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Contributed by Robert G. Webster, August 10, 2017 (sent for review March 14, 2017; reviewed by Wendy Barclay, Todd T. Davis, and Jeffrey K. Taubenberger)
Significance
Airborne transmission of influenza A viruses between mammals is not well understood. Our study of a group of similar subtype H1N1 avian influenza viruses (AIVs) isolated at Delaware Bay, USA, revealed genetic markers for airborne transmission in nonstructural genes. Viruses not containing all markers lacked airborne transmissibility in the ferret model. We revealed a role for Nonstructural Protein 1 (NS1) in facilitating airborne transmission that was associated with viral replication in the upper respiratory tract (URT). Non–airborne-transmissible NS1 mutants showed delayed viral maturation, resulting in limited tissue pathology and little virus at the mucosa. Overall, our study has revealed insights into viral replication in the URT associated with the airborne transmissibility of AIVs in mammals.
Abstract
North American wild birds are an important reservoir of influenza A viruses, yet the potential of viruses in this reservoir to transmit and cause disease in mammals is not well understood. Our surveillance of avian influenza viruses (AIVs) at Delaware Bay, USA, revealed a group of similar H1N1 AIVs isolated in 2009, some of which were airborne-transmissible in the ferret model without prior adaptation. Comparison of the genomes of these viruses revealed genetic markers of airborne transmissibility in the Polymerase Basic 2 (PB2), PB1, PB1-F2, Polymerase Acidic-X (PA-X), Nonstructural Protein 1 (NS1), and Nuclear Export Protein (NEP) genes. We studied the role of NS1 in airborne transmission and found that NS1 mutants that were not airborne-transmissible caused limited tissue pathology in the upper respiratory tract (URT). Viral maturation was also delayed, evident as strong intranuclear staining and little virus at the mucosa. Our study of this naturally occurring constellation of genetic markers has provided insights into the poorly understood phenomenon of AIV airborne transmissibility by revealing a role for NS1 and characteristics of viral replication in the URT that were associated with airborne transmission. The transmissibility of these viruses further highlights the pandemic potential of AIVs in the wild bird reservoir and the need to maintain surveillance.
Aquatic birds are a major reservoir for influenza A viruses in nature, such that 16 of the 18 hemagglutinin (HA) subtypes and 9 of the 11 neuraminidase (NA) subtypes can be found in this reservoir (1, 2). The North American wild bird reservoir is important as the causative viruses of two of the four novel human influenza pandemics that have occurred in the past century have origins in this reservoir (3⇓–5). Further, influenza viruses containing genes similar to one of these viruses, the 1918 pandemic strain, continue to circulate in aquatic birds (6). Despite their importance, knowledge about the potential of the viruses in the North American wild bird reservoir to transmit and cause disease in mammals is limited. Our recent studies of subtype H1N1 avian influenza viruses (AIVs) isolated at Delaware Bay, USA, over a period of more than 25 y showed that a majority of the viruses studied caused severe disease in the mouse model (7). More concerning and atypical for AIVs was that all of the viruses we studied were capable of contact transmission in the ferret model of human influenza virus transmission (8). We also identified viruses that could transmit between ferrets via the airborne route (8). This was concerning as these viruses did not appear to require prior adaptation to become transmissible. To investigate airborne transmission, we compared the genomes of viruses that were or were not airborne-transmissible in the ferret model and identified genetic markers that correlated with transmission. These residues were in Polymerase Basic 2 (PB2), PB1, PB1-F2, Polymerase Acidic-X (PA-X), Nonstructural Protein 1 (NS1), and the Nuclear Export Protein (NEP) (8) (Table S1). Our objective was to study these subtype H1N1 AIVs to better understand the airborne transmission of AIVs in mammals.
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Table S1.
Genetic markers for airborne transmission of avian H1N1 influenza viruses isolated in Delaware Bay, USA
Results
Putative Transmission Markers in Avian H1N1 Influenza Viruses Isolated at Delaware Bay, USA, Were Important for Airborne Transmission.
