Ionic Strength-Dependent, Reversible Pleomorphism of Recombinant Newcastle Disease Virus

Oncolytic viruses are being developed for cancer therapy, as they selectively target, infect, and kill cancer cells. NDV is particularly attractive because while it is pathogenic to avians (e.g., chickens), it does not cause significant viremia in humans. We have developed a genetically modified recombinant NDV (rNDV) that has much reduced pathogenicity in chickens but is highly oncolytic. The morphology of rNDV transitions from spherical at very low salt concentrations to a heterogeneous population of spherical and elongated virions in isotonic (physiologic salt concentration) and hypertonic solutions. The infectivity (cell-killing activity by infecting cells) of rNDV is unaltered by changes in salt concentration despite morphological changes. These observations are significant for purification and formulation of rNDV, as exposure to different salt concentrations may be needed. Importantly, at physiological salt concentration, relevant to clinical testing, infectivity and, therefore, oncolytic activity will not be compromised despite morphological heterogeneity.

ABSTRACT

A genetically modified, recombinant form of Newcastle disease virus (rNDV) undergoes ionic strength-dependent changes in morphology, as observed by cryo-electron microscopy (cEM). In hypotonic solutions with ionic strengths ranging from < 0.01 to 0.02 M, rNDV virions are spherical or predominantly spherical. In isotonic and hypertonic solutions, rNDV displays pleomorphism and contains a mixed population of spherical and elongated particles, indicating that a change from spherical to elongated shape is induced with increasing salt concentration. This ionic strength-dependent transition is largely reversible, as determined by cEM. Concomitantly, we measured infectious titers of these same rNDV samples at different ionic strengths using a fluorescent focus assay (FFA). The infectivity of oncolytic rNDV was found to be independent of ionic strength, ranging from 0.01 M to approximately 0.5 M. These structural and functional observations, in combination, suggest that infectivity (and, by inference, oncolytic activity) of rNDV virions is fully maintained in their pleomorphic forms.

IMPORTANCE Oncolytic viruses are being developed for cancer therapy, as they selectively target, infect, and kill cancer cells. NDV is particularly attractive because while it is pathogenic to avians (e.g., chickens), it does not cause significant viremia in humans. We have developed a genetically modified recombinant NDV (rNDV) that has much reduced pathogenicity in chickens but is highly oncolytic. The morphology of rNDV transitions from spherical at very low salt concentrations to a heterogeneous population of spherical and elongated virions in isotonic (physiologic salt concentration) and hypertonic solutions. The infectivity (cell-killing activity by infecting cells) of rNDV is unaltered by changes in salt concentration despite morphological changes. These observations are significant for purification and formulation of rNDV, as exposure to different salt concentrations may be needed. Importantly, at physiological salt concentration, relevant to clinical testing, infectivity and, therefore, oncolytic activity will not be compromised despite morphological heterogeneity.

INTRODUCTION

Newcastle disease virus (NDV) is an enveloped, negative-sense, single-stranded, RNA-based avian paramyxovirus (1). It is particularly attractive as an oncolytic agent due to its ability to selectively kill cancer cells combined with a lack of preexisting immunity against this avian virus in a majority of the human population. In addition, a phase I clinical study demonstrated a favorable safety profile following intravenous administration in human patients (2, 3). A recombinant form of NDV has been developed (referred to as rNDV in this paper), starting with a lentogenic strain, introducing genetic modifications to further reduce avian pathogenicity, and adding the granulocyte macrophage colony-stimulating factor (GM-CSF) transgene to enhance its immunotherapeutic potential (4, 5). The therapeutic efficacy of oncolytic viruses is believed to be directly related to their infectivity, as infecting the cancer cells triggers oncolysis. Therefore, accurate measurements of infectious titer under formulation and physiologic conditions are critical to dose determinations. We recently reported development of a high-throughput, semiautomated fluorescent focus assay (FFA) for rNDV (6). Infectious titers, measured by this validated assay as focus-forming units (FFU), were fully concordant with those measured as PFU by a validated plaque assay (6).

