Deletions of neuraminidase and resistance to oseltamivir may be a consequence of restricted receptor specificity in recent H3N2 influenza viruses
© Gulati et al; licensee BioMed Central Ltd. 2009
Received: 07 January 2009
Accepted: 14 February 2009
Published: 14 February 2009
Influenza viruses attach to cells via sialic acid receptors. The viral neuraminidase (NA) is needed to remove sialic acids so that newly budded virions can disperse. Known mechanisms of resistance to NA inhibitors include mutations in the inhibitor binding site, or mutations in the hemagglutinin that reduce avidity for sialic acid and therefore reduce the requirement for NA activity.
Influenza H3N2 isolates A/Oklahoma/323/03 (Fujian-like), A/Oklahoma/1992/05 (California-like), and A/Oklahoma/309/06 (Wisconsin-like) lost NA activity on passage in MDCK cells due to internal deletions in the NA-coding RNA segment. The viruses grow efficiently in MDCK cells despite diminished NA activity. The full length NA enzyme activity is sensitive to oseltamivir but replication of A/Oklahoma/323/03 and A/Oklahoma/309/06 in MDCK cells was resistant to this inhibitor, indicating that NA is not essential for replication. There was no change in HA activity or sequence after the NA activity was lost but the three viruses show distinct, quite restricted patterns of receptor specificity by Glycan Array analysis. Extensive predicted secondary structure in RNA segment 6 that codes for NA suggests the deletions are generated by polymerase skipping over base-paired stem regions. In general the NA deletions were not carried into subsequent passages, and we were unable to plaque-purify virus with a deleted NA RNA segment.
H3N2 viruses from 2003 to the present have reduced requirement for NA when passaged in MDCK cells and are resistant to NA inhibitors, possibly by a novel mechanism of narrow receptor specificity such that virus particles do not self-aggregate. These viruses delete internal regions of the NA RNA during passage and are resistant to oseltamivir. However, deletions are independently generated at each passage, suggesting that virus with a full length NA RNA segment initiates the first round of infection.
Influenza viruses have two membrane bound surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA is involved in virus attachment to cell surface receptors and mediates entry of the virus into the cell by a membrane fusion process. NA is required for virus release. The enzyme catalyses cleavage of the α-ketosidic linkage between a terminal sialic acid and an adjacent sugar residue. The removal of sialic acid from the carbohydrate moiety of newly synthesized hemagglutinin and neuraminidase is necessary to prevent aggregation of the virions at the cell surface [1, 2]. This receptor-destroying role assumes similar specificity of HA and NA, and there are several reports describing reciprocal changes in HA affinity and NA activity [3–5]. However, the specificities of HA and NA are not always matched [2, 6, 7]. We previously showed that a Fujian-like virus, A/Oklahoma/323/03, does not elute from red blood cells by its own NA activity or even with Vibrio cholerae sialidase , indicating that NA activity does not cleave the receptor bound by the HA. Efficient growth of A/OK/323/03 in tissue culture suggested that either the non-cleavable receptor of red blood cells is not present, or the virus is not dependent on receptor destroying activity. We have now shown that A/Oklahoma/323/03 and several subsequent isolates accumulate large internal deletions of the neuraminidase coding sequence. The resulting loss in NA activity has no detrimental effect of growth of the viruses in MDCK cells.
We used four H3N2 influenza viruses that were isolated in primary rhesus monkey kidney (RMK) cells from throat swabs in the winters of 2003, 2005, 2006 and 2008. All the isolates grew to high yield (HA titer = 16–64) in the first passage when transferred to Madin-Darby canine kidney (MDCK) cells. The HA and NA sequences showed that A/Oklahoma/323/03 is similar to the A/Fujian/411/02 vaccine strain  while A/Oklahoma/1992/05, A/Oklahoma/309/06 and A/Oklahoma/483/08 are closely related to H3N2 vaccine strains A/California/7/04, A/Wisconsin/67/05 and A/Brisbane/10/07 respectively.
NA activity of A/OK/323/03 decreased on passage
We passaged A/Oklahoma/1992/05 five times under the same conditions and found ~50% decrease in NA activity but no change in HA titer or in the titer of released virus during the passages. With this virus, the decrease in NA activity occurred under both limiting dilution and moi 0.01 conditions.
