WO2010055184A1

WO2010055184A1 - Method and kit for hepatitis e virus (hev) detection

Info

Publication number: WO2010055184A1
Application number: PCT/ES2009/070492
Authority: WO
Inventors: Marta Fogeda Moreno; José Manuel ECHEVARRÍA MAYO; Ana AVELLÓN CALVO; Francisco POZO SÁNCHEZ; Jesús Enrique ROYUELA CASAMAYOR
Original assignee: Instituto De Salud Carlos Iii
Priority date: 2008-11-11
Filing date: 2009-11-10
Publication date: 2010-05-20
Also published as: ES2338853A1; ES2338853B1

Abstract

In the present invention there is described a combination of primers and a method for molecular detection of the different circulating strains of the hepatitis E virus (HEV) in humans and animal reservoirs distributed geographically throughout the world. Amplification of the HEV genome is realised by nested RT-PCRs wherein the primers employed amplify two fragments of the ORF1 (Open Reading Frame) and ORF2 regions of the genome of this virus. Furthermore the present invention includes a kit including the combination of the aforementioned primers and other components.

Description

METHOD AND KIT FOR THE DETECTION OF THE HEPATITIS E VIRUS

(VHE)

In the present invention a combination of primers and a method for the molecular detection of the different circulating strains of hepatitis E virus (HEV) in humans and animal reservoirs distributed geographically throughout the world are described. The amplification of the HEV genome is performed by nested RT-PCRs where the primers used amplify two fragments of the ORF1 (Open Reading Frame) and ORF2 regions of the genome of this virus. In addition, the present invention includes a kit with the combination of the mentioned primers and other components.

STATE OF THE PREVIOUS TECHNIQUE

Hepatitis E is a disease caused by the VHE virus. This virus has not been able to recognize specifically until recent dates. Even when it is really a virus that was well established in the human populations of the Old World in the past, the truth is that HEV is today an agent that only circulates significantly in the most economically depressed regions , in which it constitutes a frequent cause of epidemic outbreaks of acute viral hepatitis. HEV infection is endemic in central and southeast Asia, and has caused epidemics in the Middle East, in North and West Africa, and in Mexico. The route of fecal-oral transmission is the predominant one, and the majority of known epidemics have been related to the consumption of water contaminated with fecal matter (Ippagunta et al. (2007) J. Med. Virol. 79 (12): 1827 -31). Overall mortality is low, but it increases very significantly in pregnant women. To this day, it can be postulated that, in Western Europe, HEV is a zoonotic agent that circulates in some species of farm animals, and that is transmitted to humans with a frequency that is difficult to specify and, due to circumstances, still little known. Laboratory tests used to diagnose HEV infection include electronic immunomicroscopy (IME), serological tests for the identification of anti-HEV of the IgM and IgG classes, and molecular techniques for the detection of viral RNA in feces. and in serum.

The IME allows to detect viral particles directly in the feces of the infected patient. This technique is very specific, but it is expensive, laborious and not very sensitive, so it is not used for diagnostic purposes.

The development of sensitive serological tests for the detection of anti-HEV has allowed a more complete analysis of the epidemiology of this infection worldwide. The serological diagnosis of the acute infection is made by detection of specific IgM by enzyme immunoassay (EIA), which can be confirmed by recombinant immunoblot (IBR). Prevalence studies are carried out by means of the detection of anti-HEV of the IgG class, which is also carried out by EIA with confirmation by IBR.

However, the degree of agreement between the results obtained by different tests for detection of anti-HEV on the same group of samples is low (Mast et al. (1997) J. Infect. Dis. 176 (1): 34 -40), and it is not clear to what extent the reactivities obtained in EIA tests in non-endemic areas reflect the real presence of antibodies against the virus. The recent availability of IBR methods introduces a valuable confirmation instrument, and its use has already shown that part of the reactivities recorded by EIA are not specific.

Acute HEV infection can also be diagnosed by detection of viral RNA in feces and serum by genomic amplification procedures (patents WO9919732, WO2001046696, JP2005102689 and CN1300771). These tests yield positive results between 6 and 40 days, in this type of samples, after exposure to the virus, some days before the elevation of serum transaminase levels and Ia is observed. appearance of specific antibodies. Sometimes, these molecular diagnostic techniques yield positive results in cases that are not detected by serological diagnosis, which opens up the possibility that the latter may not be adequate to detect infections due to certain variants of HEV. Finally, genomic amplification techniques facilitate, in addition to the etiological diagnosis, a useful material to obtain other information that serology cannot provide, such as the identification of the viral genotype involved in the infection and the possibility of carrying out phylogenetic studies that compare between yes the strains from different cases.

