- Email: [email protected]
Influenza virus A(H3N2) strain isolated from cerebrospinal fluid from a patient presenting myelopathy post infectious
Journal of Clinical Virology 58 (2013) 283–285
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Influenza virus A(H3N2) strain isolated from cerebrospinal fluid from a patient presenting myelopathy post infectious T.M. Paiva a,∗ , G. Theotonio b , R.S. Paulino a , M.A. Benega a , D.B.B. Silva a , S.E.T. Borborema a , T.I. Ikeda c , J.J. Kisielius d , M. Ueda d , M.I. Oliveira a , C.L.S. Santos a a
Núcleo de Doenc¸as Respiratórias, Instituto Adolfo Lutz, São Paulo, SP, Brazil Hospital Regional Taguatinga, Brasília, Distrito Federal, Brazil c Núcleo de Cultura de Células, Instituto Adolfo Lutz, São Paulo, SP, Brazil d Núcleo de Microscopia Eletrônica, Instituto Adolfo Lutz, São Paulo, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 29 January 2013 Received in revised form 2 May 2013 Accepted 28 May 2013 Keywords: Influenza A(H3N2) strain Cerebrospinal fluid Laboratory diagnosis Real time RT-PCR Virus sequencing
a b s t r a c t Background: Neurological involvement during influenza infection has been described during epidemics and is often consistent with serious sequelae or death. Objective: To investigate the etiologic agent involved in myelopathy post influenza-like syndrome. Study design: This investigation focuses on virus isolation from the cerebrospinal fluid (CSF) collected from a 19-year-old male student presenting with clinical diagnosis of myelopathy post influenzalike syndrome. To achieve this goal, different cell cultures and molecular methodologies were carried out. Results: Influenza virus A(H3N2) strain was isolated in MDCK cell culture; virus particles were observed under electron microscopy. Phylogenetics analyses showed that the Brazilian influenza A(H3N2) strains were closely related to the A/Perth/16/2009-like. Conclusion: This study demonstrates that influenza virus A(H3N2) strain was the cause of illness of the students. According to the Brazilian influenza virus sentinel surveillance data A/Perth/16/2009-LIKE (H3N2) strain has predominated during the 2010 influenza virus season in Brasília – DF. © 2013 Elsevier B.V. All rights reserved.
1. Background Influenza viruses are important human pathogens that throughout history have caused epidemics and pandemics. Neurological involvement during influenza infection has been described during epidemics and is often consistent with serious sequelae or death. A variety of clinical manifestations of central nervous system (CNS) infections, such as Reye’s syndrome, acute necrotizing encephalopathy, and myelitis as well as autoimmune conditions, such as Guillain–Barré syndrome, may occur during the course of influenza infection [1–7].7 Virological diagnosis is
essential and based on virus isolation, antigen detection, RNA detection by RT-PCR, and serological analyses. In October 2010, a 19-year-old male student presenting with fever, cough, sore throat and paresthesia of the lower limbs was admitted to the Intensive Therapy Unit (ITU) of a hospital located in Brasília – DF, Midwest region of Brazil. Facing a clinical diagnosis of myelopathy post influenza-like syndrome, a sample of cerebrospinal fluid (CSF) was collected and sent to Institute Adolfo Lutz on October 11, 2010 to investigate influenza A(H1N1) pdm09.
2. Objective
∗ Corresponding author. Tel.: +55 11 30682913; fax: +55 11 30853505. E-mail address: [email protected] (T.M. Paiva). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.05.021
The objective of the present study was to investigate the etiologic agent involved in myelopathy post influenza-like syndrome by using virus isolation in cell cultures and molecular methodologies.
284
T.M. Paiva et al. / Journal of Clinical Virology 58 (2013) 283–285
Fig. 1. Electromicroscopy influenza virus from infected MDCK cell culture.
3. Study design Only a cerebrospinal fluid sample was sent to the laboratory to investigate the etiologic agent involved in the myelopathy post influenza-like syndrome. The CSF sample identified as (DF 86727) was inoculated into HEp-2, Vero, MRC-5 and MDCK cell cultures. Electron microscopy (EM) [8] and RT-qPCR [9] were performed from the infected cell culture to identify the viral infection. Viral RNA was extracted from the infected cell culture in order to investigate influenza viruses by using the Centers for Disease Control and Prevention (CDC) RT-qPCR. Following the complete HA gene amplification by RT-PCR, the products were directly sequenced. Phylogenetic trees were constructed by using neighbor-joining method (MEGA 4) [10] based on the continuous nucleotide sequences aligned with Clustal X. The reliability of the trees was evaluated by the bootstrap method with 1000 replications.
