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Development and optimization of a direct plaque assay for trypsin-dependent human metapneumovirus strains
Accepted Manuscript Title: Development and optimization of a direct plaque assay for trypsin-dependent human metapneumovirus strains Authors: Jiuyang Xu, Yu Zhang, John V. Williams PII: DOI: Reference:
S0166-0934(18)30116-2 https://doi.org/10.1016/j.jviromet.2018.05.012 VIRMET 13471
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
4-3-2018 24-5-2018 24-5-2018
Please cite this article as: Xu J, Zhang Y, Williams JV, Development and optimization of a direct plaque assay for trypsin-dependent human metapneumovirus strains, Journal of Virological Methods (2018), https://doi.org/10.1016/j.jviromet.2018.05.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
HMPV plaque assay
Xu et al.
Development and optimization of a direct plaque assay for trypsin-dependent human metapneumovirus strains
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Jiuyang Xua,b, Yu Zhanga, and John V. Williamsa,‡
Department of Pediatrics, University of Pittsburgh School of Medicine, Children’s
Hospital of Pittsburgh of UPMC, Pittsburgh, PA 15224, USA b
Department of Basic Medical Sciences, Tsinghua University School of Medicine, Beijing
Corresponding Author:
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‡
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100084, China
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John V. Williams, Children’s Hospital of Pittsburgh, Division of Pediatric Infectious
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Diseases, 4401 Penn Ave, Rangos 9122, Pittsburgh, PA 15224, Phone: (412) 692-8298
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Fax: (412) 692-7636, Email: [email protected]
Highlights
A direct plaque assay for HMPV trypsin-dependent strains was developed.
This assay yields similar sensitivity and results with immuno-staining assay.
This assay is economical, timing-saving, and does not require use of antibodies.
This assay is applicable to HMPV from all subgroups and animal tissue samples.
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Summary
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Human metapneumovirus (HMPV) is a non-segmented, negative strand RNA virus belonging to the family Pneumoviridae, previously a subfamily of Paramyxoviridae. It is a leading cause of lower respiratory tract infection in infants, children, and adults with
underlying medical conditions. HMPV grows poorly in cell culture and requires trypsin to cleave and mature the virus particles, which adds to the challenge of HMPV research.
Currently, an indirect immuno-staining assay is commonly used to titrate HMPV, which is time-consuming and costly. In order to simplify virus quantification for HMPV, a direct
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plaque assay was developed. By optimizing trypsin concentration and other
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supplements in the agarose overlay, it was found that HMPV strains from all four
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subgroups formed clear and countable plaques 5-7 days post-infection. Animal tissue homogenate can also be directly titrated with this assay. Compared with the traditional
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assay, the direct plaque assay yields similar titer result, but saves time and eliminates the use of antibodies. Potentially, it can also be applied to plaque purification for HMPV
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clinical isolates. The direct plaque assay will be a valuable tool in HMPV research.
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Keywords: Human metapneumovirus; Plaque assay; Quantification
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1. Introduction
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Human metapneumovirus (HMPV) belongs to the family Pneumoviridae, previously a member of Paramyxoviridae (Adams et al., 2016; Kuhn et al., 2015). Although HMPV was discovered in the year 2001, antibodies against HMPV can be detected in human
serum samples collected more than 60 years ago (van den Hoogen et al., 2001). HMPV is a leading cause of lower respiratory tract infection in children (Williams et al., 2004).
Almost all children have been exposed to HMPV by the age of five (van den Hoogen et al., 2001). HMPV is also among the most commonly detected pathogens in pneumonia-
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associated hospitalization for both adults and children (Jain et al., 2015a; Jain et al.,
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2015b). Despite the burden of HMPV, no antiviral treatment or vaccine is commercially
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available.
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HMPV grows poorly in cell culture, posing a challenge to basic research. Trypsin is usually supplemented in cell culture medium to facilitate virus growth (Tollefson, Cox,
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and Williams, 2010; van den Hoogen et al., 2001). The quantification assays to measure virus titer for HMPV are limited. Most groups use an indirect immuno-staining-
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based plaque assay to titrate infectious virus particles in virus stocks, in which either HMPV anti-sera (Biacchesi et al., 2004; Guerrero-Plata et al., 2006; Williams et al.,
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2005) or monoclonal antibodies targeting F or N proteins (Deffrasnes et al., 2008; Zhang et al., 2012) are used to detect viral antigens. Plaques (virus foci) are visualized
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by staining with conjugated secondary antibodies and substrates. However, the immuno-staining procedure is time-consuming and expensive. Alternatively, viral genomic RNA copy number determined by RT-qPCR is a sensitive method for viral
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RNA detection, but it cannot provide information on the viability of virus. Indeed, it has been reported that HMPV RNA can be detected in mice lungs months after infectious virus is cleared (Erickson et al., 2012). Other quantification methods, including cytopathogenic-effect (CPE)-based TCID50 assays (Hamelin et al., 2005; Zhang et al., 2012) and recombinant viruses encoding reporting genes (de Graaf et al., 2007; Zhou et
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al., 2013), are either less sensitive or limited to specific recombinant strains. An easier, more cost-effective, and universal quantification assay to titrate HMPV is needed. Previously, Zhang et al (Zhang et al., 2012) developed a direct agarose plaque assay for avian metapneumovirus (AMPV), the other member of subfamily
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Metapneumovirinae. The assay can be applied to certain HMPV strains that have selfcleavage sites in F protein and are independent of trypsin. However, most HMPV strains require trypsin to grow, and therefore cannot be titrated with this assay.
