This work reports a method for rapid amplification of the complete genome of equine influenza virus subtype 2 (H3N8).
A ThermoScriptTM reverse transcriptase instead of the avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase was used. This enzyme has demonstrated higher thermal stability and is described as suitable to make long cDNA with a complex secondary structure. The product obtained by this method can be cloned, used in later sequencing reactions or nested-PCR with the purpose of achieving a rapid diagnosis and characterization of the equine influenza virus type A. This detection assay might be a valuable tool for diagnosis and screening of field samples as well as for conducting molecular studies.En este trabajo comunicamos un método rápido que permite la amplificación del genoma completo del subtipo 2 (H3N8) del virus de la influenza equina. Se utilizó la enzima transcriptasa reversa ThermoScriptTM en lugar de la transcriptasa reversa del virus de la mieloblastosis aviar o la transcriptasa reversa del virus de la leucemia murina de Moloney. Esta enzima ha demostrado tener una alta estabilidad térmica y la capacidad de hacer largas copias de ADN con una estructura secundaria compleja. El producto obtenido por esta técnica puede ser clonado y utilizado posteriormente en reacciones de secuenciación o de PCR anidada con la finalidad de lograr un diagnóstico rápido y la caracterización del virus de la influenza equina tipo A. Este ensayo de detección puede llegar a ser una valiosa herramienta para el diagnóstico y el análisis de muestras de campo, así como para la realización de estudios moleculares

Fuentealba, N. A.

Galosi, Cecilia Mónica

Pecoraro, M. R.

Sguazza, G. H.

