Pathogenesis and Transmission Assessment of 3 Swine-Origin Influenza A(H3N2) Viruses With Zoonotic Risk to Humans Isolated in the United States, 2017-2020.

The sporadic occurrence of human infections with swine-origin influenza A(H3N2) viruses and the continual emergence of novel A(H3N2) viruses in swine herds underscore the necessity for ongoing assessment of the pandemic risk posed by these viruses. Here, we selected 3 recent novel swine-origin A(H3N2) viruses isolated between 2017 to 2020, bearing hemagglutinins from the 1990.1, 2010.1, or 2010.2 clades, and evaluated their ability to cause disease and transmit in a ferret model. We conclude that despite considerable genetic variances, all 3 contemporary swine-origin A(H3N2) viruses displayed a capacity for robust replication in the ferret respiratory tract and were also capable of limited airborne transmission. These findings highlight the continued public health risk of swine-origin A(H3N2) strains, especially in human populations with low cross-reactive immunity.

An integrated system of air sampling and simultaneous enrichment for rapid biosensing of airborne coronavirus and influenza virus.

Point-of-care risk assessment (PCRA) for airborne viruses requires a system that can enrich low-concentration airborne viruses dispersed in field environments into a small volume of liquid. In this study, airborne virus particles were collected to a degree above the limit of detection (LOD) for a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This study employed an electrostatic air sampler to capture aerosolized test viruses (human coronavirus 229E (HCoV-229E), influenza A virus subtype H1N1 (A/H1N1), and influenza A virus subtype H3N2 (A/H3N2)) in a continuously flowing liquid (aerosol-to-hydrosol (ATH) enrichment) and a concanavalin A (ConA)-coated magnetic particles (CMPs)-installed fluidic channel for simultaneous hydrosol-to-hydrosol (HTH) enrichment. The air sampler's ATH enrichment capacity (EC) was evaluated using the aerosol counting method. In contrast, the HTH EC for the ATH-collected sample was evaluated using transmission-electron-microscopy (TEM)-based image analysis and real-time qRT-PCR assay. For example, the ATH EC for HCoV-229E was up to 67,000, resulting in a viral concentration of 0.08 PFU/mL (in a liquid sample) for a viral epidemic scenario of 1.2 PFU/m3 (in air). The real-time qRT-PCR assay result for this liquid sample was "non-detectable" however, subsequent HTH enrichment for 10 min caused the "non-detectable" sample to become "detectable" (cycle threshold (CT) value of 33.8 ± 0.06).

Retrospective Assessment of the Antigenic Similarity of Egg-Propagated and Cell Culture-Propagated Reference Influenza Viruses as Compared with Circulating Viruses across Influenza Seasons 2002-2003 to 2017-2018.

Suboptimal vaccine effectiveness against seasonal influenza is a significant public health concern, partly explained by antigenic differences between vaccine viruses and viruses circulating in the environment. Haemagglutinin mutations within vaccine viruses acquired during serial passage in eggs have been identified as a source of antigenic variation between vaccine and circulating viruses. This study retrospectively compared the antigenic similarity of circulating influenza isolates with egg- and cell-propagated reference viruses to assess any observable trends over a 16-year period. Using annual and interim reports published by the Worldwide Influenza Centre, London, for the 2002-2003 to 2017-2018 influenza seasons, we assessed the proportions of circulating viruses which showed antigenic similarity to reference viruses by season. Egg-propagated reference viruses were well matched against circulating viruses for A/H1N1 and B/Yamagata. However, A/H3N2 and B/Victoria cell-propagated reference viruses appeared to be more antigenically similar to circulating A/H3N2 and B/Victoria viruses than egg-propagated reference viruses. These data support the possibility that A/H3N2 and B/Victoria viruses are relatively more prone to egg-adaptive mutation. Cell-propagated A/H3N2 and B/Victoria reference viruses were more antigenically similar to circulating A/H3N2 and B/Victoria viruses over a 16-year period than were egg-propagated reference viruses.

