- Email: [email protected]
Sequence analysis of haemagglutinin and neuraminidase of H1N1 strain from a patient coinfected with Mycobacterium tuberculosis
Accepted Manuscript Sequence analysis of haemagglutinin and neuraminidase of H1N1 strain from a patient coinfected with Mycobacterium tuberculosis Ahmed N. Alghamdi, Mohammad E. Mahfouz, Fahd A. Hamdi, Daifullah Al Aboud, Tawfiq Z. Al-laylah, Mohammed I. Alotaibi, Khalid W.A. Al-Thomali, Ahmed S. AbdelMoneim PII:
S0890-8508(17)30042-7
DOI:
10.1016/j.mcp.2017.05.002
Reference:
YMCPR 1288
To appear in:
Molecular and Cellular Probes
Received Date: 7 March 2017 Revised Date:
20 April 2017
Accepted Date: 8 May 2017
Please cite this article as: Alghamdi AN, Mahfouz ME, Hamdi FA, Al Aboud D, Al-laylah TZ, Alotaibi MI, Al-Thomali KWA, Abdel-Moneim AS, Sequence analysis of haemagglutinin and neuraminidase of H1N1 strain from a patient coinfected with Mycobacterium tuberculosis, Molecular and Cellular Probes (2017), doi: 10.1016/j.mcp.2017.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Sequence analysis of haemagglutinin and neuraminidase of H1N1 strain from a patient coinfected with Mycobacterium tuberculosis
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Ahmed N. Alghamdi1, Mohammad E. Mahfouz 1,2, Fahd A. Hamdi2, Daifullah Al Aboud1, Tawfiq Z. Al-laylah1, Mohammed I. Alotaibi1, Khalid W. A. Al-Thomali2,
College of Medicine, Taif University, Al-Taif 21944, Saudi Arabia, 2King Faisal
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Ahmed S. Abdel-Moneim1,3§
Hospital, Al-Taif, Saudi Arabia, 3Virology Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt.
Correspondence author: Ahmed S. Abdel-Moneim
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Email: [email protected] / [email protected]
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Abstract The 2009 H1N1 pandemic (H1N1pdm09) was associated with a considerable influenza-related
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morbidity and mortality. Among the complications, Mycobacterial tuberculosis was recorded as a coinfection with influenza in rare cases. The full-length sequences of the viral haemagglutinin and neuraminidase of H1N1pdm09 influenza A virus were analyzed from a recently infected patient. The patient was chronically infected with Mycobacterium tuberculosis. Molecular
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modelling and in-silico docking of the virus, and other selected strains with the drug oseltamivir
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were conducted and compared. Sequence analysis of the viral haemagglutinin revealed it to be closely related to the 6B.1 clade, with high identity to the circulating H1N1pdm09 strains, and confirmed that the virus still harbouring high affinity to the α-2,6-sialic acid human receptor. The viral neuraminidase showed high identity to the neuraminidase of the recently circulating strains of the virus with no evidence of the development of oseltamivir-resistant mutants. Regular
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Introduction
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resistant strains.
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monitoring of the circulating strains is recommended to screen for a possible emergence of drug-
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Since the first detection of the H1N1 in Mexico in March of 2009 [1], the virus began to circulate worldwide in a pandemic manner, hence the name H1N1pdm09. There have been more than 18,500 laboratory-confirmed deaths from infection with the H1N1pdm09 strain. Most of the lethal cases of H1N1pdm09 have been recorded in young and middle-aged adults [2] rather than in young children and old people as with the seasonal influenza epidemics. The influenza, hemagglutinin (HA), is the principal surface antigen and the main target of vaccine-induced neutralizing antibodies. The virus frequently mutates vulnerable HA epitopes,
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with subsequent changes in its antigenic structure, to escape recognition and virus elimination by the immune system[3]. Determining the changes in the antigenic sites provide structural insights
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into the rationale for optimizing vaccines to match circulating variants. Viral neuraminidase (NA) is a receptor destroying enzyme that helps to liberate the viral particles from the infected cells, which renders it an attractive target for anti-influenza drugs. Although many neuraminidase inhibitors exists, including oseltamivir, zanamivir, laninamivir
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and peramivir [4], oseltamivir is considered to be the most commonly used. The early use of
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oseltamivir can reduce the mortality rate in hospitalized persons infected with H1N1pdm09 by half [5]. Oseltamivir resistance typically emerges in immune-compromised patients, but is much less frequently found in immunocompetent people [6] and it constitutes a major challenge to the effectiveness of the drug for the treatment of influenza and highlights the necessity for clinical
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assessment of antiviral resistance.
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Tuberculosis (TB) is caused by Mycobacterium tuberculosis. Active tuberculosis occurs in up to 10% of the infected individuals, while the remaining cases develop a latent infection lasting for
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years [7]. Reactivation of latent infection may occur, with the subsequent development of active
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disease in most cases pulmonary TB disease [8]. The current study describes the full-length sequence analysis of both haemagglutinin and neuraminidase of H1N1pdm09 strain from TB chronically infected patient. In addition, the virtual docking of the neuraminidase against oseltamivir of the detected strain in comparison with selected pdm09 classical and H275Y mutant strains was assessed.