4. Results Seasonal influenza A(H3N2) was identified by RT-qPCR in the MDCK infected cell culture corresponding to the first cell culture passage; a pleomorphic virus particle with a higher thickness diameter of 63.6 nm and 295.4 nm of length presenting in the surface typical influenza spikes was also identified; in addition, typical influenza granulations were observed in the inner particle under EM investigations. This characteristic morphology of orthomyxovirus by EM is shown in the Fig. 1. Virus replication in cell culture revealed by cytopathic effect development has not observed into Hep-2, Vero and MRC-5 infected cell cultures; in addition, infected cell supernatants submitted to RT-PCR presented influenza virus A and B negative results. Phylogenetics analyses showed that the Brazilian influenza A(H3N2) strains were closely related to the A/Perth/16 2009-LIKE (H3N2), the vaccine strain for the 2010 Southern Hemisphere (Fig. 2).
Fig. 2. Phylogenetic relationships of the HA1 nucleotide sequences of influenza A(H3N2) strains circulating in Brazil, 2010–2011. The tree constructed with the NJ method was evaluated by the bootstrap method with 1000 replications using MEGA package version 4.0.1. Only the bootstrap values of >80 are shown at the corresponding nodes.
5. Conclusion The present study shows that influenza virus A(H3N2) strain was the etiologic agent involved in myelopathy post influenzalike syndrome. Unfortunately, information regarding the potential correlations of the number of myelopathic occurrences with the H3N2 diagnosis has not been described in Brazil. According to the medical information, no other disease or chronic myelopathy was diagnosed and the student had not been vaccinated during the 2010 influenza vaccination campaign.
T.M. Paiva et al. / Journal of Clinical Virology 58 (2013) 283–285
National influenza virus sentinel surveillance data have shown the circulation of influenza A/Perth/16 2009-LIKE (H3N2) strain in the Southeast, Midwest and Northeast regions of Brazil; this strain has predominated during the 2010 influenza virus season in Brasília – DF, located at Midwest region. Funding This investigation integrates the influenza virus surveillance sponsored by Brazilian Ministry of Health (MoH); we receive reagents and materials from our Institution, MoH, and Centers for Disease Control and Prevention. Competing interests The authors have no conflicts of interest to declare. Ethical approval Our Institution is a World Health Organization (WHO) National Influenza Centre since 1950 decade; in addition influenza virus
285
surveillance follow up project has been approved by the Conselho Nacional de Ética em Pesquisa (CONEP)-135/2002. References [1]. Studahl M. Influenza virus and CNS manifestation. J Clin Virol 2003;28:225–32. [2]. Hayase Y, Tobita K. Influenza virus and neurological diseases. Psychiatry Clin Neurosci 1997;51:181–4. [3]. Rothberg MB, Haessler SD, Brown RB. Complications of viral influenza. Am J Med 2008;121:258–64. [4]. Kwon S, Kim S, Cho MH, Seo H. Neurological complications and outcomes of pandemic (H1N1) 2009 in Korean children. J Korean Med Sci 2012;27(April (4)):402–7. [5]. Wiwanitkit V. Neurological manifestation of swine flu: a brief note. Acta Neurol Taiwan 2010;19:145–7. [6]. Sangle SA, Vadgaonkar G, Kadam DB, Chadha M. Influenza A(H3N2) associated acute necrotising encephalopathy. J Assoc Physicians India 2011;January (59):52–4. [7]. Salonen O, Koskiniemi M, Saari A, Myllylä V, Pyhälä R, Airaksinen L, et al. J Neurovirol 1997;3:83–5. [8]. Brenner S, Horne RW. A negative staining method for high resolution electron microscopy of viruses. Biochem Biophys Acta 1959;34:103–10. [9]. Manual for the laboratory diagnosis and virological surveillance of influenza. http://whqlibdoc.who.int/publications/2011/9789241548090 eng.pdf [10]. Tamura K, Dudley J, Nei M, Kumar S. MEGA 4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Influenza virus A(H3N2) strain isolated from cerebrospinal fluid from a patient presenting myelopathy post infectious T.M. Paiva a,∗ , G. Theotonio b , R.S. Paulino a , M.A. Benega a , D.B.B. Silva a , S.E.T. Borborema a , T.I. Ikeda c , J.J. Kisielius d , M. Ueda d , M.I. Oliveira a , C.L.S. Santos a a
