Cell culture adaptation of H3N2 influenza virus impacts acid stability and reduces ferret airborne transmission

Airborne transmission of seasonal and pandemic influenza viruses is responsible for their epidemiological success and public health burden in humans. Efficient airborne transmission of H1N1 influenza virus relies on receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examine the role of HA pH of fusion on transmissibility of a cell culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decrease the acid stability of the virus from a pH of 5.5 (WT) to 5.8 (mutant). In addition, we observed that this mutant H3N2 virus replicated to higher titers in cell culture but had reduced Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 March 2021 doi:10.20944/preprints202103.0297.v1 © 2021 by the author(s). Distributed under a Creative Commons CC BY license. airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, we demonstrate that the NA segment noncoding regions can impact the pH of fusion of reassortant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA


Introduction
Influenza A viruses cause acute respiratory disease in mammals and birds whereas aquatic avian species and bats act as zoonotic reservoirs. Host tropism is determined by a combination of viral proteins and host factors, which allow for efficient replication and transmission within a given species. Influenza virus particles have two major surface antigens, hemagglutinin (HA) and neuraminidase (NA). The trimeric membrane-bound HA binds sialic acid on receptors and is a major host tropism determinant. Avian HA proteins preferentially bind to a sialic acid with an 2,3-linkage, whereas human viruses an 2,6-linkage [1][2][3]. HA is functionally balanced by the tetrameric receptor-destroying NA. At later stages of infection, NA plays a major function in removing sialic acids from host cell receptors as well as from newly synthesized HA and NA on nascent virions, which are sialylated during the cellular glycosylation process [4,5]. Sialic acid removal by NA prevents virion aggregation and promotes spread to new target cells by preventing binding to the same dying host cell via HA [5]. The functional balance between HA and NA is important to maintain viral fitness as NA needs to be active enough to disaggregate virions upon release but not so much that HA receptor attachment is decreased [6].
Proteolytic cleavage of the HA0 precursor by host proteases produces HA1 and HA2 subunits and reveals the fusion peptide, which is required for membrane fusion [7,8]. Once bound to sialic acids, influenza virus is internalized via receptormediated endocytosis and HA mediates escape from early endosomes in a pHdependent manner. At a fixed pH, HA undergoes an irreversible conformational change, which facilitates fusion of the viral and cellular membranes and releases viral genomes into the host cytoplasm [9]. The stability of HA at low pH varies by host origin of the virus strain. Avian influenza viruses with less acid stable HA proteins undergo this conformational change at a higher pH between 5.5 and 6.2 [10][11][12][13][14][15]. The HA proteins from circulating human influenza viruses are more acid stable at pH 5.0-5.4 [16][17][18]; whereas swine isolates have a higher pH of HA activation between 5.6-5.7 [18,19]. Stability of the HA protein is an important factor in host adaptation infectivity, transmissibility and pandemic potential [18,[20][21][22].  [25].
Plasmid-based reverse genetics. The viruses used in this study were generated by reverse genetics using bi-directional reverse genetics plasmids based of pHW2000 [26]. The plasmids were cloned from cDNA reverse transcribed from the for single A nucleotide inserted after 21 st nucleotide in noncoding region at 5′ end of the NA vRNA. We also used the variant HA and NA plasmids described in [23] as pHW-Perth09-HA-G78D-T212I and pHW-Perth09-NA. These variant plasmids differ in two ways from the initial set of eight: they have noncoding regions from the lab-adapted X-31 strain, and the HA has two amino-acid mutations that were selected after passaging in cell culture (see [23] for details). The virus termed "rPerth WT" was generated from the pHW-Perth09-*-SL series of plasmids, whereas the virus termed "rPerth mutant" used the pHW-Perth09-HA-G78D-T212I and pHW-Perth09-NA plasmids and the other six genes from the pHW-Perth09-*- Transmission studies. Our transmission caging setup is a modified Allentown ferret and rabbit bioisolator cage similar to those used in [27][28][29]. For each study, three ferrets were anesthetized by isoflurane and inoculated intranasally with 10 6 TCID50/500uL of A/Perth/16/2009 WT or mutant virus to act as donor animals.