We began by confirming that the genetic markers we previously identified are important for the airborne transmissibility of H1N1 AIVs between mammals using the ferret model (Table S1). It should be noted that we restricted these studies to wild-type viruses due to the current pause on gain-of-function research by the US Government. A/ruddy turnstone/Delaware/300/2009 (H1N1) (DE300), which contained all of the genetic markers and was airborne-transmissible in ferrets in our previous studies (7, 8), again transmitted via the airborne route. Another virus that contained all of the genetic markers, A/ruddy turnstone/New Jersey/AI09_256/2009 (H1N1) (NJ256), also transmitted via the airborne route. A/shorebird/Delaware Bay/558/2009 (H1N1), which did not contain any of these markers, did not transmit via the airborne route (Fig. 1 A–C and G). Two other viruses contained some, but not all, markers. These were A/shorebird/Delaware/170/2009 (H1N1) (DE170) and A/ruddy turnstone/New Jersey/AI09_1299/2009 (H1N1) (NJ1299). Neither of these viruses showed airborne transmission, highlighting the importance of the identified markers (Fig. 1 D, E, and G). NJ1299 lacked genetic markers in the PB2, NS1, and NEP genes, and DE170 only lacked those in the NS1 and NEP genes (Fig. 1G). Because both DE170 and NJ1299 lacked markers in NS1 and NEP and neither was airborne-transmissible, we were interested in these genes, particularly as they have not previously been shown to be important in mammalian transmissibility. As such, we then studied the transmissibility of a virus containing markers in the NS1 and NEP genes but lacking them in PB2, PB1, and PB1-F2: A/ruddy turnstone/New Jersey/AI_09-841/2009 (H1N1) (NJ841). This virus did not transmit via the airborne route, suggesting that markers in NS1 and NEP are important, but not sufficient, for mammalian airborne transmissibility (Fig. 1 F and G).
Fig. 1.
Only those viruses containing all of the putative genetic markers for airborne transmission were transmissible via the airborne route in ferrets. A/ruddy turnstone/Delaware/300/2009 (A) and A/ruddy turnstone/New Jersey/AI09_256/2009 (B) contained all genetic markers for airborne transmission and transmitted via AC. (C) A/shorebird/Delaware/558/2009, which lacked all putative genetic markers, transmitted via DC but not via AC. A/shorebird/Delaware/170/2009 (D) and A/ruddy turnstone/New Jersey/AI09_1299/2009 (E), which contained all putative genetic markers with the exception of those in NS1 or NS1 and PB2, respectively, transmitted by DC but not via AC. (F) A/ruddy turnstone/New Jersey/AI09_841/2009 contained putative genetic markers only in PA-X and NS1, and transmitted by DC but not via AC. Each bar represents the viral titer measured in the nasal wash from a single ferret on the indicated DPI. (G) Summary of transmission data showing putative transmission markers in orange. Viruses that transmitted by AC are shaded in orange, while those that did not are shaded in green (n = 2 inoculated, DC, and AC ferrets per virus). I, inoculated.
To determine if any mutations were present in viruses that transmitted by direct contact (DC) or by airborne contact (AC), we sequenced viruses isolated from DC and AC ferrets infected with DE300 or NJ256. We found no amino acid changes in viruses isolated from infected DE300 DC or AC ferrets or NJ256 AC ferrets. In one DC ferret infected with NJ256, we observed E204D in HA, and in the other NJ256-infected DC ferret, we observed V111L in NS1, K71N in HA, and S559L in HA. Therefore, it would appear that these viruses did not accumulate mutations that may have contributed to airborne transmission. There was also no evidence that differences in airborne transmissibility were due to differences in viral load in either inoculated or DC ferrets, as there were no significant differences in the magnitude or duration of detection of infectious virus titers in nasal washes collected every other day beginning at 2 d postinoculation (DPI). Further, there were no obvious differences in the symptoms exhibited by ferrets inoculated with different viruses in terms of body temperature and weight loss (Fig. S1).
Fig. S1.
There were no significant differences in the body temperature or weights of ferrets in experiments detailed in Fig. 1. Overall there were no significant differences in body temperatures (A–C and G–I) or weights (D–F and J–L) between groups of ferrets involved in the experiments whose results are shown in Fig. 1. (A and D) A/ruddy turnstone/Delaware/300/2009 (H1N1) (DE300). (B and E) A/ruddy turnstone/New Jersey/AI09_256/2009 (H1N1) (NJ256). (C and F) A/shorebird/Delaware/558/2009 (DE558). (G and J) A/shorebird/Delaware/170/2009 (H1N1) (DE170). (H and K) A/ruddy turnstone/New Jersey/AI09_1299/2009 (H1N1) (NJ1299). (I and L) A/ruddy turnstone/New Jersey/AI09_841/2009 (H1N1) (NJ841). Each data point represents the weight or body temperature of a single ferret at each time point [n = 2 inoculated (blue), DC (green), and AC (red) ferrets per virus].
NS1 Residue 213 Was Important for Inhibition of the IFN Response.