In viruses whose genetic material is RNA (as is the case with HEV), genomes replicate with an error rate several orders of magnitude greater than that of cellular DNA.

In the present invention, when designing degenerate primers to carry out genomic amplification, the great variability of strains existing worldwide described since 1987 until the current date, including strains representing the 4 genotypes described in humans and animals, as well as strains of different geographical origin for each of them.

This is an important difference with respect to other methods that could use primers similar to those of the present invention, since using primers designed from so few sequences, and especially not updated, makes it very likely that some of the new variants that they are generated over time may not be detected with the methods described until today.

EXPLANATION OF THE INVENTION In the present invention a combination of primers and a method for the molecular detection of the different circulating strains of hepatitis E virus (HEV) in humans and animal reservoirs distributed geographically throughout the world are described. The amplification of the HEV genome is performed by nested RT-PCRs where the primers used amplify two fragments of the ORF1 (Open Reading Frame) and ORF2 regions of the genome of this virus. The design of the primers has been based on the creation of degenerate primers of the ORF1 / ORF2 regions of the HEV genome. In the design of the primers, the different circulating strains of HEV described from 1987 to the current date have been taken into account, both in humans and in the different reservoirs detected. For this, an exhaustive analysis of the sequences deposited in the GenBank database has been performed, identifying areas conserved in the ORF2 and variable zones within the ORF1 region that allow a genotypic characterization of the detected HEV strain.

Therefore, in the design of the primers the great variability of existing strains throughout the world has been taken into account. Strains representing the 4 genotypes described in humans have been included, as well as strains of different geographical origin for each of them. In viruses whose genetic material is RNA, genomes replicate with an error rate several orders of magnitude greater than that of cellular DNA, which gives populations of these agents a high genetic diversity. On the other hand, it is important to note that the evolution rates measured in different RNA viruses are in the range 10 ^{~ 1} to 10 ^{~ 4} cumulative substitutions in the genomic RNA per nucleotide and year. This is very important, since using primers designed from so few sequences, and especially not updated, it is very likely that some of the new variants that are generated over time may not be detected with the methods described until today .

The present invention therefore allows to determine the prevalence of the virus HEV in a representative sample of the general population, determine the clinical spectrum of acute infection in patients from rural and urban areas, evaluate the performance of serological diagnosis and molecular diagnosis in the identification of such cases, study the presence of HEV in pig farms and in environmental samples and obtain sequences of all fragments and conduct studies aimed at assessing their phylogenetic relationships.

Thus, a first aspect of the present invention is the use of the direct primers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 and the reverse primers SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 to detect hepatitis E virus (HEV) in an isolated biological sample.

A primer is a sequence of a specific oligonucleotide complementary to a target nucleotide sequence that is capable of hybridizing with the target nucleotide sequence and serving as a starting point for a nucleotide polymerization catalyzed by RNA polymerase, DNA polymerase or reverse transcriptase.

To amplify a fragment by PCR, two primers are necessary, one of them will be attached to the template strand (direct primer) and the other to the complementary strand (reverse primer), in a position that allows obtaining, through successive amplifications with an enzyme Heat-resistant RNA / DNA polymerase, a fragment that can be detected and / or quantified. A previous step can be the retrotranscription of the sequence located upstream of the reverse primer by means of the use of a retrotranscriptase enzyme.

The primers that are part of the described uses have been designed following criteria detailed in the examples. First, all the virus strains described from 1987 to the current date were chosen. After the regions of the viral genome that allowed amplification in all types of known viruses were selected. The two regions selected were the ORF1 and the ORF2 of the HEV genome. The ORF1 codes for the non-structural proteins of this virus (methyltransferase, RNA polymerase, protease and RNA helicase), the primers of the present invention are designed within the methyltransferase region, which allows the construction of robust phylogenetic trees with the identification of the viral genotype involved in the infection. The ORF2 region codes for the viral capsid protein, which plays a very important role in the evasion of the immune system and in the formation of the virion. For the choice of the target areas in the amplification, an exhaustive analysis of the entire genome of this viral agent (7200 base pairs) was performed, always looking for a highly conserved area to cover all the natural variants of the agent (ORF2) and a conserved but variable enough area (ORF1) that would allow genotypically characterize circulating strains. The sequences of the primers have degenerate nucleotides in specific positions that allow the detection of the different circulating strains of HEV in humans and animal reservoirs distributed geographically throughout the world. In this first aspect of the invention, primers with degenerations in other different nucleotide sites could be used so that they hybridize in the same nucleotide sequence to the primers proposed in the present invention or a different one within the ORF described in the present invention, which would allow amplification fragments of HEV DNA.