[Genetic characterization of influenza A virus and assessment of vaccine efficacy in Yantai from 2014 to 2017].

In this study, the swabs were collected among patients with an influenza-like illness (ILI) admitted to 2 sentinel surveillance hospitals of Yantai from April 2014 to August 2017. All specimen were cultured and identified by hemagglutination inhibition assay. Complete sequences of Hemagglutinin (HA) of influenza A were amplified, sequenced and analyzed using molecular and phylogenetic methods. The potential vaccine efficacy were calculated using Pepitope model. The results showed that the antigenicity of A (H3N2) had changed greatly. 8 strains of influenza A (H1N1) pdm09 belonged to subclade 6B.1 and 14 strains clustered in 6B.2. 12 strains of influenza A (H3N2) fell into subgroup 3C.3a and 33 strains clustered in 3C.2a. Several residues at antigen sites and potential glycosylation sites had changed in influenza A strains. Vaccine efficacy of influenza A (H1N1) pdm09 in 2015/2016 and 2016/2017 seasons were 77.29% and 79.11% of that of a perfect match with vaccine strain, meanwhile vaccine efficacy of influenza A (H3N2) in 2014/2015, 2015/2016 and 2016/2017 were-5.18%, 16.97% and 42.05% separately. In conclusion, the influenza A virus circulated in Yantai from 2014 to 2017 presented continual genetic variation. The recommended vaccine strains still afforded protection against influenza A (H1N1) pdm09 strains and provided suboptimal protection against influenza A (H3N2) strains.

Update: Influenza Activity in the United States During the 2018-19 Season and Composition of the 2019-20 Influenza Vaccine.

Influenza activity* in the United States during the 2018-19 season (September 30, 2018-May 18, 2019) was of moderate severity (1). Nationally, influenza-like illness (ILI)† activity began increasing in November, peaked during mid-February, and returned to below baseline in mid-April; the season lasted 21 weeks,§ making it the longest season in 10 years. Illness attributed to influenza A viruses predominated, with very little influenza B activity. Two waves of influenza A were notable during this extended season: influenza A(H1N1)pdm09 viruses from October 2018 to mid-February 2019 and influenza A(H3N2) viruses from February through May 2019. Compared with the 2017-18 influenza season, rates of hospitalization this season were lower for adults, but were similar for children. Although influenza activity is currently below surveillance baselines, testing for seasonal influenza viruses and monitoring for novel influenza A virus infections should continue year-round. Receiving a seasonal influenza vaccine each year remains the best way to protect against seasonal influenza and its potentially severe consequences.

Epidemiology and burden of influenza in healthy children aged 6 to 35 months: analysis of data from the placebo arm of a phase III efficacy trial.

BACKGROUND: Despite World Health Organization recommendations, in many countries young children are not targeted for influenza vaccination. To help inform influenza vaccination policy, we examined the occurrence and burden of influenza in healthy children aged 6 to 35 months using data from a recent phase III placebo-controlled influenza vaccine trial conducted in countries in the Northern and Southern Hemispheres. METHODS: This was an analysis of data from participants included in the placebo arm of a phase III clinical trial in healthy children aged 6 to 35 months (EudraCT no. 2013-001231-51). Included children had never been vaccinated for influenza and were observed for one influenza season. Outcome measures included the occurrence of influenza-like illness (ILI), laboratory-confirmed influenza, virus types/subtypes, severe symptoms and complications of confirmed influenza, and healthcare use associated with confirmed influenza. RESULTS: Data from 2210 participants were analysed. ILI was reported for 811 participants (36.7%). Of these, 255 participants (31.4%) had 263 virologically confirmed episodes of influenza. The overall influenza attack rate was 11.5%. The most common influenza virus detected was A(H3N2) (40.7%), followed by B/Yamagata (23.6%), A(H1N1) (18.6%), and B/Victoria (8.0%). Grade 3 fever was reported in 24.3% of confirmed episodes, acute lower respiratory infection in 8.7%, acute otitis media in 6.1%, and pneumonia in 1.9%. In most influenza episodes (93.2%), antipyretics, analgesics, or non-steroidal anti-inflammatory drugs were taken. Antibiotics were prescribed for 41.4% of influenza episodes. More than half of the influenza episodes (57.0%) resulted in outpatient visits. Influenza resulted in overnight hospitalisation in 1.1% of episodes. CONCLUSIONS: Influenza is associated with a significant burden of disease in healthy children. This analysis also revealed that antibiotics continue to be frequently used for young children with influenza. TRIAL REGISTRATION: EudraCT no. 2013-001231-51 .

