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    Molecular and phylogenetic analysis of influenza A H1N1 pandemic viruses in Cuba - Article 9

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    Molecular and Phylogenetic Analysis of Influenza A H1N1 Pandemic Viruses in Cuba - Article 9

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    ‘ternational journal of infectious Diseases 17 (2013) e555-¢567 Contents lists available at SciVerse ScienceDirect International Journal of Infectious Diseases ELSEVIER journal homepage: www. Isevier.com/locate/ijid Short Communication Molecular and phylogenetic analysis of influenza A H1N1 pandemic Dene viruses in Cuba, May 2009 to August 2010 Alexander Pifén Ramos”, Belsy Acosta Herrera, Odalys Valdés Ramirez, ‘Amely Arencibia Garcia, Mayra Muné Jiménez, Clara Savon Valdés, Angel Goyenechea Fernandez, Grehete Gonzalez, Suset |. Oropesa Fernandez, Guelsys Gonzalez Baez, Barbara Hernandez Espinosa Tropa edn ste Po Ror tops Nov del Mea Kt La Us Cid a Hab, 17100 cuba ARTICLE INFO SUMMARY Artie soy ‘The influenza A(HIN1)pdin09 virus was detected in Cuba in May 2008, The introduction ofa new virus Receive 8 January 2013, with increased transmissibility into a population makes surveillance of the pandemic siain to the ecelved in rviod for 9 January 2013 ‘Accepted 3 January 2013 Corresponding Editor: shld Petersen ‘molectlar level necessary. The aim af the present study was the molecular and phylogenetic analysis of pandemic influenza A(HIN1pdm09 strains that circulated in Cuba between May 2009 and August 2010, ‘Seventy clinical samples were included in the study. Nucleotide sequences from the hemagglutinin HAT region segment were obtained directly from clnial samples, Genetic distances were caleulated using MEGA ¥.5.05. A phylogenetic tree was constructed using MrBayes v3.1.2 software, Potential N. eyo lyosylation sites were predicted using NeINGiye server 1.0. The 48 Cuban sequences of influenza iene ste "AIINT)pdmo obtained were similar to the Aalfornia(07/2009 (HIN) vaccine strain, Most of the yen {cuban stains belonged to cade 7. Cuban viruses showed aminoacid changes, some of them located at Eee three antigenic sites Ca, Sa, and Sb. Two dominant mutations were detected P835 (1003) and $2037 eee (85.79, Glycosylation site analysis eveaed the gan of one site at position 162 in 13 sequences. The Findings inthis study contribute to our understanding of the progress ofthe influenza A(HIN1)pdin09) virus, since this virus is atthe starting point ofits evolution in humans. 1©°2015 Internationa Society for Infectious Diseases, Published by Elsevier Ld, Al rghts reserve. 1. Introduction Published protocol! Nucleotide sequences of PCR products were determined using the BigDye GenomeLab Dye Terminator Cycle The pandemic virus influenza A(HINI}pdm09 was detected in Sequencing Kit (Beckman Coulter, USA). Genetic distances were Cuba in May 2009. Since then, the virus has spread across the calculated in MEGA v.5.05 using the Kimura two-parameter model, country. The aim of the present study was the molecular and A phylogenetic tree was constructed using MrBayes v3.1.2 phylogenetic analysis of influenza AHINI)pdm09 strains that software.* The tree was visualized using Dendroscope v.2.7.4, Circulated in Cuba between May 2009 and August 2010. Potential N-glycosylation sites were predicted using NetNClye server 1,0, Cuban sequences were deposited in the GenBank 2. Methods database under accession numbers: HM159409.1-HM159418.1 HM176606.1-HM176611.1; HQ190937-HQ190939, 10190941: A total of 70 clinical samples positive for influenza and 9922272-19922304, AHIND}pdm09 virus were included in the study, representing, initial (n= 15), frst wave (n= 20), second wave (n= 20), and final period (n=15) samples. Total viral RNA was automatically 3 Results and discussion extracted from clinical samples using the QlAamp Viral RNA. Mini. Kit (Qiagen, USA) in a QlAcube extractor (Qiagen, USA). Sequence analysis of the 48 obtained partial HAI segment Amplification reactions were achieved following a previously hemagglutinin of Cuban influenza ALIN1)pdm09 sequences showed an amino acid similarity of 98.8% with vaccine strain influenza ACalifornia/07/2009 (HIN1). ~ corresponding autor Phylogenetic analysis ofthe Cuban sequences showed that G7 al adres slexgreipksc (AP, Ramos. ‘was the most represented, harboring the S203T mutation located 1201-9712)835.00- se root mater ©2015 international Society for afections Diseases. Published by Ebvir Ln. All ight seserved np ddotong! 