Molecular subtyping of European swine influenza viruses and scaling to high-throughput analysis

Nasal swabs (MW950Sent2mL Virocult®, Kitvia, Labarthe-Inard, France) were collected from herds in France by veterinary practitioners or ANSES personnel, in the context of virological diagnosis passive surveillance programs or specific epidemiological investigations. They were taken from pigs during outbreaks of acute respiratory disease. The swabs were mixed vigorously and supernatants were stored at −70 °C until analysis. Some lung samples were also obtained from necropsy of fatal cases. Total viral RNAs were extracted from 200 μL of nasal swab supernatants or 20–30 mg of lung homogenate using the NucleoSpin RNA or Nucleospin 8 RNA kits (Macherey-Nagel, Hoerdt, France) or the RNeasy Mini Kit (Qiagen, Courtaboeuf, France). IAV genome detection was performed by M gene RT-qPCR using one of the two ready-to-use commercial kits previously validated by the French National Reference Laboratory (NRL), i.e. the LSI VetMAX™ Swine Influenza A-A/H1N1/2009- included kit (Life technologies, Carlsbad, CA, USA) or the ADIAVET™ SIV REALTIME kit (Bio-X Diagnostics, Rochefort, Belgique). Both steps, i.e. viral RNA purification and IAV genome detection, followed the instructions provided by RT-qPCR manufacturers [21].

Virus isolation was attempted from positive clinical samples in Madin Darby canine kidney (MDCK) cells, according to standard procedures [22]. Reference strains, previously selected as representative of the main viral subtypes and lineages encountered in European pigs over the last few years, were also retrieved from the collection of the French NRL or kindly provided by the European surveillance network for influenza in pigs (ESNIP) [8, 23]. A/California/04/2009 (H1N1pdm) was obtained from the French National Reference Center for Influenza, Institut Pasteur, Paris.

Sets of primers (forward and reverse) and the probe were designed to specifically amplify the different HA and NA genes from European swIAVs, on the basis of alignments of nucleotide sequences of viruses isolated since 2000 and retrieved from the IRD, GISAID (Global Initiative on Sharing All Influenza Data) or Genbank databases. Melting temperatures and/or basic properties of oligonucleotides were approximated using OligoCalc [26] or Vector NTI Advance^® sequence analysis and design software (Thermo Fischer Scientific, Waltham, MA, USA) (Table 1). Whereas ready-to-use commercial kits have previously been validated for H1pdm and N1pdm detection [21], new sets of primers and the probe specific to these two genes were designed in our study in order to include H1pdm and N1pdm RT-qPCRs in the high-throughput PCR array. Thus, the H1_av, H1_hu, H1pdm and H3 sets specifically amplify HA genes from the European H1_avN1, H1_huN2, H1N1pdm and H3N2 lineages, respectively. The N1 set matches with NA genes from both the H1_avN1 and the H1N1pdm lineages whereas the N1pdm set specifically amplifies the NA gene from the H1N1pdm lineage. The N2 primers and probe can detect NA genes from both the H1_huN2 and the H3N2 lineages. In addition, an H1_{huΔ146–147} set was manually designed to identify the novel antigenic variant that have emerged among H1_huN2 viruses following antigenic drift [18]. Alignments comprised full length HA sequences of H1_huN2 and H1_huN2_Δ146–147, as well as various H1_huN1 reassortant viruses. Thus, the H1_{huΔ146–147} set design took into account a deletion of 6 nucleotides and a mutation located in the receptor binding site of the H1_huN2_Δ146–147 variant (manuscript in preparation). Finally, M and β-actin sets were included, since in-house RT-qPCRs for swIAV and reference gene detection were used, respectively, as controls in high-throughput PCR assays. All RT-qPCRs used standard TaqMan DNA probes except those for H1pdm, N1pdm and H1_{huΔ146–147} which used highly specific DNA probes with conjugated minor groove binder (MGB) groups at the 3‘-end. The fluorophore covalently attached to the 5’-end of the oligonucleotide probe was 6-carboxyfluorescein (FAM), hexachlorofluorescein (HEX) or cyanine5 (Cy5) (Table 1). All standard TaqMan probes used black hole quencher (BHQ1) at the 3′-end. Primers and labelled standard TaqMan probes were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). MGB-labelled probes were from Life technologies (Carlsbad, CA, USA).