Twenty-four hours later, a recipient ferret was placed into the cage but separated from the donor animal by two staggered perforated metal plates welded together one inch apart. Recipients were exposed for 14 days. Nasal washes were collected  Table 1.
Serology assays. Analysis of neutralizing antibodies from ferret sera was performed as previously described [27]. Briefly, the microneutralization assay was Agglutination was read and HAI titers were expressed as the inverse of the highest dilution that inhibited four agglutinating units of virus.
Tissue sample collection. Euthanized ferrets were dissected aseptically.
Collection of respiratory tissue were performed in the following order: entire right middle lung, left cranial lung (a portion equivalent to the right middle lung lobe), one inch of trachea cut lengthwise, entire soft palate, and nasal turbinates. Tissues were harvested as described in [28] and frozen at -80 C. Tissue samples were weighed and Leibovitz's L-15 medium (Invitrogen) was added to make a 10% w/v homogenate. Tissues were dissociated using an OMNI GLH (OMNI International Inc) homogenizer and cell debris was removed by centrifugation at 1500 RPMs for 10 minutes. Infectious virus was quantified by TICD50 by the endpoint method [25].
In vitro HA pH inactivation assay. Virus stocks were incubated in PBS adjusted to the indicated pH values for 1 hour at 37 C. The remaining virus was titered by TCID50 by the endpoint method [25]. The pH that reduces the titer by 50% (EC50) was calculated by regression analysis of the dose-response curves. Each experiment was performed in triplicate at least twice.

Results
Viruses with cell culture-adaptive HA mutations replicate better in MDCK cells. A previous study using a 6 Figure 1A). However, the mutant strain (rPerth mutant) was generated from plasmids in which the HA and NA were cloned into reverse genetic plasmids with the noncoding regions from the lab-adapted H3N2 reassortant strain X-31, with the HA also containing the two lab-adaptation mutations G78D and T212I ( Figure 1A).   The pH of inactivation rPerth mutant is higher than that of WT. Cell culture adaptation has been associated with a broader pH range at which HA fuses with the host endosomal membrane early in the life cycle [31][32][33]. Treatment of virions with acidic pH causes HA to undergo a conformational change in HA, which results in premature activation of HA and an irreversible loss of viral infectivity [19]. To determine whether the HA changes associated with cell adaptation impacted HA acid stability, we tested the titer of virus incubated in pH-adjusted PBS after 1 hour ( Figure 3A). The pH of inactivation was expressed as the EC50, which is the pH that reduces the titer by 50%. The EC50 pH of inactivation for rPerth WT was   Figure 3B). These data are consistent with human seasonal H1N1 viruses, which have been shown to have a pH of inactivation <5.5 [34]. The two H3N2 swine viruses tested had EC50 values that were higher (EC50 = 5.88 and 5.68) and more similar to rPerth mutant ( Figure 3B).  H3N2 mutant virus with a higher pH of fusion has reduced airborne transmission to naïve ferrets. HA stability is an important factor that impacts transmission efficiency [18,20,21]. To determine whether pH stability of H3N2

Replication of the rPerth mutant in ferrets is
confers an airborne transmission disadvantage the impact of prolonged MDCK cell passage on the transmission efficiency of the rPerth mutant, we performed two sequential transmission studies with each virus. Three donor ferrets were infected intranasally with either rPerth WT or rPerth mutant and 24 hours later a naïve recipient ferret was placed in the adjacent cage, which has directional air flow from the donor to recipient [27,29]. The naïve recipient was exposed for 14 days. Viral titers in nasal washes were collected every other day and seroconversion was determined on day 14 post-infection. rPerth WT transmitted to 3/3 recipients while the transmission efficiency of the rPerth mutant was reduced to 1/3 recipients (Table 1). All donors and recipients that shed virus in their nasal washes also seroconverted ( Table 1). These results indicate that the cell-adapted rPerth mutant virus has reduced airborne transmission as compared to WT. indicating that X-31 UTR flanked NA segment is necessary but not sufficient to alter the pH of inactivation. These results show that both HA (G78D and T212I) and NA X-31 UTR segments are required but not sufficient alone to increase the pH of inactivation for the rPerth virus.  [20,21]. Evolution of HA is an important player in interspecies transmission and host range expansion. Mutations that stabilize the H5 HA can enhance replication in the upper respiratory tract of mice and ferrets [36][37][38], but result in a concomitant decrease in replication, virulence and transmissibility in its natural avian host [39,40]. Conversely, adaptation of human influenza viruses to a murine host for use as an animal model for influenza virus research, requires adaptive changes in the HA protein to alter receptor preference and decreased acid stability from 5.2-5.3 to 5.6-5.8 [41][42][43].