Evolutionary study and phylodynamic pattern of human influenza A/H3N2 virus in Indonesia from 2008 to 2010.

Influenza viruses are by nature unstable with high levels of mutations. The sequential accumulation of mutations in the surface glycoproteins allows the virus to evade the neutralizing antibodies. The consideration of the tropics as the influenza reservoir where viral genetic and antigenic diversity are continually generated and reintroduced into temperate countries makes the study of influenza virus evolution in Indonesia essential. A total of 100 complete coding sequences (CDS) of Hemagglutinin (HA) and Neuraminidase (NA) genes of H3N2 virus were obtained from archived samples of Influenza-Like Illness (ILI) surveillance collected from 2008 to 2010. Our evolutionary and phylogenetic analyses provide insight into the dynamic changes of Indonesian H3N2 virus from 2008 to 2010. Obvious antigenic drift with typical 'ladder-like' phylogeny was observed with multiple lineages found in each year, suggesting co-circulation of H3N2 strains at different time periods. The mutational pattern of the Indonesian H3N2 virus was not geographically related as relatively low levels of mutations with similar pattern of relative genetic diversity were observed in various geographical origins. This study reaffirms that the existence of a particular lineage is most likely the result of adaptation or competitive exclusion among different host populations and combination of stochastic ecological factors, rather than its geographical origin alone.

Rapid detection and subtyping of multiple influenza viruses on a microfluidic chip integrated with controllable micro-magnetic field.

Influenza viruses have threatened animals and public health systems continuously. Moreover, there are many subtypes of influenza viruses, which have brought great difficulties to the classification of influenza viruses during any influenza outbreak. So it is crucial to develop a rapid and accurate method for detecting and subtyping influenza viruses. In this work, we reported a rapid method for simultaneously detecting and subtyping multiple influenza viruses (H1N1, H3N2 and H9N2) based on nucleic acid hybridization on a microfluidic chip integrated with controllable micro-magnetic field. H1N1, H3N2 and H9N2 could be simultaneously detected in 80min with detection limits about 0.21nM, 0.16nM, 0.12nM in order. Moreover, the sample and reagent consumption was as low as only 3µL. The results indicated that this approach possessed fast analysis and high specificity. Therefore, it is expected to be used to simultaneously subtype and detect multiple targets, and may provide a powerful technique platform for the rapid detection and subtyping analysis of influenza viruses.

Whole genome sequencing identifies influenza A H3N2 transmission and offers superior resolution to classical typing methods.

OBJECTIVES: Influenza with its annual epidemic waves is a major cause of morbidity and mortality worldwide. However, only little whole genome data are available regarding the molecular epidemiology promoting our understanding of viral spread in human populations. METHODS: We implemented a RT-PCR strategy starting from patient material to generate influenza A whole genome sequences for molecular epidemiological surveillance. Samples were obtained within the Bavarian Influenza Sentinel. The complete influenza virus genome was amplified by a one-tube multiplex RT-PCR and sequenced on an Illumina MiSeq. RESULTS: We report whole genomic sequences for 50 influenza A H3N2 viruses, which was the predominating virus in the season 2014/15, directly from patient specimens. The dataset included random samples from Bavaria (Germany) throughout the influenza season and samples from three suspected transmission clusters. We identified the outbreak samples based on sequence identity. Whole genome sequencing (WGS) was superior in resolution compared to analysis of single segments or partial segment analysis. Additionally, we detected manifestation of substantial amounts of viral quasispecies in several patients, carrying mutations varying from the dominant virus in each patient. CONCLUSION: Our rapid whole genome sequencing approach for influenza A virus shows that WGS can effectively be used to detect and understand outbreaks in large communities. Additionally, the genomic data provide in-depth details about the circulating virus within one season.

