Conceived and designed the experiments: EvdV EJVK KJS MS ADMEO RAMF CABB SH. Performed the experiments: EvdV EJVK KJS ML AvdL EJAS LML GvA. Analyzed the data: EvdV EJVK TK RAMF CABB SH. Contributed reagents/materials/analysis tools: MS CABB. Wrote the paper: EvdV EJVK TK RAMF CABB SH.
I have read the journal's policy and have the following conflicts. KJS, EJBVK, MS and ADMEO are employees of ViroClinics Biosciences BV. ADMEO is chief scientific officer of ViroClinics Biosciences BV. ADMEO, MS, EVDV and CABB participate in the IRIS study sponsored by Hoffmann-La Roche, Inc.
Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602).
Recently, a 2009 pandemic A/H1N1 influenza virus was isolated from an immune compromised patient, with antiviral resistance to the neuraminidase inhibitor class of drugs. This virus had an amino acid change in the viral neuraminidase enzyme; an isoleucine at position 223 was substituted for an arginine (I223R). Patients infected with a pandemic virus that is resistant to all neuraminidase inhibitors, would leave physicians without antiviral treatment options, since these viruses are naturally resistant to the other class of antivirals, the adamantanes. To date, it is unknown if this I223R mutant virus is affected in its ability to cause severe disease and to transmit to other humans. Therefore, we have addressed this question by comparing the I223R mutant virus with a wild type reference virus in a ferret pathogenicity and transmission model. We found that the I223R mutant virus was not severely affected in its pathogenicity, although fewer lung lesions and alveolitis scores were found for the I223R mutant virus. In addition, we demonstrated that this virus transmitted efficiently to naïve ferrets. Consequently, we conclude that this I223R mutant virus has the potential to cause disease and may spread among humans. Therefore, influenza surveillance for this resistance pattern is advised.
Two classes of antiviral drugs are approved for prophylaxis and therapy of influenza virus infected patients
To date, the incidence of NAI resistant 2009 pandemic A/H1N1 viruses is very low. Nevertheless, 565 cases of patients infected with an (H275Y, N1 numbering) oseltamivir (OS) resistant virus have been reported to the World Health Organization
Within the first years after approval of the NAIs in 1999, antiviral resistance in influenza viruses at a population level was rare (0.4%). In clinical trials, the incidence of resistant viruses was higher, varying from 0.4 to 1% in adults and up to 18% in young children
Recently, the identification of a novel multidrug resistant 2009 pandemic A/H1N1 virus was reported, isolated from an immune compromised child with reduced susceptibility to all NAIs
A pandemic 2009 influenza virus with reduced susceptibility to all NAIs that was isolated from a Dutch immune compromised child was studied here. Full genome sequencing of this clinical isolate A/NL/2631_1202/2010 (NL/2631-R223, GenBank accession numbers JF906180-906187) harboring an I223R mutation in the neuraminidase was performed. Since no drug susceptible virus had been isolated from this patient before start of antiviral therapy, the well-characterized NAI-susceptible virus isolate A/NL/602/2009 (NL/602, GenBank accession numbers CY046940-046945 and CY039527-039528) was used as a reference virus in all experiments. This reference virus is a representative of pandemic H1N1 viruses that circulated in 2009, with only amino acid changes I108V and V407I (N1 numbering) in NA being unusual among the deposited sequences in the Influenza Research Database
Virus replication was studied
MDCK (panel A) or MDCK-SIAT1 (panel B) cells were inoculated with 0.001 TCID50 virus per cell of recNL/602 (black circles), isolate NL/2631-R223 (black triangles) and recNL/602-I223R (open circles). Supernatants were harvested after 6, 12, 24, and 48 hours post infections and were titrated in MDCK cells. Geometric mean titers and standard deviations were calculated from two independent experiments. The lower limit of detection is indicated by the dotted line.