In this study, we developed and optimized a direct agarose plaque assay for HMPV trypsin-dependent strains. This assay yields comparable titer results with the
conventional indirect immuno-staining assay. Viral plaques can be visualized and
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counted 5-7 days post-infection by staining with crystal violet. Actinomycin-D, an
inhibitor of cellular but not viral RNA transcription (Reich et al., 1961; Reich et al., 1962),
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can be supplemented in the overlay medium to increase plaque size. We have tested
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2. Materials and methods
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this assay in titrating animal tissue homogenates and various clinical isolates of HMPV.
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2.1 Cells
Rhesus monkey kidney cells LLC-MK2 (ATCC CCL-7) were maintained in OPTIMEM reduced serum medium (Invitrogen) containing 2% heat-inactivated fetal bovine
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serum (FBS). African green monkey kidney cells Vero E6 (ATCC CRL-1586), human bronchial epithelial cells BEAS-2B (ATCC CRL-9609), and human lung epithelial cell
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A549 (ATCC CCL-185) were maintained in DMEM (Corning) containing 10% FBS. The cell culture medium was also supplemented with 2 mM glutamine, 2.5 µg/ml
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amphotericin B, and 50 µg/ml gentamicin. All cells were routinely tested for mycoplasma contamination with a PCR-based detection kit according to manufacturer’s directions (Venor GeM mycoplasma detection kit, Sigma). 2.2 Viruses The HMPV clinical isolates TN/1501/A1, TN/94-49/A2, OH/202/B1, and TN/91320/B2 were originally obtained from nasopharyngeal specimens collected from children 4
with acute respiratory illness in Nashville, TN, and Cincinnati, OH. All viruses were grown in LLC-MK2 cells in serum-free OPTI-MEM supplemented with 1 µg/ml TPCKtreated trypsin (Invitrogen) at 37℃. All clinical isolates were used at <10 passages in cell culture. Purified virus working stocks (around passage 10) were generated by ultra-
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centrifuge on a 20% sucrose cushion as described previously (Williams et al., 2005). The infectious cDNA clone and supporting plasmids of HMPV NL/1/00 and NL/1/99 were kindly provided by Dr. Bernadette G. van den Hoogen (Erasmus Medical Center). Recombinant viruses (rNL/1/00 and rNL/1/99) were generated as previously described (Herfst et al., 2004; van den Hoogen and Fouchier, 2017). Successful virus recovery was confirmed by immunostaining and were propagated in LLC-MK2 cells.
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The recombinant virus rNL/1/00-F bearing a trypsin-independent cleavage site in the fusion (F) protein and rNL/1/00-F-GFP, which contains a green fluorescent protein
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(GFP) insertion at the MluI site in the leader of NL/1/00 strain, were kindly provided by
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Dr. Jianrong Li and generated as described previously (Zhang et al., 2012). The
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recombinant virus rCAN97-83-GFP was purchased from ViraTree (MPV-GFP1) and amplified in LLC-MK2 cells.