Materials and Methods
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Ethical approval The study was ethically approved by the King Faisal Hospital Ethical and Research Committee.
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Informed written consent was obtained from the patient. Subject
An 86-year-old Pakistani female resident in Al-Taif, Saudi Arabia, infected with Mycobacterium
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tuberculosis was admitted to King Faisal hospital at the end of November 2015. The patient was
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confirmed to have a recent infection of H1N1pdm09 by Makkah Regional Laboratory, Saudi Ministry of Health Laboratory. Oseltamivir was used for the treatment of the patient after laboratory confirmation of H1N1pdm09. RNA extraction
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Nasopharyngeal, throat swabs and sputum samples were collected from the patient at day 3 of
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admission in a virus transport medium. Total viral RNA was extracted from 300 μl of the sample using viral RNA extraction kit (Promega). Extracted RNAs were eluted into a final volume of 60
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One-step RT-PCR
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μl in an elution buffer.
One-step RT-PCR was conducted using Script™ RT (Promega) and in-house specific primer sets that flank the HA and NA genes of H1N1pdm09. Three overlapping primer sets were used for the
HA
gene:
pH1-F1:CAAAAGCAGGGGAAAACAAA,
R1:TTGATGTCCCCACAAAAACA,pH1-F2:GCTCCGCCAATCCTACATTA,
pH1pH1-
R2:GATAACCGTACCATCCATCTACC, pH1-F3: CCAGCCTCCCATTTCAGAATA, pH1-
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R3:
GTAGAGACCCATTAGAGCACATC
ACCATTGGTTCGGTCTGTATG,
pN1-R1
and
two
for
the
NA
genes:
pN1-F1
CACTTGGTCCATCGGTCATTAT,
pN1-F2
GGCTGTGTTAAAGTACAATGGC and pN1-R2 AAGACCAACCCACAGTGTC. Reverse
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transcription was conducted at 45 °C for 45 minutes followed by 5 min denaturation with an initial denaturation step at 95 °C and 35 cycles of 95 °C for 30 sec., 52 °C for 30 sec. (54°C for pH1-F1/R1 and 50°C pN1-F1/R1) and 72 °C for 1.5 min. This was followed by a final
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elongation step for 10 min at 72 °C. PCR amplicons were gel extracted and sequenced directly in
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both forward and reverse directions using the same primers. Sequence analysis
The raw sequences were assembled with the MEGA 5.2 package and the same program was used to perform multiple sequence alignment using representative HA and NA sequences of
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H1N1pdm09 viruses obtained from the NCBI flu database and the EpiFlu database of the Global
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Initiative on Sharing Avian Influenza Data (GISAID). The Maximum Likelihood method based on the Tamura-Nei substitution model was used to build a phylogenetic tree [9]. The bootstrap
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consensus tree from 1000 replicates represented the evolutionary history of the analyzed taxa. All positions containing gaps and missing data were eliminated. Evolutionary analyses and
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deduced amino acid sequences of HA and NA proteins were conducted in MEGA5[10]. Molecular modelling and in-silico molecular docking The NA protein of the A/Saudi Arabia /TU-1/2015 and the NA amino acid sequences of A/CzechRepublic/11/2016_H274Y, A/California/07/2009, A/Boston/678/2009 were obtained from the NCBI-flu database. Modelling of each protein sequence was conducted online using Swiss-model
free
online
software
(http://swissmodel.expasy.org/).
Minimization
and
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equilibration of steric clashes caused by the addition of hydrogen atoms, and alleviation of water and ions were performed by Molsoft (Molsoft ICM 3.5–8C) prior to performing the molecular dynamics. In-silico molecular docking was conducted for the NA protein of different H1N1
8C). The comparison was based on the best binding free energy.
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Results and Discussion
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strains against the oseltamivir ligand using ‘Internal Coordinate Mechanics (Molsoft ICM 3.5–
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The patient was treated from the first day of admission with oseltamivir 75 mg oral capsule twice daily, ceftriaxone 1 g by intravenous injection twice daily and azithromycin 500 mg once daily (orally) as an empirical treatment for pneumonia and suspected H1N1pdm09. After obtaining the
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result of the bacteriological investigation, administration of antimycobacterial therapy (isoniazid 300 mg, rifampicin 450 mg, ethambutol 1.2 g and pyrazinamide 1.5 g once daily) was initiated along with trimethoprim/sulfamethoxazole 800 mg with discontinuing the use of ceftriaxone,
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azithromycin and oseltamivir. The patient improved during the 7 days of treatment in ICUisolation and was transferred back to a regular bed in the isolation unit. By the 11th day of
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hospitalization, her hepatic enzymes have increased and antimycobacterial therapy stopped until the hepatic enzymes become normal, which was achieved 3 days later. The anti-tuberculosis agents were then resumed with daily liver function tests. Patient treatment was continued with both the anti-tubercular and the anti-viral agents for 14 days. Symptoms of lethargy, cough and
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fever gradually improved within 1 week. After three consecutive negative sputum samples for
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regular basis.