Fitness cost of reassortment in human influenza.

Reassortment, which is the exchange of genome sequence between viruses co-infecting a host cell, plays an important role in the evolution of segmented viruses. In the human influenza virus, reassortment happens most frequently between co-existing variants within the same lineage. This process breaks genetic linkage and fitness correlations between viral genome segments, but the resulting net effect on viral fitness has remained unclear. In this paper, we determine rate and average selective effect of reassortment processes in the human influenza lineage A/H3N2. For the surface proteins hemagglutinin and neuraminidase, reassortant variants with a mean distance of at least 3 nucleotides to their parent strains get established at a rate of about 10-2 in units of the neutral point mutation rate. Our inference is based on a new method to map reassortment events from joint genealogies of multiple genome segments, which is tested by extensive simulations. We show that intra-lineage reassortment processes are, on average, under substantial negative selection that increases in strength with increasing sequence distance between the parent strains. The deleterious effects of reassortment manifest themselves in two ways: there are fewer reassortment events than expected from a null model of neutral reassortment, and reassortant strains have fewer descendants than their non-reassortant counterparts. Our results suggest that influenza evolves under ubiquitous epistasis across proteins, which produces fitness barriers against reassortment even between co-circulating strains within one lineage.

Burden of influenza among hospitalized febrile children in Ghana.

BACKGROUND: Influenza surveillance data from Africa indicate a substantial disease burden with high mortality. However, local influenza data from district hospitals with limited laboratory facilities are still scarce. OBJECTIVES: To identify the frequency and seasonal distribution of influenza among hospitalized febrile children in a rural hospital in Ghana and to describe differential diagnoses to other severe febrile infections. METHODS: Between January 2014 and April 2015, all children with a temperature of ≥38°C admitted to a district hospital in Ghana were screened for influenza A and B by RT-PCR and differentiated to subtypes A(H1N1)pdm09 and A(H3N2). Malaria microscopy and blood cultures were performed for each patient. RESULTS: A total of 1063 children with a median age of 2 years (IQR: 1-4 years) were recruited. Of those, 271 (21%) were classified as severe acute respiratory infection (SARI) and 47 (4%) were positive for influenza, namely 26 (55%) influenza B, 15 (32%) A(H1N1)pdm09, and 6 (13%) A(H3N2) cases. Influenza predominantly occurred in children aged 3-5 years and was more frequently detected in the major rainy season (OR = 2.9; 95% CI: 1.47-6.19) during the first half of the year. Two (4%) and seven (15%) influenza-positive children were co-diagnosed with an invasive bloodstream infection or malaria, respectively. CONCLUSION: Influenza contributes substantially to the burden of hospitalized febrile children in Ghana being strongly dependent on age and corresponds with the major rainy season during the first half-year.

Assessment of Molecular, Antigenic, and Pathological Features of Canine Influenza A(H3N2) Viruses That Emerged in the United States.

Background: A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods: In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results: A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions: Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health.

Reassortment of high-yield influenza viruses in vero cells and safety assessment as candidate vaccine strains.