Similarly to virus detection by M gene RT-qPCR, the HA/NA subtyping methods were assessed on RNA extracted from amplified viruses or clinical samples using the NucleoSpin RNA kit (Macherey-Nagel, Hoerdt, France) or the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) following the manufacturer’s instructions. Real-time RT-PCR assays were run either on Chromo4 (Bio-Rad Laboratories, Hercules, CA, USA) or MX3005P (Stratagene, Agilent Technologies, La Jolla, CA, USA). Each RT-qPCR mixture contained 5 μL of RNA extract and 20 μL of master mix containing 1X GoTaq Probe qPCR Master Mix, dUTP (2X) (Promega, Fitchburg, WI, USA), 2X GoScript RT Mix for 1-step RT-qPCR (50X) (Promega, Fitchburg, WI, USA) as well as primers and probe in different concentrations depending on the target gene. Thus, the master mix contained 800 nM of each primer and 100 nM of probe for H1_av, H3, N1 and N2, 800 nM of each primer and 140 nM of probe for H1_hu, 400 nM of each primer and 500 nM of probe for H1_{huΔ146–147}, and 800 nM of each primer and 250 nM of probe for H1pdm and N1pdm. All RT-qPCRs were run as singleplex assays, except N1 and N2 that were run as a duplex assay. The cycling conditions used for H1_av, H1_hu, H3 and N1/N2 were: 15 min reverse transcription at 45 °C, 2 min of denaturation at 95 °C and 42 cycles of 15 s at 95 °C and 1 min at 56 °C. The program used for H1_{huΔ146–147}, H1pdm and N1pdm (with MGB probe) was 15 min reverse transcription at 45 °C, 2 min of denaturation at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

Several panels incorporating virus strains and/or clinical samples were constituted to evaluate the performance of the RT-qPCRs developed for the amplification of the H1_av, H1_hu, H1_{huΔ146–147}, H3, N1 and N2 genes.

A first panel (panel 1) of 42 swIAV isolates was set up to assess the analytical specificity of the RT-qPCRs designed for H1_av, H1_hu, H3, N1 and N2 gene amplifications. Among these virus strains, 32 belong to one of the four European enzootic lineages, i.e. 12 H1_avN1, 10 H1_huN2, 8 H1N1pdm and 2 H3N2, whereas 10 others are isolates originating from reassortments between enzootic viruses, i.e. 5 H1_huN1 and 5 H1_avN2 (Table 2). RNA extracts were diluted in RNAse free-water to adjust the quantification cycle (Cq) value to 24–27 when tested with one of the M gene RT-qPCRs described above, in order to reduce the risk of cross-reactions between H1_av and H1_hu.

Panel 2 was assembled to test the specificity of the H1_{huΔ146–147} gene RT-qPCR, i.e. its ability to differentiate the H1 gene of the antigenic variant H1_huN2_Δ146–147 virus among HA genes previously identified as belonging to the H1_hu lineage. This panel consisted of RNA extracts obtained from 46 swIAV strains previously identified as H1_huN2 viruses (44/46) or H1_huN1 viruses (2/46) (Additional file 1).

The analytical sensitivity of the assays was evaluated by testing 10-fold serial dilutions (from 10⁻² to 10⁻⁹) of RNA extracted from reference strains (Table 3). The range of linearity, the coefficient of linear regression (R²) and the efficacy were then calculated to evaluate the performance (Table 4). The repeatability of each RT-qPCR was estimated by testing, in 5–6 independent assays, the RNA extract dilution immediately below the last dilution in which the target gene was detected by the assay, and calculating the inter-assay coefficient of variation (CV) (Table 5).