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AFB, the patient was discharged with instructions to continue taking anti-tubercular agents on a
The primer sets developed for amplification of the HA and NA genes successfully amplified the
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target genes from the clinical sample and gave good sequencing results (Accession numbers: NCBI: KX458011-KX458012). Eight genetic groups of H1N1pdm09 have existed since 2009, and the phylogenetic tree produced in this study revealed that the recently detected Saudi strain is related to clade 6B (Fig. 1). It is worth mentioning that most of the influenza isolates from 2014 and onwards are related to clade 6B.1. On the other hand, the NA gene of the current Saudi strain was clustered with 2015 and 2016 H1N1pdm09 strains (Fig.1).
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The amino acid sequence identity of the HA to A/California/7/2009 was 97%, the same as with previously reported Saudi strains in 2009 and 2010 that related to clade 1, and 99% with the strains in clade 6B. The NA protein of the current Saudi strain showed 96% identity with the
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A/California/7/2009 strain and to the previously isolated strains from Saudi Arabia. Meanwhile, it showed 99% identity to other 2015 and 2016 strains (Data not shown).
Xu et al. studied the HA sequence variations in the natural isolates, along with HA mutations in
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viruses grown with monoclonal antibodies and described five antigenic sites named Sa, Sb, Ca
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(Ca1 and Ca2) and Cb [11]. These antigenic sites found to be conserved in the Saudi strain with the exception of two sites in the Sa site: Ser 162 to Asn and Lys 163 to Gln. The first of these (Ser 162 to Asn) results in an additional N-glycosylation site in the Saudi strain and other isolates of the 6B clade. This additional site was found in the Saudi strain and also in some of the other published 2015-2016 sequences. Lys 163 to Gln, meanwhile, was detected in the recent
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Saudi strain and the majority of 2015 and 2016 isolates. Lys 163 to Gln, and Ala 256 to Thr
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amino acid substitutions, were detected in all isolates of the 6B clade including A/Saudi
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Arabia/TU-1/2015, while K283E and E499K were detected in both the 6B and 6C clades. Ala 13 to Thr in the signal peptide was detected only in 2015 and 2016 strains of the 6B clade. A unique
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Ile 286 to Met amino acid substitution was detected only in the A/Saudi Arabia/TU-1/2015 (Fig.2). Ile 32 to Leu, Asp 97 to Asn, Ser 185 to Thr, Glu to 374 Lys, and Ser 451 to Asn amino acid substitutions were found in A/Saudi Arabia/TU-1/2015 (Fig.2).These mutations were also reported in H1N1pdm09 from different geographical regions [12-15]. A phenotypic changes in the HA protein of the circulating H1N1pdm09 indicated a relative increase in affinity to α-2,6linked sialic acids [15]. In addition, both Asp 187 and Asp 222 increase the affinity to the upper
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Oseltamivir is a neuraminidase inhibitor typically administered in Saudi Arabia for the treatment of influenza. Amino acid substitution that induce resistance to oseltamivir is dependent on the NA Group. NA groups 1 (N1, N4, N5, N8), the His 275 to Tyr amino acid substitution (N1 numbering) is most common, while in N2 group viruses (N2, N3, N6, N7, N9), Glu 119 to Val,
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Arg 293 to Lys, Asn 295 to Ser or Ile 223 to Arg (N1 numbering) are most common resistant
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related mutation [17-19]. His 275 to Tyr resulted in resistance among 2.8% of the 2010-2011 H1N1pdm09 patients [20]. No relevant mutations were found in the current A/Saudi Arabia/TU1/2015 (Fig.3). In the neuraminidase protein both the catalytic residue sites (Arg 118, Asp 151, Arg 152, Arg 225, Glu 277, Arg 293 and Arg 368, Tyr 402) and the framework residues (Glu 119, Arg 156, Trp 179, Ser 180, Asp199, Ile223, Glu 228, His 275, Asn295 and Glu 425) (N1
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numbering) [21] were found to be conserved in the Saudi strain (Fig.3). The virtual binding to oseltamivir of the current strain, the 2009 original strain, together with the
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His 274 Tyr mutant strain from 2009 and 2015 was screened, but the docking score (Kcal/ mol) showed no major differences between the different viruses (Fig. 4, Table 1). Interestingly, the
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virtual binding activities of oseltamivir, peramivir and laninamivir to pH1N1, H7N9, H5N1 were compared, the peramivir was superior in binding to different viruses followed by laninamivir and then oseltamivir [22]. Although the current strain shows neither altered virtual sensitivity to oseltamivir nor changes in amino acid residues that are associated with increased virulence, regular monitoring of the circulating strains is recommended to screen for possible emergence of possible virus mutants.
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Conflict of interest
All authors declare no conflict of interest.