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HPIV5 (ATCC-VR288) was propagated in LLC-MK2 cells in OPTI-MEM containing
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2% FBS. HPIV3 (obtained from a nasopharyngeal specimen collected from a child with acute respiratory illness in Nashville, TN) was grown in LLC-MK2 cells in OPTI-MEM supplemented with 1 µg/ml TPCK-treated trypsin for 5 passages before generating the
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sucrose-purified working stock. Recombinant RSV with red fluorescent protein rRSVA2-K-Line19F was kindly provided by Dr. Martin L. Moore (Hotard et al., 2012) and was
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amplified in HEp-2 cells in DMEM containing 10% FBS. 2.4 Indirect immuno-staining assay for HMPV
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The immuno-staining assay was described previously (Williams et al., 2005). In brief,
confluent LLC-MK2 cells in 24-well plates were infected in triplicate with 10-fold serially diluted virus. After 1h incubation at room temperature on a rocking platform, the cells were overlaid with 1 ml overlay medium containing 0.75% methylcellulose in OPTIMEM, supplemented with 5 µg/ml trypsin-EDTA (Invitrogen). After 4-5 days’ incubation
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at 37℃, the monolayers were formalin fixed, rinsed, and stained with guinea pig antiHMPV serum prepared by infecting guinea pigs with sucrose-purified HMPV TN/9449/A2 (Williams et al., 2005) (1:1000 dilution), followed by peroxidase-labeled goat antiguinea pig immunoglobulin (KPL) (1:1000 dilution). Plaques were developed using
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TrueBlue substrate (KPL). 2.5 Direct plaque assay for HMPV
LLC-MK2 cells were seeded in 6-well plates at a density of 1.2x106 cells per well. After overnight incubation at 37℃, the monolayer was washed three times with PBS containing 1mM CaCl2 and inoculated with 10-fold serially diluted virus. After 1h
incubation at room temperature with constant shaking, the monolayer was overlaid with
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2 ml overlay medium containing 0.25% low-melting-point (LMP) agarose (GenePure),
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1.5 µg/ml TPCK-treated trypsin, and other ingredients listed in Table 1. Where indicated, the overlay medium was supplemented with 0.1 µg/ml Actinomycin-D
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(Sigma). All ingredients in Table 1 were prepared in stock solution stored at -20℃ and
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mixed before each assay. The plates were incubated at 4 ℃ for 30 min to allow the agarose to solidify. After incubation at 37℃ for 6 days, the cells were fixed with 10%
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formalin (v/v) for 1h. The agarose overlay was discarded, and the plaques were
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visualized by staining the monolayer with 0.1% crystal violet (w/v) in 20% ethanol (v/v). Alternatively, the monolayer was topped and stained with 2 ml agarose containing
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0.03% neutral red at 37℃ 16h before fixation. Plaque images were taken by FUJIFILM LAS-3000 and quantified by ImageJ FIJI software (Schindelin et al., 2012).
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2.6 Growth kinetics of HMPV LLC-MK2 cells were inoculated with HMPV strain TN/94-49/A2 at a multiplicity of
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infection (MOI) of 0.01 in T25 flasks. The inoculum was removed after adsorption at 37℃ for 1 h. Cells were cultured at 37℃ with OPTI-MEM containing 0.5 µg/ml TrypsinTPCK. Every day, 400 µl supernatant fluid was collected, snap-frozen and stored at 80℃. Fresh medium with trypsin was replenished. Virus titer was determined using either indirect immunostaining assay or direct agarose plaque assay as described above.
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2.7 Animal experiments C57BL/6 mice were purchased from The Jackson Laboratory, and were maintained under specific-pathogen-free environment at the University of Pittsburgh under the approved IACUC protocol number 15025585. Seven-week old female mice were
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anesthetized with 0.5% isoflurane and infected with HMPV strain TN/94-49/A2 at 1.0x106 PFU per animal via intra-tracheal (I.T.) or intra-nasal (I.N.) route. Four days
post-infection, the lung and nasal turbinate (NT) were harvested, homogenized in OPTIMEM, and clarified by centrifuging at 1,200 rpm for 5 min before titration. 2.8 Statistical analysis
Statistical analysis was performed by student’s t-test or one-way ANOVA followed by
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multiple comparison test using GraphPad Prism 6 software. A value of P<0.05 was
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considered statistically significant.
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3. Results
3.1 HMPV formed plaques in an agarose overlay plaque assay in LLC-MK2 cells
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First, we wanted to determine whether HMPV can form plaques under agarose
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overlay. Previously Zhang et al (Zhang et al., 2012) reported a direct plaque assay for a recombinant HMPV strain with self-cleavable F protein. However, they did not observe
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plaque formation by a wild-type HMPV strain with trypsin-dependent F cleavage site. Since higher trypsin concentration has been reported to enhance HMPV growth in cell culture (Tollefson et al., 2010), we hypothesized that higher concentration of trypsin
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would also facilitate HMPV plaque formation. To test this, we performed a direct HMPV plaque assay in LLC-MK2 cells with trypsin-TPCK concentration ranging from 0.2 µg/ml
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to 5.0 µg/ml in overlay medium. BSA and CaCl2 were supplemented in the overlay medium to stabilize the cell monolayer (Table 1). An HMPV A2 strain TN/94-49/A2 was used in this assay. As Fig.1A shows, although viral plaques were barely visible with low trypsin concentration (< 0.4 µg/ml), clear countable plaques were observed with 1.0-2.0 µg/ml trypsin after 7 days incubation. Further increase of trypsin concentration (5.0 µg/ml) caused disruption of the cell monolayer and made it difficult to count the plaques
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on day 7. Similarly, 5.0 µg/ml trypsin also reduced the monolayer integrity at earlier time points (Fig.2A), and thus did not facilitate earlier enumeration although plaque sizes were larger under this condition. We also tested BEAS-2B, A549, and VeroE6 cells for HMPV plaque formation. However, the cell monolayers were all disrupted with 1.0 µg/ml trypsin in overlay medium and no plaques were observed in these cell lines (data not
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shown).