Deletions in the NA gene
Single internal deletions in the NA genes of H3N2 viruses isolated from 2003 to 2008.
1 to ...
... to 1467
Length of fragment
Mosaic fragments of the NA gene
5' end cRNA
After 1 passage in MDCK cells
1 – 63
1 – 81
1290 – 1467
1 – 108
194 – 324
120 – 190
1227 – 1467
After 3 passages in the presence of oseltamivir
1 – 191
1210 – 1287
1301 – 1411
1419 – 1467
1 – 70
1 – 74
130 – 204
1253 – 1290
1310 – 1447
We plaque purified viruses from several stocks containing deleted NA gene segments but were unable to isolate any virus that lacked a full length NA RNA.
We attempted to follow the generation of the deletions by real-time PCR with SYBR green detection using primer sets that amplified (a) the conserved 3' and 5' end sequences or (b) the middle portion that was deleted. We hoped to see an increase in "a" product as the ends came closer together and a decrease in "b" product as the NA became deleted, either during a single cycle experiment or over multiple cycles, but there was no significant change in the ratios at 6 hr, 18 hr or 72 hr. A better experiment would have used a primer set within the retained 5' or 3' regions rather than spanning the whole segment, but no suitable primers were identified by the ABI software, probably because of the high A (or U) content (33.4%) of influenza mRNA (or vRNA) strands.
A/OK/323/03 and A/Oklahoma/309/06 are sensitive to Tamiflu in the enzymatic assay but resistant in MDCK culture
We passaged A/Oklahoma/323/03 and A/Oklahoma/309/06 three times in the presence of 10 μM oseltamivir. RT-PCR of viral RNA from the last passage showed no trace of the full length NA band and very weak shorter products (Figure 5B). We sequenced some short bands from each virus and found multiple junctions in A/Oklahoma/309/06 similar to those seen after passage without oseltamivir (Table 2). A/Oklahoma/323/03 contained a long internal deletion (192–1209) but also showed two other small deletions (1288–1300 and 1412–1418, Table 2).
Deletions are not randomly occurring in other gene segments
The PCR products shown in Figure 3 were obtained using NA-specific primers, and all the sequences we obtained of subgenomic PCR products were derived from the NA gene segment. Growth of influenza at high multiplicity is known to generate defective (DI) particles containing internally deleted fragments of RNA segments, particularly from the polymerase genes. Therefore it was possible that the deletions we found in NA merely reflected increasing proportions of randomly generated DI viruses. To investigate if the deletions were occurring in all RNA segments we amplified RNA from selected passages by RT-PCR using common primers that will amplify all 8 segments of influenza virus. There were several small bands obtained from the universal primers in addition to those amplified by NA-specific primers but sequences obtained indicated that most were the result of mis-priming. We found only one internal deletion from another gene, which was an internal deletion of 1394 nucleotides from segment 5 RNA, coding for the NP. We conclude that there were few deletions occurring in the other gene segments and that the NA was specifically lost when A/OK/323/03 was passaged at an moi of ~0.01.
Properties and sequence of the HA did not change when NA gene was deleted
The NA-deficient virus stock of A/OK/323/03 shown in Figure 1 yielded no visible full-length NA PCR product and has only background NA activity. To see if the loss of requirement for NA activity was accompanied by a change in HA, we sequenced the HA segment in virus stocks that showed no full length NA segment. There were no changes in the HA sequences when compared to HA of viruses passaged at low multiplicity.