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References
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[1] C. Fraser, C.A. Donnelly, S. Cauchemez, W.P. Hanage, M.D. Van Kerkhove, T.D. Hollingsworth, J. Griffin, R.F. Baggaley, H.E. Jenkins, E.J. Lyons, T. Jombart, W.R. Hinsley, N.C. Grassly, F. Balloux, A.C. Ghani, N.M. Ferguson, A. Rambaut, O.G. Pybus, H. Lopez-Gatell, C.M. Alpuche-Aranda, I.B. Chapela, E.P. Zavala, D.M. Guevara, F. Checchi, E. Garcia, S. Hugonnet, C. Roth, Pandemic potential of a strain of influenza A (H1N1): early findings, Science (New York, N.Y.) 324(5934) (2009) 1557-61. [2] E. Bautista, T. Chotpitayasunondh, Z. Gao, S.A. Harper, M. Shaw, T.M. Uyeki, S.R. Zaki, F.G. Hayden, D.S. Hui, J.D. Kettner, A. Kumar, M. Lim, N. Shindo, C. Penn, K.G. Nicholson, Clinical aspects of pandemic 2009 influenza A (H1N1) virus infection, The New England journal of medicine 362(18) (2010) 1708-19. [3] T. Han, W.A. Marasco, Structural basis of influenza virus neutralization, Annals of the New York Academy of Sciences 1217 (2011) 178-90. [4] N. Spanakis, V. Pitiriga, V. Gennimata, A. Tsakris, A review of neuraminidase inhibitor susceptibility in influenza strains, Expert review of anti-infective therapy 12(11) (2014) 1325-36. [5] A.C. Hurt, H. Kelly, Debate Regarding Oseltamivir Use for Seasonal and Pandemic Influenza, Emerging infectious diseases 22(6) (2016) 949-55. [6] J.L. McKimm-Breschkin, Resistance of influenza viruses to neuraminidase inhibitors--a review, Antiviral research 47(1) (2000) 1-17. [7] WHO, World Health Organization: WHO report 2009 : Global tuberculosis control - epidemiology, strategy, financing, 2009. who int/tb/publications/global_report/2009/en/index html. [8] P.J. Cardona, A dynamic reinfection hypothesis of latent tuberculosis infection, Infection 37(2) (2009) 80-6. [9] K. Tamura, M. Nei, Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees, Mol Biol Evol 10(3) (1993) 512-26. [10] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, S. Kumar, MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods, Molecular Biology and Evolution 28 (2011) 2731-2739. [11] R. Xu, D.C. Ekiert, J.C. Krause, R. Hai, J.E. Crowe, Jr., I.A. Wilson, Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus, Science (New York, N.Y.) 328(5976) (2010) 35760. [12] J. Ellis, M. Galiano, R. Pebody, A. Lackenby, C. Thompson, A. Bermingham, E. McLean, H. Zhao, S. Bolotin, O. Dar, J.M. Watson, M. Zambon, Virological analysis of fatal influenza cases in the United Kingdom during the early wave of influenza in winter 2010/11, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 16(1) (2011). [13] V. Veljkovic, H.L. Niman, S. Glisic, N. Veljkovic, V. Perovic, C.P. Muller, Identification of hemagglutinin structural domain and polymorphisms which may modulate swine H1N1 interactions with human receptor, BMC structural biology 9 (2009) 62. [14] N. Kosoltanapiwat, U. Boonyuen, P. Pooruk, S. Iamsirithaworn, A. Mungaomklang, K. Chokephaibulkit, P. Auewarakul, P. Puthavathana, Amino acid substitutions in hemagglutinin of the 2009
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Figure 1
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Figure legends
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pandemic influenza A(H1N1) viruses that might affect the viral antigenicity, BMC research notes 7 (2014) 951. [15] R.A. Elderfield, S.J. Watson, A. Godlee, W.E. Adamson, C.I. Thompson, J. Dunning, M. FernandezAlonso, D. Blumenkrantz, T. Hussell, M. Zambon, P. Openshaw, P. Kellam, W.S. Barclay, Accumulation of human-adapting mutations during circulation of A(H1N1)pdm09 influenza virus in humans in the United Kingdom, Journal of virology 88(22) (2014) 13269-83. [16] V. Baldo, C. Bertoncello, S. Cocchio, M. Fonzo, P. Pillon, A. Buja, T. Baldovin, The new pandemic influenza A/(H1N1)pdm09 virus: is it really "new"?, Journal of preventive medicine and hygiene 57(1) (2016) E19-22. [17] J.A. Ives, J.A. Carr, D.B. Mendel, C.Y. Tai, R. Lambkin, L. Kelly, J.S. Oxford, F.G. Hayden, N.A. Roberts, The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo, Antiviral research 55(2) (2002) 307-17. [18] F.Y. Aoki, G. Boivin, N. Roberts, Influenza virus susceptibility and resistance to oseltamivir, Antiviral therapy 12(4 Pt B) (2007) 603-16. [19] M. Samson, A. Pizzorno, Y. Abed, G. Boivin, Influenza virus resistance to neuraminidase inhibitors, Antiviral research 98(2) (2013) 174-85. [20] I.A. Leneva, E.I. Burtseva, S.B. Yatsyshina, I.T. Fedyakina, E.S. Kirillova, E.P. Selkova, E. Osipova, V.V. Maleev, Virus susceptibility and clinical effectiveness of anti-influenza drugs during the 2010-2011 influenza season in Russia, International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 43 (2016) 77-84. [21] P.M. Colman, J.N. Varghese, W.G. Laver, Structure of the catalytic and antigenic sites in influenza virus neuraminidase, Nature 303(5912) (1983) 41-4. [22] A.F. Eweas, A.S. Abdel-Moneim, In-silico structural analysis of the influenza A subtype H7N9 neuraminidase and molecular docking with different neuraminidase inhibitors, Virusdisease 26(1-2) (2015) 27-32.