A recombinant derivative of NL/602 with the I223R mutation in NA (recNL/602-I223R) replicated to lower peak titers in both cell lines compared to recNL/602 and NL/2631-R223. In addition, initial virus replication of recNL/602-I223R was delayed by 6 to 12 hours in MDCK-SIAT1 cells.
The pathogenicity of clinical isolate NL/2631-R223 was compared with NL/602 in the ferret model that was previously established to study the ability of influenza viruses to cause pneumonia
Ferrets were inoculated intratracheally with 1×106 TCID50 of NL/602 or NL/2631-R223. Body weights for NL/602 (Panel A) and NL/2631-R223 (Panel B) inoculated animals are depicted as percentage of body weight relative to the time of inoculation. Data are shown for individual animals until the animals were euthanized at day 4 or 7 p.i.. Virus detection in throat (panel C) and nose swabs (panel D) is indicated for NL/602 (white bars), and NL/2631-R223 (black bars). Geometric mean titers from 6 (day 1 to 4) or 3 animals (day 5 to 7) are displayed and the error bars indicate the standard deviations. The number of influenza virus positive animals per day is depicted in each bar. The lower limit of detection is indicated by the dotted line.
Nose and throat swabs were collected daily from the inoculated animals and virus titers were determined by end-point titration in MDCK cells. Infectious virus shedding from the throat was detected from day 1 p.i. onwards in all ferrets, with similar patterns of virus shedding from the throat of the animals in the two groups (
At day 4 and 7 p.i., three animals of each group were euthanized and lungs were collected for virological and pathological examination. At day 4 p.i., no marked differences were found between the virus titers for both groups of ferrets (
Lung virus titers (panel A), percentage of affected lung tissue (panel B) and relative lung weights (panel C) were determined for lungs of ferrets inoculated with NL/2631-R223 (triangles) or NL/602 (circles) that were euthanized at day 4 or 7 p.i.. Semi-quantitative assessment of the extent and severity of the tracheitis (panel D), bronchiolitis (panel E) and alveolitis (panel F) are shown. Individual values are displayed. In panel A, the lower limit of detection is indicated by a dotted line.
Gross pathology of the lungs of all animals revealed pulmonary lesions at day 4 and 7 p.i. (
Virus titers in throat (black bars) and nose swabs (grey bars) are displayed for inoculated (Panel A and C) and exposed ferrets (Panel B and D). The geometric mean titers of positive samples are displayed and the error bars indicate the standard deviations. The number of positive exposed animals per day is depicted. The lower limit of detection is indicated by the dotted line.
The bronchial and bronchiolar epithelium from ferrets in both groups showed slight multifocal necrosis with moderate intra-epithelial infiltrates of neutrophils and multifocal peribronchiolar infiltration of macrophages, lymphocytes, neutrophils and plasma cells. The lumina contained moderate amounts of mucus mixed with cellular debris and few neutrophils. The tracheal epithelium in both groups showed mild neutrophilic infiltrates. The severity of both bronchiolitis and tracheitis increased from day 4 to 7 p.i. in ferrets infected with both viruses, but the differences in scores between groups were minimal (
Individually housed ferrets were inoculated with virus isolate NL/2631-R223 or NL/602 and naïve animals were placed in a cage adjacent to each inoculated ferret at day 1 p.i. to allow aerosol or respiratory droplet transmission. All inoculated ferrets started to shed virus at day 1 p.i. with virus titers up to 106 TCID50/ml in throat and nose swabs (
The naïve ferrets became infected, because of aerosol or respiratory droplet transmission, 1, 2 or 3 days p.e. In the naïve animals, virus was detected in 4 (NL/602), or 3 (NL/2631-R223) out of 4 animals (
When the multi-cycle replication kinetics were studied of viruses with or without the I223R substitution in MDCK cells, it was noticed that the recombinant virus in which the I223R mutation was introduced, recNL/602-I223R, replicated to lower titers than its parental virus recNL/602 (
Virus titers in throat (black bars) and nose swabs (grey bars) are displayed for inoculated (Panel A and C) and exposed ferrets (Panel B and D). The geometric mean titers of positive samples are displayed and the error bars indicate the standard deviations. The number of positive exposed animals per day is depicted. The lower limit of detection is indicated by the dotted line.