101016) 201301.028, AP. Ramos eal nemaional journal of eto Disenss 17 (2013) e565-e567, aera as a0 ja 053 a0 aan 15005 200 pn 38 0 [Satjase sna 0 (austin) ———a ate 07732030 nee [sate ae too [seas 04 0 are re) [sr se ao [sewn se 208 ar-spaat ios a tn 8290 ls 000% 200 [panna tssnani0 |__ as — stim fate 5820 i 2090208 jaa 520 Air 0230) Ap 92009 A 9,208 hie 808 ans 57,200. ee) A 97852 ‘es 957 0 Figure 1. Phylogenetic elation of he HAI peptide ofthe nftenza AHIN)pd2008 vite detected in Cub The phylogenetic ie was constructed wing Mayes 3:12 software, incororating the HRY = G model of nuceotde sobsttton The generation number and sample reueney sampling wer ed to 5 000000 and 1000, respectively Every lade was supported by Bayesian posterior probabilities (BPP atthe antigenic site Ca (Figure 1), which has been widely reported.” {A further observation was the subgroup located in clade 7 (Figure 1), integrated with sequences obtained from clinical samples collected during the last months of the epidemic. This subgroup included 12 Cuban sequences and showed the A186T ‘mutation located at antigenic site Sb. The other Cuban sequences ‘were not included in any of the defined clades, maybe because of the studies describing seven clades completed the phylogeny with a whole concatenated genome. The most common change in Cuban sequences was P83, (100%), Other important changes located at antigenic sites were detected: S203T and D222E (Ca antigenic site); NISBK and SI62N (Sa antigenic site); At86T (Sb antigenic site). The frequencies of ‘occurrence of the mutations mentioned above are shown in Figure 2, Mutation $2031 found in 85.7% of Cuban sequences was located in the vicinity ofthe receptor binding domain. It has been reported that this change emerged in a transient way in sequences of influenza A HIN] viruses collected from humans in 1934 and from swine in 1976-77." showing that this amino acid change could affect the infectivity and transmissibility of influenza A(HIN1Jpcim09 virus in humans The mutation S162N located at the antigenic site Sa, and detected in Cuban sequences during the final period of the ‘AP. Rams eo nteratonal our of nets senses 17 (2013) 565-2567 oer Matton at antigen ster Matton equenet) Figure 2 Frequencies of occurence ofthe man mutations detected in Cuban sequences when compared withthe vaccine strain inuenza ACalori07/2009 (HINT) epidemic, represents the acquisition of an N-glycosylation site. It ‘was confirmed that glycans added to positions 127, 155, and 162 could effectively block the immune reaction of antibodies elicited against pandemic strains from the years 1918 and 2008.° The mutation ALS6T was another change detected in Cuban sequences. This mutation is located at antigenic site Sb in the receptor binding site and appears in combination with mutations, [N31D, S162N, and 1172 V in six sequences. The same observation ‘was reported in sequences from the USA and Ecuador, wth $162N and A186T modulating antibody attachment Thisis an important finding since it could be an important step in the evolution of influenza AHIN1 jpd09, In one Cuban sequence, the mutation N156K located at antigenic site Sa was also detected. A previous report described [NI56K andfor N125D as mutations associated with a diminished Acalifornia/07/2009 vaccine strain antibody recognition in vaccinated individuals.” ‘The aspartic acid to glycine change at position 222 (D222) allows the virus to gain double specificity in receptor binding. This change allows the virus to reach the lower respiratory tract, causing an increase in severity of disease.® None of the Cuban sequences showed the D222G change, however a virus from a patient suffering a mild disease harbored the D222 mutation located at antigenic site Sa, a mutation also detected by others” Some authors have associated the D222G change with an increased severity ofthe disease caused by influenza virus AHINI}pdm09."° On the other hand, substitution D226 was not associated with severity of the disease.* This research constitutes the first molecular study of the influenza pandemic viruses circulating in Cuba in 2009-2010. Cuban sequences were similar to vaccine strain influenza A/ California07/2009 (H1N1). However, further antigenic analysis is, needed 10 assess the characteristics of sequenced viruses, specifically those that possess changes at antigenic and glycosyla- tion sites of the hemagglutinin. Our findings, like those of other studies, confirm the genetic variability of the influenza ACHINT)pdm09 viruses and show the importance of systematic ‘molecular surveillance for the effective management of influenza epidemics and pandemics. Conflict of interest: No conflict of interest to declare, References 1. Riz-Carrscoso G, Casas | Por F Peres-Gonzaler€, Rena J Perez-ronaP. cal. Development and imprementatin of Influenza A viru subyping and ‘ecection of genotypic rentance to neuraminace beer. Med Vol 2, Ronit F.Huelsenbeck JP Mayes 3: Bayesian phylogenetic inference under nied moves Brnormenks 2003:19:1572-4 4. Nelson Mh. spiro. Wentworth D, Beck E an J. GhedinE,et a. The early

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