Three residues in NS1 were predicted to be important for transmission; 7, 213, and 227. These were located in the RNA binding domain and the C-terminal “disordered tail’ region of the effector domain. Residues 7 and 227 have been associated with viral pathogenicity in mice, but, to our knowledge, no role has been ascribed to residue 213 (9, 10). Segment eight of the influenza virus genome encodes NS1 and NEP in different reading frames (11, 12). As the first 10 residues of NS1 and NEP are shared, NS1 7 and NEP 7 were identical. NS1 213 was within the overlapping coding sequences of NS1 and NEP, but in these viruses, changes at NS1 213 did not alter NEP due to the shifted reading frame. NS1 227 was also in the NS1/NEP overlapping coding sequence. In these viruses, NS1 G227 corresponded to NEP G70 and NS1 E227 corresponded to NEP S70 (Table S1).
NS1 has several known functions, including enhancing the function of the influenza virus polymerase complex and interfering with the mammalian IFN response, an important cellular antiviral response (13). As such, we studied these activities of DE300 and DE170 NS1. We performed polymerase assays using the polymerase complex of DE300 and the NS1 of DE300 or DE170 to determine if there was a difference between the NS1 genes of airborne-transmissible and non–airborne-transmissible viruses. There were no significant differences between the polymerase activity of this complex coexpressed with DE170 or DE300 NS1 (Fig. S2). Interestingly, the NS1 protein of DE300 was less able to inhibit the retinoic acid-inducible gene 1 (RIG-I)–mediated activation of the IFN-β promoter in vitro compared with the DE170 NS1 (Fig. 2). We mutated the residues at these positions in DE300 NS1 to those in DE170 (i.e., L7S, S213P, G227E). L7S and G227E did not have any effect on the activity of DE300 NS1; however, S213P alone reverted the activity of DE300 to that of DE170 (Fig. 2). Therefore, residue 213 appeared critical for this function of NS1.
Fig. 2.
Residue 213 in the NS1 of DE300 was critical for the inhibition of the IFN pathway. The NS1 of DE170, which was not airborne-transmissible in ferrets, inhibited the IFN-β promoter significantly more than the NS1 of DE300, which was airborne-transmissible in ferrets. Residue 213 was critical for this difference, as L7S and G227E did not alter the activity of DE300 NS1 but S213P changed the activity of DE300 NS1 to that of DE170 NS1. The mean ± SEM is shown. Values are normalized to the activity of the IFN-β promoter in the absence of NS1 (i.e., IFN-β promoter activity without inhibition). ***P < 0.001.
Fig. S2.
Activity of the polymerase complex of A/ruddy turnstone/Delaware/300/2009 (H1N1) (DE300) with NS1 from DE300 or A/shorebird/Delaware/170/2009 (H1N1) (DE170) was similar. In an in vitro polymerase activity assay, the NS1 of A/ruddy turnstone/DE/300/2009 (DE300), which was airborne-transmissible in ferrets, did not enhance the activity of the DE300 polymerase complex compared with the NS1 of A/shorebird/DE/170/2009 (H1N1) (DE170), which was not airborne-transmissible in ferrets. The DE300 polymerase complex showed significantly less activity compared with the polymerase complex of A/California/04/2009 (H1N1) (CA/04) and A/Puerto Rico/8/1934 (H1N1) (PR8). Data points show the ratio of firefly and Renilla luciferase activity. The mean ± SEM is shown. ***P < 0.001.
Mutations in NS1 Abrogated Airborne Transmissibility and Altered Viral Replication and Pathology in the Upper Respiratory Tract.
Our transmission studies revealed that markers in NS1/NEP were required, but not sufficient, for airborne transmission (Fig. 1). As we were restricted from conducting gain-of-function research, we used a loss-of-function strategy to study the role of NS1/NEP in airborne transmission using viruses produced via reverse genetics (rg) (14). We constructed an rg version of DE300 (rgDE300), which was airborne-transmissible (Fig. 1A). As position 213 in NS1 appeared important for the suppression of the IFN pathway (Fig. 2), we introduced NS1 S213P into rgDE300 as a loss-of-function mutation to produce the rgDE300 P213 virus. We also studied the transmissibility of A/gull/Delaware/AI09_438/2009 (H1N1) (DE438), which contained all of the markers in PB2, PB1, PB1-F2, and PA-X. In NS1/NEP, DE438 only contained the marker S213 in NS1 (Fig. 3D). Therefore, we assessed the importance of position 213 in NS1 for airborne transmission using a virus containing all transmission markers except NS1 213 (rgDE300 P213) and a virus containing no transmission markers in NS1/NEP with the exception of NS1 S213 (DE438).