Next, we wanted to characterize the plaque-forming kinetics of HMPV in LLC-MK2 cells. LLC-MK2 cells were infected with serially diluted TN/94-49/A2 and incubated in overlay medium containing 2.0 µg/ml trypsin-TPCK at 37℃ for 3 to 7 days. Visible
HMPV plaques were detected as early as on day 3 post-infection, and clear countable
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plaques began to form on day 5 (Fig. 1B). Increased plaque size and decreased
background were observed over time (Fig.1B-E). The plaque formation was examined
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by light microscopy and multinucleated giant cells were observed around the edge of
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plaques (Fig.1C), suggesting that these plaques were formed by HMPV-induced cell
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fusion. We also counter-stained the plaques by immuno-staining assay and confirmed that cells around the plaques were positive for HMPV antigen (data not shown).
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Although varying incubation time changed plaque sizes greatly, it was found not to alter virus titers calculated on day 5, 6, or 7 post-infection (Fig.1F). This suggests that
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the plaque numbers are consistent after clear countable plaques are formed. Therefore, it is possible to optimize assay time for better plaque morphology without affecting
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titration results.
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3.2 Optimization of agarose plaque assay The conditions of the plaque assay were then optimized to obtain clear plaques in
the shortest time. LLC-MK2 cells were infected by the same amount of virus in each
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well, overlaid with medium containing trypsin-TPCK from 0 µg/ml to 5.0 µg/ml, and incubated for 3 to 7 days (Fig. 2A). Although trypsin supports HMPV plaque formation, high concentrations of trypsin-TPCK (3.0-5.0 µg/ml) disrupted the cell monolayer and increased background. Trypsin-TPCK within 1.0-2.0 µg/ml was found to be optimal because the plaques were clear and the cell monolayer was intact. In addition, there was no significant difference among titers observed within this trypsin concentration
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range (Fig.2B). Therefore, we incubated cells with 1.5 µg/ml trypsin for 6 days, or 2.0 µg/ml trypsin for 7 days in subsequent experiments. Previously, Actinomycin-D (AcD) has been reported to enhance plaque formation for AMPV and trypsin-independent HMPV strains (Zhang et al., 2012). We wanted to test
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whether AcD also accelerates plaque development of trypsin-dependent HMPV strains. AcD at a final concentration of 0, 0.1, or 0.2 µg/ml were included in overlay medium and plaques were fixed and developed under the previously optimized trypsin conditions
(Fig. 3A). Although AcD significantly increased plaque size at 0.1 µg/ml, it disrupted the cell monolayer at 0.2 µg/ml (Fig.3C-D). In addition, this enhancement by AcD did not affect plaque number (Fig.3B). We conclude that AcD at low concentration (e.g. 0.1 µg/ml) can be used to further increase plaque sizes or potentially reduce the
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development time.
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Methylcellulose has also been tested for direct HMPV plaque assay. Previously,
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0.75% of methylcellulose in overlay medium has been used in immuno-staining, and the
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ready-to-use medium is easy to handle. However, 0.75% methylcellulose did not support HMPV plaque formation at conditions optimized for the agarose plaque assay
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(Fig.4A). CPE was present with 5.0 µg/ml trypsin-TPCK, but no plaques were observed.
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As an alternative to low-melting-point (LMP) agarose, non-LMP agarose also facilitated clear HMPV plaque formation at the optimized concentration for LMP agarose (0.1%-0.5%) (Fig.4B). However, non-LMP agarose solidified very quickly at room
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temperature and therefore was more difficult to handle than LMP agarose, especially if multiple plates were processed at the same time. The recipe of 0.25% LMP agarose
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was adopted because it supported clear plaque formation and had a reasonable solidification time (about 30 min at 4℃), compared with more than 1h for 0.1% LMP.
