PL220139B1 - Oligonucleotides for the detection and differentiation of the resistant and susceptible strains of the Oseltamivir influenza - Google Patents

Abstract

The invention is a nucleotide sequence of primers and probes for amplifying fragments of the NA protein gene approximately 172 nucleotides long containing a mutation site responsible for acquiring resistance to oseltamivir (Fig. 1). The invention also includes the use of the nucleotide sequence of primers and probes for amplifying a fragment of the NA protein gene approximately 172 nucleotides long containing a mutation site responsible for acquiring resistance to an antiviral drug.

Description

The subject of the invention is a qualitative diagnostic method based on the real-time PCR technique, used to detect influenza virus (Influenza Virus, IV) and determine its resistance or susceptibility to the popular antiviral drug - oseltamivir, as well as nucleotides suitable for virus analysis. The invention relates to the use of nucleotides (primers and probes) for the detection of the mutation responsible for oseltamivir sensitivity.

Influenza A virus (lAV) belongs to the Orthomyxoviridae family and is one of the most common pathogens infecting millions of people and animals (especially birds) each year. Virions take a spherical or filamentous shape and the genetic material of the virus is single-stranded segmented negative-strand RNA. This feature is conducive to high genetic variability of the virus, which occurs through point mutations (antigenic drift) and reassortation (antigenic jump).

Due to the high variability of the virus, it has a great potential to cause local epidemics and pandemics on a global scale. Three such events took place in the 20th century, claiming tens of millions of lives.

Influenza A viruses are widespread among various bird populations around the world - whether wild, domestic or even ornamental [Esterday et al., 1998]. However, a virus that is pathogenic to one species can cause asymptomatic disease transmission in other organisms [Webster, 1997].

The most important reservoir of all virus strains are wild, aquatic and migratory birds in which viral infections pass asymptomatically. They are the main source of infection in domestic birds and other animals [Webster, 2002; Esterday et al., 1998]. It is suspected that all strains infecting mammals, including humans, come from strains of avian viruses and, through adaptation and mutation, became capable of infecting other organisms [Webster, 2002]. Infections in wild birds usually occur asymptomatically through direct contact with another sick animal or through contact with material containing the virus, eg faeces [Webster, 2002].

The bird flu virus lives in the environment and its survival is prolonged by the organic particles that surround it, e.g. faeces. The infectivity of the virus contained in feces at 4 ° C is about 30-35 days, at 20 ° C even 7 days. One gram of faeces containing over 1 billion copies of viral RNA is capable of infecting approximately 1 million birds. However, the disinfectants used destroy the virus with 100% efficiency [ECHCPD, 2003]. Infection in humans is generally via the droplet route [WHO, 2008a].

The variety of virus serotypes is associated with the different virulence of individual strains. However, it is possible to divide it into Highly Pathogenic Avian Influenza (HPAl) and Low Pathogenic Avian Influenza (LPAI) viruses. The first group includes viruses of the H5 and H7 serotype, the appearance of which in the herd most often leads to 100% mortality. All other virus subtypes are included in LPAI. There is little or no presence of this type of virus in the body.

Whether or not a given strain is highly virulent depends on the ease of proteolytic cleavage of hemagglutinin during infection at the endosomal stage. Viruses become virulent by mutation. This is what happened, for example, with the H7N1 virus, which changed from a weakly pathogenic to a highly malignant. This virus was first identified as highly pathogenic on Italian farms in 1999-2000 [ECHCPD, 2003].

Clinical signs in birds vary with the virus type and species of animal. HPAI virus infections are characterized by acute and severe course, respiratory, digestive and nervous system symptoms. Mortality is very high and often reaches 100% [Subbarao et al., 1998].

Low pathogenicity viruses (LPAI) cause mild respiratory symptoms, sometimes reduce egg laying, and many times the birds show no disease symptoms [Alexander, 1993].

In humans, the clinical manifestations of influenza are high fever and respiratory symptoms. Viral infections are often associated with tracheitis and bronchitis. The first symptoms are visible already 1-4 days after the virus has entered [WHO, 2008a].

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Each year, influenza affects about 5-15% of the human population and causes between 250,000 and 500,000 deaths as a result of complications, especially in children and the elderly. The two most important strains circulating in the human population are H3N2 and H1N1 [WHO, 2008a]. In Poland in 2005, over 730,000 people fell ill with influenza. people [GUS, 2007].

The flu virus infects between 300 and 900 million people worldwide each year. Most cases do not require specialized treatment as the disease is dealt with by the host's immune system. However, complications from the influenza virus and numerous deaths occur every year. The elderly and children, as well as people with a weakened immune system, are exposed to the greatest risk [WHO, 2008a].

