Influenza B viruses isolated in Uruguay during the 2002–2005 seasons: Genetic relations and vaccine strain match

Virus Research 123 (2007) 100–104

Short communication

Influenza B viruses isolated in Uruguay during the 2002–2005 seasons: Genetic relations and vaccine strain match Natalia Go˜ni a,b , Mariana Baz b , Dora Ruchansky b , Leticia Coppola b , Jos´e Russi b , Juan Cristina a,∗ a

Laboratorio de Virolog´ıa Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Igu´a 4225, 11400 Montevideo, Uruguay b Centro Nacional de Referencia de Influenza, Servicio Nacional de Laboratorios de Salud P´ ublica, Ministerio de Salud P´ublica, Av. 8 de Octubre 2720, 11200 Montevideo, Uruguay Received 6 June 2006; received in revised form 1 August 2006; accepted 2 August 2006 Available online 20 September 2006

Abstract Monitoring antigenic and genetic variations of circulating influenza viruses is critical for the selection of annual vaccine strains. In order to gain insight into the molecular evolution of Influenza B viruses (IBV) isolated in Uruguay in 2002 and 2005 outbreaks, antigenic and phylogenetic studies were carried out for the Hemagglutinin (HA) gene. Antigenic relations among Uruguayan and reference strains isolated elsewhere were performed by means of hemagglutination inhibition assays (HAI). Genetic relations of HA genes from Uruguayan as well as 41 IBV strains isolated elsewhere were established by means of the construction of phylogenetic trees. HAI assays showed a distant antigenic relationship among the 2002 Uruguayan isolates and the 2002 vaccine strain B/Sichuan/379/99. Phylogenetic analysis also revealed a distant genetic relationship among Uruguayan and 2002 vaccine strains. All 2005 IBV Uruguayan strains were both antigenically and genetically related to B/Victoria lineage-viruses. The results of these studies revealed that 2002 IBV Uruguayan strains have a distant antigenic and genetic relation with the 2002 IBV vaccine strain used in Uruguay. The high rate of susceptible individuals in the youngest cohort (<25 years) might be related to the fact that the B/Victoria lineage-viruses were not previously circulating in Uruguay. © 2006 Elsevier B.V. All rights reserved. Keywords: Influenza B virus; Evolution; Vaccine match

Influenza B virus (IBV) is a member of the family Orthomyxoviridae and contains a single-stranded, negative sense, segmented genome (Neumann et al., 2004). The segmented genome of influenza viruses allows genetic exchange to occur by a process called reassortment, where gene segments from different viruses infecting the same host can mix (Cox and Bender, 1995). Reassortment of IBV has been observed between cocirculating IBV virus strains of different lineages (Chi et al., 2002; Shaw et al., 2002; Yamashita et al., 1988). Recent isolates of IBV fall into two major phylogenetic lineages: B/Victoria/2/87 and B/Yamagata/16/88 (Rota et al., 1992). HA genes that were grouped together before the divergence into the two currently circulating genetic lineages are designated lineage I, B/Victoria/2/87-like HA genes are designated

∗

Corresponding author. Tel.: +5982 525 09 01; fax: +5982 525 08 95. E-mail address: [email protected] (J. Cristina).

0168-1702/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2006.08.002

lineage III, and B/Yamagata/16/88 HA genes are designated lineage II (Lindstrom et al., 1999). Increased incidence of B/Victoria/2/87 viruses in the regions beyond Asia in the 2001–2002 season resulted in the World Health Organization recommendation of B/Hong Kong/330/01 (a member of the B/Victoria/2/87 lineage) for the vaccine strain for both the northern and southern hemispheres in the 2002–2003 season (World Health Organization, 2002a,b). The Yamagata-lineage viruses were represented by B/Sichuan/379/99 in 2001–2002 vaccine composition (Alsandi et al., 2003). Influenza B viruses were predominantly isolated in Uruguay during the 2002 and 2005 winter seasons (southern hemisphere), where 18 and 27% of influenza isolates were identified as IBV, respectively. By comparison, IBV accounted for only 4 and 16% during 2003 and 2004, respectively. To gain insight into the antigenic and genetic relations among IBV isolated in Uruguay during the 2002 and 2005 IBV out-