HAs of 2003–2008 H3N2 viruses have different receptor specificities
Minimal oligosaccharides bound by recent H3N2 viruses
Minimal binding motifs
Neu5Acα2–6GalNAc in any context
Secondary structures in NA gene segments
During influenza infection, viruses that lack NA activity usually fail to spread to new cells because they aggregate at the infected cell surface due to binding of the HA to sialic acid on the surface glycoproteins of neighboring virus particles [1, 2]. We previously isolated a mutant of NWS-G70c (H1N9) virus that lacked NA activity due to a large internal deletion in the NA gene, but this virus was selected by adding bacterial sialidase to the growth medium and was dependent on exogenous sialidase for multicycle growth . The low-NA H3N2 viruses reported here do not require added sialidase for efficient growth in MDCK cells. The viruses isolated in 2003, 2005 and 2006 have different susceptibilities to deletions in the NA segment. A/OK/323/03 retained full NA segment length and full NA activity when passaged strictly under limiting dilution conditions (moi ~10-5), but generated internal deletions of the NA gene when passaged at higher moi of 0.01, with different deletions at each passage. A/OK/1992/05 generated deleted NA segments that increased with passage number, but there was only slight difference if the passages were made at limiting dilution or at 0.01 moi. A/OK/309/06 and A/OK/483/08 showed deletions even after a single passage in MDCK cells. The 5' and 3' ends were always retained, in accord with these being the location of specific packaging signals [13, 14]. We were unable to plaque-purify viruses with deleted NA gene; there was always full length NA present. This suggested there was some advantage of either full length NA gene or NA activity. All these viruses were resistant to oseltamivir in replication assays but the NA was sensitive in the enzyme assay, suggesting NA activity is not required and therefore the requirement might be for the full-length NA-coding RNA segment.
The deletions are targeted to the NA gene segment
The PCR reactions in Figures 1 and 3 were carried out with NA-specific primers, so there was a possibility that the deletions we saw in NA were just a subset of general genome-wide deletions. It is well known that high-multiplicity passage of influenza virus leads to accumulation of defective-interfering (DI) particles containing sub-genomic RNAs resulting from internal deletions, most commonly in the polymerase gene segments [15–19]. We looked for subgenomic RNAs derived from other gene segments by using PCR primers with the common 3' and 5' gene segment sequences. We found one non-NA subgenomic RNA with an internal deletion in the NP RNA segment. Other sub-genomic bands resulted from mis-priming, presumably due to the short sequences (12 nt and 13 nt) used to start the amplification. We conclude that the large number of NA deletions we found result from lack of selective pressure to retain NA when these viruses replicate in MDCK cells.
Do H3N2 viruses delete NA because they have low receptor affinity or narrow specificity?
The 2003–2008 viruses used for these experiments all agglutinate human red blood cells with a similar avidity, giving titers of 16–64 when grown in MDCK cells. The specific HA titers of purified virus preparations were not significantly different (3–4 log2HAU per μg viral protein). However, glycan array analysis showed different specificities. A/OK/309/06 had the broadest specificity, followed by the 2003–5 viruses, while A/OK/483/08 was restricted in its binding to sialylated polylactosamines of at least 5 sugars (Figure 6 and Table 3). There was no correlation between the variety of glycans bound and the ease of generating deletions in NA. The signal strength is similar when similar amounts of virus are applied to the array. If the diversity of glycans on the surface of an MDCK cell is similar to that on the array we might expect that affinity, and NA dependence, would correlate with diversity of binding, but there is little information on the glycans present on MDCK cells. The greatest loss in NA was seen in A/OK/323/03, which is intermediate in binding specificity. The HA sequence did not change in viruses as they deleted the NA segment.
NA is not always a receptor-destroying enzyme
If the only function of NA is to cleave receptors bound by the HA, then HA and NA activity of a viable virus should be matched quantitatively (Kd of HA and Kcat of NA) and qualitatively (same specificity). Several studies have found that lower NA activity can be compensated by low HA avidity [4–7, 20, 21] leading to conclusions that the activities are balanced. However, these studies did not consider specificity of binding and release. Many recent H3N2 viruses do not elute from red cells by their NA activity, showing that the specificities of HA and NA are mismatched . The NA activity of A/OK/323/03 can cleave α2–6-linked sialic acid from the trisaccharide sialyllactose but does not release the virus from red cells, indicating that the HA binds to high affinity ligands that have a structure that is resistant to viral NA activity but sensitive to sialidase from M. viridifaciens . The hemagglutinating site on N9 NA shows similar resistance to cleavage by viral NA activity .
