National Institute for Research Cantacuzino National Influenza Centre, Laboratory for Respiratory Viruses Spl.Independentei 103, sector 5, 050096 Bucuresti Romania Report on virological surveillance of influenza activity in the Romania 2014/15 season Summary In Romania, the influenza epidemic of 2014/15 season started in the week 4/2015, when more than 10% of samples tested were positive for influenza. The influenza activity was higher than in previous season and characterised by co-circulation of influenza A(H3N2), A(H1N1)pdm09 and B viruses. First influenza virus from sentinel system was detected in week 49/2014, a significant rise of positive specimens started in week 3/2015, and the highest number of influenza positive specimens were detected in week 9. Last positive detections were registered during week 18/ 2015. In ILI sentinel and non-sentinel system, 931 specimens collected were tested by real-time RT- PCR out of which 500 (54.8%) were positive for influenza virus: 290 (58%) influenza A and 210 (42%) influenza B, including two coinfections of A(H3N2) and B influenza. Of the influenza A cases subtyped, 110 (38%) were H1N1 pdm09 and 180 (62%) were H3N2. In SARI sentinel system 407 specimens collected were tested by real-time RT-PCR and 164 (41%) were positive for influenza virus: 99 (60.4%) influenza A and 65 (39.6%) influenza B, including two coinfections (H3N2 and B; H1N1 and B). Of the influenza A cases, 52 (52.5%) were subtyped as H1N1 pdm09 and 47 (47.5%) as H3N2. From a total of 243 negative influenza virus samples, 55 were tested for other respiratory viruses and 34 of them were positive (including four coinfections). A total of 111 strains were isolated on MDCK or MDCK SIAT1 cells (31 strains H1N1 pdm09, 18 strains H3N2 and 62 strains type B Yamagata lineage). With available antisera all A(H1N1)pdm09 viruses were antigenically characterized as A/California/7/2009, but a higher heterogeneity was observed for subtype H3N2 and type B, indicating a mismatch between circulating and vaccine strains. Thirty five strains were tested for antiviral sensitivity, oseltamivir and zanamivir, by phenotypic method. Genetically, all A(H1N1)pdm09 viruses fall in group 6, subgroup B (A/South Africa/3626/2013). All B type viruses belonged to clade 3 (B/Phuket/3073/2013). The A(H3N2) strains belonged to clades 3C.2 and 3C.3. 1
Methods In Romania, the influenza surveillance is coordinated by the National Institute for Public Health (INSP) in close collaboration with the National Influenza Centre located in Cantacuzino Institute in Bucharest. In this season the sentinel system was based on 193 general practitioner doctors (GPs) distributed across 15 counties and Bucharest. Severe acute respiratory infection (SARI) surveillance was based on twenty hospitals (emergency hospitals, infectious diseases, pulmonary and paediatric hospitals) from 6 counties and Bucharest Municipality. From week 46 until the onset of influenza activity samples from the first 3 detected SARI cases in each sentinel hospital were tested for influenza; after that, one sample from the first detected SARI case. Throat swabs were collected randomly from ILI/SARI patients. These specimens were examined for the presence of influenza viruses (IV). The following genes are targeted by rrt-pcr tests: type A and B influenza matrix gene; haemagglutinin gene specific for influenza A subtypes: H1N1 pdm09 virus (formerly pandemic A(H1N1) and A(H3N2). When influenza A viruses were detected, a second rrt-pcr analysis was performed on the HA gene for determination of H1N1 pdm09 or H3N2 subtype. When influenza B viruses were detected, a second rrt-pcr analysis was performed for lineage determination (Yamagata or Victoria lineages). Other respiratory viruses were tested by Multiplex RT-PCR SEEPLEX RV 15 ONE-STEP Kit (Seegene.Inc., Seoul, Koreea), based on classic RT-PCR according with the manufactured work protocol. In house classic RT-PCR will be used as an alternative test for CoV, ADV or RSV. The selection method for virus isolation/sequencing use as criteria: Specimens in balance number of each type/subtype during all influenza season. Selected influenza vaccinated and nonvaccinated cases. Ct value under 30 Influenza virus isolation was performed on MDCK (ATCC/CCL 34) and MDCK SIAT1 for influenza virus A(H3N2). The presence of multiplied virus is revealed by hemagglutination with 0.5% turkey red blood cells (type A, subtype H1N1 pdm09 and type B) and 0.75% guinea pig erythrocytes (type A, subtype H3N2). Influenza virus isolates were characterized antigenically with haemagglutination inhibition (HI) assay using turkey/guinea pig red blood cells. For influenza A(H3N2) characterization we employ a modified HI assay with guinea pig erythrocytes in the presence of 20nM oseltamivir. HI test is performed in U bottom 96-well microtiter plates. Two-fold serial dilutions of standard ferret sera (25 µl system) are mixed with an equal volume of diluent containing each influenza virus isolate or standard antigen at 4 HA units/25 µl. After incubation minutes at room temperature (15-30ºC), 50 µl of erythrocytes suspension is then added to every well on all plates 2