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Table 1 HAI assays of IBV strains isolated in Uruguaya Virus

Reference antisera Lineageb

B/Sichuan/379/99 B/Shangai/361/02 B/H. Kong/1434/02 B/Brisbane/32/02 B/Hawaii/33/04 B/H. Kong/330/01 B/Shizouka/15/02 B/H. Kong/1351/02 B/Uruguay/19/05 B/Uruguay/69/05 B/Uruguay/33/05 B/Uruguay/28/05 B/Uruguay/75/04 B/Uruguay/4/02 B/Uruguay/19/02 B/Uruguay/5/02 B/Uruguay/2/02 B/Uruguay/7/02 B/Uruguay/NG/02 a b c

Y Y V V V V Y V

B/Sic/379

B/Shan/361

B/HK/1434

B/Bris/32

B/HI/33

B/HK/330

B/SHIZ/15

B/HK/1351

1280 640

640 640

c

c

c

c

c

c

c

c

c

c

c

c

c

c

c

nd nd

c

c

c

c

c

c

c

c

640 640 320 320 nd nd 80 80 40 40

80

320 nd nd nd nd nd nd

2560 2560 1280 nd nd nd 640 640 320 320 nd nd nd nd nd nd nd

nd nd nd nd nd nd 1280

nd nd nd nd nd nd

640

2560 1280 640 nd nd nd 80 160 80 80 nd nd nd nd nd nd nd

nd nd nd nd nd 1280

c c c c c c

c

nd nd nd nd nd nd

c

320 nd nd nd nd c

320 80 320 320 80 160

c

nd nd nd nd nd c c c c c c

c

640 nd nd nd nd nd 80 320 80 80 320 320

Hemagglutination inhibition titers against reference antisera is shown. Reference viruses and homologous titers are shown in bold, nd means not done. Y means Yamagata-lineage; V means Victoria-lineage. Indicates a titer of <40. Reference strains titers data was obtained by CDC, USA. Table 2 (Continued )

Table 2 Influenza B virus strains Name

HA accession no.

NP accession no.

B/Lee/40 B/Russia/69 B/Memphis/12/97 B/Nagano/2038/98 B/Yamanashi/166/98 B/Lusaka/270/99 B/Lusaka/432/99 B/Sagan/S172/99 B/Sichuan/379/99 B/Vienna/1/99 B/Kouchi/193/99 B/Hong Kong/330/01 B/Victoria/504/2000 B/Nebraska/1/01 B/Nebraska/2/01 B/Maryland/1/01 B/Yamagata/270/01 B/Yamagata/320/01 B/Los Angeles/1/02 B/Yamagata/222/02 B/Shangai/361/02 B/Uruguay/NG/02 B/Uruguay/7/02 B/Uruguay/5/02 B/Uruguay/19/02 B/Uruguay/4/02 B/Yamagata/115/03 B/Memphis/13/03 B/Sydney/4/04 B/Sydney/6/04 B/Malaysia/2506/04 B/Christchurch/7/04 B/Christchurch/22/04 B/Christchurch/27/04

NC002207 AB027387 AY200945 AF100350 AF100355 AB059242 AB059250 AB036863 AF319590 AF387494 AB036864 AY504610 AY504602 AY581966 AY581967 AY581963 AB120488 AB120490 AY581968 AB120506 ISDN80784 AM295324 AM295321 AM295323 AM295320 AM295322 AB120507 AY581969 ISDN110430 ISDN110483 ISDN124776 ISDN69105 ISDN110429 ISDN110625

NC002208 ISDNCHB031 AY260953 AF100370 AF100373 AB059247 AB059255 AB036874 AJ784062 ISDNCHB013 NI AY504611 AY504603 NI NI AY582027 AB120222 AB120223 AY582030 AB120224 AJ784078 AM295331 AM295332 AM295333 NA AM295330 AB120337 AY582031 NA NA NA NA NA NA

Name

HA accession no.