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Phylogenetic trees of the haemagglutinin and neuramindase genes of the H1N1pdm09. Maximum likelihood phylogenetic tree with 1000 bootstrap replicates of the different H1N1pdm09 strains in comparison to the A/Saudi Arabia/TU-1/2015 (red in colour). G symbol denotes strains obtained from GISAID. a) Haemagglutinin, b) Neuraminidase.
Figure 2
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Deduced amino acid sequence of the haemagglutinin protein of A/Saudi Arabia/TU1/2015 and selected H1N1pdm09 strains. Signal peptide is boxed. N-glycosylations sites, NXS/T sequences (X is any amino acid except P), are underlined. Available Saudi strains
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and closely related strains in the current study in addition to were included in the
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comparison.
Figure 3
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Deduced amino acid sequence of the neuraminidase protein of selected H1N1pdm09 strains. Catalytic sites are in red while the framework-sites are in blue colour. Nglycosylation sites are underlined. Available Saudi strains and closely related strains in the current study in addition to 2009 and 2016 strains with H275Y amino acid substitution
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were included in the comparison.
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Figure 4
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Molecular docking of the A/Saudi Arabia/TU-1/2015 neuraminidase with oseltamivir.
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Table 1. Comparison of the docking score and amino acid involving in binding to oseltamivir A/Saudi Arabia/TU-
A/Czech Republic/11/2016
1/2015
H275Y
Docking score*
-86.10
-84.26
-86.12
R118
2.01 A
2.18 A
1.99 A
E119
2.46 A
2.09 A
2.24 A
D151
2.71 A
2.23 A
2.00 A
R152
1.97 A
2.00 A
R293
2.52 A,2.39A
2.13 A
R368
2.71 A/2.30 A/1.60 A
1.79 A/2.56 A/2.27 A
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-85.31 2.55 A/2.57 A
-
-
2.04 A
-
2.53 A/2.40 A
-
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A/Boston/678/2009
H275Y
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*Kcal/mol
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A/California/07/2009
2.71 /2.26 A/1.60 A
2.64 A/2.71 A
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Highlights No recent H1N1pdm09 sequences are available from Saudi Arabia.
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The viral HA is related to the 6B clade with two amino acid substitution in the Sa antigenic site.
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The viral NA showed unique amino acid substitution without an evidence of
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oseltamivir-resistance based on sequence analysis.
S0890-8508(17)30042-7
DOI:
10.1016/j.mcp.2017.05.002
Reference:
YMCPR 1288
To appear in:
Molecular and Cellular Probes
Received Date: 7 March 2017 Revised Date:
20 April 2017
Accepted Date: 8 May 2017
Please cite this article as: Alghamdi AN, Mahfouz ME, Hamdi FA, Al Aboud D, Al-laylah TZ, Alotaibi MI, Al-Thomali KWA, Abdel-Moneim AS, Sequence analysis of haemagglutinin and neuraminidase of H1N1 strain from a patient coinfected with Mycobacterium tuberculosis, Molecular and Cellular Probes (2017), doi: 10.1016/j.mcp.2017.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Sequence analysis of haemagglutinin and neuraminidase of H1N1 strain from a patient coinfected with Mycobacterium tuberculosis
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Ahmed N. Alghamdi1, Mohammad E. Mahfouz 1,2, Fahd A. Hamdi2, Daifullah Al Aboud1, Tawfiq Z. Al-laylah1, Mohammed I. Alotaibi1, Khalid W. A. Al-Thomali2,
College of Medicine, Taif University, Al-Taif 21944, Saudi Arabia, 2King Faisal
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Ahmed S. Abdel-Moneim1,3§
Hospital, Al-Taif, Saudi Arabia, 3Virology Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt.