So far, no fully effective drug against the influenza virus has been developed. Two types of drugs have been developed to inhibit the growth of the virus in the body. The first group is the M2 ion channel inhibitors. These are mainly amantadine, the methyl derivative of rimantidine and the less commonly used ribavirin. These drugs are used not only for therapeutic purposes, but also for prophylaxis. Unfortunately, the virus mutates very quickly and becomes insensitive to drugs of this generation. It is estimated that 80% of isolated viruses are completely immune to the action of this drug [Beigel, 2008].

The second group includes drugs that are competitive inhibitors of neuramimdase. Examples are zanamivir (Relenaza®) and oseltamivir (Tamiflu®) [McKimm-Breschkin et al., 2001]. These are new generation drugs recommended by the Advisory Committee on Vaccinations (ACIP). These drugs are effective both in the fight against influenza virus type A and type B, however, they must be administered up to 48 hours after infection with this pathogen is identified [de Clercq, 2006]. The effectiveness of these drugs has been repeatedly confirmed, inter alia, through their use in the fight against the H7N7 virus in China and Belgium, and in the fight against the mutant H5N1 virus infecting people in China in March 2003 [Brydak, 2004]. However, more and more strains are characterized by resistance to oseltamivir, which makes treatment of patients infected with this strain much more difficult. Drug resistance has been shown that the virus obtains a single mutation in the gene encoding the protein neuraminidase, resulting in a mutation at position 275 - the conversion of Histidine into tyrosine (H275Y).

Vaccination is the most effective way to fight the virus and its complications. Unfortunately, due to the high variability of the virus, it is necessary to vaccinate each year with a preparation against the virus active in a given season. This involves the need to produce a new vaccine each year. The time needed to produce a working preparation is about 6 months, and the production capacity is estimated at about 250 million doses per year, with the demand about 2-3 times greater. Currently, two types of vaccines are used; inactivated and attenuated. The challenge is to create an active recombinant vaccine, the production of which will be much faster and less labor-intensive [Kamble, et al. 2003].

In Poland, influenza vaccination has been included in the vaccination calendar since 1994, but as recommended, not obligatory [Gerdil, 2003].

Diagnostic methods for determining the susceptibility of influenza virus to oseltamivir have been described in many publications.

Chutinimitkul et al. (Chutinimitkul, S., K. Suwannakarn, T. Chieochansin, Q. Maile, S. Damrongwatanapokin, A. Chaisingh, A. Amonsin, O. Landt, T. Songserm, A. Theamboonlers, and Y. Povorawan. 2007. H5N1 oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled

TaqMan probes. J. Virol. Methods 139: 44-49.), Hindiyeh et al. (Hindiyeh M, Ram D, Mandelboim M, Meningher T, Hirsh S, Robinov], Levy V, Orzitzer S, Azar R, Grossman Z, Mendelson E. 2010. Rapid detection of influenza A pandemic (H1N1) 2009 virus neuraminidase resistance mutation H275Y by real-time reverse transcriptase PCR J Clin Microbiol. 48 (5): 1884-7.) Or Payungporn et al. (Payungporn, S., Poomipak, W., Makkoch, J., Rianthavorn, P., Theamboonlers, A., Poovorawan, Y. 2011. Detection of oseltamivir sensitive / resistant strains of pandemic influenza A virus (H1N1) from patients admitted to hospitals in Thailand. J Virol Methods. 177 (2): 133-139.) describe a real-time RT-PCR method using two TaqMan probes. The probes were designed for both the wild type (Oseltamivir resistant) and the resistant variant. However, the need to use two probes increases the cost of the analysis.

Tong et al. (Tong, S.Y.C., Dakh, F., Hurt, A.C., Deng, Y-M., Freeman, K., Fagan, P.K., Barr,

I.G., Giffard, F.M. 2011. Rapid Detection of the H275Y Oseltamivir Resistance Mutation in Influenza A / H1N1 2009 by Single Base Pair RT-PCR and High-Resolution Melting. PLoS One. 6 (6): e21446.) Presented a method based on the HRM technique, however, as the authors themselves state, this method is prone to false results and it is difficult to distinguish between susceptible and resistant strains in the case of low

Virus titers (at Ct> 30). Similarly, Lee et al. Lee, HK, Lee, CK, Loh, TP, Tang, JW-T, Tambyah, PA, Siew-Chuan Koay, E. 2011. High-Resolution Melting Approach to Efficient Identification and Quantification of H275Y Mutant Influenza H1N1 2009 Virus in Mxed -Virus-Population Samples. J Clin Microbiol 2011 October; 49 (10): 3555-3559.) Presented a method based on HRM. However, the disadvantages of this method remained.

It is an object of the present invention to provide tools that could be used to rapidly analyze virus susceptibility and treatment compliance with an antiviral drug, preferably oseltamivir.