Fig. 3.
NS1 was critical for AC, but not DC, transmission in ferrets. (A) The rg virus A/ruddy turnstone/Delaware/300/2009 (H1N1) (rgDE300) transmitted to DC and AC ferrets, as per the wild-type DE300 virus. (B) Introduction of S213P into the NS1 of rgDE300 abrogated AC, but not DC, transmission. (C) NS1 of A/gull/Delaware/AI09_438/2009 (H1N1), which contained S213 in conjunction with S7 and E227, transmitted to DC ferrets, but not to AC ferrets. Each bar represents the viral titer measured in the nasal wash from a single ferret on the indicated DPI. (D) Summary of transmission data showing putative transmission markers in orange. Viruses that transmitted by AC are shaded in orange, while those that did not are shaded in green (n = 2 inoculated, DC, and AC ferrets per virus). I, inoculated.
There were no significant differences in the infectious virus titers in nasal washes obtained from ferrets inoculated with rgDE300, rgDE300 P213, or DE438 and rgDE300 transmitted via the airborne route in a similar fashion to DE300 (Fig. 3A). However, both rgDE300 P213 and DE438 were not airborne-transmissible (Fig. 3 B and C). DE438 was similar to rgDE300 P213 in that there was a lack of transmission to AC ferrets, and the nasal washes of only one DC ferret contained infectious virus titers until 6 DPI (Fig. 3C). Again there were no obvious differences in the symptoms exhibited by these ferrets in terms of body temperature or weight loss (Fig. S3). Further, these viruses showed similar in vitro growth kinetics (Fig. S4). Therefore, NS1 S213 was clearly important for airborne transmission; however, in the context of NS1/NEP, NS1 S213 alone was insufficient for airborne transmissibility.
Fig. S3.
There were no significant differences in the body temperature or weights of ferrets in experiments detailed in Fig. 3. Overall, there were no significant differences in body temperatures (A, C, and E) or weights (B, D, and F) between groups of ferrets involved in the experiments whose results are shown in Fig 3. (A and B) The rg virus A/ruddy turnstone/Delaware/300/2009 (H1N1) (rgDE300). (C and D) The rgDE300 containing NS1 S213P (rgDE300 P213). (E and F) A/gull/Delaware/AI09_438/2009 (H1N1) (DE438). Each data point represents the weight or body temperature of a single ferret at each time point [n = 2 inoculated (blue), DC (green), and AC (red) ferrets per virus].
Fig. S4.
Growth kinetics of A/ruddy turnstone/Delaware/300/2009 (H1N1) (DE300), DE300 generated by rg (rgDE300), rgDE300 containing S213P in NS1 (rgDE300 P213), and A/gull/Delaware/AI09_438/2009 (H1N1) (DE438) were similar. Data points show the mean and SEM of two independent experiments (n = 2 in each experiment). TCID50, tissue culture infectious dose, 50%.
We next studied the respiratory tract of ferrets inoculated with rgDE300, rgDE300 P213, or DE438 to determine if there were any correlations between viral spread and/or pathology that correlated with airborne transmission. We studied these tissues at 5 DPI, 1 d after infectious virus titers were detected in the nasal washes of rgDE300 ACs (Fig. 3A). In the lower respiratory tract, we observed clusters of infected bronchiolar epithelial cells in many lobes of the lung in all inoculated ferrets, with limited spread to alveoli. There were no significant differences between ferrets inoculated with different viruses. However, influenza virus staining of the nasal respiratory epithelium and olfactory neuroepithelium appeared to be more persistent and extensive following inoculation with rgDE300 P213 or DE438, neither of which was airborne-transmissible, compared with rgDE300 (Fig. 4 A–C). Moreover, rgDE300 P213 and DE438 were found predominantly in the nucleus of infected cells in contrast to rgDE300, which was more evenly distributed in both the nucleus and cytoplasm. Immunostaining also revealed less virus antigen at the mucosal surface in ferrets inoculated with rgDE300 P213 or DE438 compared with ferrets inoculated with rgDE300 (Fig. 4 A–C). The rgDE300 P213- and DE438-infected cells appeared rounded up and exfoliated; however, apoptosis/necrosis was rare in comparison to infected cells in the rgDE300-infected mucosa, in which they were numerous (Fig. 4 D–I). NS1 has been shown to influence apoptosis in vitro, although both apoptotic and antiapoptotic roles have been observed (15, 16). Overall, these data suggest that differences in intracellular maturation in infected mucosa may have resulted in less virus at the nasal mucosal surfaces in ferrets inoculated with rgDE300 P213 or DE438, compared with ferrets inoculated with rgDE300. However, infectious virus titers in nasal washes from ferrets inoculated with these viruses were similar. This is suggestive of a disconnect between airborne transmissibility and infectious viral titers in nasal washes.