Historic observations, although semiquantitative, were as follows. Wild-type NDV (wtNDV) particles appeared roughly spherical in allantoic fluid and when reconstituted in water (7, 8). On the other hand, upon reconstitution of the semipurified virus in normal saline, a significant population of filamentous (elongated) particles was visible (9, 10). Subsequently, a mixed population of filamentous and spherical forms of wtNDV was reported in chicken embryonic cells infected with this virus (11). Pleomorphism has since been reported for other viruses such as influenza (12–15). However, the salt concentration (i.e., ionic strength) dependence of morphology of NDV virions was a unique observation that raised the question of its functional significance, if any. In the context of oncolytic applications of our genetically engineered rNDV, structure-infectivity correlation was especially important. First, we asked if our rNDV underwent the morphological change as a function of ionic strength as was reported for wtNDV. The second question was to determine if infectivity of rNDV was, in turn, impacted by such a change in morphology. The answer to this second question was significant in the context of optimizing process and formulation conditions for maximal oncolytic potential.

We addressed these two questions by measurements of morphology and infectious titer employing, respectively, (i) cryo-electron microscopy (cEM), and (ii) FFA of rNDV under conditions of relatively low, medium (isotonic), and high ionic strength. Ionic strength dependence of rNDV was quantitatively and reproducibly established. Morphology and infectivity were then examined in parallel in exactly the same solutions of identical ionic strengths. These studies indicate that rNDV undergoes an ionic strength-dependent change in morphology, which is reversible, with no significant impact on the in vitro infectivity of the virus.

RESULTS

Determination of morphology as a function of ionic strength.

The dependence of rNDV morphology on ionic strength was evaluated for two independently purified lots (lot 1 and lot 2) as described in “rNDV sample preparation” in Materials and Methods. Morphology of rNDV virions was examined for each lot under three ionic strength (μ) conditions of 0.181 M, 0.018 M, and 0.004 M while maintaining the pH at 7.0 ± 0.1. For each μ, the numbers of elongated and spherical virions counted from multiple high magnification images are listed in Table 1. For both lots, the percentage of spherical virions was 100% at the lowest μ of 0.004. With increasing μ, elongated virions were observed, and they constituted approximately 8 to 10% of the total number of virions at μ of 0.018 M and to approximately 32 to 34% at μ of 0.181 M for both of these two lots of rNDV. We also determined independently that the presence of various amounts of sucrose at a constant ionic strength did not have any effect on morphology (data not shown). The effects reported above were entirely attributable to variations in salt concentration or ionic strength.

TABLE 1.

Ionic strength dependence of rNDV morphology as determined quantitatively by cEM for two independently purified rNDV lots

rNDV lot no.	Shape of virions counted in cEM images	No. of rNDV virions ina (% of total):
		0.4 mM phosphate plus 3.0 mM NaClb	2 mM phosphate plus 15 mM NaClc	20 mM phosphate plus 150 mM NaCld
1	Elongated	1 (0.3)	30 (10.2)	98 (33.7)
	Spherical	301 (99.7)	264 (89.8)	193 (66.3)
	Total counted	302	294	291
2	Elongated	0 (0)	16 (7.7)	88 (31.9)
	Spherical	93 (100)	191 (92.3)	188 (68.1)
	Total counted	93	207	276

^aIonic strength values were calculated for pH 7.0 using a pK_a of 7.2 for monobasic to dibasic ionization for sodium phosphate.

^bpH 7.0; μ = 0.004 M.

^cpΗ 7.0; μ = 0.018 M.

^dpH 7.0; μ = 0.181 M.

Evaluation of reversibility of morphology change of rNDV.

Another independently purified lot (lot 3) of rNDV was used to examine if the ionic strength dependence of morphology was reversible when rNDV ionic strength environment was returned from hypotonic to isotonic or a higher salt concentration. This same lot was also used to test the effect of ionic strength on infectious titer (see “Infectious titer measurements by FFA at different ionic strengths” below). The results of cEM measurements for rNDV samples incubated in pH 7.0, 5-mM sodium phosphate buffer containing different NaCl concentrations are summarized in Table 2. It should be noted that the distribution of sizes of virions, as measured by diameters of spherical particles and area-equivalent diameters of elongated particles, was consistent with the range of sizes reported for NDV virions (16). A progressively higher percentage of elongated virions was observed with increasing ionic strength values ranging from 0.011 M to 0.475 M. A concomitant decrease in the percentage of spherical virions was observed, thereby producing increasing heterogeneity in virion morphology with increasing ionic strength. The percentage of elongated particles increased to 25% and 34% at μ of 0.160 M and μ of 0.475 M, respectively. Although the exact percentages of elongated virions counted (25%) at physiologic ionic strength (μ = 0.160 M) differed to a small extent from those reported in Table 1 for two other rNDV lots at a slightly higher μ of 0.181, the morphological heterogeneity in isotonic (or slightly hypertonic) solution was quantitatively reproduced, and a further increase in transition from spherical to elongated virions with increasing μ was demonstrated. Figure 1A shows an example of mixed morphology under isotonic conditions (μ = 0.16 M).