HEV is a non-enveloped icosahedral symmetry RNA virus, approximately 32-34 nm in diameter. That genome is a single-stranded RNA of positive polarity, with a size of 7.2 kb. HEV has been classified within the Hepeviridae family, according to the latest report of the International Committee on Viral Taxonomy (2004), and is the only member of the genus Hepevirus. Through these primers it is possible to detect strains of the VHE virus that belong to any of the four known genotypes: genotype 1 is subdivided into five subtypes and includes epidemic strains characteristic of some endemic regions, such as the prototype strain isolated in Burma and the related strains of Africa and other regions of Asia. Genotype 2 has two subtypes, and includes the prototype isolated in Mexico and several strains detected from Nigeria. Genotype 3 is subdivided into ten subtypes, has a wide geographical distribution, and includes strains of human or swine origin isolated from native cases in Europe, USA. UU, Argentina and New Zealand. Finally, genotype 4 is subdivided into seven subtypes, and includes strains that come, above all, from the Far East.

Through the primers of the present invention it is possible to detect HEV in endemic areas such as central and southeast Asia, north and west Africa and in Mexico. In non-endemic countries such as Spain, they can be used to identify those non-affiliated cases (of unknown origin) and to be able to characterize the origin of the strain causing the infection, that is, to know if it is imported or native.

This use facilitates, in addition to the etiological diagnosis, a useful material to obtain other information that the serology cannot provide, such as the viral genotype involved in the infection and the possibility of carrying out phylogenetic studies that compare strains from different cases.

In addition, the present invention allows the molecular characterization of the circulating strains, aiding in the investigation of the epidemic outbreaks produced by this virus. These primers can, therefore, be used in the study of hepatic pathologies of unknown etiology (acute non-affiliated hepatitis), in epidemiological studies, and in the field of animal health.

Another aspect of the present invention is a method for the detection of hepatitis E virus (HEV) comprising: to. obtain a biological sample and isolate its nucleic acids, b. Simultaneously amplify two fragments of the ORF1 and ORF2 regions of the nucleic acid of section (a) by means of the primer pair SEQ ID NO: 1 / SEQ ID NO: 2 and the primer pair SEQ ID NO: 5 / SEQ ID NO: 6, c. Simultaneously amplify two internal fragments of the fragments obtained in section (b) by means of the pair of primers SEQ ID NO: 3 / SEQ ID NO: 4 and the pair of primers SEQ ID NO: 7 / SEQ ID NO: 8 d. determine the deviation of sections (b) and (c) with respect to controls.

The biological sample is isolated from the mammalian body to carry out the rest of the steps of the in vitro method. The sample can come from a physiological fluid such as blood, plasma, serum, urine, faecal samples and / or any cellular tissue from an organism. Preferably the sample is serum or feces.

In order to carry out the detection of the VHE virus as described in section (b) of the method, amplifications are carried out comprising at least the back transcription of the corresponding fragment of the VHE RNA followed by a polymerase chain reaction. (PCR) that is, an RT-PCR. Thus, a quantitative or real-time RT-PCR can also be carried out, or any other technique in which the combination of primers described is required to carry out the amplification.

