Molecular Markers of Influenza B Lineages and Clades

Co-circulation of two influenza B virus lineages, B/Yamagata and B/Victoria, has been recognized since the late 1980s. The assessment of the prevalent lineage and the group of viruses in circulation is of importance in order to decide on the vaccine composition and evaluate its efficacy. The molecular characterization of influenza B viruses in circulation has been the aim of this study; this was approached by identifying and locating nucleotide substitutions in the influenza B virus hemagglutinin (HA) and neuraminidase (NA), specific for the lineage and/or clade. By the alignment of 3456 sequences from the influenza GISAID EpiFlu database, a high number of lineage- and group-specific nucleotide positions have been observed in the HA gene, but not in the NA gene. Additionally, an RT-PCR method has been developed, applicable directly to clinical specimens, which amplifies a short HA region that includes a group of unique molecular signatures. Twenty eight influenza B virus-positive respiratory specimens, collected in Tuscany in the seasons 2012–2013 and 2013–2014, were analyzed. The results revealed two clearly distinguishable patterns: one, more frequent, was characterized by all of the nucleotide changes associated with the B/Yamagata lineage (in most cases of Group 2), whereas the other exhibited all of the changes associated with the B/Victoria lineage. It can be concluded that the analysis of this short HA sequence can permit a rapid, highly sensitive determination of influenza B virus lineages and clades.


Introduction
Currently, influenza B viruses in circulation belong to two lineages distinct by their genetic and antigenic characteristics, which are referred to as the Yamagata and Victoria lineages, designated after their original isolates, B/Yamagata/16/88 and B/Victoria/2/87 [1,2]. These two lineages have co-circulated since the late 1980s [2]. Since 2008, most B/Victoria/2/87 lineage viruses have belonged to the B/Brisbane/60/2008 genetic clade (Group 1) [3] based on the hemagglutinin (HA) gene sequences. Instead, since 2007, the majority of B/Yamagata lineage viruses have been distributed into two main groups, with distinct genetic and antigenic characteristics, Group 2, represented by B/Brisbane/3/2007, and Group 3, represented by B/Bangladesh/3333/2007, which also contains the vaccine strain B/Wisconsin/01/2010 recommended for the season of 2012-2013 [4].
The assessment of the lineage and the group prevalent in circulation is of importance, in order to select the virus to be included in influenza vaccines and to evaluate the efficacy of vaccination. However, influenza B characterization is not commonly performed due to the underestimation of this information and to the lack of rapid and specific typing assays; the latter is performed by hemagglutination inhibition (HI) using suitable sera panels and/or by full-length sequencing of the HA gene.
As expected, by sequencing the HA and/or the NA genes of influenza B viruses, several amino acid substitutions, insertions and deletions can be detected [5][6][7][8][9][10]. HA protein sequences within each lineage show an identity higher than 97%, whereas inter-lineage sequence identity is on average 88%-90% [11,12]. However, there are few data in the literature reporting the detection of the lineage or group-specific molecular signatures [3,6].
The aim of this study was to identify and locate lineage-and/or clade-specific nucleotide substitutions in the HA and NA genes of influenza B viruses, to be used for the molecular characterization of the viruses in circulation. For this purpose, 3456 sequences of HA or NA genes of both the B/Victoria and B/Yamagata lineage viruses isolated between 2000 and 2013 have been aligned. A high number of lineage or clade characteristic nucleotide substitutions, located along the entire sequence of the HA gene, was identified. Our attention has been focused on a group of six nucleotide substitutions, located around an AAA insertion/deletion, occurring in a short sequence of about 60 nts.

Detection of Target Sequences in the HA and NA Gene for Influenza B Characterization
Two thousand nine hundred and fifty-six HA gene sequences and 500 NA gene sequences of influenza B viruses isolated between 2000 and 2013 have been downloaded from the influenza GISAID (Global Initiative in Sharing Avian Influenza Data) EpiFlu database, which reports also the lineage and, in many cases, the clade. Altogether, the 2956 HA sequences were 1460 from the B/Victoria and 1496 from the B/Yamagata lineage. These sequences were aligned using ClustalW v1.4 included in BioEdit v7.0.0.