Tizzano, Marco Antonio

Centro de Servicios en Gestión de Información

Genome amplification of Influenza virus 207ISSN 0325-7541Revista Argentina de Microbiología (2009) 41: 207-211INFORME BREVEComplete genome amplification of Equineinfluenza virus subtype 2G. H. SGUAZZA1*, N. A. FUENTEALBA1, 2, M. A. TIZZANO1, 3, C. M. GALOSI1, 3, M. R. PECORARO1.1Cátedra de Virología, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata;2Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); 3Comisión de Investigaciones Científicas de laProvincia de Buenos Aires (CIC), Argentina.*Correspondence. E-mail: sguazza@fcv.unlp.edu.arABSTRACTThis work reports a method for rapid amplification of the complete genome of equine influenza virus subtype 2 (H3N8).A ThermoScriptTM reverse transcriptase instead of the avian myeloblastosis virus reverse transcriptase or Moloneymurine leukemia virus reverse transcriptase was used. This enzyme has demonstrated higher thermal stability and isdescribed as suitable to make long cDNA with a complex secondary structure. The product obtained by this methodcan be cloned, used in later sequencing reactions or nested-PCR with the purpose of achieving a rapid diagnosis andcharacterization of the equine influenza virus type A. This detection assay might be a valuable tool for diagnosis andscreening of field samples as well as for conducting molecular studies.Key words: Equine influenza virus, genome, diagnosis, RT-PCRRESUMENAmplificación del genoma completo del subtipo 2 del virus de la influenza equina. En este trabajo comunicamosun método rápido que permite la amplificación del genoma completo del subtipo 2 (H3N8) del virus de la influenzaequina. Se utilizó la enzima transcriptasa reversa ThermoScriptTM en lugar de la transcriptasa reversa del virus de lamieloblastosis aviar o la transcriptasa reversa del virus de la leucemia murina de Moloney. Esta enzima ha demostradotener una alta estabilidad térmica y la capacidad de hacer largas copias de ADN con una estructura secundariacompleja. El producto obtenido por esta técnica puede ser clonado y utilizado posteriormente en reacciones desecuenciación o de PCR anidada con la finalidad de lograr un diagnóstico rápido y la caracterización del virus de lainfluenza equina tipo A. Este ensayo de detección puede llegar a ser una valiosa herramienta para el diagnóstico y elanálisis de muestras de campo, así como para la realización de estudios moleculares.Palabras clave: Virus de la influenza equina, Genoma, Diagnóstico, RT-PCRCátedra de Virología, Facultad de Ciencias Veterinarias, Universidad Na-cional de La Plata 60 & 118, La Plata, CP 1900, Buenos Aires, ArgentinaTelephone and fax number: 54 221 4257980Influenza viruses belong to the Orthomyxoviridae familyand are classified into three great types: A, B, and C,according to antigenic differences in their nucleoprotein(NP) and matrix (M) proteins. Influenza viruses types Band C are predominantly human pathogens that have alsobeen isolated from seals and pigs, respectively. On theother hand influenza viruses type A have been isolatedfrom many species including humans, pigs, horses, mar-ine mammals and a wide range of domestic and wild birds(10). Influenza viruses type A can be further subdiv-ided in different serologically differentiated subtypesaccording to the structure and composition of two antigenicglycoproteins located in the surface of the virion: haemag-glutinin (HA) involved in binding of the virus to host cellsand neuraminidase (NA) implicated in release of virusfrom infected cells. At present, 16 HA and 9 NA subtypeshave been identified among influenza A viruses. All thesesubtypes are found in avian species. However, only twoequine influenza virus subtypes have been associatedwith the disease in horses, the H7N7 subtype (equi-1)and the H3N8 subtype (equi-2). The H7N7 subtype wasfirst isolated from horses in Czechoslovakia in 1956 (pro-totype strain: A/equine1/Prague/56); later it was isolatedin many countries of Europe and America. Although thissubtype has not been isolated since 1980, it may still cir-culate in subclinical form and persist at very low levels insome parts of the world (10). The H3N8 subtype of equineinfluenza virus was first isolated in 1963 during an out-break of this disease in Miami (prototype strain: A/equine2/Miami/63) and can be found in different parts of the world,except in Australia, New Zealand, and Iceland. Recentstudies of the H3N8 subtype of equine influenza viruseshave demonstrated that these strains have diverged intotwo distinct evolutionary lineages (2).208 Revista Argentina de Microbiología (2009) 41: 207-211The genome of equine influenza A virus contains eightlinear segments of negative stranded RNA (about 13.6kb). Six segments code for single proteins: the three viralpolymerases (PA, PB1 and PB2), HA, NA and NP. Theother two segments codify two proteins, one segment formatrix 1 and 2 (M1 and M2) and the other segment fornon-structural protein 1 (NS1) and nuclear export protein(NEP) (5).The analysis of virion RNA from influenza A strainsindicates that all eight RNA segments contain a commonsequence of 13 nucleotides at the 3' terminus and an-other common sequence of 12 nucleotides at the 5' ter-minus (3, 9). No homologies can be found among seg-ments. On these bases, the aim of this work was to de-velop a rapid method that allows the genetic characteriz-ation of the whole genome of influenza virus using a proce-dure of reverse transcription followed by the amplificationof all viral segments through polymerase chain reaction(RT-PCR).The standard method for