The first step is to amplify two fragments simultaneously using primer pairs SEQ ID NO: 1 / SEQ ID NO: 2 (direct primer / reverse primer) and SEQ ID NO: 5 / SEQ ID NO: 6 (direct primer / reverse primer). In the first place, a DNA sequence coding (cDNA) from the region is synthesized upstream of the hybridization site of the reverse primer with the RNA by means of the retrotranscription reaction (RT). The cDNA synthesis is performed by means of any enzyme (retrotranscriptase; RT or reverse transcriptase) that uses the RNA isolated from the biological sample and the reverse primers SEQ ID NO: 2 and SEQ ID NO: 6, which will act as initiators to obtain the single chain or single stranded cDNA complementary to the RNA sequence of the VHE virus. Secondly, double-stranded DNA fragments obtained by PCR (Polimerase Chain Reaction) are obtained using the cDNA described and the primer pairs SEQ ID NO: 1 / SEQ ID NO: 2 and SEQ ID NO: 5 as template. / SEQ ID NO: 6. This PCR is called RT-PCR. For the synthesis of cDNA and subsequent amplifications, the presence of dNTPs nucleotides is necessary, that is, dATP, dTTP, dCTP and dGTP, thermo-resistant DNA polymerase, magnesium chloride in a given concentration as well as buffers that create the appropriate physico-chemical conditions for the operation of all the components and thus the amplification is achieved (see examples). In this way a reaction mixture is obtained containing the amplified fragments of the ORF1 and ORF2 (if the sample contains VHE virus) and serves as a template for the second amplification.

In the second step, described in section (c) of the method, the template DNA used is the product of the PCR reaction obtained in the first step of

The amplification (section (b) of the method). In this step the primer pairs SEQ ID NO: 3 / SEQ ID NO: 4 (direct primer / reverse primer) and

SEQ ID NO: 7 / SEQ ID NO: 8 (direct primer / reverse primer). With these primers two subfragments are amplified taking as a template the two amplified fragments in the first PCR reaction. The pair of primers

SEQ ID NO: 3 / SEQ ID NO: 4 amplifies a sub-fragment of the fragment of

ORF1 of a size of 171 base pairs, and SEQ ID NO: 7 / SEQ ID NO: 8 amplifies a subfragment of the ORF2 fragment of 220 base pairs.

This type of PCR is known with the term nested PCR where the product of an amplification is used as a template to perform a second amplification with primers that are located within the first amplified sequence. To determine the detection of the VHE virus, it is necessary to measure the deviation of sections (b) and (c) with respect to a control. In this case, the controls are those amplifications that come both from samples in which the HEV virus is absent (negative control) and from samples in which it is present in a certain way (positive control). The measurement of the deviation of the amplifications obtained from the problem samples with respect to the controls can be carried out by comparing the presence or absence of bands, as well as the measurement of the band intensity or the measurement of the expression can be taken to carried out by real-time or quantitative PCR. This deviation can be attributed to the presence or absence of the VHE virus in the analyzed samples.

In a preferred embodiment of the method, the isolated biological sample is obtained from a mammal. The mammal is selected from the list comprising human or non-human. Non-human mammals are selected from the list comprising pig, wild boar, mongoose or primate.

It has been shown that the VHE virus is transmitted not only by water (contaminated by the feces of these animals) but also by the consumption of food contaminated with this viral agent. On the other hand, it may be of special interest to carry out epidemiological studies and / or basic experimentation studies in other non-domestic animals such as mongoose or primates as possible zoonotic reservoirs of HEV.

In Spain, studies conducted on pig farms show that HEV is present in the vast majority of them, that its prevalence is very high, and that the majority of pigs acquire the infection within the first two months of life

In addition to the detection of the VHE virus in Spanish pig farms, it has also been found in wastewater from urban environments. On the other hand, in this class of virus the Ia is frequent appearance of viral variants with the ability to cross the barrier between species and infect new hosts that can cause diseases.

All of the above has important implications not only in Public Health, but also in Veterinary Health. Therefore, the detection of HEV in these animals carrying the virus has a clear application in the prevention of infection of both animals and humans.

In another preferred embodiment, the method includes an internal control to evaluate the presence of amplification inhibitors. This control consists of a DNA based on the sequence of a plasmid of known sequence. In addition, the primers are included for the amplification of a fragment that allows to verify, by the amplification conditions described above, that the amplification reaction has not been inhibited. Preferably primers SEQ ID NO: 9 (Direct primer. RTS primer for chimeric DNA) and SEQ ID NO: 10 (Reverse primer. RTA primer for chimeric DNA) for the first PCR amplification, SEQ ID NO: 11 (Direct primer. NS3 primer for chimeric DNA) and SEQ ID NO: 12 (Reverse primer. NA2 primer for chimeric DNA) for the second PCR amplification.