Primer Design
The primers specific for the HA and NA target sequence of influenza B viruses were designed using Primer3, in order to amplify a short region encompassing the characteristic positions. The HA sequence primers were pF 5'GAC CCT ACA RAM TTG GAA CC'3 and pR 5'ART GGA ACC CCC AAA CRG TA'3, which amplify a 185-bp sequence, and for the NA gene, the forward and reverse primers were pF 5'GGG CAA AAT CCC AAC WGT Ag'3 and pR 5'GCA ATT GCA GGC ACT TTC TT'3, which amplify a 210-bp sequence.

Clinical Samples
During the influenza season of 2012-2013, a total of 376 respiratory samples were analyzed by RT real-time PCRs to detect influenza viruses A(H1N1) 2009, A(H3N2) and influenza virus type B [13]. Thirty-seven patients were positive for A(H1N1) 2009 pandemic virus; 3 were positive for A(H3N2); and 36 were positive for influenza B virus. In addition, during the last influenza season, a total of 311 respiratory samples were analyzed with the same objective. Twenty-nine patients were positive for A(H1N1) 2009; twenty-eight were positive for A(H3N2); and only 4 were positive for influenza B virus. The influenza type B isolates were further analyzed to verify if they could be clustered in discrete lineages and groups in circulation.

Viral RNAs Extraction and One-Step RT-PCR
Extraction of viral RNAs from clinical samples was carried out using a commercially available kit (E.Z.N.A. Viral RNA kit, Omega bio-tek, Norcross, GA, USA) according to the manufacturer's instructions. Two one-step RT-PCRs have been developed, one for the amplification of target sequences on the HA gene and the other one for the amplification of target sequences on the NA gene. The QuantiTect virus kit, without ROX dye (Qiagen, Valencia, CA, USA), was used for each RT-PCR according to the manufacturer's instructions.
After retro-transcription and denaturation, 40 cycles of amplification were performed (95 °C for 15 s, 55 °C for 30 s, 72 °C for 1 min), followed by a final extension at 72 °C for 7 min. The reaction volume was 25 µL (5 µL of 5X QuantiTect virus master mix, 0.25 µL of 100X QuantiTect virus RT mix, 0.5 µL of each primer [10 µM], 5 µL of extracted RNA and H2O to reach the final volume).

Sanger Sequencing and Phylogenetic Analysis
The PCR products were analyzed by dideoxy Sanger sequencing. Sequencing was carried on an ABI Prism 377 automatic sequencer (Applied Biosystems, Milan, Italy), using the ABI Prism Dye Terminator cycle sequencing Ready Reaction kit. Then the sequences obtained were aligned using ClustalW v1.4 included in BioEdit v7.0.0. A group of 18 influenza B viruses were cultured in MDCK cells, allowing us to perform the full sequencing of the HA1 gene. The phylogenetic analysis of these HA sequences was carried out using the neighbor joining (NJ) method (MEGA software, version 6.0). The parameter employed was p-distance, and the robustness of the internal branches was determined by 1000 bootstrap replications.

Results
Two thousand nine hundred and fifty-six sequences of HA and 500 sequences of NA genes of influenza B viruses of both the B/Victoria and B/Yamagata lineage, isolated between 2000 and 2013, have been downloaded and aligned. About 100 lineage-specific molecular signatures were observed in the HA gene, and none in the NA gene. We focused our attention on a short region of the HA gene, encompassing seven lineage-specific molecular signatures, quite close to each other ( Figure 1); in fact, using the numbering of B/Brisbane/60/2008, starting from the ATG codon, all of the viruses belonging to the B/Victoria lineage had an AAA insertion at the position 538-540 (corresponding to the amino acid position 180), which is deleted in the B/Yamagata lineage; moreover, B/Victoria lineage viruses at position 547-549 had the triplet ACA coding for threonine (T 183) and at position 558-560 the triplet TCC coding for serine (S 187) instead of AAT coding for asparagine (N) and of CCC coding for proline (P), respectively, which are characteristic of the B/Yamagata lineage viruses. Furthermore, most B/Victoria lineage viruses had a C at nucleotide position 522 (4% only had a T) and an A at nucleotide positions 555 and 568; instead the B/Yamagata lineage had, always, T, G and G in the same positions. As a group-specific signature, the triplet AAC at position 541-543 (coding for N at position 181) characterized the B/Yamagata lineage, Group 2, and the TAC (coding for tyrosine, Y) in the same position characterized the viruses of Group 3. As regards the NA gene, it was observed that at nucleotide position 600, in the triplet AGT, which codes for S at amino acid position 198, a G was associated mainly with the B/Yamagata lineage, whereas an A (AAT, which codes for N) was more frequently detected in the B/Victoria lineage. Near nucleotide position 600, at nucleotide position 596, a G to A modification creates the amino acid substitution D197N: this may be associated with reduced susceptibility to neuraminidase inhibitors (NAIs). A total of 28 out of the 40 clinical samples (collected during the two influenza seasons) positive for type B influenza viruses could be amplified by the two one-step RT PCRs targeting short sequences of the HA and the NA gene. These reactions were unable to amplify specimens positive in the diagnostic RT real-time PCR (targeting the M gene) with a threshold cycle (Ct) > 37 (corresponding, approximately, to 12 copies/µL). The same sensitivity characterized, in our hands, the RT-PCR suggested by WHO (World Health Organization) [14], used as a reference method to establish the influenza B lineage. These 28 samples, with a Ct ranging between 20 and 37, were from both inpatients (11 adults and one child) and outpatients (14 children and two adults).