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We also tested neutral red dye as an alternative to crystal violet staining. To stain
the plaques with neutral red, another layer of agarose containing 0.03% neutral red was topped on the overlay agarose plug one day before fixation. After incubation at 37℃ overnight (about 16 h), cells were fixed with 10% formalin and plaques were directly counted without removing the agarose. Neutral red staining was also able to produce
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good contrast between HMPV plaques and background (Fig.4C), thus serving as a good alternative to crystal violet in HMPV plaque assay. 3.3 Comparing direct plaque assay with indirect immuno-staining assay To evaluate its accuracy and reliability, we applied the direct plaque assay to
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measuring the growth kinetics of TN/94-49/A2 in LLC-MK2 cells in parallel with the conventional indirect immuno-staining assay. As shown in Fig.5A, TN/94-49/A2 had an exponential growth phase from day 1 to day 3, and the virus titer dropped on day 4
when massive CPE appeared and cells began to detach (Fig.5C). The growth curve generated by the direct plaque assay was consistent with that by the conventional
indirect assay (Fig.5A). Virus titers from three independent experiments were combined
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to evaluate if the two methods produced equivalent results. The linear regression fit
curve showed a strong correlation between the two assays (slope =0.96±0.03, R2=0.98)
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with the indirect immuno-staining assay.
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(Fig.5B). This shows that the direct plaque assay is accurate, reliable, and equivalent
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3.4 Testing viral burden in mice tissue homogenates To further explore application of the plaque assay, we titrated HMPV in animal
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samples. Seven-week old C57BL/6 mice were infected with 1.0 x 106 PFU TN/94-49/A2
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via intra-nasal (I.N.) or intra-tracheal (I.T.) route. Lung and nasal turbinate (NT) were harvested on day 4 post-infection, and the tissue homogenate was titrated with direct
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plaque assay and indirect immunostaining assay in parallel. As shown in Fig.6A, HMPV strain TN/94-49/A2 was still able to form plaques under agarose overlay despite the presence of inflammatory cytokines in animal samples, although plaques in the lowest
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dilution were smaller in size. Virus titers determined by the direct plaque assay was comparable to those by the indirect immunostaining assay in samples from all four
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groups (Fig.6B). Therefore, the direct plaque assay can be used to titrate animal tissue homogenates. 3.5 Direct plaque assay is applicable to HMPV common laboratory strains and clinical isolates
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Next, we wanted to study whether plaque formation under agarose overlay was strain-specific or universal for HMPV. We chose clinical isolate strains TN/1501/A1, OH/202/B1, and TN/91-320/B2 apart from the reference strain TN/94-49/A2, together representing all four HMPV subgroups. All clinical isolates tested could form countable plaques under the agarose overlay (Fig.7A). Light microscopic images of the plaques
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showed the presence of syncytia around the edge of plaques, suggesting that the
plaques were formed by viral infection (Fig.7C). Recombinant viruses based on the
genomic sequence of several commonly-used HMPV strains were also generated. All recombinant viruses, rNL/1/99, rNL/1/00-F with trypsin-independent cleavage site, rNL/1/00-F-GFP, and rCAN97-83-GFP, formed clear plaques under the agarose
overlay, and syncytia were observed around the plaques (Fig.7B, D). We concluded that
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this direct plaque assay can be applied to multiple strains of HMPV.
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3.6 HPIV3 and HPIV5 but not HRSV interferes with the HMPV direct plaque assay
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Some closely related viruses in the pneumovirus and paramyxovirus families were
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also tested for plaque formation under this assay. We found that human parainfluenza virus type 3 and type 5 (HPIV3 and HPIV5), but not a recombinant strain of human
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respiratory syncytial virus (rRSV-A2-K-Line19F), were able to form clear plaques under the agarose overlay optimized for HMPV (Fig.8A-B). This suggests that this plaque
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assay cannot differentiate HMPV from other closely related pathogens if co-infection
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occurs.
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4. Discussion
Here we reported a direct plaque assay for trypsin-dependent HMPV strains.
Previously, a direct plaque assay was developed for AMPV (Zhang et al., 2012) and
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was applicable to trypsin-independent strains of HMPV. However, most HMPV laboratory strains and clinical isolates have trypsin-dependent F protein cleavage sequences (Yang et al., 2009). In the current study, by increasing trypsin concentration and modifying ingredients in overlay medium, we have developed and optimized a direct agarose plaque assay for HMPV trypsin-dependent strains.
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The incubation time (5-7 days) in direct plaque assay is similar to that of traditional indirect immuno-staining (4-5 days), but the staining time is much shorter; staining the cell monolayer with crystal violet can be finished within one minute, compared with hours of blocking and antibody staining in the indirect assay. Moreover, the direct
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plaque assay conserves resources for antibody/animal anti-sera. The direct plaque assay has wide applications to titrate HMPV in both cell culture and animal tissue homogenates. The assay can detect recombinant viruses derived from several commonly used lab strains, as well as clinical isolates from all four
subgroups. Since the virus is not inactivated in this assay, the direct agarose plaque
assay can also be applied for traditional plaque purification of virus. The main limitation of the direct plaque assay is that it cannot differentiate HMPV from other closely-related
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viruses, thus the indirect antibody-based method is still necessary in cases of co-
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infection.