Another preferred embodiment is the method that also allows to determine the genotype of the VHE virus by means of the nested RT-PCR of the ORF1 region. In this case, to determine the genotype of the VHE strain detected, the sequencing of the amplified fragments is carried out by means of the nested RT-PCR as described in the present invention, of the ORF1 region. The sequences obtained can be assigned to a given genotype by comparing them with the VHE sequences of the available databases. Another preferred embodiment is the method described in the preceding paragraphs as a tool for monitoring the evolution of HEV infection, where serial samples of affected mammals are taken. The analysis of the presence or absence of the expected fragments or of the ascent or decrease of the quantification of the amplified DNA allows to evaluate whether the response to the treatment is negative or positive.

The monitoring procedure comprises a series of steps that begin by taking serial samples. Serial sampling is understood as the extraction of the biological samples mentioned in the present invention. The sampling is carried out at different times since the treatment is administered, so that the amplification of the fragments of the ORF1 and ORF2 of the VHE virus in each of the samples from the same patient, indicate the efficacy thereof. Thus, a decrease in the deviation of the amplification values with respect to a control, the latter represented, for example, by amplification values in the same individual, prior to the treatment, assumes that the treatment is taking effect in the sense of decreasing The amount of HEV virus causing hepatitis E. This example would not be limited only to the use of this type of control.

Another aspect of the present invention is a kit for determining the HEV genotype in a biological sample isolated by nested RT-PCR of the ORF1 region comprising, at least, primers SEQ ID NO: 1 / SEQ ID NO: 2 and SEQ ID NO: 3 / SEQ ID NO: 4.

Through this kit, fragments of the ORF1 region amplified by nested RT-PCR are obtained which, after being sequenced, allow the determination of the detected HEV genotype, as described in a previous paragraph. Another aspect of the present invention is a kit for the detection of the VHE virus in an isolated biological sample by nested RT-PCR comprising, at least, the direct primers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO : 5 and SEQ ID NO: 7 and reverse primers SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

In this kit, a combination of primers with degenerations at other different nucleotide sites could be used so that the primers hybridized in the same or other different nucleotide sequence within the ORFs described in the present invention that allowed amplifying fragments of HEV RNA.

In addition to the above described aspects, the kit could include the reagents necessary for amplification by RT-PCR, DNA polymerase, retrotranscriptase, nucleotides, or other components. It could also include instructions for carrying out the amplification by means of the appropriate conditions; alignment temperature, hybridization, elongation, number of amplification cycles, etc.

According to a preferred embodiment, the kit for the detection of the VHE virus is used for monitoring the response to a hepatitis E treatment, as described in a previous paragraph.

In another preferred embodiment, the above kits also comprise an internal control necessary to evaluate the presence of amplification inhibitors.

This control consists of a DNA based on the sequence of a plasmid of known sequence. In addition, the primers are included for the amplification of a fragment that allows to verify, by the amplification conditions described above, that the amplification reaction has not been inhibited. Preferably primers SEQ ID NO: 9 (direct) and SEQ ID

NO: 10 (reverse) for the first PCR amplification, SEQ ID NO: 11

(direct) and SEQ ID NO: 12 (reverse) for the second PCR amplification. Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

FIG 1. Shows the results of the sensitivity of nested PCRs for the ORF1 and ORF2 regions of HEV.

A) ORF1: 171 base pair band. B) ORF2: 220 bp band. Serial dilutions of 10 in 10 of the reference strain of HEV SAR-55 (genotype 1, 10 ^{4 5} PID50 / ml).

FIG. 2. shows the results obtained from the phylogenetic analysis performed with 13 sequences obtained from the PCR products of the ORF1 region.

The phylogenetic tree was constructed with the sequences obtained from the amplification products of the ORF1 region of HEV (nucleotides 26-197) together with the sequences deposited in the GeneBank of the same viral fragment, of genotypes 1, 2, 3 and 4.

FIG 3. Shows the results of the specificity of the method.

A) ORF1 and B) ORF2. CN: negative control; C +: positive control of ORF1 for HEV (171 base pairs); and positive control of ORF2 for HEV (220 bp). Cl: internal control (350 bp). Panel of tested samples:

A: positive HCV serum B: positive HCV serum C: positive HCV serum D: positive HCV serum E: positive HBV serum F: positive HBV serum G: positive HBV serum H: positive HBV serum I: positive HCV serum J: positive HBV serum

HCV: hepatitis C virus HBV: hepatitis B virus

EXAMPLES

The invention is illustrated below by means of examples that describe the design of the primers of the invention and the method for the detection of the different HEV genotypes.

EXAMPLE 1. Design of the primers of the invention.