Here, a 2009 pandemic influenza A/H1N1 virus isolate, harboring an I223R multidrug resistance mutation, was characterized by studying its replication capacity in MDCK cells and its pathogenicity and transmissibility in the ferret model. This I223R mutant virus is not attenuated for replication in the ferret respiratory tract and transmitted as well as NAI susceptible reference virus NL/602. Furthermore, it was demonstrated here that compensatory mutations for the I223R mutation are not required, since recombinant NL/602 with a single I223R change transmitted as efficiently as its parental virus in ferrets.
To date, 2009 pandemic viruses with an amino acid substitution at position 223 have only sporadically been isolated from patients. A I223V/H275Y double mutant was detected in two closely residing patients who were treated with OS
In a pathogenesis experiment, no statistical significant differences were found when weight loss was compared of ferrets inoculated with clinical isolates NL/2631-R223 or NL/602 (
Both macroscopic and microscopic evaluation of the lungs of the ferrets at day 4 p.i., revealed no major differences in the percentage of affected lung tissue and relative lung weights between NL/2631-R223 and NL/602 (
Despite the moderate pathogenicity of NL/2631-R223, this virus transmitted to 3 out of 4 exposed animals via aerosols or respiratory droplets (
When the impact of the single I223R mutation in the recombinant NL/602 backbone on
These results suggested that compensatory mutations may be required to accommodate the isoleucine to arginine substitution at position 223 in NA and emphasizes the importance of the viral backbone used to study resistance-associated mutations. However, when recNL/602-I223R was tested in the ferret transmission model, the virus transmitted to 2 out of 2 exposed animals (
Although these results suggest that introduction of the I223R does not attenuate the virus, it cannot be ruled out that other mutations than 223R in NL/2631-R223 may have compensated for the initial loss of fitness due to the I223R mutation. Sequence comparison revealed 5 amino acid differences between NL/2631-R223 and NL/602. The only amino acid substitution that is located near the active site of the neuraminidase is at position 248, where NL/602 harbors an aspartic acid and NL/2631-R223 an asparagine. Interestingly, neighboring residue 247 has been linked to NAI resistance in combination with the H275Y mutation
To note, small differences between NL/602 and recNL/602 could be observed in replication capacity and transmission patterns in ferrets (
The different inoculation routes and inoculation doses used for influenza research is subject of debate. The intratracheal route of inoculation is often used to study pathogenicity or to study the efficacy of vaccines to prevent lower respiratory tract infection. In contrast, the intranasal route of inoculation is used when transmissibility is studied. Unfortunately, these inoculation routes and inoculation doses do not accurately mimic the natural way of infection and may mask the fitness differences between the drug-resistant and drug sensitive viruses.
However, the recipient animals in the transmission experiment are infected via the natural route; aerosols or respiratory droplets shed by the donor ferret. The virus secretion pattern, which is the combination of the amount of virus secreted and the duration of virus shedding from the upper respiratory tract, of animals exposed to recNL/602 and rec/NL602-I223R are similar. This suggests that no marked differences in viral fitness are introduced by the single I223R mutation.
The present study demonstrates for the first time that a 2009 pandemic A/H1N1 clinical isolate containing a resistance mutation at position 223 in the NA is not attenuated in its replication capacity and transmissibility in a ferret model. Although the pathogenicity of this virus seems less severe compared to a relevant reference virus in the ferret model, it is unclear whether this moderate pathogenicity has implications for infections with multidrug-resistant viruses in humans. Continuous surveillance is needed to monitor the emergence of (novel) influenza viruses with reduced susceptibility to the NAIs or mutations that may facilitate the emergence of circulating multi drug resistant influenza viruses.