Discussion
The differentiation between the two lineages of influenza B viruses is traditionally performed by HI. This assay is time-consuming, first requiring virus isolation and cultivation. Molecular assays aimed at detecting lineage-and/or clade-specific molecular markers could achieve the same result in less time, with high sensitivity. The comparison of 2,956 HA gene sequences from either the B/Victoria or the B/Yamagata lineage (clade 2 or clade 3) showed a high number of possible lineage-and/or clade-specific molecular markers, at variance with the NA gene sequences. The first difference observed was an AAA insertion/deletion at position 538-540 in the HA sequence able to distinguish between the two lineages. In a short sequence, 60 nts long, including this AAA ins/del, six other nucleotide substitutions, lineage-and/or clade-specific, were identified. The RT-PCR provides the amplification of the part of the HA gene, (185 nts long, encompassing the 60 nts-long target sequence), whose sequence can identify the lineage and clade of the influenza B viruses in circulation. During these epidemic seasons, 66 out of 639 patients were shown to be positive for influenza A(H1N1) 2009. Forty four were from inpatients (21 (47%) hospitalized in intensive care units, ICUs) and 22 from outpatients. Another 40 were positive for influenza B virus, 20 from inpatients, (two hospitalized in ICU (10%)) and 20 from outpatients. These data suggest that influenza B viruses may be frequently the cause of hospitalization. However, more severe forms, requiring hospitalization in ICUs, occur less frequently than following influenza A(H1N1) 2009 virus infection.
Forty six percent of influenza B virus infections, in this study, required hospitalization, independently of the lineage or clade. It is possible that the pathogenicity of type B influenza virus has been underestimated in the past. After the 2009 pandemic, patients hospitalized in different clinical settings are more frequently controlled for all types of influenza viruses using RT real-time PCR assays of high sensitivity, which could be the reason for the detection of a higher percentage of influenza B severe infections. Different molecular methods have been developed for the characterization of influenza B viruses. These include mainly real-time PCR, which amplifies HA sequences using primers and probes specific for the two lineages [14][15][16], and pyrosequencing analysis of target regions in the HA and NA genes. Pyrosequencing analysis has been used also to detect simultaneously, in the NA gene, markers that distinguish between B/Yamagata and B/Victoria lineages and several mutations associated with NAIs [5]. In this study, two one-step RT-PCRs followed by sequencing were developed with the aim of detecting simultaneously molecular markers, both in the HA gene and NA gene, allowing one to distinguish and characterize the influenza B viruses. However, only the analysis of the HA gene sequence has given reliable results, in agreement with data in the literature [6]. In comparison with other published assays [5,6,[14][15][16], the method described here has the advantage of simultaneously distinguishing the two main lineages of circulating influenza B viruses and the different groups within the lineages by a one-step RT-PCR targeting a small HA sequence. Only one method reported in the literature, based on pyrosequencing [6], is also able to distinguish the clades. However, it requires the use of a specific instrument not available in all laboratories. The RT-PCR described here allowed also the analysis of clinical samples with a low viral load (12 copies/µL).
The importance of the characterization of influenza B viruses, and not only of influenza A viruses in circulation, is evident. In fact, on this basis, the WHO recommended for Northern Hemisphere influenza vaccine composition ( [4]. Moreover, WHO has proposed to include in the quadrivalent vaccine an influenza B/Victoria virus related to the B/Brisbane/60/2008 virus.
As a consequence, there is the need for sensitive and suitable methods able to identify not only the lineage, but also the group of the type B influenza viruses in circulation, as with the assay described here, for diagnostic and epidemiological analyses.