Panels 3 and 4 were set up to evaluate the diagnostic abilities of the various RT-qPCRs, i.e. their potential to identify HA and NA genes directly from clinical samples previously shown to contain swIAV genome (M gene RT-qPCR positive). Panel 3 included 60 RNA extracts obtained from 59 nasal swab supernatants and one lung tissue homogenate (Table 6). The swIAV genome was previously subtyped in 37 nasal swabs and the lung sample (11 samples were positive for H1_avN1, 4 for H1_avN2, 15 for H1_huN2, 2 for H1_huN1, 1 for H3N2, 4 for H1N1pdm, and 1 for both H1_av and H1_hu genes mixed with N2), whereas in 22 nasal swab supernatants the IAV genome was only partially subtyped using conventional RT-PCRs (18 samples tested positive for H1_av but not for N1 or N2; 3 samples tested negative for any HA but were found positive for N2, and 1 sample was found to be negative for HA and NA). Panel 4 was specifically assembled to assess the diagnostic ability of the H1_{huΔ146–147} RT-qPCR. It was composed of 78 RNA extracts, i.e. 56 obtained from nasal swab supernatants and 22 from swIAV strains, that were previously found either positive for H1_hu but not sequenced, or positive for N2 but in which HA was not identified, or in which neither HA nor NA were identified (see Additional file 2).

High-throughput real-time RT-PCR amplifications (M, β-actin, H1_av, H1_hu, H1_{huΔ146–147,} H1pdm, H3, N1, N1pdm and N2) were conducted on a LightCycler^®1536 real-time PCR system (Roche, Meylan, France). A Bravo automated liquid handling platform equipped with a chiller and a PlateLoc thermal microplate sealer (Agilent Technologies, La Jolla, CA, USA) was used as a microplate dispenser. Each RT-qPCR mixture contained 1 μL of RNA extract and 1 μL of master mix containing 1X RealTime ready DNA Probes Master (Roche, Meylan, France), 2X GoScript RT Mix for 1-step RT-qPCR (50X) (Promega, Fitchburg, WI, USA), 800 nM of each concerned primer, with the exception of the M gene forward primer that was included at a final concentration of 400 nM, and 250 nM of standard or MGB-labelled TaqMan probe. The one-step real-time RT-PCR program involved a 30 min reverse transcription of RNA at 45 °C, followed by a 2 min denaturation step at 95 °C, and lastly 40 cycles of 0 s (i.e. a pulse) at 95 °C and 1 min at 58 °C.

Panel 5 was established to evaluate the analytical specificity of the real-time RT-PCRs (including new in-house H1pdm and N1pdm RT-qPCRs) when using the high-throughput RT-qPCR procedure (see Additional file 3). This panel included of RNA extracts obtained from 88 swIAVs and 30 nasal swab supernatants. The isolates as well as the viruses present in clinical samples were first subtyped using RT-qPCRs run under low-throughput conditions. Serial 10-fold dilutions (10⁻¹ to 10⁻⁸) of RNA obtained from 8 reference strains were prepared in water to evaluate the analytical sensitivity of each RT-qPCR when run on the LightCycler^®1536 (Table 7). The M gene copy number present in each dilution point was measured using a duplex M/β-actin gene RT-qPCR as described separately [27]. Each dilution point was tested in duplicate.

The analytical performances of the novel RT-qPCRs developed for European swIAV subtyping in addition to the H1pdm and N1pdm commercial kits previously validated were first evaluated in low-throughput analyses.

Nasal swabs from animals naturally infected with H1_avN1, H1_huN2 or H3N2 swIAVs, as well as with H1_avN2 or H1_huN1 reassortant viruses (panel 3), were detected positive for both HA and NA corresponding genes in 100% of cases tested for both genes (Table 6). Additional samples tested either only in the simplex HA assays or in the N1/N2 multiplex assay were also qualified positive according to the expected HA or NA gene. Inclusion of 4 samples positive for H1N1pdm confirmed N1 amplification without any H1_av gene amplification. Sample 130103–2, which was previously known to contain both the H1_av and H1_hu genes together with the N2 gene, was also proved here to contain the N1 gene, enabling us to confirm the hypothesis of a mixture of H1_avN1 and H1_huN2 viruses in this sample. Analyses of 18 samples previously partially subtyped as “H1_avN?” confirmed the H1_av subtype in 9/18 cases. Others did not give a positive signal, probably due to limiting genome amounts (7/11 were samples with M gene RT-qPCR Cq-values >32, while the H1_av RT-qPCR detection limit was estimated around M gene Cq-value = 33). Conversely, the N1 gene was identified in 10/18 H1_avN? samples, even in samples where M gene Cq-value >27, which was estimated as the detection limit. The N2 gene was not amplified in any of these samples. Panel 3 also comprised 3 H?N2 samples. The N2 gene was detected in all of them, whereas the H1_hu gene was identified in 2/3. The last one (sample 130277–3) was shown to have an H1_hu gene after propagation in cell culture (data not shown). Finally, a sample (130133–5) that was left fully unsubtyped using conventional RT-PCRs was here classified as containing the H1_av, H1_hu and N2 genes, confirming the ability to detect virus mixtures using these RT-qPCRs.