Animals were housed and experiments were conducted in strict compliance with European guidelines (EU directive on animal testing 86/609/EEC) and Dutch legislation (Experiments on Animals Act, 1997). All animal experiments were approved by the independent animal experimentation ethical review committee ‘stichting DEC consult’ (Erasmus MC permit number EUR1821) and were performed under animal biosafety level 3+ conditions. Animal welfare was observed on a daily basis, and all animal handling was performed under light anesthesia using ketamine to minimize animal suffering. Influenza virus seronegative 6-month-old female ferrets (
Madin-Darby Canine Kidney (MDCK) cells were obtained from American Type Culture Collection. MDCK-SIAT1 cells, constitutively expressing the human 2,6-sialyltransferase (SIAT1), were kindly provided by Professor H.D. Klenk, Philipps University Marburg
Virus titers in nasal and throat swabs, homogenized tissue samples, or samples for replication curves were determined by endpoint titration in MDCK cells. MDCK cells were inoculated with 10-fold serial dilutions of each sample, washed 1 hour after inoculation with phosphate-buffered saline (PBS), and grown in 200 µl of infection medium, consisting of EMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1.5 mg/ml sodium bicarbonate, 10 mM HEPES, nonessential amino acids, and 20 µg/ml trypsin (Lonza). Three days after inoculation, the supernatants of inoculated cell cultures were tested for agglutinating activity using turkey erythrocytes as an indicator of virus replication in the cells. Infectious-virus titers were calculated from 4 replicates by the method of Spearman-Kärber
Multi-cycle replication curves were generated by inoculating MDCK or MDCK-SIAT1 cells at a multiplicity of infection (MOI) of 0.001 50% tissue culture infectious dose (TCID50) per cell. One hour after inoculation, at time point 0, the cells were washed once with PBS, and fresh infection medium was added. The supernatants were sampled at 6, 12, 24, and 48 h post infection and the virus titers in these supernatants were determined by means of endpoint titration in MDCK cells.
The pathogenesis experiment was done as described previously with some minor changes in the protocol
Necropsy was done by opening the thoracic and abdominal cavities and examining all major organs. Whilst inflated, all lung lobes (left cranial lobe, left caudal lobe, right cranial-, middle- and caudal lobes and accessory lobe) were evaluated. The extent of consolidation was estimated by visual assessment. The lungs were weighed after the trachea was removed at its bifurcation. The relative lung weights were calculated as proportion of the body weight on day of death (lung weight/body weight x 100). Tissues (∼0.4 g) from the right lung were collected for determination of lung virus titers at day 4 and 7 p.i. The left lung and trachea were collected for histological examination, and immersed for fixation in 10% neutral-buffered formalin. All samples were sectioned in a standardized way (a total of 4 lung sections per animal; 1 cross section and 1 longitudinal section from both the left cranial and left caudal lobe, and 1 central tracheal cross section) and routinely processed, paraffin embedded and cut to 4 µm hematoxylin and eosin (H&E) stained slides. The samples were histologically examined for the character and severity of influenza virus–associated lesions without knowledge of the identity of the animals. The extent of alveolitis/alveolar damage (0 = 0%, 1 = <25%, 2 = 25-50%, 3 = >50% of a section) and the severity of alveolitis, bronchi(oli)tis (including bronchial submucosal glands) and tracheitis (0 = none, 1 = few, 2 = moderate number, 3 = many inflammatory cells) were scored per slide. The overall histology score for alveolitis is the sum of the scores for the extent and severity of the alveolitis (score 0 to 6).
The transmission experiments were done as described previously
For the pathogenesis experiment, statistical analysis was done for each time point, until 4 days after inoculation (when there were still 6 animals present in each group). The Mann-Whitney-U test was used to compare weight losses and virus shedding of the six animals in both groups.
We thank Salin Chutinimitkul, Erin Sorrell, Dennis de Meulder and Peter van Run for excellent technical assistance.