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Although this assay was developed for trypsin-dependent strains of HMPV, it can
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also be applied to the trypsin-independent strain rNL/1/00-F. The virus with selfcleavage site formed plaques in trypsin-free conditions (data not shown), but the
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addition of trypsin increased the plaque sizes and yielded clearer plaques. This suggests that trypsin should be a routine ingredient in the overlay recipe. We used
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trypsin-TPCK rather than trypsin-EDTA because EDTA chelates divalent cations required for integrin function, and RGD-binding integrins serve as receptors for HMPV
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(Cox et al., 2012; Cox et al., 2015; Cseke et al., 2009). We have noticed a few other factors for HMPV plaque formation. LLC-MK2 cells
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supported HMPV plaque formation in a wide range of initial seeding density (0.5 x 106 to 2.0 x 106 cells/well in a 6-well plate). Within this range, we observed that lower seeding
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density may increase plaque sizes, but it also causes higher background (data not shown). The 1.2 x 106 cells/well density was chosen in all experiments in balance of the plaque size and background signal. We also tested tissue culture treated 6-well plates from three suppliers (TPP, Greiner Bio-one, and Corning Costar). While all three plates supported HMPV plaque formation, we observed differences in plaque sizes and background signal noise among different brands -- HMPV plaques were slightly larger
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and clearer in TPP brand plates (data not shown). Therefore, TPP plates were used in all subsequent experiments. Viral factors also likely contribute to plaque formation, as different HMPV strains form plaques of different sizes under the same conditions. We hypothesize that the ability to form better plaques is correlated with better syncytium formation and in vitro replication. The viral factors that determine plaque size are yet to
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be identified. HMPV TN/94-49/A2 is the best plaque-forming strain tested in this assay, and also grows to higher titer and induces more CPE than other strains in cell culture (data not shown). Therefore, like other common HMPV strains, TN/94-49/A2 is likely partly cell-adapted but may serve as a good tool for HMPV research.
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Conflicts of interest statement
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The authors have no conflicts of interest to declare.
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Acknowledgement
This work was supported by NIH/NIAID grant R01 AI085062-06 to JVW. YZ received
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a Research Advisory Council (RAC) fellowship award from the Children’s Hospital of
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Pittsburgh of UPMC. JX received funding as visiting scholar to the University of Pittsburgh from China Scholarship Council ([2016]3097). We thank Dr. Bernadette G.
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van den Hoogen, Dr. Martin L. Moore, and Dr. Jianrong Li for the HMPV and RSV reverse genetics systems, and we thank Dr. Ursula Buchholz for BSR-T7/5 cells to
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generate recombinant viruses.
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Reference
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Biacchesi, S., Skiadopoulos, M.H., Tran, K.C., Murphy, B.R., Collins, P.L. and Buchholz, U.J., 2004. Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes. Virology 321, 247-59. Cox, R.G., Livesay, S.B., Johnson, M., Ohi, M.D. and Williams, J.V., 2012. The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins. J Virol 86, 12148-60. Cox, R.G., Mainou, B.A., Johnson, M., Hastings, A.K., Schuster, J.E., Dermody, T.S. and Williams, J.V., 2015. Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes. PLoS Pathog 11, e1005303. Cseke, G., Maginnis, M.S., Cox, R.G., Tollefson, S.J., Podsiad, A.B., Wright, D.W., Dermody, T.S. and Williams, J.V., 2009. Integrin alphavbeta1 promotes infection by human metapneumovirus. Proc Natl Acad Sci U S A 106, 1566-71. de Graaf, M., Herfst, S., Schrauwen, E.J.A., van den Hoogen, B.G., Osterhaus, A.D.M.E. and Fouchier, R.A.M., 2007. An improved plaque reduction virus neutralization assay for human metapneumovirus. Journal of Virological Methods 143, 169-174. Deffrasnes, C., Hamelin, M.E., Prince, G.A. and Boivin, G., 2008. Identification and evaluation of a highly effective fusion inhibitor for human metapneumovirus. Antimicrob Agents Chemother 52, 279-87. Erickson, J.J., Gilchuk, P., Hastings, A.K., Tollefson, S.J., Johnson, M., Downing, M.B., Boyd, K.L., Johnson, J.E., Kim, A.S., Joyce, S. and Williams, J.V., 2012. Viral acute lower respiratory infections impair CD8+ T cells through PD-1. J Clin Invest 122, 2967-82. Guerrero-Plata, A., Casola, A., Suarez, G., Yu, X., Spetch, L., Peeples, M.E. and Garofalo, R.P., 2006. Differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus. Am J Respir Cell Mol Biol 34, 320-9. Hamelin, M.E., Yim, K., Kuhn, K.H., Cragin, R.P., Boukhvalova, M., Blanco, J.C., Prince, G.A. and Boivin, G., 2005. Pathogenesis of human metapneumovirus lung infection in BALB/c mice and cotton rats. J Virol 79, 8894-903. Herfst, S., de Graaf, M., Schickli, J.H., Tang, R.S., Kaur, J., Yang, C.F., Spaete, R.R., Haller, A.A., van den Hoogen, B.G., Osterhaus, A.D. and Fouchier, R.A., 2004. Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA. J Virol 78, 8264-70. Hotard, A.L., Shaikh, F.Y., Lee, S., Yan, D., Teng, M.N., Plemper, R.K., Crowe, J.E., Jr. and Moore, M.L., 2012. A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis. Virology 434, 129-36. Jain, S., Self, W.H., Wunderink, R.G., Fakhran, S., Balk, R., Bramley, A.M., Reed, C., Grijalva, C.G., Anderson, E.J., Courtney, D.M., Chappell, J.D., Qi, C., Hart, E.M., Carroll, F., Trabue, C., Donnelly, H.K., Williams, D.J., Zhu, Y., Arnold, S.R., Ampofo, K., Waterer, G.W., Levine, M., Lindstrom, S., Winchell, J.M., Katz, J.M., Erdman, D., Schneider, E., Hicks, L.A., McCullers, J.A., Pavia, A.T., Edwards, K.M., Finelli, L. and Team, C.E.S., 2015a. Community-Acquired Pneumonia Requiring Hospitalization among U.S. Adults. N Engl J Med 373, 415-27. Jain, S., Williams, D.J., Arnold, S.R., Ampofo, K., Bramley, A.M., Reed, C., Stockmann, C., Anderson, E.J., Grijalva, C.G., Self, W.H., Zhu, Y., Patel, A., Hymas, W., Chappell, J.D., Kaufman, R.A., Kan, J.H., Dansie, D., Lenny, N., Hillyard, D.R., Haynes, L.M., Levine, M., Lindstrom, S., Winchell, J.M., Katz, J.M., Erdman, D., Schneider, E., Hicks, L.A., Wunderink, R.G., Edwards,
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K.M., Pavia, A.T., McCullers, J.A., Finelli, L. and Team, C.E.S., 2015b. Community-acquired pneumonia requiring hospitalization among U.S. children. N Engl J Med 372, 835-45. Kuhn, J.H., Dietzgen, R.G., Easton, A.J., Kurath, G., Nowotny, N., Rima, B., Rubbenstroth, D., Vasilakis, N., Walsh, J., Collins, P.L., Fouchier, R.A., Lamb, R., Maisner, A., Randall, R., Rota, P., Wang, L.-F. and Kondo, H., 2015. ICTV taxonomic proposal 2015.011a-gM.A.v2.Pneumoviridae. Elevate subfamily Pneumovirinae (family Paramyxoviridae) to the taxonomic rank of family, named Pneumoviridae, in the order Mononegavirales; change the name of genus Pneumovirus to Orthopneumovirus. Reich, E., Franklin, R.M., Shatkin, A.J. and Tatum, E.L., 1961. Effect of actinomycin D on cellular nucleic acid synthesis and virus production. Science 134, 556-7. Reich, E., Franklin, R.M., Shatkin, A.J. and Tatumel, 1962. Action of actinomycin D on animal cells and viruses. Proc Natl Acad Sci U S A 48, 1238-45. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A., 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-82. Tollefson, S.J., Cox, R.G. and Williams, J.V., 2010. Studies of culture conditions and environmental stability of human metapneumovirus. Virus Res 151, 54-59. van den Hoogen, B.G., de Jong, J.C., Groen, J., Kuiken, T., de Groot, R., Fouchier, R.A. and Osterhaus, A.D., 2001. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 7, 719-724. van den Hoogen, B.G. and Fouchier, R.A.M., 2017. Recovery of a Paramyxovirus, the Human Metapneumovirus, from Cloned cDNA. Methods Mol Biol 1602, 125-139. Williams, J.V., Harris, P.A., Tollefson, S.J., Halburnt-Rush, L.L., Pingsterhaus, J.M., Edwards, K.M., Wright, P.F. and Crowe, J.E., 2004. Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children. N Engl J Med 350, 443-450. Williams, J.V., Tollefson, S.J., Johnson, J.E. and Crowe, J.E., Jr., 2005. The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity. J Virol 79, 10944-51. Yang, C.F., Wang, C.K., Tollefson, S.J., Piyaratna, R., Lintao, L.D., Chu, M., Liem, A., Mark, M., Spaete, R.R., Crowe, J.E., Jr. and Williams, J.V., 2009. Genetic diversity and evolution of human metapneumovirus fusion protein over twenty years. Virol J 6, 138. Zhang, Y., Wei, Y., Li, J. and Li, J., 2012. Development and optimization of a direct plaque assay for human and avian metapneumoviruses. J Virol Methods 185, 61-8. Zhou, M., Kitagawa, Y., Yamaguchi, M., Uchiyama, C., Itoh, M. and Gotoh, B., 2013. Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase. J Clin Virol 56, 31-36.