Vaccination is the practiced and accessible measure for preventing influenza infection. Because chicken embryos used for vaccine production have various insufficiencies, more efficient methods are needed. African green monkey kidney (Vero) cells are recommended by the World Health Organization (WHO) as a safe substitute for influenza vaccine production for humans. However, the influenza virus usually had low-yield in Vero cells, which limits the usage of Vero cellular vaccines. This study used 2 high-yield influenza viruses in Vero cells: A/Yunnan/1/2005Va (H3N2) and B/Yunnan/2/2005Va (B) as donor viruses. It used 3 wild strain viruses to reassort new adaptation viruses, including: A/Tianjin/15/2009(H1N1), A/Fujian/196/2009(H3N2), and B/Chongqing/1384/2010(B). These three new viruses could maintain the characteristic of high-yield in Vero cells. Furthermore, they could keep the immunogenic characteristics of the original wild influenza viruses. Importantly, these viruses were shown as safe in chicken embryo and guinea pigs assessment systems. These results provide an alternative method to produce influenza vaccine based on Vero cells.

Estimation of influenza-attributable medically attended acute respiratory illness by influenza type/subtype and age, Germany, 2001/02-2014/15.

BACKGROUND: The total burden of influenza in primary care is difficult to assess. The case definition of medically attended "acute respiratory infection" (MAARI) in the German physician sentinel is sensitive; however, it requires modelling techniques to derive estimates of disease attributable to influenza. We aimed to examine the impact of type/subtype and age. METHODS: Data on MAARI and virological results of respiratory samples (virological sentinel) were available from 2001/02 until 2014/15. We constructed a generalized additive regression model for the periodic baseline and the secular trend. The weekly number of influenza-positive samples represented influenza activity. In a second step, we distributed the estimated influenza-attributable MAARI (iMAARI) according to the distribution of types/subtypes in the virological sentinel. RESULTS: Season-specific iMAARI ranged from 0.7% to 8.9% of the population. Seasons with the strongest impact were dominated by A(H3), and iMAARI attack rate of the pandemic 2009 (A(H1)pdm09) was 4.9%. Regularly the two child age groups (0-4 and 5-14 years old) had the highest iMAARI attack rates reaching frequently levels up to 15%-20%. Influenza B affected the age group of 5- to 14-year-old children substantially more than any other age group. Sensitivity analyses demonstrated both comparability and stability of the model. CONCLUSION: We constructed a model that is well suited to estimate the substantial impact of influenza on the primary care sector. A(H3) causes overall the greatest number of iMAARI, and influenza B has the greatest impact on school-age children. The model may incorporate time series of other pathogens as they become available.

Influenza A Viruses of Swine (IAV-S) in Vietnam from 2010 to 2015: Multiple Introductions of A(H1N1)pdm09 Viruses into the Pig Population and Diversifying Genetic Constellations of Enzootic IAV-S.

Active surveillance of influenza A viruses of swine (IAV-S) involving 262 farms and 10 slaughterhouses in seven provinces in northern and southern Vietnam from 2010 to 2015 yielded 388 isolates from 32 farms; these viruses were classified into H1N1, H1N2, and H3N2 subtypes. Whole-genome sequencing followed by phylogenetic analysis revealed that the isolates represented 15 genotypes, according to the genetic constellation of the eight segments. All of the H1N1 viruses were entirely A(H1N1)pdm09 viruses, whereas all of the H1N2 and H3N2 viruses were reassortants among 5 distinct ancestral viruses: H1 and H3 triple-reassortant (TR) IAV-S that originated from North American pre-2009 human seasonal H1, human seasonal H3N2, and A(H1N1)pdm09 viruses. Notably, 93% of the reassortant IAV-S retained M genes that were derived from A(H1N1)pdm09, suggesting some advantage in terms of their host adaptation. Bayesian Markov chain Monte Carlo analysis revealed that multiple introductions of A(H1N1)pdm09 and TR IAV-S into the Vietnamese pig population have driven the genetic diversity of currently circulating Vietnamese IAV-S. In addition, our results indicate that a reassortant IAV-S with human-like H3 and N2 genes and an A(H1N1)pdm09 origin M gene likely caused a human case in Ho Chi Minh City in 2010. Our current findings indicate that human-to-pig transmission as well as cocirculation of different IAV-S have contributed to diversifying the gene constellations of IAV-S in Vietnam. IMPORTANCE: This comprehensive genetic characterization of 388 influenza A viruses of swine (IAV-S) isolated through active surveillance of Vietnamese pig farms from 2010 through 2015 provides molecular epidemiological insight into the genetic diversification of IAV-S in Vietnam after the emergence of A(H1N1)pdm09 viruses. Multiple reassortments among A(H1N1)pdm09 viruses and enzootic IAV-S yielded 14 genotypes, 9 of which carried novel gene combinations. The reassortants that carried M genes derived from A(H1N1)pdm09 viruses became predominant, replacing those of the IAV-S that had been endemic in Vietnam since 2011. Notably, one of the novel reassortants likely caused a human case in Vietnam. Given that Vietnam is the second-largest pig-producing country in Asia, continued monitoring of IAV-S is highly important from the viewpoints of both the swine industry and human public health.