and incubated for 30-45 minutes at room temperature (15-30ºC). Plates are then tilted and read for agglutination. We are using the traditional sequencing technique ( the Sanger reaction ) to monitor influenza evolution for at least 10% of original specimens detected positive, for whole HA gene. The phylogenetic trees are constructed using FastTree version 2.1.7with GTRGAMMA substitution model and 10000 bootstraps. Trees were drawn using FigTree and Inkscape as scalable vector graphics editor. SNP genotyping is used to screen for H275Y substitution in NA gene of H1N1 pdm09. Results Influenza Virus (IV) detections in ILI cases Since the beginning of the season, 913 clinical specimens, representing ILI suspected cases, collected from sentinel and non-sentinel sources, have been tested by real-time RT-PCR. Thirty eight percent of specimens were collected from non-sentinel system. The positivity rate began to rise in the second week of January (27%), while the highest rates were in weeks 7-9 (more than 70%). Last positive detection was at the end of April, albeit with very few specimens after early April and May. Second part of the epidemic was characterized by a dominance of type B. Distributions by type/subtype in ILI cases from sentinel and non-sentinel systems is presented in table 1. Table 1. Distributions of influenza virus detections by type/subtype in ILI cases sentinel and non-sentinel systems. 559 ILI from sentinel system primary care (61.2%) 354 ILI from nonsentinel system (38.8%) Total samples N = 913 ILI % Total pos A&B 500 54.8 303 (54.2%) 197 (55.6) type A 290 58 189 (62.4) 101 (51.3) A/H1 pdm09 110 37.9 72 (38.1) 38 (37.6) A/H3 180 62.1 117 (61.9) 63 (62.4) type B 210 42 114 (37.6) 96 (48.7) Neg 413 45.2 256 (45.8) 157 (44.4) A mixed virological aspect was noticed, with a slight dominance of type A over type B and a dominance of subtype H3N2 over H1N1 pdm09, both in the primary care system and among non-sentinel patients (generally hospitalized patients). Two mixed infections were detected: A (H3N2) and B. The weekly distribution of positive samples are presented figure 1. 3
40 43 46 49 52 3 6 9* 12 15 18 42 45 48 51 2 5 8 11 14 17 20 42 45 48 51 2 5 8 11 14 17 20 Influenza and non-influenza viruses detections in SARI cases Since the beginning of the season, 407 clinical specimens collected from sentinel sources have been tested by real-time RT-PCR, out of which 164 (40.3%) were positive for influenza virus: 99 specimens type A (52 H1N1 pdm09, 47 H3N2) and 65 type B. Weekly distribution and their proportion weight are presented in figure 2 and 3. From a total of 243 influenza virus negative samples, 55 (22.6%) were tested for other respiratory etiology. Thirty four of them were positive for other respiratory viruses, of which 18 (53%) for RSV. We found four non influenza virus coinfections, as follows: RSV&EV, RSV&AdV, RSV&RhV and AdV&EV. Figure 1. Weekly distribution of influenza virus detection in 2014-2015 in ILI and SARI 60 50 40 30 ILI GPs: Total = 559 - Neg: 256 (45.8%) - Pos: 303 (54.2%) - H3: 117 (38.6%) - H1: 72 (23.8%) - B: 114 (37.6%) ILI hospitalized: Total = 354 - Neg: 157 (44.4%) - Pos: 197 (55.6%) - H3: 63 (32%) - H1: 38 (19.3%) - B: 96 (48.7%) SARI: Total = 407 - Neg: 209 (51.4%) Total Pos: 198 (48.6%) Influenza: 164 - H3: 47 (28.7%) - H1: 52 (31.7%) - B: 65 (39.6%) - Pos Non-influenza : 34 20 10 0 H3 H1pdm09 B Others 4
# detections 40 Figure 2. Weekly distribution of SARI cases 2014-2015 35 30 25 20 15 10 5 0 40 42 44 46 48 50 52 2 4 6' 8*# ^ 10 12 14 16 18 20 H1N1pdm09 A/H3N2 B RSV hmpv PIV 1 PIV3 PIV 2 PIV 4 Corona ADV Rhino Boca HEV Figure 3. Distribution of respiratory viruses in SARI samples 9% 8% 26% 33% 24% IV H1pdm09 IV H3 IV type B RSV Other Regarding groups of age, the smallest percentage of positive influenza virus specimens was registered for 0-4 years old (16.2%) but a high percentage of other respiratory viruses was registered within this group (Table 2 and Figure 4). All positive specimens for other respiratory viruses were from children 5 year old. Type A influenza virus was dominant, except groups 5-14 and 30-64 years, where 50% were type B. Subtype H3 was dominant in extreme age groups (0-4 and 64) and subtype H1 pdm09 in subjects belonging to 30-64 years. 