Fig. 4.
Virus at the mucosal surfaces of the respiratory epithelium and olfactory neuroepithelium was critical for airborne transmission. More virus staining was observed at the mucosal surfaces of the nasal respiratory epithelium and olfactory neuroepithelium of ferrets 5 DPI with the rg virus A/ruddy turnstone/Delaware/300/2009 (H1N1) (rgDE300) (A) compared with ferrets inoculated with rgDE300 containing P213 in NS1 (rgDE300 P213) (B) or A/gull/Delaware/AI09_438/2009 (H1N1) (DE438) (C). The rgDE300 P213 and DE438 were found predominantly in the nucleus of infected cells, while rgDE300 was found in the cytoplasm and the nucleus. Apoptotic/necrotic cells were numerous in these tissues in ferrets inoculated with rgDE300 (D and G), while they were rare in ferrets inoculated with rgDE300 P213 (E and H) or DE438 (F and I). Sections were stained with anti-H1N1 influenza virus antibody (brown staining) (A–C), with hemotoxin and eosin (D–F), or with caspase 3 (brown staining) and hematoxylin (blue staining) (G–I) (n = 2 ferrets per virus). (Scale bars: 50 μm.)
As NS1 is known to modulate the immune response, we examined the expression of cytokines and chemokines in the respiratory tract of inoculated ferrets using quantitative RT-PCR. There appeared to be greater expression of IFN-α and IFN-β mRNA in the nasal turbinates of ferrets inoculated with rgDE300 compared with ferrets inoculated with rgDE300 P213 or DE438 (Fig. S5 A and B). The expression of IFN-γ mRNA also appeared greater in the nasal turbinates of rgDE300- and rgDE300 P213-inoculated ferrets compared with DE438-inoculated ferrets (Fig. S5C). The expression of these IFNs in the tracheas of these animals appeared similar (Fig. S5 D–F). Further, there were also no significant differences in the expression of selected cytokines/chemokines in four different lobes of the lung (Fig. S6). While these data are suggestive of a correlation between IFN expression in the upper respiratory tract (URT) and airborne transmission, variability and small sample sizes (n = 2) limited analysis of these data.
Fig. S5.
No significant differences were evident in cytokine gene expression in the URTs of inoculated ferrets. Quantitative real-time PCR on RNA isolated from the nasal turbinates (A–C) or tracheas (D–F) of ferrets inoculated with A/ruddy turnstone/Delaware/300/2009 (H1N1) derived by rg (rgDE300), A/gull/Delaware/AI09_438/2009 (H1N1) (DE438), or rgDE300 containing S213P in NS1 (rgDE300 P213) did not reveal any significant differences in the expression of IFN-α (A and D), IFN-β (B and E), or IFN-γ (C and F) genes at 5 DPI (n = 2 ferrets per virus). RQ, relative quantitation.
Fig. S6.
No significant differences were evident in cytokine gene expression in four different lobes of the lungs of inoculated ferrets. Quantitative real-time PCR on RNA isolated from four different lobes of the lungs (shown as black, gray, orange, and red data points) of ferrets inoculated with A/ruddy turnstone/Delaware/300/2009 (H1N1) derived by rg (rgDE300), A/gull/Delaware/AI09_438/2009 (H1N1) (DE438), or rgDE300 containing S213P in NS1 (rgDE300 P213) did not reveal any significant differences in the expression of the following cytokine genes at 5 DPI: IFN-α, IFN-β, and IFN-γ (A–C); TNF-α (D); or IL-2, IL-6, IL-8, and IL-10 (E–H). (E) Note that data were not obtained for IL-2 gene expression from one lobe (n = 2 ferrets per virus). RQ, relative quantitation.
H1N1 AIVs Containing All Putative Genetic Markers for Transmission Were Only Isolated at Delaware Bay, USA, in 2009.