TABLE 2.

Reversibility of ionic strength dependence of rNDV morphology as determined quantitatively by cEM^a

Shape of virions counted in cEM images	No. of rNDV virions inb (% of total):
	pH 7.0 phosphate-NaCl buffer A0c	pH 7.0 phosphate-NaCl buffer A1d	pH 7.0 phosphate-NaCl buffer B1e	pH 7.0 phosphate-NaCl buffer C1f	rNDV transferred from μ = 0.011 M to μ = 0.275 M buffer, pH 7.0
Elongated	1 (0.4)	5 (1.9)	68 (25.4)	91 (34.0)	59 (22.4)
Spherical	261 (99.6)	253 (98.1)	200 (75.6)	177 (66.0)	204 (77.6)
Total counted	262	258	268	268	263

^aSamples were prepared using a different rNDV lot (lot 3) from those used to collect cEM data reported in Table 1.

^bIonic strength values were calculated for pH 7.0 using a pK_a of 7.2 for monobasic to dibasic ionization for sodium phosphate.

^cμ = 0.011 M.

^dμ = 0.025 M.

^eμ = 0.160 M.

^fμ = 0.475 M.

FIG 1.

Cryo-EM images (×52,000 magnification) of rNDV samples incubated in different salt concentrations and ionic strengths (μ) while maintaining the pH at 7.0. (A) 6.5 mM sodium phosphate, 150 mM NaCl, μ = 0.160 M; (B) 5.3 mM sodium phosphate, 3.0 mM NaCl, 0.2% sucrose, μ = 0.011 M; (C) after incubation at μ = 0.011 M. As in panel B, the rNDV sample was incubated in 5.8 mM sodium phosphate, 258 mM NaCl, 0.06% sucrose, μ = 0.275 M. The purpose was to test if the ionic strength-dependent change in morphology was reversible between spherical and elongated shapes.

To test if the change from heterogeneous to ∼100% spherical morphology observed by transferring rNDV from isotonic to a hypotonic environment is reversed by moving it back to a higher-ionic-strength solution, we made a 1:1 dilution of rNDV in buffer A₀ with buffer C. This dilution resulted in a buffer composition of 5.75 mM sodium phosphate and 0.258 M NaCl (μ = 0.275 M). After incubating the rNDV for 1 h, cEM images were collected, and particles were counted and characterized. The percentage of elongated particles increased from <1% to 22% upon increasing the ionic strength from 0.011 M to 0.275 M, suggesting that the ionic strength-dependent change in rNDV morphology is largely reversible (Table 2, last column). Figure 1B shows a representative image of rNDV morphology (spherical) at μ of 0.011 M. Figure 1C is a representative image of heterogeneity developing in rNDV morphology after the ionic strength of the sample was raised from 0.011 M to 0.275 M. Table 2 shows the quantitative results for morphological transitions in terms of percentages of spherical and elongated virions at all ionic strengths examined. In addition to confirming the ionic strength dependence of morphology, these results (Table 2 and Fig. 1) also indicate that the morphological transition is largely reversible with respect to changes in ionic strength.

Infectious titer measurements by FFA at different ionic strengths.