diagnosis of influenza is theisolation of influenza virus particles using embryonatedchicken eggs or Madin-Darby canine kidney cells (MDCK).This system has the disadvantage of requiring 4-6 daysfor completion. The complete genome amplification byRT-PCR reported in this paper followed by subsequentamplicon sequencing is a more versatile method becauseit allows obtaining more detailed information about circul-ating strains and it will help in the virus surveillance.To amplify the influenza virus genome, two primersbased on the highly conserved non-coding sequences atthe 5´ and 3´ end present in all viral genomic segments ofinfluenza A virus were used, according to the consensussequence regions previously reported (3, 9) and widelyused to amplify the complete genome of human and avianinfluenza A virus (4) and equine influenza A virus subtype1 (1). To confirm the presence of those conserved regionsin the genome of equine influenza subtype 2, 50 differentsequences of all segments from the viral genome retrievedfrom the GenBank at the National Center of Biotechnol-ogy Information (http://www.ncbi.nlm.nih.gov) were com-pared by multiple alignment using the CLUSTAL X (v1.8)software.The consensus regions were identified as suitablesequences for use as primers in RT-PCR, and the oligo-nucleotide primers were subsequently commercially syn-thesized (Integrated DNA Technologies, Coralville, IA,USA). The primers used in this work were a 12-mer primerUFLU-S 5´AGCAAAAGCAGG 3´ and a 13-mer primerUFLU-AS 5´AGTAGAAACAAGG 3´ (Fig. 1).An equine influenza virus strain, A/Equine-2/La Plata/2001 (H3N8), previously isolated in our laboratory duringthe year 2001, was used. The virus was propagated inthe allantoic cavity of 11-day-old embryonated hen’s eggsderived from a healthy flock.Total RNA was extracted using Trizol reagent (GIBCOBRL, Gaithersburg, MD, USA). In brief, 500 µl of allantoicFigure 1. Alignment of non-coding terminal regions of the eight segments of Influenza A virus (A/equine-2/Kentucky/5/02. GenBankaccession numbers AY855340, AY855339, AY855338, AY855341, AY855342, AY855343, AY855344 and AY855345 respectively).The 5´ terminus of each segment has 12 conserved nucleotides (A), and the 3´ terminus has 13 conserved nucleotides (B).Genome amplification of Influenza virus 209fluid (haemagglutination titre 1:128) were mixed with 500µl of Trizol reagent. The mixture was extracted with 220µl of chloroform. After centrifugation at 10000 x g for 10minutes, the RNA in the aqueous solution was precipitatedby adding an equal volume of isopropanol. The precipi-tated RNA was collected by centrifugation at 10000 x gfor 20 minutes, washed by 70% ethanol, dissolved in 50µl of RNAse-free water and measured by spectrophotom-eter (SmartSpec 3000, BIO-RAD, Hercules, CA, USA).RNA extracted from uninfected allantoic fluid was usedas negative control. Total RNA and control RNA (375.06ng/µl and 225.52 ng/µl respectively) were used for fol-lowing steps.One microgram of each RNA was used to produce asingle-strand copy of DNA (ssDNA). This reaction wascarried out using the avian myeloblastosis virus (AMV)reverse transcriptase (Promega Corporation, Madison,WI, USA), the Moloney murine leukemia virus (M-MMLV)reverse transcriptase (Promega) and the ThermoScriptRT-PCR System kit (Invitrogen-Life Technologies, Bue-nos Aires, Argentina) under conditions specified by thesuppliers, using a concentration 0.5 µM of the universalprimer complementary to the conserved 3’ end of allvirus segments (UFLU-S).The single-strand DNA (ssDNA) obtained with AMV,M-MLV and RT-ThermoScript was used directly as tem-plate in the PCR reaction whereas the cDNA generatedby RT-ThermoScript was additionally employed for mak-ing a double-strand DNA copy (dsDNA) in agreement withthe supplier’s suggestion.The second chain of DNA was synthesized using 3 µlof ssDNA previously obtained (approximately 100 ng) ina reaction mixture containing: 75 mM Tris-HCl (pH 8.8),20 mM (NH4)2SO4, 0.01% Tween 20, 200 µM of eachdNTPs, 1.5 mM of MgCl2 and 0.5 µM of UFLU-AS primerin a final volume of 24.5 µl. The mixture was incubated at95 °C during 4 minutes, followed by the addition of 0.5 µl(2.5 U) of Taq DNA polymerase (Fermentas Inc, GlenBurnie, MD, USA) and it was incubated at 65 °C during20 minutes.The PCR reaction was performed by adding 5 µl fromthe ssDNA or dsDNA to a reaction mixture containing afinal concentration of 75 mM Tris-HCl (pH 8.8), 20 mM(NH4)2SO4, 0.01% Tween 20, 200 µM of each one ofdNTPs, 0.5 µM of each one of primers (UFLU-S andUFLU-AS), a variable concentration of MgCl2 (1, 1.5, 2and 2.5 mM) and variable percentages of dimethil sulfoxide(0, 5 and 10% of DMSO) in a final volume of 49.5 µl.The mixture was heated at 95°C during 4 minutes andincubated with variable annealing temperatures (38, 40,42 and 44 °C) for 2 minutes, then 0.5 µl (2.5 U) from TaqDNA polymerase (Fermentas) were added. The amplificationprogram started with 1 cycle at 65 °C for 10 minutes, fol-lowed by 40 cycles at 92 °C for 2 minutes, the same an-nealing temperature initially used for 2 minutes, and 65 °Cfor 10 minutes; the program ended with one cycle at 72 °Cfor 10 minutes. The PCR product was run on agarose gel(1.5%) at 50 V during 6 hours, and the gel was stainedwith ethidium bromide and visualized with a UV transillu-minator.To evaluate the efficiency among reverse transcrip-tases AMV, M-MLV and ThermoScript, a protocol wasused to amplify the whole genome of equine influenzavirus. Improved results were observed when using dsDNAgenerated by ThermoScript-RT. When the ssDNA templateobtained from AMV, M-MLV and