We isolated 36 H1N1 AIVs from shorebirds during our surveillance at Delaware Bay, USA, during the period of 1990–2015, but the majority of these viruses were isolated in 2009 (17 H1N1 AIVs). Further, putative transmission markers were only common in H1N1 AIVs isolated in 2009 (Table S1). Only one marker, G31 in PB1-F2, was present in H1N1 AIVs isolated after 2009. To investigate the origins of these markers, we conducted phylogenetic analysis on these 36 H1N1 AIVs to determine if gene segments in airborne-transmissible viruses had different origins compared with those in non–airborne-transmissible viruses. Overall, the gene segments found in airborne-transmissible AIVs did not cluster distinctly from those found in non–airborne-transmissible AIVs (Fig. S7). Further, there were no indications that gene segments associated with airborne transmission were phylogenetically related to those of prior pandemic H1N1 viruses (Fig. S7).
Fig. S7.
Influenza virus gene segments in airborne-transmissible viruses did not appear to have unique origins. Gene segments in airborne-transmissible viruses did not appear to have origins unique from those in non–airborne-transmissible viruses, and they were not more closely related to prior pandemic H1N1 viruses or putative swine precursor viruses to the 2009 pandemic viruses. (A–H) Neighbor-joining trees were constructed using the full nucleotide sequences of the PB2, PB1, PA, HA, NP, NA, matrix (M) and nonstructural (NS) gene segments, respectively. Branches show the percentage of replicate trees in which associated taxa clustered together in the bootstrap test using 1,000 replicates, and evolutionary distances were calculated using the Poisson correction method. The 36 H1N1 avian influenza A viruses (AIVs) isolated in Delaware Bay, USA, from 1990 to 2015, inclusive, as shown in Table S1, are labeled in red in these trees. Viruses that were airborne-transmissible are labeled with orange dots, and viruses that were not airborne-transmissible are labeled with green dots.
Discussion
Airborne transmission between humans is an important phenotype of pandemic influenza viruses that is incompletely understood. Our study has shown that AIVs in the wild bird reservoir are capable of airborne transmission in mammals without prior adaptation in an intermediate host, indicating that the pandemic potential of these viruses is greater than currently appreciated. We have also studied a role for NS1 in facilitating mammalian airborne transmission that is linked to aspects of viral replication in the URT that, to our knowledge, have not been previously characterized.
Several physiological differences, or species barriers, in avian species that limit AIV infection and transmission in humans have been identified. These include the higher temperature and the expression of different sialic acid linkages in the avian enteric tract, the primary site of viral replication in avian species, compared with the human respiratory tract (17⇓–19). Well-characterized adaptations in the influenza virus surface glycoprotein genes HA and NA and in the internal PB2 gene have been linked with overcoming these species barriers (20⇓⇓⇓–24). These include E627K in PB2, associated with replication in the mammalian respiratory tract, and E190D and G225D in HA (H3 numbering), associated with preferential binding to human-type sialic acid linkages (25⇓⇓–28). The viruses studied here did not contain these mutations. Further, the transmission markers identified in these viruses were not in the surface glycoproteins of the virus and did not include other residues previously linked to mammalian transmissibility of AIVs. Further, mutations known to be associated with changes in HA sialic acid binding preferences were not detected in the consensus sequence of the HA gene of DE300 viruses isolated from infected AC ferrets. We previously demonstrated that DE300 showed binding to both α2,3- and α2,6-linked sialic acids and that selection for α2,6-linked sialic acid binding seemed to occur with airborne transmission (8). Surprisingly, we did not detect any mutations in HA indicative of changes in sialic acid binding preferences using Sanger sequencing. Therefore, it is possible that changes in the minor variant populations may have impacted the overall sialic acid binding preferences of DE300 isolated from AC ferrets. Future studies using deep sequencing are likely to reveal if this is the case. Overall, the determinants of airborne transmissibility of these viruses appeared to be outside those classically associated with this phenotype.
NS1 was a critical component of the airborne transmission phenotype of these viruses, such that a single residue change was sufficient to abrogate airborne transmission. Owing to their immunomodulatory activities, NS1, PB1-F2, and PA-X have been linked to improving replication in mammals, but not directly linked to airborne transmission (29⇓⇓–32). Here, we have shown that NS1 had a clear impact on viral replication and pathology in the URT that was likely important for airborne transmission. Overall, there appeared to be some impediment to the intracellular maturation of non–airborne-transmissible viruses that resulted in less virus at the mucosal surface in the URT. However, these differences were not reflected in the magnitude or duration of detection of infectious virus titers in the nasal washes of inoculated ferrets, suggesting that another aspect of viral shedding may have been affected.