Oseltamivir inhibits the NA activity of A/OK/323/03 and A/OK/309/06 but does not inhibit virus growth in MDCK cells (Figure 5). Mechanisms that allow virus propagation in the presence of NA inhibitors include mutations in the drug binding site of NA, as in the resistance of recent H1N1 human isolates that carry the H274Y mutation, but in the 2003–2008 viruses studied here the NA enzyme activity is fully sensitive to oseltamivir inhibition. A/OK/309/06 was resistant to oseltamivir after a single passage in MDCK cells, suggesting the potential exists for resistance in humans by a mechanism in which NA activity is not required. It has been noted that aggregation of virus particles seen in electron micrographs when influenza virus is grown without NA activity is due to virus-virus interactions rather than the virus-cell interactions [1, 2] and it seems likely that it is more important for NA to cleave sialic acids from the viral glycoproteins (only N-linked glycans) than from the cell surface (N-linked and O-linked glycans and glycolipids). Thus the specificities of HA and NA do not need to be matched and, furthermore, NA is not required if the HA does not bind to sialic acid structures on other HAs. NAs of human viruses have a marked preference for α2–3 linked sialic acid, in contrast to the strict requirement of HA for α2–6 sialylated glycans.
We previously showed that a reassortant H1N9 virus, NWS-G70c, progressively lost NA coding capacity when passaged in the presence of bacterial sialidase and anti-NA antiserum [2, 12, 23]. The result was a group of viruses that had no full-length NA segment and therefore no coding capacity for the active NA enzyme. Each virus had a single internal deletion in the NA gene with retention of at least 100 nucleotides at the 3' end of the genomic RNA and 200 nucleotides at the 5' end . These viruses were dependent on added sialidase for multi-cycle replication and we did not succeed in adapting them to grow without exogenous sialidase. Kawaoka and colleagues replicated these NWS-G70c NA deletions by passaging the virus in MDCK cells that constitutively expressed N2 NA. The resulting NA-deficient virus was gradually weaned off its sialidase requirement and eventually variants were obtained that could grow without added sialidase in MDCK cells, eggs or mice. These viruses had low or zero ability to agglutinate chicken red cells and they had multiple mutations in the HA gene . The authors concluded that adaptation to grow without NA activity required a reduction in receptor binding as occurs in NA inhibitor-resistant mutants that can be released from their receptors by thermal motion and so are not dependent on NA activity [24–27].
Deletions in NA but with no change in HA were reported by Hughes et al.  when an H3N2 virus, A/Tottori/872/94, was passaged in MDCK cells that had reduced surface sialic acid. The authors concluded that the lower density of sialic acids on the cell surface and on the viral glycoproteins resulted in lower avidity, thus allowing virus spread in the absence of NA activity. However, the infectivity was reduced by several orders of magnitude.
The NA-deficient stocks of A/OK/323/03 reported here have very different properties. They were generated by passaging virus in normal MDCK cells without any exogenous sialidase. There was no decrease in infectious titer during these passages and no change in HA activity. Ferraris et al.  also reported H3N2 isolates from 2002 to 2005 that had no detectable NA activity. The 2003–2004 isolates that were further studied were resistant to NA inhibitors and yielded no full-length NA PCR product. The properties that we have described may be common among recent H3N2 viruses. The concern is that these viruses can replicate in the absence (or near absence) of NA activity, and so are resistant to oseltamivir (Figure 5). The OK/323/03 virus only acquired this trait after several passages in MDCK cells, but the other viruses were resistant after a single passage.
What is the selection for full length NA RNA?
The full-length NA gene segment of A/OK/323/03 was never completely lost and we were unable to plaque-purify a virus with no full-length NA segment. This is in accord with the observation that different deletions were found at each passage (Figure 4), suggesting the deletions were generated anew during each passage.
It is important to note that the two conditions of passage used for A/OK/323/03 were not strikingly different. While one was strictly limiting dilution, the "high-multiplicity" passages were inoculated with an moi of about 0.01. We saw no accumulation of deleted polymerase gene fragments, as are routinely found in infections when the moi is >1 [18, 19]. Cairns and Fazakas noted many years ago that if cells are infected at high multiplicity, the virus yield is much lower than if the infection is initiated with one infectious particle, despite the fact that the final cycle of a multi-cycle replication from a single infectious unit occurs at high multiplicity . They also pointed out that the total yield of virus at the end of a multi-cycle replication has an upper limit that is determined by the number of cells, not by the number of infectious particles that initiated the infection, except when the infection is poor due to a high moi. We found that the virus yield, whether measured as infectious units (TCIU) or as particles (HA titration), is the same from 12 passages under 0.01-moi conditions (1000 TCIU per well) as from limiting dilution (~3 TCIU). Therefore replication is not impaired by the higher moi or by the loss of NA activity. Our results could be interpreted either as a positive selection for shorter NA segments, with little or no requirement for NA activity, or as negative selection against NA activity but a requirement for at least the 3' and 5' NA RNA sequences. The total yield of virus per well (105 cells) was 2–3 × 106 TCIUs, so if NA deletions occur at a frequency of 10-4 or 10-5, positive or negative selection may occur during higher multiplicity multi-cycle infections but would not be observed at limiting dilution.