Correspondence author: Ahmed S. Abdel-Moneim
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Email: [email protected] / [email protected]
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Abstract The 2009 H1N1 pandemic (H1N1pdm09) was associated with a considerable influenza-related
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morbidity and mortality. Among the complications, Mycobacterial tuberculosis was recorded as a coinfection with influenza in rare cases. The full-length sequences of the viral haemagglutinin and neuraminidase of H1N1pdm09 influenza A virus were analyzed from a recently infected patient. The patient was chronically infected with Mycobacterium tuberculosis. Molecular
SC
modelling and in-silico docking of the virus, and other selected strains with the drug oseltamivir
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were conducted and compared. Sequence analysis of the viral haemagglutinin revealed it to be closely related to the 6B.1 clade, with high identity to the circulating H1N1pdm09 strains, and confirmed that the virus still harbouring high affinity to the α-2,6-sialic acid human receptor. The viral neuraminidase showed high identity to the neuraminidase of the recently circulating strains of the virus with no evidence of the development of oseltamivir-resistant mutants. Regular
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Introduction
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resistant strains.
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monitoring of the circulating strains is recommended to screen for a possible emergence of drug-
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Since the first detection of the H1N1 in Mexico in March of 2009 [1], the virus began to circulate worldwide in a pandemic manner, hence the name H1N1pdm09. There have been more than 18,500 laboratory-confirmed deaths from infection with the H1N1pdm09 strain. Most of the lethal cases of H1N1pdm09 have been recorded in young and middle-aged adults [2] rather than in young children and old people as with the seasonal influenza epidemics. The influenza, hemagglutinin (HA), is the principal surface antigen and the main target of vaccine-induced neutralizing antibodies. The virus frequently mutates vulnerable HA epitopes,
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with subsequent changes in its antigenic structure, to escape recognition and virus elimination by the immune system[3]. Determining the changes in the antigenic sites provide structural insights
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into the rationale for optimizing vaccines to match circulating variants. Viral neuraminidase (NA) is a receptor destroying enzyme that helps to liberate the viral particles from the infected cells, which renders it an attractive target for anti-influenza drugs. Although many neuraminidase inhibitors exists, including oseltamivir, zanamivir, laninamivir
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and peramivir [4], oseltamivir is considered to be the most commonly used. The early use of
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oseltamivir can reduce the mortality rate in hospitalized persons infected with H1N1pdm09 by half [5]. Oseltamivir resistance typically emerges in immune-compromised patients, but is much less frequently found in immunocompetent people [6] and it constitutes a major challenge to the effectiveness of the drug for the treatment of influenza and highlights the necessity for clinical
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assessment of antiviral resistance.
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Tuberculosis (TB) is caused by Mycobacterium tuberculosis. Active tuberculosis occurs in up to 10% of the infected individuals, while the remaining cases develop a latent infection lasting for
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years [7]. Reactivation of latent infection may occur, with the subsequent development of active
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disease in most cases pulmonary TB disease [8]. The current study describes the full-length sequence analysis of both haemagglutinin and neuraminidase of H1N1pdm09 strain from TB chronically infected patient. In addition, the virtual docking of the neuraminidase against oseltamivir of the detected strain in comparison with selected pdm09 classical and H275Y mutant strains was assessed.
Materials and Methods
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Ethical approval The study was ethically approved by the King Faisal Hospital Ethical and Research Committee.
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Informed written consent was obtained from the patient. Subject
An 86-year-old Pakistani female resident in Al-Taif, Saudi Arabia, infected with Mycobacterium
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tuberculosis was admitted to King Faisal hospital at the end of November 2015. The patient was
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confirmed to have a recent infection of H1N1pdm09 by Makkah Regional Laboratory, Saudi Ministry of Health Laboratory. Oseltamivir was used for the treatment of the patient after laboratory confirmation of H1N1pdm09. RNA extraction
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Nasopharyngeal, throat swabs and sputum samples were collected from the patient at day 3 of
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admission in a virus transport medium. Total viral RNA was extracted from 300 μl of the sample using viral RNA extraction kit (Promega). Extracted RNAs were eluted into a final volume of 60
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One-step RT-PCR
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μl in an elution buffer.