The diagnostic ability of the H1_{huΔ146–147} RT-qPCR was evaluated by testing 78 samples of mainly H1N2 or H?N2 subtypes included in panel 4. The H1_{huΔ146–147} gene was identified in 3/15 H1_huN2 isolates (not sequenced previously), in 16/46 nasal swab supernatants in which the H1_huN2 virus was identified, as well as in 1/6 H1_huN? samples (Additional file 2). Although the specificity of the H1_{huΔ146–147} was previously demonstrated, these results were further validated by sequencing the HA genes of the three isolates that tested positive for the H1_{huΔ146–147} gene as well as the HA gene of strain A/Sw/France/29–150034/15 that tested negative. Sequencing confirmed that the three positive strains contained the 6 nt deletion by contrast to the other one that contained a H1_hu gene without this deletion (Genbank accession numbers KJ128334, KY241154, KY241155 and KY241115). Thus, 20/78 viruses or clinical samples previously identified as H1_hu-positive were rapidly identified as having an H1_{huΔ146–147} gene.

Real-time RT-PCRs for H1_av, H1_hu, H1_{huΔ146–147}, H3, N1 and N2 gene amplifications, as well as RT-qPCRs designed to amplify the H1pdm and N1pdm genes specifically, were evaluated in high-throughput analyses. Real-time RT-PCRs targeting the IAV M gene and the porcine β-actin genes were included in order to check the amount and quality of the genetic material and/or demonstrate any potential PCR inhibition. These last RT-qPCRs were previously validated in low-throughput analyses [21, 27]. Thus, 10 RT-qPCR assays were run together on a LightCycler®1536 to analyze the 118 samples from panel 5, i.e. 83 swIAVs, 5 virus mixtures and 30 nasal swab supernatants, all previously analyzed in low-throughput PCR assays (see Additional file 3). As expected, all samples were detected positive for the M gene. RNA extracts from virus strains exhibited M gene Cq-values ranging from <5 to 16.69, whereas extracts from clinical samples exhibited higher Cq-values, which mostly ranged between 14 and 30. Only 2/118 samples (1.7%) (both RNA extracts from MDCK cell culture supernatants) were found negative for the β-actin gene.

All HA and NA RT-qPCRs exhibited 100% specificity, as no undesirable cross-reaction or false-positive results were obtained, irrespective of the nature of the sample, i.e. RNA extracts from virus stock containing high amounts of genetic material, or RNA extracts from nasal swab supernatants. As expected, the H1_av RT-qPCR amplified H1 genes originating from the H1_avN1 lineage, i.e. H1 genes from H1_avN1 enzootic viruses and from H1_avN2 reassortants. The H1_hu RT-PCR amplified H1 genes from H1_huN2 viruses, H1_huN2_Δ146–147 variants and H1_huN1 reassortants. By contrast, the H1_{huΔ146–147} RT-PCR only detected the H1_huN2_Δ146–147 antigenic variants. In accordance with the results obtained in low-throughput analyses, it did not detect H1_huN1 reassortants bearing H1_hu genes with one or two amino acid deletions close to the receptor-binding site (RBS) (called H1_huN1_Δ146–147 and H1_huN1_Δ147). The H1pdm RT-qPCR amplified HA genes of viruses from the H1N1pdm lineage but also any other H1 gene, and the H3 RT-qPCR amplified HA from H3N2 viruses only. The N1 RT-qPCR amplified N1 genes from both the H1_avN1 and the H1N1pdm lineages, whereas the N1pdm RT-qPCR amplified the N1 genes from H1N1pdm viruses specifically. The N2 RT-qPCR amplified NA genes from all HxN2 viruses. Thus, all samples were fully subtyped according to expected results, including the virus mixtures, except one of them in which the N2 gene was not amplified in parallel to the H1_av, H1_hu and N1 genes, as expected. As observed for the M gene, the Cq-values obtained for the HA and NA RT-qPCRs were lower on RNAs extracted from virus isolates as compared to clinical samples. In several cases, the Cq-value was too low to be precisely determined (< 5). A sample without any viral RNA but rather consisting of water was added in each PCR array as a negative control and no unspecific detection was observed (data not shown).