The stock rNDV, assayed using our standard protocol (method 1) without preincubation in a buffer condition yielded an infectious titer of 9.7-log₁₀ FFU/ml. When rNDV samples prediluted in buffers A₀, A₁, B₁, and C₁ were tested with method 1, titers were identical to the stock rNDV (9.7 ± 0.1 log₁₀ FFU/ml). When stock rNDV was run as a positive control and tested in method 3 (Table 3, bottom row), a reduction in infectious titer to 9.2-log₁₀ FFU/ml was observed. This decrease relative to 9.7-log₁₀ FFU/ml can be attributed to the removal of inoculum from cells after 90 min. Normally in method 1, the inoculum is left on the cells for the entire 16 h and not removed after 90 min. We see the same effect in the samples tested in Vero cell growth media (VCGM) (method 3) as a positive control (Table 3, rows 5 to 8) for each of the buffer conditions evaluated in method 2. Samples tested in method 2 were exposed to their respective ionic strength conditions for 90 min to allow binding of virus before the inoculum was removed and VCGM was added. The change in titer from positive control (Table 3, bottom row) and from each respective buffer sample diluted in VCGM instead of buffer solution (Table 3, rows 1 to 4 versus rows 5 to 8) ranged from 0.0- to 0.5-log₁₀ FFU/ml, which is within the expected variability of 0.5-log₁₀ FFU/ml that was previously established for the FFA assay (6). This study demonstrated that there was no significant loss in infectious titer due to the buffer condition itself or the change in virus morphology. While the titer differences were within the variability of the assay, a small extent of cell death during the 90-min incubation of cell with a hypotonic solution could have also contributed to a small titer drop relative to isotonic solutions.

TABLE 3.

Infectious titers of rNDV incubated in pH 7.0 buffers of different ionic strengths as measured by FFA^a

Method no.	Predilution concn of stock rNDVa in bufferb	Final buffer compositionc ,d	Ionic strength (M)	Serial dilution buffer for FFA set up with initial 90-min incubation	Final titer (log10 FFU/ml) corrected for dilutione	Difference from stock titer (log10 FFU/ml)
2	1:50 in buffer A	Buffer A0	0.011	Buffer A	8.89 ± 0.06	−0.43
	1:10 in buffer A	Buffer A1	0.025	Buffer A	8.93 ± 0.08	−0.40
	1:10 in buffer B	Buffer B1	0.160	Buffer B	9.27 ± 0.12	−0.06
	1:10 in buffer C	Buffer C1	0.475	Buffer C	9.06 ± 0.11	−0.27
3	1:50 in buffer A	Buffer A0	0.011	VCGM	9.21 ± 0.06	−0.11
	1:10 in buffer A	Buffer A1	0.025	VCGM	9.25±0.11	−0.08
	1:10 in buffer B	Buffer B1	0.160	VCGM	9.29 ± 0.10	−0.04
	1:10 in buffer C	Buffer C1	0.475	VCGM	9.18 ± 0.07	−0.15
	None (stock)	VCGM	0.181	VCGM	9.33 ± 0.10

^aStock rNDV was stored frozen at −80^°C in 20 mM phosphate containing 150 mM NaCl and 10% (wt/vol) sucrose at pH 7.0 and thawed shortly prior to dilution and sample preparation for FFA.

^bCompositions of dilution buffers A, B, and C are described in “rNDV sample preparation” in Materials and Methods.

^cpH 7.0.

^dCompositions of final buffers A₀, A₁, B₁, and C₁ (resulting from dilution of stock rNDV into buffers A, B, and C) are described in “rNDV sample preparation” in Materials and Methods.

^eMean and standard deviation values obtained from four independent incubation of samples (inoculum) with cells, including two samples incubated at 75 μl and the other two at 100 μl; n = 4.

DISCUSSION

The data described above allowed us to understand various aspects of the observed shift in virus morphology and its impacts on infectious titers. To further clarify the FFA methods, the objective of infectious titer method 2 (described in “Determination of infectious titer of rNDV by FFA” in Materials and Methods) was to ensure that rNDV maintained its respective ionic strength-dependent morphological state while binding to cells. This procedure would allow us to obtain infectious titer measurements of the virus in the representative morphological population (Table 2). In method 3 (described in “Determination of infectious titer of rNDV by FFA” in Materials and Methods), serial dilutions of rNDV samples prior to incubation with cells were done in VCGM. The purpose was to avoid exposing Vero cells to a nonisotonic environment and, thus, serve as a control for method 2. It is likely that, for all samples using methods 1 and 3, the cells would be in contact with the same rNDV morphology (as they were all diluted into VCGM prior to infection). Based on cEM observation, the ionic strength-dependent change in morphology is largely reversible. The infectious titers observed by all methods were nearly indistinguishable. It is worth noting that within the small range of variability we have seen (within assay error), the highest titer has been reproducibly observed at a physiologic ionic strength, where rNDV exists in a mixed state of spherical and elongated morphology.