Núcleo de Doenc¸as Respiratórias, Instituto Adolfo Lutz, São Paulo, SP, Brazil Hospital Regional Taguatinga, Brasília, Distrito Federal, Brazil c Núcleo de Cultura de Células, Instituto Adolfo Lutz, São Paulo, SP, Brazil d Núcleo de Microscopia Eletrônica, Instituto Adolfo Lutz, São Paulo, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 29 January 2013 Received in revised form 2 May 2013 Accepted 28 May 2013 Keywords: Influenza A(H3N2) strain Cerebrospinal fluid Laboratory diagnosis Real time RT-PCR Virus sequencing
a b s t r a c t Background: Neurological involvement during influenza infection has been described during epidemics and is often consistent with serious sequelae or death. Objective: To investigate the etiologic agent involved in myelopathy post influenza-like syndrome. Study design: This investigation focuses on virus isolation from the cerebrospinal fluid (CSF) collected from a 19-year-old male student presenting with clinical diagnosis of myelopathy post influenzalike syndrome. To achieve this goal, different cell cultures and molecular methodologies were carried out. Results: Influenza virus A(H3N2) strain was isolated in MDCK cell culture; virus particles were observed under electron microscopy. Phylogenetics analyses showed that the Brazilian influenza A(H3N2) strains were closely related to the A/Perth/16/2009-like. Conclusion: This study demonstrates that influenza virus A(H3N2) strain was the cause of illness of the students. According to the Brazilian influenza virus sentinel surveillance data A/Perth/16/2009-LIKE (H3N2) strain has predominated during the 2010 influenza virus season in Brasília – DF. © 2013 Elsevier B.V. All rights reserved.
1. Background Influenza viruses are important human pathogens that throughout history have caused epidemics and pandemics. Neurological involvement during influenza infection has been described during epidemics and is often consistent with serious sequelae or death. A variety of clinical manifestations of central nervous system (CNS) infections, such as Reye’s syndrome, acute necrotizing encephalopathy, and myelitis as well as autoimmune conditions, such as Guillain–Barré syndrome, may occur during the course of influenza infection [1–7].7 Virological diagnosis is
essential and based on virus isolation, antigen detection, RNA detection by RT-PCR, and serological analyses. In October 2010, a 19-year-old male student presenting with fever, cough, sore throat and paresthesia of the lower limbs was admitted to the Intensive Therapy Unit (ITU) of a hospital located in Brasília – DF, Midwest region of Brazil. Facing a clinical diagnosis of myelopathy post influenza-like syndrome, a sample of cerebrospinal fluid (CSF) was collected and sent to Institute Adolfo Lutz on October 11, 2010 to investigate influenza A(H1N1) pdm09.
2. Objective
∗ Corresponding author. Tel.: +55 11 30682913; fax: +55 11 30853505. E-mail address: [email protected] (T.M. Paiva). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.05.021
The objective of the present study was to investigate the etiologic agent involved in myelopathy post influenza-like syndrome by using virus isolation in cell cultures and molecular methodologies.
284
T.M. Paiva et al. / Journal of Clinical Virology 58 (2013) 283–285
Fig. 1. Electromicroscopy influenza virus from infected MDCK cell culture.
3. Study design Only a cerebrospinal fluid sample was sent to the laboratory to investigate the etiologic agent involved in the myelopathy post influenza-like syndrome. The CSF sample identified as (DF 86727) was inoculated into HEp-2, Vero, MRC-5 and MDCK cell cultures. Electron microscopy (EM) [8] and RT-qPCR [9] were performed from the infected cell culture to identify the viral infection. Viral RNA was extracted from the infected cell culture in order to investigate influenza viruses by using the Centers for Disease Control and Prevention (CDC) RT-qPCR. Following the complete HA gene amplification by RT-PCR, the products were directly sequenced. Phylogenetic trees were constructed by using neighbor-joining method (MEGA 4) [10] based on the continuous nucleotide sequences aligned with Clustal X. The reliability of the trees was evaluated by the bootstrap method with 1000 replications.