In summary, we have demonstrated the importance of these markers for airborne transmission. We have also identified a role for NS1 as a facilitator of airborne transmission in these viruses and characterized how mutations in NS1 impacted viral replication in the URT. That this naturally occurring constellation of gene segments conferred airborne transmissibility to these viruses without adaptation further reinforces the pandemic potential of AIVs in the wild bird reservoir and the need to maintain surveillance.
Experimental Procedures
Viruses and Cells.
Viruses used to inoculate ferrets were minimally passaged in 10-d-old embryonated chicken eggs (7). Virus titers were determined by measuring the egg infectious dose, 50% (EID50). The viruses generated by rg in this study were produced using the eight-plasmid rg system and propagated in eggs as described previously (14). Viruses generated by rg were sequenced to ensure that there were no sequence differences compared with the wild-type virus. The A549 cells were maintained in Kaighn’s Modification of Ham’s F-12 Medium (American Type Culture Collection) containing 10% FBS (Invitrogen), l-glutamine (Invitrogen), and penicillin/streptomycin (Invitrogen). The 293T cells were maintained in OptiMEM containing 5% FBS (Invitrogen). Sanger sequencing was used to determine if any mutations were present in viruses isolated from ferrets compared with the virus stock used for inoculation.
Ferret Experiments.
Three- to four-month-old outbred male ferrets (Mustela putorius furo) were purchased from Triple F Farms. All experiments were conducted in an animal biosafety level 2+ (ABSL2+) facility in compliance with the policies of the National Institutes of Health and the Animal Welfare Act and with the approval of the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee (protocol no. 428, approved September 2015). Inoculated ferrets were anesthetized using 3% isoflurane (2% oxygen) and inoculated intranasally with 106 EID50 of virus in 1 mL of PBS. At 1 DPI, one DC ferret was placed in the same cage with each inoculated ferret and one AC ferret was placed in an adjacent cage separated by a wire grill. Six ferrets were used per virus: two inoculated, two DCs, and two ACs. All ferrets were monitored daily for weight loss, body temperature, and clinical signs of influenza for 16 DPI. Every second day, ferrets were lightly anesthetized with 40 mg/kg ketamine administered intramuscularly, and nasal wash specimens were collected using 1 mL of sterile PBS as described previously (7). Viral titers in nasal wash specimens were determined by EID50.
Histology and Immunohistochemistry.
Ferrets were euthanized according to institutional protocols. Whole lungs, tracheas, and nasal turbinates were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. Sections were stained using hematoxylin and eosin for histology or an antiinfluenza virus H1N1 polyclonal antibody (US Biologicals) for immunohistochemistry. In vitro immunohistochemistry performed on Madin–Darby canine kidney cells infected with these viruses did not reveal any differences in the specificity of this antibody. Cleaved caspase 3 was detected following antigen retrieval at 100 °C for 32 min on a Discovery Ultra immunostainer (Ventana Medical Systems). Slides were stained with a rabbit polyclonal anti–caspase-3 antibody (Biocare Medical), followed by goat anti-rabbit polyclonal antibodies labeled with a proprietary hapten nitropyrazole (OmniMap anti-Rb HRP; Ventana Medical Systems). Color was then developed with ChromoMap 3,3′-diaminobenzidine tetrahydrochloride chromogen, and slides were then counterstained with hematoxylin (all from Ventana Medical Systems). Sections were scored in a blinded fashion.
IFN-β Promoter Activity Assay.
Plasmids expressing the IFN-β promoter, firefly luciferase, the Renilla luciferase plasmid reporter containing a thymidine kinase promoter (Promega), RIG-I, and the NS1 gene in question were cotransfected in 293T cells using XtremeGene HP (Promega) (33). Cells were harvested at 24 h posttransfection, and luciferase activity was measured using the Dual-Luciferase Reporter Assay kit (Promega) as per the manufacturer’s instructions. Firefly luciferase activity was normalized to that of Renilla luciferase. Experiments were performed in triplicate.
Quantitative PCR.
RNA was extracted from ferret tissues or cells in TRIzol (Invitrogen) using the Direct-zol RNA MiniPrep Kit (Zymo Research). One microgram of RNA was used for RT using SuperScript III reverse transcriptase (Invitrogen) and random primers (Invitrogen). Quantitative PCR was performed using SYBR Green PCR master mix (QIAGEN) and a 7500 Fast Quantitative PCR System (Applied Biosystems). The housekeeping gene β-actin was used as an endogenous control, and relative quantification values were calculated by comparing inoculated groups with uninoculated groups.