Our initial testing of samples incubated in various buffer solutions involved using these buffers as a replacement for VCGM in the FFA. This change resulted in cells being exposed to the buffer solutions for the entire 16-h duration of the assay, leading to various degrees of cell death. Therefore, we introduced the 90-min incubation to allow for initial virus binding to occur while in the respective buffers and to then add VCGM to maintain cell health. We wanted to evaluate if we could use a reduced sample (inoculum) volume to ensure that the virus had an adequate contact duration with cells since we introduced the 90-min incubation. This initial incubation of virus with cells also led us to utilize method 3 as a control for this exposure of cells to buffer solution. We wanted to be certain that any change in potency, if observed, would not be due to an artifact caused by exposure of cells to low or high ionic strength. All sample volumes resulted in titers within 0.5-log₁₀ FFU/ml of the control. However, within this range, the difference in titer compared to control was greater for the 50-μl sample volume than either 75 or 100 μl. We determined that there was no benefit in reducing sample volume. The results we have reported in Table 3 are those for 75- and 100-μl sample volumes, which were indistinguishable within the precision established for our validated FFA, reported earlier (6).

We have also found that gene copy numbers, as measured by quantitative reverse transcriptase PCR (RT-PCR), remain unchanged after incubation at these different ionic strengths (data not shown). Furthermore, the reversibility of ionic strength-dependent change in morphology suggests that the nature of this change is physical and not chemical or genetic. This reversibility permits variations in salt concentrations that may be exploited during the purification process or formulation.

In this study, we have used NaCl to vary ionic strength to be consistent with physiological as well as our formulation conditions. In a recent study of another enveloped RNA virus of a different family (bunyavirus), a potassium ion-dependent change in in vitro infectivity (using A549 cells) was observed (17). This finding was attributed to a conformational change of spike proteins (extension on virus surface) with increased potassium concentration, apparently leading to increased penetration of the virus into host cells. No potassium ion or ionic strength-dependent change in the morphology of the virion was reported. In our study, we observed both elongated and spherical particles with spikes, suggesting a lack of a sodium ion (or ionic strength) effect on spike formation or density. The resolution of our cEM images would not allow detection of small differences (in the low nanometer range) of spike dimensions. The lack of any detectable difference in infectious titers of elongated (higher sodium) and spherical particles (low sodium) is our primary finding, which is consistent with the lack of an ionic strength- or sodium ion-related impact on the ability of NDV to infect cells.

Overall, the conclusion from these results is that the in vitro infectivity of rNDV remains unchanged by exposure to various ionic strengths ranging from nearly 0 to 0.5 M. This result is significant considering that the morphology of rNDV transitions from nearly 100% spherical virions at a very low ionic strength to a heterogeneous population of spherical and elongated particles in isotonic and hypertonic solutions, with elongated morphology being induced as a function of increasing ionic strength. This change in morphology is largely reversible, as suggested by cEM performed after varying the ionic strength environment of the same rNDV sample from 0.011 M to 0.275 M. The ionic strength-dependent morphological transition possibly reflects a physical or mechanical characteristic of the virus. Changes in mechanical properties of viral capsids in response to environmental stress have been discussed on the basis of experimental observations and mathematical modeling (18–20).

Infectious titers of rNDV were unaltered (within the standard deviation of our validated assay) at all of these ionic strengths. Furthermore, reversal of ionic strength of the same rNDV sample in both directions did not affect the infectious titer. The insensitivity of the infectious titer of rNDV to ionic strength implies that elongated and spherical forms of the virus are equally infective. This finding is of particular significance as rNDV is being developed for oncolytic virotherapy. Under the physiological condition of isotonicity, both morphological forms of rNDV are likely to coexist with equal infectivity. Another significance of our finding is that variations in salt concentration that may be required between different bioprocessing steps of the purification process should not affect infectivity of rNDV even though morphological transitions may happen. This reversibility allows for some flexibility in process development and obviates the need for purifying or formulating rNDV in exclusively one morphological form or the other. With this flexibility, one can modify the virus population (by various salt concentrations) without concerns of a negative impact on drug product potency and clinical testing, especially since morphology for clinical testing cannot be adjusted and will revert to the physiological state. This also ensures to us that the potency testing method being utilized in vitro is relevant to the in vivo human trials and is not testing a product that is in a morphology/population that will change once exposed to the physiological environment.