1.1. Choice of circulating strains of HEV that are included in the design of the primers.

The different strains of HEV described from 1987 to the current date, detected both in humans and in different animal reservoirs, were able to cause the latter, cases of clinically indistinguishable zoonoses. 1.2. Selection of viral genome regions.

After an exhaustive analysis of the HEV genome, through the use of freely distributed software, two regions were chosen (ORF1 / ORF2). The ORF1 region encodes the non-structural polyprotein, and allows the genotypic characterization of the detected HEV strain and the ORF2 region, which encodes the structural protein, confirms the microbiological detection of this pathogen.

1.3. Sequences of the primers.

Each of the regions chosen was subsequently analyzed with additional software, locating areas of approximately 20 nucleotides in length, whose efficacy as primers for amplification was also empirically estimated. In the design of the primers, nucleotides degenerated into specific positions (in some cases, more than one nucleotide in a certain position). These degenerations allow us to identify the different circulating strains of HEV in humans and animal reservoirs distributed geographically throughout the world. In all cases, the specificity of each of the regions identified as valid for primers was further verified by comparison with public access sequence databases (GenBanK) by means of public access software (Blast).

In addition, an internal control consisting of a chimeric DNA based on the sequence of a plasmid (Pozo F et al., (2007) J Clin Virol 40: 224-228) was included in the reaction mixture. as well as the primers necessary for the amplification of a 350 base pair fragment, which gives us a quality control to evaluate the presence of amplification inhibitors in each of the samples tested. The primers for amplifying the internal control are the following: SEQ ID NO: 9 (direct) and SEQ ID NO: 10 (reverse) for the first PCR amplification, SEQ ID NO: 11 (direct) and SEQ ID NO: 12 (reverse) for the second PCR amplification.

Example 2. Clinical samples

In the present investigation, a retrospective study was carried out with the serum samples of 14 patients selected from another work previously performed (Echevarría, (2006) Med Clin 126 (6): 234-6). On the one hand, these samples were previously selected from a total of 129 samples that had been received in the hepatitis laboratory of the National Center for Microbiology to be diagnosed by HEV between January 2000 and December 2004. The 14 samples analyzed presented IgG antibodies by enzyme immunoassay (EIA) and six of them showed IgM antibodies by recombinant immunoblot (RIBA). On the other hand, in these 14 samples analyzed, the samples received between January 2007 and June 2008 to be diagnosed by HEV, from a total of 81 samples that arrived in that period of time, were also included prospectively. In total 95 serum samples were included in the investigation. In all samples the presence of HEV RNA was studied, as described below.

All patients at the time of admission had symptoms and signs of acute hepatitis when they were treated in their corresponding hospitals. Other possible causes of liver damage were ruled out in these patients (hepatic drug poisoning, autoimmune hepatitis, infection by other liver viruses A, B and C, cytomegalovirus infection and Epstein-Barr infection).

Example 3. Detection of RNA-HEV

A new design of primers was developed to be used in the polymerase chain reaction (PCR) in order to detect the HEV genome. The objective of this new design is to be able to detect all the new circulating variants of HEV both in humans and in the different reservoirs detected. To achieve this objective, an alignment of a total of 76 complete VHE genome sequences deposited in the GenBank, both human and animal, was performed. The design of the method was based on the creation of degenerate primers of the ORF1 / ORF2 regions of the HEV genome. The nucleotide sequence of the primers is shown in Table 1. For this, an exhaustive analysis of the sequences was carried out, identifying areas conserved in the ORF2 and variable areas in ORF1 that allow a genotypic characterization of the detected HEV strain. The composition of the reaction mixture, and the relative concentration of the primers used in the amplification phase, have been optimized by experimental tests until the best sensitivity / specificity parameters are obtained.