ThermoScript was used,only a few bands could be observed in agarose gel (Fig. 2)corresponding to small size fragments of the genome.The optimum PCR reaction was obtained by adding 5µl from the dsDNA to a reaction mixture containing a finalconcentration of 75 mM Tris-HCl (pH 8.8), 20 mM(NH4)2SO4, 0.01% Tween 20, 200 µM of each  dNTP, 1.5µM of MgCl2, 0.5 µM of each primer, 10% of DMSO and2.5 U of Taq DNA polymerase.An improved PCR amplification was carried out byheating the PCR mixture at 95 °C during 4 minutes, an-Figure 2. RT-PCR using ssDNA as template: Lanes 3, 4 and 6:amplicon obtained from AMV, M-MLV and RT-ThermoScript;Lanes 1, 2 and 7: negative controls (RNA from uninfected allantoicfluid) obtained from AMV, M-MLV and ThermoScript RT,respectively. Lane 5: molecular size marker (λ EcoRI-HindIIIMarker - Promega).210 Revista Argentina de Microbiología (2009) 41: 207-211Figure 3. RT-PCR using dsDNA as template: Lane 1: molecularsize marker (λ EcoRI-HindIII Marker - Promega); Lane 2: RT-PCR product.nealing at 40 °C for 2 minutes and then adding 0.5 µl (2.5U) from Taq DNA polymerase (Fermentas). The amplifi-cation program started with 1 cycle at 65 °C for 10 min-utes, followed by 40 cycles at 92 °C for 2 minutes, 40 °Cfor 2 minutes, and 65 °C for 10 minutes and a final exten-sion at 72 °C for 10 minutesThe eight segments of equine influenza subtype 2 wereamplified simultaneously using primers UFLU-S andUFLU-AS (Fig. 3). No amplified DNA was observed fromthe RNA negative control. Bands corresponding to basicpolymerases (PB2 and PB1, segments 1 and 2 respec-tively) could not be clearly differentiated from each otherbecause they were the same size (2341 bp each). It wasfollowed by a band belonging to the acid polymerase (PA)of 2233 bp, segment 4 (HA) with 1778 bp, segment 5(NP) of 1565 bp, segment 6 (NA) with 1413 bp, segment7 (M) of 1027 bp and, at the end, a band belonging tosegment 8 (NS) which showed an increased PCR prod-uct amount probably due to its smaller size.In this study, the amplification conditions for the com-plete EIV genome were improved. The data presented inthis paper demonstrate that this method could be a help-ful tool in the surveillance of the equine influenza virusand appropriate for cloning the eight genomic segments,which will facilitate large-scale EIV genome sequencingand greatly ease systematic genetic analyses of thevirus. Adeyefa et al. (1) have described a multiplex RT-PCR method in which 12-mer and 13-mer oligonucleotidescomplementary to the conserved regions were used,resulting in simultaneous amplification of all of the eightRNA segments of Equine Influenza virus subtype 1. Inthis work, a ThermoScript RT instead of AMV or M-MLVRT was used to amplify the full length genome of equineinfluenza virus subtype 2 (H3N8). This enzyme has higherthermal stability and is described as suitable to make longcDNA with complex secondary structure.The product obtained by this method can be clonedand used in later sequencing reactions  or nested-PCRwith the purpose of achieving a rapid diagnosis and char-acterization of the equine influenza virus type A (1, 8).This test is much faster than the isolation of the virusfrom nasopharyngeal swabs/washings using embryonatedeggs or cell cultures and the possibility of automation willallow handling a large number of clinical samples moreconveniently than with serological tests (7, 8).RT-PCR might be useful in detecting inapparent, sub-clinical infection in animals that contribute to the spreadof the virus. Accurate identification of infected animalsand inapparent carriers is essential for the control of thisdisease.REFERENCES1. Adeyefa CA, Quayle K, McCauley JW. A rapid method forthe analysis of influenza virus genes: application to thereassortment of equine influenza virus genes. Virus Res1994; 32: 391-9.2. Daly JM, Lai AC, Binns MM, Chambers TM, BarrandeguyM, Mumford JA. Antigenic and genetic evolution of equineH3N8 influenza A viruses. J Gen Virol 1996; 77: 661-71.3. Desselberger U, Racaniello VR, Zazra JJ, Palese P. The 3’and 5’-terminal sequences of influenza A, B and C virusRNA segments are highly conserved and show partialinverted complementarity. Gene 1980; 8: 315-28.4. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR.Universal primer set for the full-length amplification of allinfluenza A viruses. Arch Virol 2001; 146: 2275-89.5. McCauley JW, Mahy BW. Structure and function of the in-fluenza virus genome. Biochem J 1983; 211: 281-94.Genome amplification of Influenza virus 211Recibido: 28/04/09 – Aceptado: 22/09/096. Oxburgh L, Klingeborn B. Cocirculation of two distinctlineages of equine influenza virus subtype H3N8. J ClinMicrobiol 1999; 37: 3005-9.7. Oxburgh L, Hagström A. A PCR based method for theidentification of equine influenza virus from clinical samples.Vet Microbiol 1999; 67: 161-74.8. Quinlivan M, Cullinane A, Nelly M, Van Maanen K, HeldensJ, Arkins S. Comparison of sensitivities of virus isolation,antigen detection, and nucleic acid amplification for detectionof equine influenza virus. J Clin Microbiol 2004; 42: 759-63.9. Skehel JJ, Hay AJ. Nucleotide sequences at the 5' terminiof influenza virus RNAs and their transcripts. Nucleic AcidsRes 1978; 5: 1207-19.10. Webster RG, Bean WJ, Gorman OT, Chambers TM,Kawaoka Y. Evolution and ecology of influenza A viruses.Microbiol Rev 1992; 56: 152-79.

Complete genome amplification of Equine influenza virus subtype 2

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Complete genome amplification of Equine influenza virus subtype 2

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