In HA is an important determinant of influenza virus transmissibility, which needs to remain stable as it travels in the environment between hosts [18,44]. HA proteins that are stable at acidic pH have an advantage because they are less prone to being inactivated within the environment [45][46][47]. Yet, striking a fine pH balance for influenza virus is important because within the host cell a replicative advantage is conferred to viruses that encode a less acid stable HA as this facilitates efficient viral uncoating in endosomes [48]. However, avian virus HA proteins with a higher activation pH value have some advantages over their more acid stable humanadapted HA counterparts. During macrophage infection, less acid stable HA proteins release avian viruses earlier from the endosome to escape lysosomal degradation and allow continued replication [49]. Furthermore, a higher membrane fusion pH has been shown to help avian virus HA proteins avoid detection by the interferon-inducible transmembrane proteins IFITM2 and IFITM3, which restrict virus fusion [50]. Mutations in the head region of rPerth mutant, which arose during repeated passage in MDCK cells, lead to destabilization of HA and raised its activation pH. Similarly, MDCK cell culture adaption of egg-grown H3N2 X-31 virus caused mutations in or near the fusion peptide, which resulted in higher pH of fusion mutants within a few passages [51]. Improving virus growth in cell culture is important for producing high yields of virus for vaccines. This enhanced growth is often associated with a broader pH range of virus-host fusion. Site-directed mutagenesis indicates that mutations in HA2 fusion peptide [32,33,52] and the transmembrane domain [31] can stabilize HA to produce a virus that replicates to higher titers in cell culture. Taken together, these data suggest that cell-adaptation correlates with decreased acid stability.
HA receptor binding and stability are important determinants of transmission but others including polymerase activity, resistance to host countermeasures that restrict influenza virus replication and virus morphology [53]. Influenza viruses are pleiomorphic structures with the two major surface glycoproteins HA and NA being packed closely but irregularly distributed on the surface of the virus particle [54][55][56]. Typically, NA is present in much smaller quantities that HA and it has estimated that influenza virus particles have roughly 300 HA and 20-40 NA proteins [55,57]. HA and NA have opposite functions and a fine balance is required for efficient virus replication as HA binds to sialic acid containing receptors and NA removes sialic acid from host cells. Mutations that alter the NA enzymatic active site or stalk length have been linked to unbalancing the HA/NA relationship [58][59][60]. The activation pH of the rPerth mutant, which carries mutation G78D and T212I in HA and X-31 UTRs flanking the NA segment ( Figure 1A) was altered, however, the HA mutations alone were insufficient to raise the pH of activation.
Similarly, the X-31 UTRs flanked NA segment was insufficient on its own to increase the pH of fusion and needed to be expressed in combination with the mutant HA. The viral gene segment UTRs are essential promoter elements required for initiation of viral replication and transcription. Decreased incorporation of NA in virions has been observed to negatively impact NA activity and compensate for functional differences in HA [61,62]. The balance between HA and NA is critical for influenza virus fitness and a future avenue of research will be determining how this balance is affected by different UTRs.