The HEV RNA was extracted from a total of 100 μl of serum with the MagNA Puré automatic extractor (ROCHE, Mannheim, Germany). The RNA obtained was resuspended in an elution buffer at a final volume of 50 μl and subsequently stored at -8O ⁰ C until use. RT-PCR was performed with 5 μl of RNA extract in a final volume of 50 μl. The reaction mixture contains 25 μl of Master Mix (Taq polymerase: 50U / ml; dNTPs: 40OuM; MgCI2: 3mM; Promega, Co., Madison, Wisconsin, USA), 1 μl of the enzyme AMV Reverse Transcripse (Promega Co .), and 50 pmol of each primer. The PCR reaction consists of 39 cycles of denaturation at 94 ⁰ C for 35 sec, banding at 51 ⁰ C for 45 sec (ORF1 test), and 48 ⁰ C for 45 sec (ORF2 test), and an extension at 72 ⁰ C for 1 min, with a final elongation at 72 ⁰ C for 7 min. The amplification of the 2 ^to round PCR (nested-PCR) was performed with 2μl of PCR product of the first round amplification, and consisted of 29 cycles of denaturation at 94 ⁰ C for 35 sec, annealing at 48 ⁰ C for 45 sec (ORF1 test), and at 5O ⁰ C for 45 sec (ORF2 test), extension at 72 ⁰ C for 1 min, with a final incubation at 72 ⁰ C for 7 min and 50 pmol of each primer. The analytical sensitivity of the nested PCR assays for ORF1 and ORF2 were calculated with the prototype strain Sar-55 (Tsarev, 1992), which contained a known infectious titer of 10 ^{4 5} infectious doses in mono (PID50) per ml (provided by Dr. R Purcell, CDC, Atlanta, Georgia, USA) (FIG 1). With this strain, serial dilutions were made based on 10 in a saline phosphate buffer. The RNA extracted from each of these dilutions was tested for each assay. In addition, the specificity of the method was analyzed using serum samples from patients infected with other types of hepatitis virus, HCV and HBV, demonstrating the amplification of a size band waiting only in those positive controls for HEV and not for the rest of the samples, both for the ORF1 and for the ORF2 (FIG 3).

Table 1. Degenerated primers used for amplification by nested RT-PCR.

Primer Sense Region Sequence Genome Position *

Amplification

ORF1 F ORF1 Direct 1 ^to PCR SEQ ID NO: 1 9-29

ORF1 R - Reverse 1 ^to PCR SEQ ID NO: 2 627-643

ORF1 FN - Direct 2 ^to PCR SEQ ID NO: 3 26-44

ORF1 RN - Reverso 2 ^to PCR SEQ ID NO: 4 178-197

ORF2F ORF2 Direct 1 ^to PCR SEQ ID NO: 5 6265-6285

ORF2R - Reverse 1 ^to PCR SEQ ID NO: 6 7069-7085

ORF2FN - Direct 2 ^to PCR SEQ ID NO: 7 6314-6331

ORF2RN - Reverso 2 ^to PCR SEQ ID NO: 8 6515-6534

The position of the nucleotides is numbered according to the strain of the Mexico virus. The sequence SEQ ID NO: 4 contains a nucleotide n which is an Inosine.

Example 4. Genotyping of HEV

The positive PCR products of the amplification of the ORF1 region of HEV were sequenced. Both chains were sequenced directly with the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, California, USA), using the ABI PRISM 3700 DNA Analyzer (Applied Biosystems) sequencer. The sequences obtained were assembled with the SeqMan Lasergene 7.0 program. For the alignment of the sequences the program Clustal X method of BioEdite 7.0.9 was used. The phylogenetic tree (FIG 2) was performed with the MEGA version 4.0 program (Tamura K et al., (2007) Mol Biol Evol 24: 1596-9), and distances with the Neighbor-Joining (NJ) Kimura 2 method -parameters with 1,000 replicas.

In summary, in the present invention a new method has been developed for the detection of the hepatitis E virus (HEV), being tested in 95 serum samples corresponding to patients who presented clinical pictures of acute hepatitis A-C (Table 2). This methodology allowed to identify 13 cases of acute hepatitis produced by this viral agent, out of a total of 95 samples tested.

Table 2. Genotypes and markers of HEV infection, identified in the samples of Spanish patients with acute non-A-C hepatitis.

a) City of Spain in northern Africa; b) Country of origin and unknown birth

Nd: not determined;

Table 2 shows the results obtained from the comparison of the serological techniques and the method designed for the detection of the viral RNA of HEV in the ORF1 / ORF2 regions. Of the 95 samples studied, only 13 were positive for HEV by PCR in both regions of the genome (ORF1 / ORF2). Five cases (1-5) belonged to the retrospective study and the remaining eight correspond to the prospective study. Antibodies (Anti-HEV IgM) were analyzed in all samples, with only 11 of them. One of the patients studied was an immigrant of African origin who was treated in the health services of Spain, specifically in the city of Ceuta. The remaining 12 patients were Spanish and residents in this country. Six of them had a history of international travel with symptoms after their return, of these, four of them had returned from India, one from Bangladesh and the other from Vietnam. The patient who had returned from Bangladesh had discordant results regarding the diagnosis of the same with the combination of serological and molecular techniques. By recombinant immunoblot techniques (RIBA) it was negative and however the viral RNA of HEV was detected in the patient's serum in both PCRs (ORF1 / ORF2). When we sequenced and analyzed the product obtained in the nested PCR of the ORF1 region, it was found that it was genotype 1, which is the one that circulates in that region. The six native cases detected in Spain, corresponding to genotype 3, have been highlighted in blue, as possible cases of zoonosis.