The mechanism of deletion of internal segments of NA requires two parts; a loss of selective pressure to retain NA activity, and a mechanism to generate the deletions. Winter et al proposed that influenza polymerase can generate deletions by jumping across the ends of hairpins and even to other templates [17, 30] and the secondary structure plots generated by mFold (Figure 7) support this idea for the NA deletions, with tertiary structures also involved. The loss of NA, and the ability of the human H3N2 viruses to grow in the presence of an NA inhibitor, is not accompanied by changes in HA and appears to be a new mechanism of resistance to NA inhibitors; lack of requirement for NA activity as a consequence of a lack of binding of HA to sialylated glycans on the viral glycoproteins.
It seems likely that the retention of at least a trace full-length NA RNA segment in our experiments is due to its benefits in packaging rather than the low amount of NA activity that would be provided. The results may be best explained if virus that has packaged eight full-length RNAs is preferentially infectious but undergoes deletion during replication because NA activity is not required.
Viruses and cells
The viruses used in this study were A/Oklahoma/323/03, a Fujian-like isolate (H3N2), , A/Oklahoma/1992/05, a California-like H3N2 isolate, A/Oklahoma/309/06, a Wisconsin-like H3N2 isolate and A/NWS/33(P227H)HA – A/Memphis/31/98NA (NWS-Mem/31/98, H1N2, ). Viruses were grown in Madin-Darby canine kidney (MDCK) cells in DMEM:Ham's F12 medium (1:1) with ITS+ (BD Biosciences) and trypsin added as previously described . For RNA extraction, the virus-containing medium was cleared of cell debris (3,000 g for 5 min) then virus was concentrated by sedimentation (SW28 rotor, 25,000 rpm for 2 hr at 4°). The virus pellet was resuspended in CaMg-saline (0.25 mM CaCl2, 0.8 mM MgCl2 in borate buffered saline, pH 7.2).
Isolation of viral RNA, reverse transcription, and PCR amplification (RT-PCR)
Viral RNA was isolated from the pelleted virus using the QiAmp Viral RNA extraction mini kit (Qiagen). cDNA was synthesized using the Omniscript RT kit (Qiagen) and an oligodeoxynucleotide (5'-AGCAAAAGCAGG) that is complementary to the 12 conserved nucleotides at the 3' end of all influenza type A viral RNA segments. To amplify the N2 NA fragment a pair of N2 NA specific primers were used: 5'-GGGTCGACGCGTTTGAGCAAAAGCAGGAGTGAAAAT, complementary to nucleotides 1–21 at the 3' end of viral RNA (bolded) preceded by a T3 promoter sequence, and 5'-CGGAATTCATTAACCCTCACTAAAAGTAGAAACAAGGAG for the 5' end. The PCR conditions were 94° for 5 min, then 5 cycles of: 94° for 1 min, anneal at 32° for 1 min, extend at 72° for 2 min, followed by 25 cycles at higher temperature for specific binding of NA primers (94° 1 min, anneal at 68° 1 min, synthesis at 72° for 2 min and finally 72° for 7 min). To amplify all the 8 segments of influenza virus we used a pair of universal primers, containing the 12 and 13 nucleotides complementary to the 3' and 5' terminal sequences of all 8 RNA segments, attached to T7 promoter sequences for additional length. The primers were 5'-AATACGACTCACTATAAGCAAAAGCAGG, complementary to the 3' end of viral RNA and 5'-CGGAATTCAATACGACTCACTATAAGTAGAAACAAGG for the 5' end. The PCR conditions were 94° for 20 sec, anneal at 30° for 30 sec, extend at 72° for 5 min for 3 cycles followed by 30 cycles of 94° 20 sec, anneal at 55° for 30 sec, extend at 72° for 15 min, and finally 72° 7 min. The PCR products were separated by electrophoresis on a 1% agarose gel and extracted using the QiA Quick Gel Extraction kit (Qiagen). The purified RT-PCR products were then sequenced in the Oklahoma Medical Research Foundation DNA Sequencing Facility using an ABI 3730 Capillary Sequencer with the NA specific primers for NA or universal primers for the 8 segments.