5
No. of samples Table 2. Distribution by age groups of influenza virus detection in SARI patients. Group of Total % % pos./age % pos. # % pos. age specimens specimens # pos # H1 H1 H3 H3 # B % pos. B 0-4 118 29.3 19 16.2 4 21.1 11 57.9 4 21.1 5-14 28 7.0 8 28.6 3 37.5 1 12.5 4 50.0 15-29 34 8.5 15 44.1 5 33.3 5 33.3 5 33.3 30-64* 152 37.6 81 54.0 28 34.6 13 16.0 40 49.4 64 63 15.5 37 59.7 10 27.0 15 40.5 12 32.4 Unknown 8 2.0 4 50.0 2 50.0 2 50.0 0 0.0 Total 403 100 164 41.1 52 31.7 47 28.7 65 39.6 *one coinfection H1 pdm09&b, one coinfection H3&B Figure 4. Distribution of positive detection in SARI cases by age groups 70 60 50 40 30 20 10 0 H1 H3 B Pos. other resp. viruses 0-4 5-14 15-29 30-64* >64 Unknown Antigenic characterization: Influenza virus isolation on MDCK (ATCC/CCL 34) and MDCK SIAT1 for influenza virus A(H3N2) was performed for 16% of positive specimens, usually after 1-2 passages. The presence of multiplied virus is revealed by hemagglutination with 0.5% turkey red blood cells 6
(type A, subtype H1N1 pdm09 and type B) and 0.75% guinea pig erythrocytes (type A, subtype H3N2). The weekly distribution of influenza virus isolates is presented in Figure 5. It was very difficult to isolate influenza A (H3N2) viruses compared with type B and A(H1N1 pdm09). A (H3N2) strains have multiplied at low titer and it was difficult to be characterized by HI assay. However, a total of 111 strains were isolated: - 31 strains H1N1 pdm09, A/California/7/09-like - 18 strains H3N2: o 6 strains A/Texas/50/2012 - like and o 12 strains A/Switzerland/9715293/2013 like - 62 strains type B (B/Yamagata/16/88 lineage): o 9 strains B/Massachusetts/02/2012-like o 53 strains B/Phuket/3073/2013-like. Forty two were from hospitalized patients (ILI or SARI). Thirty three isolates were sent to WHO Collaborating Center for Reference and Research on Influenza, Medical Research Council National Institute for Medical Research (NIMR), London. A total of 35 strains of influenza virus isolates were tested by phenotypic method (NA-Star assay) to oseltamivir and zanamivir. All investigated influenza A and B strains were susceptible to neuraminidase inhibitors. Figure 5. Weekly distribution of influenza virus isolation in 2014-2015 in ILI/SARI sentinel and nonsentinel Romania 20 18 16 14 12 10 8 6 4 2 0 H3 B H1pdm09 week 7
Genetic characterization: Sequencing of HA gene was performed for 54 of original samples or isolates figures 6, 7 and 8. Full hemagglutinin H1pdm09 gene was sequenced from 19 original specimens (including an influenza B and H1N1 coinfection), out of which 14 were collected from patients hospitalized with SARI. Four of them had fatal evolution, although one patient had been vaccinated. All characterized viruses fell into clade A/California/7/2009, subgroup 6B, A/South Africa/3626/2013 (figure 6). Twenty H3 subtype strains clustered into three different clades, 3C.2a and 3C.3a or 3C.3b: o Eight strains falling in clade representative A/Perth/16/2009 A/HongKong/5738/2014 group (3C.2a) o Seven strains falling in clade representative A/Perth/16/2009 A/Switzerland/9715293/2013 (3C.3a); o Five strains falling in clade representative A/Perth/16/2009 A/Newcastle/22/2014 (3C.3b) Of note: most of the viruses sequenced last season (2013/2014) belonged to clade 3C.3 (figure 7). Sixteen type B viruses fell into clade 3 (B/Phuket/3073/2013) figure 8, four from vaccinated patients and three deaths. In addition, all influenza B positive specimens tested for lineage differentiation by SNP belonged to B/Yamagata lineage. 8
Figure 6. Phylogenetic analysis of influenza H1N1 pdm09 HA genes compared to vaccine strain and previous season strains 9
Figure 7. Phylogenetic analysis of influenza A(H3N2) HA genes compared to vaccine strain and previous season strains 10
Figure 8. Phylogenetic analysis of influenza B HA genes compared to vaccine strain and previous season strains 11
Acknowledgements. We acknowledge the technical work performed by Luiza Ustea, Emilia Dobre, Nuti Enache and Mirela Ene. Special thanks to George Necula and Sorin Dinu, who gave us valuable advice in the field of genetic analysis. Special thanks to Odette Popovici, MD, senior epidemiologist, National Institute of Public Health, Romanian CDC, focal point for SARI surveillance and Rodica Popescu, MD, senior epidemiologist, National Institute of Public Health, Romanian CDC, focal point for ILI surveillance. We acknowledge the hard work performed by sentinel physicians and their patients and other contributors in influenza surveillance. Report prepared by Alina Elena Ivanciuc, Mihaela Lazãr, Cristina Tecu, Maria Elena Mihai, Carmen Maria Cherciu, Daniela Pitigoi and Emilia Lupulescu 12