Statistical Analysis.
Data were graphed and analyzed using GraphPad Prism (GraphPad Software). Statistical analyses were performed by ANOVA. P values of <0.05 were deemed statistically significant.
SI Methods
Polymerase Activity Assay.
The 293T cells were cotransfected with pHW2000 plasmids expressing the PB2, PB1, PA, and nucleoprotein (NP) of A/ruddy turnstone/Delaware/300/2009 (H1N1) (DE300), A/California/04/2009 (H1N1) (CA/04), or A/Puerto Rico/8/1934 (H1N1) (PR8). The nonstructural gene (NS1) from DE300 or A/shorebird/DE/170/2009 (H1N1) (DE170) was also included with DE300 plasmids in some transfections (DE300 + DE300NS1 and DE300 + DE170NS1, respectively). The firefly luciferase reporter pYH-Luc and the Renilla luciferase reporter plasmid containing a thymidine kinase promoter were cotransfected in each reaction using XtremeGene HP transfection reagent (Roche). Cells were harvested 24 h posttransfection, and luciferase activity was read using the Dual-Luciferase Reporter Assay System (Promega) as per the manufacturer’s instructions. Renilla was used as a transfection control to normalize data.
Growth Kinetics.
Madin–Darby canine kidney (MDCK) cells (American Type Culture Collection) were inoculated at a multiplicity of infection of 0.001 in duplicate wells in six-well plates. Cells were washed before inoculation, and virus was allowed to adsorb for 1 h, after which cells were washed and incubated with infection medium [MEM supplemented with 5% (vol/vol) BSA (Sigma) containing 1 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone trypsin]. At specified time points, an aliquot of culture medium was removed from the cells and tissue culture infectious dose, 50% titers were determined in MDCK cells using the Reed and Muench method (34).
Quantitative PCR.
RNA was extracted from ferret tissues or cells in TRIzol (Invitrogen) using the Direct-zol RNA MiniPrep Kit (Zymo Research). One microgram of RNA was used for RT using SuperScript III reverse transcriptase (Invitrogen) and random primers (Invitrogen). Quantitative PCR was performed using SYBR Green PCR master mix (QIAGEN) and a 7500 Fast Quantitative PCR System (Applied Biosystems). The housekeeping gene β-actin was used as an endogenous control, and relative quantification values were calculated by comparing inoculated groups with uninoculated groups.
Phylogenetic Analysis.
All eight viral gene segments were aligned separately in MUSCLE. Neighbor-joining trees were constructed using the full nucleotide coding sequences of each viral gene segment using RAxML. Sequences from A/equine/Prague/1/1956 (H7N7) were used to root trees. RAxML was called as follows: ./raxmlHPC-PTHREADS-SSE3-Mac -T 4 -f a -x 486 -m GTRGAMMA -p 216 -N 100 -o A/equine/Prague/1/1956. Evolutionary distances were computed using the Poisson correction method.
Acknowledgments
We thank Professor David Stallknecht and Rebecca Poulson for generously sharing with us some of the viruses studied here. We thank Jeri-Carol Crumpton, Jennifer DeBeauchamp, and John Franks for expert technical assistance. We thank the staff of the Animal Resources Center at St. Jude Children’s Research Hospital for taking excellent care of the animals. We thank the Veterinary Pathology Core at St. Jude Children’s Research Hospital for their work on the pathology studies. This study was funded by the National Institute of Allergy and Infectious Diseases and the NIH under Contracts HHSN266200700005C and HHSN272201400006C.
Footnotes
↵1Present address: Center for Advanced Studies for Agriculture and Food, Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand.
↵2Present address: Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA 30329.
- ↵3To whom correspondence should be addressed. Email: robert.webster{at}stjude.org.
Author contributions: M.Z., S.-S.W., S.B., C.K., P.V., A.M.-P., R.J.W., and R.G.W. designed research; M.Z., S.-S.W., S.B., P.V., A.R., and A.M.-P. performed research; C.K. and S.K. contributed new reagents/analytic tools; M.Z., S.-S.W., S.B., C.K., P.V., A.R., D.D., R.J.W., and R.G.W. analyzed data; and M.Z., R.J.W., and R.G.W. wrote the paper.
Reviewers: W.B., Imperial College; T.T.D., Centers for Disease Control and Prevention; and J.K.T., NIAID/NIH.
The authors declare no conflict of interest.
See Commentary on page 11012.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1713974114/-/DCSupplemental.
References
Avian H1N1 influenza virus transmission
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