Of these 13 sequences, six corresponded to genotype 1, six to genotype 3 and one to genotype 4. Genotype 1 strains corresponded to the immigrant from African origin and two travelers who came from India and Bangladesh. Genotype 3 strains corresponded to samples from six patients who had no history of travel to endemic areas of HEV in their clinical history. The only genotype 4 found corresponded to the patient who came from Vietnam.

As shown in FIG 2, cases 7, 8 and 10 (belonging to the year 2007) were grouped together with a sequence homology of 99.2% among them. This would indicate that in that year the same strain of HEV was circulating in different provinces of Spain (Valladolid, Alicante and San Sebastián).

Thus, the results obtained from the molecular studies carried out, revealed the existence of seven imported cases of acute hepatitis due to HEV. Of these seven imported cases, six of them were genotype 1 and corresponded to patients with a history of travel to endemic areas of this genotype (India), and a patient of African origin residing in Ceuta. The other imported case belonged to genotype 4, and it was a Madrid patient who had recently been to Vietnam. The finding of genotype 3 in samples of six Spanish patients with acute hepatitis, who had no history of travel to endemic areas by this viral agent, confirmed that the origin of these infections was native. Genotype 3 has been found in humans and pigs, as well as in several species of wild animals in Europe and North America (mainly Wild Boar) (from Deus N et al., (2008). Vet Microbiol: 129: 163-170 ; Kaci S et a /., (2008). Vet Microbiol: 128: 380-385) and in urban waters (Clemente-Casares P et al., (2003). Emerα Infect Dis: 9: 448-454).

Today it is postulated that acute hepatitis due to HEV in developed countries is a disease of low incidence and of zoonotic origin.

To confirm the zonotic origin of this disease, phylogenetic studies should be performed between the sequences obtained from humans and from animals.

The results of this work through molecular methods, confirmed that the hepatitis E virus is circulating in Spain, finding native strains (genotype 3), as well as the existence of other VHE strains of imported origin, corresponding to genotype 1 and 4.

Claims

1. Use of direct primers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 and reverse primers SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 to detect hepatitis E virus (HEV) in an isolated biological sample.

2. Method for the detection of the VHE virus comprising: a. obtain a biological sample and isolate its nucleic acids, b. simultaneously amplify two fragments of the regions

ORF1 and ORF2 of the nucleic acid of section (a) by means of the pair of primers SEQ ID NO: 1 / SEQ ID NO: 2 and the pair of primers SEQ ID NO: 5 / SEQ ID NO: 6, c. Simultaneously amplify two internal fragments of the fragments obtained in section (b) by means of the pair of primers SEQ ID NO: 3 / SEQ ID NO: 4 and the pair of primers SEQ ID NO: 7 / SEQ ID NO: 8 and d. determine the deviation of sections (b) and (c) with respect to a control.

3. Method according to claim 2 wherein the biological sample is obtained from a mammal.

4. Method according to any of claims 2 or 3 wherein an internal control is included to evaluate the presence of amplification inhibitors.

5. Method according to any of claims 2 to 4 which also allows to determine the genotype of the VHE virus by means of the nested RT-PCR of the ORF1 region.

6. Method according to any of claims 2 to 4, for monitoring the response to a hepatitis E treatment.

7. Kit to determine the HEV genotype in a biological sample isolated by nested RT-PCR of the ORF1 region comprising, at least, primers SEQ ID NO: 1 / SEQ ID NO: 2 and SEQ ID NO: 3 / SEQ ID NO: 4.

8. Kit for the detection of the VHE virus in a biological sample isolated by nested RT-PCR comprising at least the direct primers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 and reverse primers SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

9. Kit according to claim 8 for monitoring the response to a hepatitis E treatment.

10. Kit according to any of claims 7 to 9, further comprising the internal control to evaluate the presence of amplification inhibitors.