NP accession no.

B/Christchurch/33/04 B/Auckland/1/04 B/Peru/1324/04 B/Peru/1364/04 B/Uruguay/75/04 B/Sydney/2/05 B/Sydney/3/05 B/S. Australia/6/05 B/S. Australia/9/05 B/S. Australia/19/05 B/Johannesburg/27/05 B/Auckland/14/05 B/Auckland/50/05 B/Auckland/34/05 B/Christchurch/38/05 B/Uruguay/19/05 B/Uruguay/65/05 B/Uruguay/33/05 B/Uruguay/28/05

ISDN110626 ISDN110432 DQ265728 DQ265726 AM295329 ISDN124789 ISDN125763 ISDN125755 ISDN125754 ISDN126579 ISDN127354 ISDN125747 ISDN125751 ISDN125748 ISDN125752 AM295326 AM295327 AM295325 AM295328

NA NA NA NA AM295337 NA NA NA NA NA NA NA NA NA NA NA AM295336 NA AM295335

NA means not available; NI means not included.

breaks, nasal swabs from 12 Uruguayan patients with clinical symptoms of influenza were cultured in MDCK cells. Virus isolates were typed by hemagglutination inhibition assay (HAI) with the World Health Organization (WHO) Influenza reagent kit, provided by the Center for Disease Control and Prevention, Atlanta, GA, USA. Viral antigens were detected by an immunofluorescent assay with type-specific monoclonal antibodies (Chemicon International, Inc., CA, USA) and with the Directigen Flu A + B (Beckton Dickinson Europe, Maylan, France).

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Fig. 1. Phylogenetic analysis of IBV isolated in Uruguay in 2002 and 2005. Strains in the trees are shown by their names, which also show geographic location and year of isolation, according to the World Health Organization (1980), (for accession numbers see Table 2). Vaccine strains Sichuan and Hong Kong are shown in bold and italics. Uruguayan strains are shown in bold. Numbers at each node of the trees show bootstrap percentages obtained after 1000 replicates. Bars at the bottom of the trees denote distances. IBV lineages are indicated I–III. In (A), the HA gene phylogeny is shown. (B) Phylogeny of the NP gene.

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Fig. 1. (Continued ).

HA sequences from IBV isolates circulating in the Uruguayan patients were obtained by means of total RNA extraction from infected cells using Trizol (Gibco, BRL) according to manufacturer’s instructions. Extracted RNA was eluted with 15 ␮l of UltraPure DNase/Rnase-free distilled water (Gibco, BRL, Life Technologies) and cDNA synthesis and PCR amplification of IBV HA and NP genes were carried out as previously described (Coiras et al., 2003; Donofrio et al., 1992; Ellis et al., 1997). Amplicons were purified by using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions. The primers used for amplification were also used for sequencing the PCR fragments. The sequencing reaction was carried out using a BigDye DNA sequencing kit on a 373 DNA sequencer apparatus (both from Perkin-Elmer). In order to gain insight into the antigenic relations among Uruguayan and reference strains isolated elsewhere, HAI assays were performed using reference antisera. The results of these studies are shown in Table 1. As it can be seen in the table, 2002 Uruguayan isolates were found to have HAs from the Victoria-lineage viruses. All 2002 Uruguayan strains showed a distant antigenic relation with 2002 B/Sichuan/379/99 vaccine strain (Yamagata lineage). The only virus isolated in Uruguay during 2004 season was antigenically similar to B/Shangai/361/02 (Yamagata-lineage virus), which re-emerged in many parts of the world during 2003–2004. All HAs from 2005 IBV Uruguayan isolates resulted in being related to Victoria-lineage strains, as 2005 vaccine strain B/Hong Kong/330/01.