MATERIALS AND METHODS

rNDV sample preparation.

rNDV stock solutions at infectious titers in the range of 9.0- to 10.0-log₁₀ FFU/ml were formulated in buffer solutions containing 20 mM sodium phosphate, 150 mM sodium chloride, and 10% sucrose at pH 7.0 and kept frozen at −80^°C.

Cryo-EM (cEM) experiments were performed on all purified lots of rNDV, including 5 GMP (good manufacturing practices)-produced lots, at the ionic strength provided by the formulation buffer. The purpose was to ensure lot-to-lot consistency in morphology of purified rNDV virions. Two of these lots (lot 1 and lot 2) were initially evaluated for any effect of salt concentration on virion morphology. In each case, lower salt concentrations were achieved by dilution of the freshly thawed stock rNDV in water prior to cEM measurements. Measurements were made at three different ionic strengths (μ), which included the formulation buffer condition, a 10-fold dilution, and a 50-fold dilution of this buffer in water. The pH was maintained at 7.0 ± 0.1. Contributions of dibasic and monobasic sodium phosphate to ionic strengths (μ) for these and all solutions used in cEM and FFA experiments were calculated using a pK_a value of 7.2.

After observing the dependence of morphology on ionic strength for two independently purified rNDV lots, we proceeded to study morphology and infectious titer simultaneously as a function of ionic strength for another rNDV lot (lot 3). For these experiments, samples were prepared as described below.

Frozen rNDV stock from lot 3, stored frozen at an infectious titer in the range of 9.5 ± 0.5-log₁₀ FFU/ml and formulated in 20 mM sodium phosphate, 150 mM NaCl, and 10% (wt/vol) sucrose (pH 7.0), was thawed and diluted 10-fold into either buffer A, B, or C. The compositions of these buffers were as follows: 5.0 mM sodium phosphate, pH 7.0 (buffer A); 5.0 mM sodium phosphate plus 150 mM NaCl, pH 7.0 (buffer B); and 5.0 mM sodium phosphate plus 500 mM NaCl, pH 7.0 (buffer C).

Additionally, a 50-fold dilution into buffer A alone was made to obtain the lowest ionic strength. These dilutions resulted in the following buffer compositions of different ionic strengths (μ) in which rNDV was incubated: 5.3 mM sodium phosphate plus 3 mM NaCl, 0.2% sucrose (wt/vol), pH 7.0, μ = 0.011 (buffer A₀); 6.5 mM sodium phosphate plus 15 mM NaCl, 1% sucrose (wt/vol), pH 7.0, μ = 0.025 (buffer A₁); 6.5 mM sodium phosphate plus 150 mM NaCl, 1% sucrose (wt/vol), pH 7.0, μ = 0.160 (buffer B₁); and 6.5 mM sodium phosphate plus 465 mM NaCl, 1% sucrose (wt/vol), pH 7.0, μ = 0.475 (buffer C₁).

Following this dilution step, rNDV was incubated for 60 min to ensure that any morphological change of rNDV was completed, although this extended duration was not necessary, as earlier cEM experiments suggested that these changes occurred rapidly, i.e., nearly instantaneously after transfer of samples to the respective buffers.

Determination of rNDV morphology by cryo-electron microscopy.

rNDV morphology was studied by cryo-electron microscopy (cEM) performed at Nanoimaging Services (NIS), San Diego, CA. Frozen rNDV stock samples stored in formulation buffer (see “rNDV sample preparation”) were shipped on dry ice and kept at −80°C until they were analyzed. Before imaging, samples were thawed and prepared to obtain hypotonic, isotonic, and hypertonic solution conditions as described above in “rNDV sample preparation.” rNDV concentrations were sufficiently high at these various dilutions for adequate numbers of virions to be seen and counted in the images. Samples were preserved on vitrified ice supported on holey carbon films on 400-mesh copper grids. Sample suspensions placed on grids were kept frozen in liquid nitrogen until imaging.

cEM was performed using an FEI Tecnai T12 electron microscope (serial no. D1100), operating at 120 keV equipped with an FEI Eagle 4K-by-4K-resolution charge-coupled-device (CCD) camera. Vitreous ice grids were transferred into the electron microscope using a cryostage that maintains the grids at a temperature below −170°C.