Hemagglutination and neuraminidase assays
Hemagglutinin titrations were done in 96 well plates using 50 μl of serial dilutions of virus harvested from MDCK cells, and adding 50 μl of 0.8% human red blood cells. The plate was kept at 4°C and agglutination was read at 90 min. Neuraminidase assays were done by fluorescence using 4-methylumbelliferylα-N-acetylneuraminic acid as substrate .
NA/HA ratios and tissue culture infectious units (TCIU)
To relate the NA activity to virus amount we used a ratio of NA to HA. For NA, this was the fluorescence reading generated by 5 μl virus-containing medium in 15 min divided by the log2 of the HA titer of 50 μl of virus. This arbitrary scale allowed comparison with the log(TCIU). One TCIU was estimated as the geometric mean of the last well showing infection in the 10-fold dilution series and the next well. The assays were done immediately after harvesting the virus in MDCK supernatants to minimize any instability in either HA or NA.
Virus growth in the presence of oseltamivir carboxylate
Viruses as serial 10-fold dilutions were used to infect MDCK cells in the absence or presence of 10 μM to 0.001 μM oseltamivir added to the infection medium. After 3 days incubation at 37°C the cytopathic effect (cpe) was estimated by eye and the HA titer was determined.
Cloning of deleted NA fragments of A/Oklahoma/309/06
We ligated the gel band at about 0.7 kb into pBluescript IIKS+ digested with EcoRV and transformed it into One-shot Top10 competent cells (Invitrogen). Three clones were sequenced using primers T7 Promoter 5' TAATACGACTCACTATAGGG complementary to the 3' end and M13 5' AACAGCTATGACCAT for the 5' end, as well as M13 5' GTTTTCCCAGTCACGAC complementary to 3' end and M13 5' CAGGAAACAGCTATGAC for the 5' end.
Glycan Array analysis
Viruses were purified by 5–20% sucrose gradient centrifugation and the purification checked by SDS gel electrophoresis. Labeling with Alexa-488 succinimidyl ester (Molecular Probes) was monitored by HA titration and a level chosen that did not result in reduction of HA titer; for these viruses 0.005 μg Alexa per HAU. The reaction conditions were as previously described . After dialysis the samples contained about 50 log2HAU per ml and about 1 mg/ml viral protein. The Glycan Arrays printed on glass slides were run by Core H of the Consortium for Functional Glycomics. Alexa-labeled viruses were diluted empirically and the slides incubated for 1 hour at pH 7, 4°C then washed and fluorescence measured using the standard buffers and procedures of Core H . Increasing concentrations of virus were applied to the same slide so approximate binding curves could be plotted for each glycan.
RNA secondary structure analysis
The program mFold [10, 11] was used to probe for possible secondary structures in RNA segment 6. We compared the results using the default parameters and with the following changes: Percent suboptimality 10 (default 5), Maximum interior/bulge loop size and maximum asymmetry of bulge/loop both 10 (default 30).
The Glycan Arrays were run by Core H of the Consortium for Functional Glycomics and DNA sequencing was done by the OMRF Sequencing Facility, and we thank Jamie Heimburg-Molinaro and Sheryl Christofferson respectively for their excellent service. This work was supported in part by grant AI18203 from the National Institute of Allergy and Infectious Disease to GMA and NIGMS grant GM62116 to Core H. We thank Dr Joseph Waner, OUHSC, for the virus isolates, Dr Warren Kati, Abbott Laboratories, for a gift of oseltamivir carboxylate, and Dr. Ruth M. Hall, University of Sydney, for assistance with population dynamics.
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