In order to confirm these findings at the genetic level, phylogenetic studies were carried out for the HA and NP gene in order to establish the degree of genetic heterogeneity of IBV isolates circulating in 2002 and 2005 outbreaks in Uruguay. HA sequences obtained from the Uruguayan patients were aligned with corresponding sequences from 41 IBV strains isolated elsewhere using the CLUSTAL W program (Thompson et al., 1994) (for strain names and accession numbers see Table 2). Once aligned, using the MEGA3 program (Kumar et al., 2004), phylogenetic trees were created by the neighborjoining method applied to distance matrix obtained under the Kimura two-parameter model (Felsenstein, 1993). The robustness of each node was estimated by the bootstrapping (1000 pseudo-replicas). As can be seen in Fig. 1A, all IBV strains included in these studies can be assigned to three different clusters supported by high bootstrap values. These clusters correspond to IBV lineages I–III previously described (see Fig. 1A). All strains isolated in Uruguay in 2002 are assigned to the same cluster, inside lineage III. Nevertheless, 2002 vaccine strain B/Sichuan/379/99 does not cluster with the Uruguayan strains but in a separate genetic lineage (lineage II). The results of these studies revealed that 2002 Uruguayan isolates have a distant genetic relation with the 2002 vaccine strain (see Fig. 1A). Inside lineage III, strains isolated in Uruguay in 2002 and 2005 cluster in two different groups, revealing that strains isolated during the same year have a more closed evolutionary

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relationship among themselves and a more distant genetic relation with strains isolated in another year. The only strain isolated in Uruguay in 2004 (B/Uruguay/ 75/04) was found to have a close genetic relation with a lineage II group, together with strain B/Shangai/361/02 (see Fig. 1A, upper part). In order to see if the same evolutionary relationships are observed in other IBV genes, the same studies were carried out using NP genes. As is shown in Fig. 1B, the NP phylogenetic studies revealed that Uruguayan strains are clustered together with the same strains that were identified in the HA studies (see Fig. 1B, bottom). The same phylogenetic relationships can be found using HA or NP genes (see Fig. 1A and B). Interestingly, the strain isolated in Uruguay in 2004 (B/Uruguay/75/04), assigned to lineage II for the HA gene, is now assigned to lineage III with the rest of the Uruguayan strains (see Fig. 1B, bottom). This speaks of a reassortment event among Victoria and Yamagata lineages in the case of this strain. Reassortment of IBV NP genes has been previously observed and the selective advantage of gene reassortment for IBV evolution has been previously suggested (McCullers et al., 1999). Taking all together, the results of these studies revealed that 2002 IBV Uruguayan strains have a distant antigenic and genetic relationship with the 2002 vaccine strain B/Sichuan/379/99 used in 2002 IBV vaccine in Uruguay. This speaks of the need of a more in-depth analysis of IBV strains circulating in South America, in order to observe their antigenic and genetic relationship with season vaccine strains for the southern hemisphere. The average age of all IBV Uruguayan patients enrolled in these studies was 10, compared with an average age of 40 for the IAV patients. The high rate of susceptible individuals of this cohort might be related to the fact that the B/Victoria lineageviruses were not previously circulating in Uruguay. Acknowledgements Juan Cristina acknowledges support from PEDECIBA, Uruguay. We deeply thank Dr. Kanta Subbarao from Laboratory of Infectious Diseases, NIAID, NIH, USA, for critical reading of this work and very helpful suggestions. References Alsandi, F., D’Agaro, P., De Florentis, D., Puzelli, S., Lin, Y.P., Gregory, V., Bennett, M., Donatelli, I., Gasparini, R., Crovari, P., Hay, A., Campillo, C., 2003. Molecular characterization of influenza B viruses circulating in northern Italy during the 2001–2002 epidemic season. J. Med. Virol. 70, 463–469.

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