Images of each grid were acquired at multiple scales to assess the overall distribution of the specimen. After identifying potentially suitable target areas for imaging at lower magnifications, high magnification images were acquired at nominal magnifications of ×110,000 (0.10 nm/pixel), ×52,000 (0.21 nm/pixel), and ×21,000 (0.50 nm/pixel). The images were acquired at a nominal underfocus of −5 μm to −2 μm and electron doses of ∼10 to 25 e⁻/Ų.

A large number of virus particles (virions) from multiple high-magnification cEM images were counted under each buffer condition to determine the percentage of spherical and elongated virions with statistical significance. An elongated particle was defined as one for which the major axis to minor axis ratio was at least 2:1.

Determination of infectious titer of rNDV by FFA.

Vero cells (ATCC CCL-81) were maintained in Vero cell growth media (VCGM) comprised of Dulbecco’s modified Eagle medium (DMEM) containing l-glutamine (Invitrogen; catalog no.11965092) supplemented with 10% fetal bovine serum (FBS; Gibco; catalog no. 26140-087) and 1% penicillin-streptomycin solution (Gibco; catalog no. 10378-016). Ninety-six-well plates were seeded with Vero cells and were grown to > 90% confluence in VCGM. Seeded plates were incubated at 37°C under 5% CO₂. A day later, rNDV samples were prepared (predilutions in buffers A₀, A₁, B₁, and C₁), as described in “rNDV sample preparation.” FFA was continued as described below, depending on the method being used.

Our goal was to determine if the infectious titer of rNDV was affected by salt concentration, i.e., by exposure to hypotonic, isotonic, and hypertonic conditions under which we studied the morphology of the virus. To ensure that any observed effect on virus infectivity was real and did not result from potentially various degrees of cell survival at different salt concentrations, we performed the following three procedures.

(i) Method 1. This method was carried out as published previously (6) to determine the effect on infectious titer after exposure to buffer conditions. rNDV samples prediluted in buffers A₀, A₁, B₁, and C₁ were serially diluted in VCGM. One hundred microliters of each of these diluted samples was added in duplicate to the plate containing phosphate-buffered saline (PBS)-washed cells and incubated at 37°C for 16 ± 0.5 h. This method was only used to determine the stock rNDV titer under our standard assay condition.

(ii) Method 2. This method was used to determine the effect on infectious titer while the virus was maintained in various buffer conditions. rNDV samples prediluted in buffers A₀ and A₁ were serially diluted with buffer A, while those in B₁ and C₁ were serially diluted with buffers B and C, respectively, to maintain very nearly the same salt concentration for each in the next step of incubation with cells. Each of these serially diluted samples was transferred in three different volumes (50, 75, and 100 μl), in duplicate, to separate wells of the cell plate containing PBS-washed cells. The 3 volumes were used to optimize this method, as explained in Discussion. An incubation time of 90 min was allowed at 37°C for binding of the virus to cells. The sample volume was then carefully removed, and 100 μl of VCGM was added. The cells were further incubated at 37°C for another 14.5 ± 0.5 h prior to FFA measurements.

(iii) Method 3. This method was a positive control for method 2 (above) to ensure that the various buffers during the initial 90-min incubation do not impact infectious titer due to a cytotoxic effect. All steps were carried out as described in method 2 except for a change to the dilution buffer. In method 3, rNDV samples prediluted in buffers A₀, A₁, B₁, and C₁ were serially diluted in VCGM (instead of buffers A, B, or C) prior to 90-min initial incubation with cells. This step was performed with multiple rNDV sample volumes as in method 2 (50, 75, and 100 μl).

For all three methods described above, at the end of a total of 16 h of incubation, fluorescence imaging, foci counting, and analysis were performed as previously reported using the ImageXpress Micro XLS from Molecular Devices (6). Results from methods 2 and 3 are reported in Results (“Infectious titer measurements by FFA at different ionic strengths”) and in Table 3.