hepatitis E were reported by means of retrospective analyses [28]. However, they were unsuccessful in demonstrating the association of blood transfusion with hepatitis E infection by molecular approaches. We demonstrated for the first time that transfusion-transmission hepatitis E existed even in a developed country by showing 100% sequence identity of HEV RNA genome both from a donor and a recipient in 2002 [29]. In this case, two blood products, fresh frozen plasma (FFP) and red cell concentrate (RCC) were derived from the HEV-positive blood. FFP was transfused to a patient and he developed hepatitis E. The nucleotide sequences of HEV isolated from the stored plasma and from the patient were identical and belonged to genotype 4 that was indigenous to Hokkaido, Japan. In contrast, the HEV-positive RCC that was transfused to another patient under immunosuppressed conditions did not cause any hepatitis symptoms. He was negative for any HEV markers on Day 130 after transfusion. The viral load of HEV in the RCC product could be too low to cause infection [29]. In Hokkaido, high prevalence of HEV RNA was observed in the donors with elevated ALT levels higher than 500 IU/l (Fig. 1). Extensive epidemiological surveys for prevalence of anti-HEV antibodies were conducted in blood donors with elevated ALT levels higher than 200 IU/l (n = 1389) and in qualified blood donors (n = 12 600) in Japan. The prevalence of IgG anti-HEV was 3·2% and 3·4% in these groups, respectively [30, 31]. The studies revealed wide spread of autochthonous HEV infection among blood donors in Japan. HEV strains of 14 genotype 3 and 1 genotype 4 were identified in blood donors with elevated ALT and most of them were shown to be indigenous to Japan by phylogenetic analysis and closely related to swine HEV strains in Japan [30]. A higher prevalence of IgG anti-HEV was observed in male donors, older donors and in donors residing in eastern Japan [31] (Fig. 2). Viruses determined among blood donors with elevated ALT levels higher than 500 IU/l. Donated blood with ALT levels higher than 500 IU/l were collected from April 2000 through March 2003 in Hokkaido, Japan (n = 74). In-house real-time PCR testing for HAV RNA, HBV DNA, HCV RNA, HEV RNA and EBV DNA and EIA (AxSYM CMV IgM; Abbott Laboratories, North Chicago, IL, US) for IgM anti-CMV were carried out. Prevalence of IgG anti-HEV in qualified Japanese blood donors. Serum samples were collected from qualified Japanese blood donors with ALT level of 60 IU/l or lower and tested negative for all the current blood screening tests for HBV, HCV, HIV-1/2, HTLV-1, syphilis and human parvovirus B19 at the seven JRC blood centres in Hokkaido, Miyagi, Tokyo, Aichi, Osaka, Okayama and Fukuoka from November 2005 through February 2006 (n = 12 600). The samples at each blood centre were collected separately by age group and by sex, resulting in 150 male and 150 female samples in each age group (a total of 1800 samples per centre). Positivity rate was summarized by age group (a) and by region (b) [31]. In 2004, we experienced the second case of transfusion-transmission hepatitis E which was associated with zoonotic food-borne infection of HEV [19] (Fig. 3). The causative donor donated blood and his blood was disqualified because of elevated ALT. By the subsequent PCR testing, the blood turned out to be HEV-positive. Look back study of the HEV-positive donor revealed that the PC donated from him 2 weeks previously also contained HEV RNA and were transfused to a patient. Three weeks before the donation, he had a barbecue party at a restaurant with his 12 family members and they cooked pig liver and intestines as well as beef and chicken. Of 13 participants, seven including the causative donor and his father, who consumed pig liver and/or intestines, were positive for IgM and/or IgG anti-HEV. The father died of fulminant hepatitis E 2 months after the dinner. However, other members were asymptomatic. HEV isolates from the donor showed 99·9% homology with that from his father and the recipient based on nearly entire HEV genome. The HEV isolates were genotype 4 that was indigenous to Hokkaido. In the recipient, the progress of HEV markers as well as liver function markers in the entire course were demonstrated; HEV RNA was detected in serum on Day 22 and reached the peak of 7·2 log copies/ml on Day 44 followed by the steep increase of ALT. AST and ALT increased at highest levels of 903 and 673 IU/l on Day 59, respectively. IgG anti-HEV emerged on Day 67; subsequently, hepatitis was resolved. HEV RNA was detectable up to Day 97 in serum, Day 85 in faeces, and Day 71 in saliva. The results for saliva suggest that besides faecal–oral route, oral–oral transmission mode might be another route of human-to-human infection of HEV, although further studies are needed. Transfusion-transmitted hepatitis E in 2004 [19]. Thereafter, nucleic acid-based screening for HEV was started experimentally, although limited in Hokkaido, (1) in order to exclude donors infected with HEV and (2) to find out asymptomatic HEV carriers among blood donors. Since January 2005, in-house real-time RT-PCR system has been implemented and nearly 300 thousands of blood donors who passed the serological tests for HBV, HCV and HIV 1/2 and with ALT levels under 60 IU/l are 20-pooled and tested for the presence of HEV RNA annually. The prevalence of HEV RNA among the blood donors in Hokkaido is approx. 0·012%. Male dominance and no seasonality are observed throughout the years. Almost half of the HEV-positive donors cause ALT elevation but most of them are asymptomatic. Phylogenetic analyses indicated that HEV isolates from the positive donors belong to genotype 3 or 4, both of which seem to be indigenous to Hokkaido or its neighbouring area. Genotype 3 is more prevalent than genotype 4. More than half of the positive donors have an eating history of animal viscera before blood donation, suggesting it may be associated with zoonotic HEV infection. Only in the first year of HEV NAT screeninng, from January 2005 through February 2006, it took about 1 week until the results came out, therefore several HEV-positive blood products such as PC and RCC having short shelf life were occasionally released and transfused before the results were available. Seven cases of transfusion of HEV-positive blood occurred in the period and two patients developed hepatitis E after transfusion. Since March 2006, HEV NAT has been applied only to qualified donors and the results come out timely together with other NAT results for HBV, HCV and HIV. Thereafter, the blood products tested positive for HEV RNA have never been released so far. In addition to our four cases in Hokkaido, two cases of HEV transmission via transfusion were reported in the different parts of Japan [7, 32]. Furthermore, three bags of source plasma sent to a manufacturer of plasma-derived medicinal products from JRC were disqualified for the presence of HEV RNA. In response to the information, JRC's look-back surveys identified the causative donors and revealed that their blood products derived from the donations had been already transfused to three patients and they caused HEV infection. In Europe, two post-transfusion HEV infections were reported in UK [33] and France [34]. In summary, subclinical HEV infection spreads worldwide not only in endemic countries but also in developed countries. Our HEV studies indicate that autochthonous HEV infection is constantly undergoing and that there was a small but significant risk of blood-borne transmission of HEV at least in Hokkaido, Japan before implementation of NAT screening for HEV. Since the prevalence of IgG antibody to HEV in other non-endemic countries in Europe and North America is comparable to that of Japan, HEV infection may be undergoing there, too. In fact, approx. 10% (4/41) of plasma fractionation pools obtained from Europe and North America were positive for HEV RNA [35]. Hepatitis E is one of emerging infectious diseases and HEV infection should not be negligible even in non-endemic countries. Further epidemiological and clinical studies are necessary to be performed. No potential conflict of interests to declare.