One-step RT-PCR was conducted using Script™ RT (Promega) and in-house specific primer sets that flank the HA and NA genes of H1N1pdm09. Three overlapping primer sets were used for the
HA
gene:
pH1-F1:CAAAAGCAGGGGAAAACAAA,
R1:TTGATGTCCCCACAAAAACA,pH1-F2:GCTCCGCCAATCCTACATTA,
pH1pH1-
R2:GATAACCGTACCATCCATCTACC, pH1-F3: CCAGCCTCCCATTTCAGAATA, pH1-
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R3:
GTAGAGACCCATTAGAGCACATC
ACCATTGGTTCGGTCTGTATG,
pN1-R1
and
two
for
the
NA
genes:
pN1-F1
CACTTGGTCCATCGGTCATTAT,
pN1-F2
GGCTGTGTTAAAGTACAATGGC and pN1-R2 AAGACCAACCCACAGTGTC. Reverse
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transcription was conducted at 45 °C for 45 minutes followed by 5 min denaturation with an initial denaturation step at 95 °C and 35 cycles of 95 °C for 30 sec., 52 °C for 30 sec. (54°C for pH1-F1/R1 and 50°C pN1-F1/R1) and 72 °C for 1.5 min. This was followed by a final
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elongation step for 10 min at 72 °C. PCR amplicons were gel extracted and sequenced directly in
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both forward and reverse directions using the same primers. Sequence analysis
The raw sequences were assembled with the MEGA 5.2 package and the same program was used to perform multiple sequence alignment using representative HA and NA sequences of
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H1N1pdm09 viruses obtained from the NCBI flu database and the EpiFlu database of the Global
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Initiative on Sharing Avian Influenza Data (GISAID). The Maximum Likelihood method based on the Tamura-Nei substitution model was used to build a phylogenetic tree [9]. The bootstrap
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consensus tree from 1000 replicates represented the evolutionary history of the analyzed taxa. All positions containing gaps and missing data were eliminated. Evolutionary analyses and
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deduced amino acid sequences of HA and NA proteins were conducted in MEGA5[10]. Molecular modelling and in-silico molecular docking The NA protein of the A/Saudi Arabia /TU-1/2015 and the NA amino acid sequences of A/CzechRepublic/11/2016_H274Y, A/California/07/2009, A/Boston/678/2009 were obtained from the NCBI-flu database. Modelling of each protein sequence was conducted online using Swiss-model
free
online
software
(http://swissmodel.expasy.org/).
Minimization
and
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equilibration of steric clashes caused by the addition of hydrogen atoms, and alleviation of water and ions were performed by Molsoft (Molsoft ICM 3.5–8C) prior to performing the molecular dynamics. In-silico molecular docking was conducted for the NA protein of different H1N1
8C). The comparison was based on the best binding free energy.
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Results and Discussion
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strains against the oseltamivir ligand using ‘Internal Coordinate Mechanics (Molsoft ICM 3.5–
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The patient was treated from the first day of admission with oseltamivir 75 mg oral capsule twice daily, ceftriaxone 1 g by intravenous injection twice daily and azithromycin 500 mg once daily (orally) as an empirical treatment for pneumonia and suspected H1N1pdm09. After obtaining the
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result of the bacteriological investigation, administration of antimycobacterial therapy (isoniazid 300 mg, rifampicin 450 mg, ethambutol 1.2 g and pyrazinamide 1.5 g once daily) was initiated along with trimethoprim/sulfamethoxazole 800 mg with discontinuing the use of ceftriaxone,
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azithromycin and oseltamivir. The patient improved during the 7 days of treatment in ICUisolation and was transferred back to a regular bed in the isolation unit. By the 11th day of
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hospitalization, her hepatic enzymes have increased and antimycobacterial therapy stopped until the hepatic enzymes become normal, which was achieved 3 days later. The anti-tuberculosis agents were then resumed with daily liver function tests. Patient treatment was continued with both the anti-tubercular and the anti-viral agents for 14 days. Symptoms of lethargy, cough and
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fever gradually improved within 1 week. After three consecutive negative sputum samples for
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regular basis.
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AFB, the patient was discharged with instructions to continue taking anti-tubercular agents on a
The primer sets developed for amplification of the HA and NA genes successfully amplified the
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target genes from the clinical sample and gave good sequencing results (Accession numbers: NCBI: KX458011-KX458012). Eight genetic groups of H1N1pdm09 have existed since 2009, and the phylogenetic tree produced in this study revealed that the recently detected Saudi strain is related to clade 6B (Fig. 1). It is worth mentioning that most of the influenza isolates from 2014 and onwards are related to clade 6B.1. On the other hand, the NA gene of the current Saudi strain was clustered with 2015 and 2016 H1N1pdm09 strains (Fig.1).
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The amino acid sequence identity of the HA to A/California/7/2009 was 97%, the same as with previously reported Saudi strains in 2009 and 2010 that related to clade 1, and 99% with the strains in clade 6B. The NA protein of the current Saudi strain showed 96% identity with the
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A/California/7/2009 strain and to the previously isolated strains from Saudi Arabia. Meanwhile, it showed 99% identity to other 2015 and 2016 strains (Data not shown).