In order to go further in evaluating the sensitivity of the methods when run as high-throughput analyses, each RT-qPCR (except the one targeting the β-actin gene) was tested against 10-fold serial dilutions (10⁻¹ to 10⁻⁸) of reference strains (Table 7). Depending on the virus stock and its initial genomic load, the M gene was still detected to the last dilution or to the 10⁻⁶ or the 10⁻⁷ dilution. In all cases, it was detected to the 10⁻⁵ dilution. The H1_av, H1_{huΔ146–147} and H1pdm genes were detected in samples exhibiting a corresponding M gene Cq-value until 32, approximately, while the H3 gene detection limit was found to exceed an M gene Cq-value >35. The detection limit for the H1_hu gene varied from 25 to 33 depending on the virus (parental virus, antigenic variant, reassortant or reassortant with deletion), that of N1 from 29 to 31, and that of N2 from 30 to 33. The N1pdm was detected until an M gene Cq-value of 31. The ranges of linearity are given in Table 8. The slopes calculated over these ranges were all (except one) comprised between −3.2 and −3.7, which means the efficacies of the RT-PCRs varied from 85% to 105%. The N2 RT-qPCR run on the reference H3N2 strain was the only one found to be outside these limits, showing an efficacy of 80.51%.

The full RT-qPCR subtyping tool began to be used routinely by the French NRL for Swine Influenza from January 2014, as part of the analytical workflow for swIAV surveillance. Thus, nasal swab supernatants previously selected as containing M gene from a swIAV were subjected to H1_av, H1_hu and H3 monoplex assays, to the N1/N2 duplex assay, as well as to commercial H1pdm and N1pdm RT-PCRs. Samples that were found to be positive for H1_hu were subjected to the H1_{huΔ146–147} RT-PCR in a second step. Considering the detection limits evaluated above, subtyping was undertaken on samples with M gene Cq-values <35 only. Samples exhibiting M gene Cq-values >35 were considered to be not typable. Looking at the proportions of HA and NA genes that were successfully subtyped in these samples from January 2014 to August 2016, it appeared that both genes were identified in almost all samples with M gene Cq-value <25 (Table 9). For 25 ≤ Cq-value <30, the proportion of unidentified HA and/or NA genes increased slightly. In these samples, the N1/N2 multiplex appeared less sensitive than HA simplex assays, as NA genes were less frequently amplified. In samples with Cq-value >30, the proportion of viruses fully or partially subtyped fell to about 50%.

Finally, the diagnostic ability of the high-throughput subtyping RT-qPCR assay was tested on RNAs extracted from 919 nasal swabs collected during a longitudinal study conducted in three pig herds located in Brittany and previously selected as M gene-positive samples [28]. These herds were known to be affected by H1_avN1 and H1_huN2 viruses only. Among them, HA and NA genes were both identified in 697 samples, whereas only the HA or NA gene was detected in 77 others, depending on the apparent amount of virus genome in the samples (Table 9). Almost all samples with an M gene Cq-value <30 were fully subtyped (Table 9). In samples with an M gene Cq-value >30, the NA gene was more frequently amplified than the HA gene (Table 9). Fourteen samples from one herd were shown to contain virus mixtures and/or reassortant viruses (data not shown).