Multiplex RT-PCR detection of H3N2 influenza A virus in dogs.

A multiplex RT-PCR (mRT-PCR) assay to detect H3N2 CIV genomic segments was developed as a rapid and cost-effective method. Its performance was evaluated with forty-six influenza A viruses from different hosts using three primer sets which amplify four segments of H3N2 CIV simultaneously. The mRT-PCR has been successful in detecting the viral segments, indicating that it can improve the speed of diagnosis for H3N2 CIV and its reassortants.

Efficient Bayesian inference under the structured coalescent.

MOTIVATION: Population structure significantly affects evolutionary dynamics. Such structure may be due to spatial segregation, but may also reflect any other gene-flow-limiting aspect of a model. In combination with the structured coalescent, this fact can be used to inform phylogenetic tree reconstruction, as well as to infer parameters such as migration rates and subpopulation sizes from annotated sequence data. However, conducting Bayesian inference under the structured coalescent is impeded by the difficulty of constructing Markov Chain Monte Carlo (MCMC) sampling algorithms (samplers) capable of efficiently exploring the state space. RESULTS: In this article, we present a new MCMC sampler capable of sampling from posterior distributions over structured trees: timed phylogenetic trees in which lineages are associated with the distinct subpopulation in which they lie. The sampler includes a set of MCMC proposal functions that offer significant mixing improvements over a previously published method. Furthermore, its implementation as a BEAST 2 package ensures maximum flexibility with respect to model and prior specification. We demonstrate the usefulness of this new sampler by using it to infer migration rates and effective population sizes of H3N2 influenza between New Zealand, New York and Hong Kong from publicly available hemagglutinin (HA) gene sequences under the structured coalescent. AVAILABILITY AND IMPLEMENTATION: The sampler has been implemented as a publicly available BEAST 2 package that is distributed under version 3 of the GNU General Public License at http://compevol.github.io/MultiTypeTree.

The effect of the MDCK cell selected neuraminidase D151G mutation on the drug susceptibility assessment of influenza A(H3N2) viruses.

Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that ∼90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of the virus isolates to zanamivir and oseltamivir were assessed using the chemiluminescent and fluorescent NA inhibition (NI) assays. In the chemiluminescent assay, which utilizes NA-Star® substrate, up to 13-fold increase in zanamivir-IC50 was detected for isolates containing a high proportion (>50%) of the G151 NA variant. However, an increase in zanamivir-IC50s was not seen in the fluorescent assay, which uses MUNANA as substrate. To investigate this discrepancy, recombinant NAs (rNAs) were prepared and tested in both NI assays. Regardless of the assay used, the zanamivir-IC50 for the rNA G151 was much greater (>1500-fold) than that for rNA D151 wild-type. However, zanamivir resistance conferred by the G151 substitution was masked in preparations containing the D151 NA which had much greater activity, especially against MUNANA. In conclusion, the presence of NA D151G variants in cell culture-grown viruses interferes with drug susceptibility assessment and therefore measures need to be implemented to prevent their emergence.

Cell culture-selected substitutions in influenza A(H3N2) neuraminidase affect drug susceptibility assessment.

Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC50) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n = 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.