Xu et al. studied the HA sequence variations in the natural isolates, along with HA mutations in
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viruses grown with monoclonal antibodies and described five antigenic sites named Sa, Sb, Ca
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(Ca1 and Ca2) and Cb [11]. These antigenic sites found to be conserved in the Saudi strain with the exception of two sites in the Sa site: Ser 162 to Asn and Lys 163 to Gln. The first of these (Ser 162 to Asn) results in an additional N-glycosylation site in the Saudi strain and other isolates of the 6B clade. This additional site was found in the Saudi strain and also in some of the other published 2015-2016 sequences. Lys 163 to Gln, meanwhile, was detected in the recent
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Saudi strain and the majority of 2015 and 2016 isolates. Lys 163 to Gln, and Ala 256 to Thr
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amino acid substitutions, were detected in all isolates of the 6B clade including A/Saudi
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Arabia/TU-1/2015, while K283E and E499K were detected in both the 6B and 6C clades. Ala 13 to Thr in the signal peptide was detected only in 2015 and 2016 strains of the 6B clade. A unique
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Ile 286 to Met amino acid substitution was detected only in the A/Saudi Arabia/TU-1/2015 (Fig.2). Ile 32 to Leu, Asp 97 to Asn, Ser 185 to Thr, Glu to 374 Lys, and Ser 451 to Asn amino acid substitutions were found in A/Saudi Arabia/TU-1/2015 (Fig.2).These mutations were also reported in H1N1pdm09 from different geographical regions [12-15]. A phenotypic changes in the HA protein of the circulating H1N1pdm09 indicated a relative increase in affinity to α-2,6linked sialic acids [15]. In addition, both Asp 187 and Asp 222 increase the affinity to the upper
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Oseltamivir is a neuraminidase inhibitor typically administered in Saudi Arabia for the treatment of influenza. Amino acid substitution that induce resistance to oseltamivir is dependent on the NA Group. NA groups 1 (N1, N4, N5, N8), the His 275 to Tyr amino acid substitution (N1 numbering) is most common, while in N2 group viruses (N2, N3, N6, N7, N9), Glu 119 to Val,
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Arg 293 to Lys, Asn 295 to Ser or Ile 223 to Arg (N1 numbering) are most common resistant
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related mutation [17-19]. His 275 to Tyr resulted in resistance among 2.8% of the 2010-2011 H1N1pdm09 patients [20]. No relevant mutations were found in the current A/Saudi Arabia/TU1/2015 (Fig.3). In the neuraminidase protein both the catalytic residue sites (Arg 118, Asp 151, Arg 152, Arg 225, Glu 277, Arg 293 and Arg 368, Tyr 402) and the framework residues (Glu 119, Arg 156, Trp 179, Ser 180, Asp199, Ile223, Glu 228, His 275, Asn295 and Glu 425) (N1
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numbering) [21] were found to be conserved in the Saudi strain (Fig.3). The virtual binding to oseltamivir of the current strain, the 2009 original strain, together with the
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His 274 Tyr mutant strain from 2009 and 2015 was screened, but the docking score (Kcal/ mol) showed no major differences between the different viruses (Fig. 4, Table 1). Interestingly, the
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virtual binding activities of oseltamivir, peramivir and laninamivir to pH1N1, H7N9, H5N1 were compared, the peramivir was superior in binding to different viruses followed by laninamivir and then oseltamivir [22]. Although the current strain shows neither altered virtual sensitivity to oseltamivir nor changes in amino acid residues that are associated with increased virulence, regular monitoring of the circulating strains is recommended to screen for possible emergence of possible virus mutants.
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Conflict of interest
All authors declare no conflict of interest.
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References
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Figure 1
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Figure legends
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Phylogenetic trees of the haemagglutinin and neuramindase genes of the H1N1pdm09. Maximum likelihood phylogenetic tree with 1000 bootstrap replicates of the different H1N1pdm09 strains in comparison to the A/Saudi Arabia/TU-1/2015 (red in colour). G symbol denotes strains obtained from GISAID. a) Haemagglutinin, b) Neuraminidase.
Figure 2
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Deduced amino acid sequence of the haemagglutinin protein of A/Saudi Arabia/TU1/2015 and selected H1N1pdm09 strains. Signal peptide is boxed. N-glycosylations sites, NXS/T sequences (X is any amino acid except P), are underlined. Available Saudi strains
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and closely related strains in the current study in addition to were included in the
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comparison.
Figure 3
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Deduced amino acid sequence of the neuraminidase protein of selected H1N1pdm09 strains. Catalytic sites are in red while the framework-sites are in blue colour. Nglycosylation sites are underlined. Available Saudi strains and closely related strains in the current study in addition to 2009 and 2016 strains with H275Y amino acid substitution
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were included in the comparison.
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Figure 4
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Molecular docking of the A/Saudi Arabia/TU-1/2015 neuraminidase with oseltamivir.
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Table 1. Comparison of the docking score and amino acid involving in binding to oseltamivir A/Saudi Arabia/TU-
A/Czech Republic/11/2016
1/2015
H275Y
Docking score*
-86.10
-84.26
-86.12
R118
2.01 A
2.18 A
1.99 A
E119
2.46 A
2.09 A
2.24 A
D151
2.71 A
2.23 A
2.00 A
R152
1.97 A
2.00 A
R293
2.52 A,2.39A
2.13 A
R368
2.71 A/2.30 A/1.60 A
1.79 A/2.56 A/2.27 A
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-85.31 2.55 A/2.57 A
-
-
2.04 A
-
2.53 A/2.40 A
-
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A/Boston/678/2009
H275Y
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*Kcal/mol
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A/California/07/2009
2.71 /2.26 A/1.60 A
2.64 A/2.71 A
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Highlights No recent H1N1pdm09 sequences are available from Saudi Arabia.
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The viral HA is related to the 6B clade with two amino acid substitution in the Sa antigenic site.
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The viral NA showed unique amino acid substitution without an evidence of
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oseltamivir-resistance based on sequence analysis.