4. Results Seasonal influenza A(H3N2) was identified by RT-qPCR in the MDCK infected cell culture corresponding to the first cell culture passage; a pleomorphic virus particle with a higher thickness diameter of 63.6 nm and 295.4 nm of length presenting in the surface typical influenza spikes was also identified; in addition, typical influenza granulations were observed in the inner particle under EM investigations. This characteristic morphology of orthomyxovirus by EM is shown in the Fig. 1. Virus replication in cell culture revealed by cytopathic effect development has not observed into Hep-2, Vero and MRC-5 infected cell cultures; in addition, infected cell supernatants submitted to RT-PCR presented influenza virus A and B negative results. Phylogenetics analyses showed that the Brazilian influenza A(H3N2) strains were closely related to the A/Perth/16 2009-LIKE (H3N2), the vaccine strain for the 2010 Southern Hemisphere (Fig. 2).
Fig. 2. Phylogenetic relationships of the HA1 nucleotide sequences of influenza A(H3N2) strains circulating in Brazil, 2010–2011. The tree constructed with the NJ method was evaluated by the bootstrap method with 1000 replications using MEGA package version 4.0.1. Only the bootstrap values of >80 are shown at the corresponding nodes.
5. Conclusion The present study shows that influenza virus A(H3N2) strain was the etiologic agent involved in myelopathy post influenzalike syndrome. Unfortunately, information regarding the potential correlations of the number of myelopathic occurrences with the H3N2 diagnosis has not been described in Brazil. According to the medical information, no other disease or chronic myelopathy was diagnosed and the student had not been vaccinated during the 2010 influenza vaccination campaign.
T.M. Paiva et al. / Journal of Clinical Virology 58 (2013) 283–285
National influenza virus sentinel surveillance data have shown the circulation of influenza A/Perth/16 2009-LIKE (H3N2) strain in the Southeast, Midwest and Northeast regions of Brazil; this strain has predominated during the 2010 influenza virus season in Brasília – DF, located at Midwest region. Funding This investigation integrates the influenza virus surveillance sponsored by Brazilian Ministry of Health (MoH); we receive reagents and materials from our Institution, MoH, and Centers for Disease Control and Prevention. Competing interests The authors have no conflicts of interest to declare. Ethical approval Our Institution is a World Health Organization (WHO) National Influenza Centre since 1950 decade; in addition influenza virus
285
surveillance follow up project has been approved by the Conselho Nacional de Ética em Pesquisa (CONEP)-135/2002. References [1]. Studahl M. Influenza virus and CNS manifestation. J Clin Virol 2003;28:225–32. [2]. Hayase Y, Tobita K. Influenza virus and neurological diseases. Psychiatry Clin Neurosci 1997;51:181–4. [3]. Rothberg MB, Haessler SD, Brown RB. Complications of viral influenza. Am J Med 2008;121:258–64. [4]. Kwon S, Kim S, Cho MH, Seo H. Neurological complications and outcomes of pandemic (H1N1) 2009 in Korean children. J Korean Med Sci 2012;27(April (4)):402–7. [5]. Wiwanitkit V. Neurological manifestation of swine flu: a brief note. Acta Neurol Taiwan 2010;19:145–7. [6]. Sangle SA, Vadgaonkar G, Kadam DB, Chadha M. Influenza A(H3N2) associated acute necrotising encephalopathy. J Assoc Physicians India 2011;January (59):52–4. [7]. Salonen O, Koskiniemi M, Saari A, Myllylä V, Pyhälä R, Airaksinen L, et al. J Neurovirol 1997;3:83–5. [8]. Brenner S, Horne RW. A negative staining method for high resolution electron microscopy of viruses. Biochem Biophys Acta 1959;34:103–10. [9]. Manual for the laboratory diagnosis and virological surveillance of influenza. http://whqlibdoc.who.int/publications/2011/9789241548090 eng.pdf [10]. Tamura K, Dudley J, Nei M, Kumar S. MEGA 4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9.