The subject of the invention is the nucleotide sequence of primers (primers) and probes used to amplify and detect fragments of the NA protein gene about 172 nucleotides long, containing the mutation site (823 nucleotide responsible for the acquisition of resistance to oseltamivir mutation H275Y (N1 numbering) - (Fig. 1).

Another object of the invention is the use of the nucleotide sequence of the primers and probes (Fig. 1A) for amplifying a fragment of the NA protein gene about 172 nucleotides in length and detecting the mutation responsible for acquiring resistance to an antiviral drug, preferably oseltamivir. Detection of mutations is carried out by a method based on the real-time PCR technique with the use of a hydrolysing probe (TaqMan type, Fig. 1B), characterized by the fact that real-time PCR is performed under the following conditions: initial denaturation 1 x: 94 ° C 10 min .; 45 cycles: denaturation 94 ° C 10 sec., Hybridization, elongation 72 ° C and the reaction products are analyzed by analyzing the plot in real time and after completion of the reaction, using the software included with the instruments. Preferably, the template DNA is influenza virus cDNA obtained by isolation of viral RNA from environmental material (e.g. throat swab) and then reverse transcribed using RT-PCR, preferably the hybridization temperature is 50 to 60 ° C and a time of 10 to 20 seconds, preferably an elongation time of 1 to 10 seconds.

A further object of the invention is the use of the nucleotide sequence of the primers (Fig. 1A) to amplify a gene segment of approximately 172 bp and detect the mutation responsible for the acquisition of resistance to an antiviral drug, preferably oseltamivir. Detection of mutations is carried out by a method based on the real-time PCR technique using a hydrolyzing probe (TaqMan type, Fig. 1B), characterized in that the reverse transcriptase Realtime PCR (RRT-PCR) reaction is performed under the conditions: reverse transcription 1 x: 61 ° C 20 min .; initial denaturation 1 x: 94 ° C 30 sec; 45 cycles: denaturation 94 ° C 10 sec., Hybridization, elongation 72 ° C and the reaction products are analyzed by analyzing the plot in real time and after completion of the reaction using the software supplied with the instrument. Preferably the template RNA is influenza virus RNA obtained by isolation from environmental material (e.g. throat swab), preferably the pre-treatment time is 2 to 10 sec, preferably the hybridization temperature is 50 to 60 ° C and the time is from 10 to 20 seconds, preferably the elongation is from 5 to 20 seconds.

The attached figures - allow for a better explanation of the essence of the invention:

Fig. 1 (a and b) - Shows the Sequences of the primers and probes used to amplify the approximately 172 nucleotide fragment of the NA gene containing the oseltamivir resistance mutation site

Fig. 2 shows a graph of the result of a real-time PCR reaction using primers and a probe (Fig. 1).

The invention is illustrated by the following non-limiting embodiment:

P r z k ł a d

Susceptibility testing of influenza virus to oseltamivir from a patient's throat swab

After the pharyngeal swab was taken, the swab was dipped in PBS buffer with antibiotics, and then viral RNA genetic material was isolated using an RNA isolation kit (eg A&A BIOTECHNOLOGY Total RNA). Subsequently, a reverse transcription reaction was performed, ie the transcription of the RNA material into cDNA, which served as a template for real-time PCR reactions. The primers shown in Figure 1A were used to amplify a specific fragment of the influenza NA gene. The probe shown in Figure 1B was used to detect the oseltamivir sensitive strain.

The PCR reaction was performed under the following conditions:

1x: 94 ° C 10 min.

45x: 94 ° C 10 sec.

56 ° C 15 sec.

72 ° C 7 sec.

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The result was observed on the computer monitor in real time. The positive result of the presence of influenza virus sensitive to oseltamivir was illustrated by an increase in the fluorescence curve. No increase in fluorescence indicated a resistant strain. The result is shown in Fig. 2.

Claims (5)

1. Nucleotide sequence of primers and probes used to amplify fragments of the NA protein gene approximately 172 nucleotides in length containing the site of the mutation responsible for the acquisition of oseltamivir resistance (Fig. 1). 2. The use of the nucleotide sequence of the primers and probes to amplify the fragment of the NA protein gene about 172 nucleotides in length containing the mutation site responsible for the acquisition of antiviral drug resistance. 3. Use according to claim 1 The method of claim 2, wherein the antiviral drug is oseltamivir. 4. Use according to claim 1 2. The method of claim 2, characterized in that real-time PCR is performed using TaqMan probes and the reaction products are analyzed in real time and after completion of the reaction, using the software provided with the apparatus. 5. Use according to claim 1 2. Process according to claim 2, characterized in that the reverse transcriptase Real-time PCR (RRT-PCR) reaction is performed with TaqMan probes and the reaction products are analyzed in real time and after completion of the reaction using the software included with the equipment.