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Fig.1 HMPV strain TN/94-49/A2 formed plaques in LLC-MK2 cells in an agarose overlay plaque assay. (A) Plaque morphology of HMPV strain TN/94-49/A2 with different trypsin concentrations. LLC-MK2 cells were infected with the same amount of virus (about 200 PFU per well) and overlaid with medium containing indicated amount of trypsin-TPCK. Plates were formalin fixed on day 7 and plaques were visualized by
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staining with 0.1% crystal violet. (B) Plaque morphology of TN/94-49/A2 in LLC-MK2
cells over time. Plaque assay was performed with 2.0 µg/ml trypsin-TPCK and plates
were fixed and stained at the indicated time post-infection. (C) Microscopic images of plaques formed by HMPV over time. Images taken under 2x stereoscope. (D-E)
Quantification of plaques. In each group, twenty single plaques were randomly selected and their sizes were quantified by ImageJ. Data were shown as mean ± SD (D) or
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median with range (E). (F) Quantification of plaque numbers over time. The same
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serially diluted virus samples were inoculated onto LLC-MK2 cells, and plaques were counted on the indicated days post-infection to determine the virus titer. Data shown as
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mean ± SD from three replicates (ns P>0.05).
Fig.2 Optimization of trypsin concentration in overlay medium. (A) LLC-MK2 cells
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were inoculated with the same amount of virus (about 30 PFU per well) on day 0 and
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were incubated with different doses of trypsin-TPCK (0-5.0 µg/ml) in overlay medium. Cells were fixed with 10% formalin at the indicated time (day 3 to day 7), and plaques were visualized by crystal violet staining. (B) Virus titer of TN/94-49/A2 was determined
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under agarose overlay with indicated amount of trypsin. Plaques were counted 6 days
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post-infection. Data shown as mean ± SD from three replicates (ns P>0.05).
Fig.3 Actinomycin-D increases plaque size without changing plaque counts. (A)
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LLC-MK2 cells were inoculated with the same amount of virus (about 30 PFU/well) and were incubated with the indicated amount of trypsin and actinomycin-D (AcD). Plaques were fixed and stained by crystal violet staining at the indicated time post-infection. (B) Virus titer of TN/94-49/A2 was determined by the direct plaque assay with or without AcD, in parallel with the indirect immuno-staining assay. Plaques were counted 6 days post-infection. Data were shown as mean ± SD from three replicates (ns P>0.05). (C-D)
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Quantification of plaques. Twenty single plaques were randomly selected and their sizes were quantified by ImageJ. Data were shown as mean ± SD (C) or median with range (D). Statistical analysis was performed only with mean ± SD (C) for simplicity (* P<0.05 by t-test). Plaques in AcD 0.2 µg/ml group were not quantified because the
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background was too light.
Fig.4 Effect of other overlay ingredients and dye. (A) Plaque morphology of TN/9449/A2 in overlay medium containing either 0.25% agarose or 0.75% methylcellulose.
The overlay medium also contained indicated concentration of trypsin-TPCK. Plaques were visualized on day 7. (B) Plaque formation under low-melting-point (LMP) or nonLMP agarose at indicated concentration. Serially diluted TN/94-49/A2 was inoculated
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onto LLC-MK2 cells and overlaid with media containing indicated amount of agarose.
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The plates were incubated at 4℃ until overlay medium completely solidified before
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being transferred to the 37℃ incubator. (C) Agarose plaque assay was performed as described in Materials and Methods. In crystal violet group, plaques were visualized on
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day 6 by staining with 0.1% crystal violet. In neutral red group, 2 ml 0.5% agarose containing 0.03% neutral red was topped on the overlay medium on day 5, and cells
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were fixed on day 6 to visualize the plaques.
Fig.5 Growth Curve of HMPV TN/94-49/A2 titrated by immunostaining and agarose
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plaque assay. (A) LLC-MK2 cells were infected with TN/94-49/A2 at MOI=0.01 in T25 flasks with 0.5 µg/ml trypsin-TPCK. Every day 400 µl supernatant fluid was collected
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and snap-frozen, and fresh medium containing trypsin was supplemented. Virus titer was determined by direct plaque assay and indirect immune-staining assay in parallel in three independent replicates. Dotted line shows the detection limit of the assay. (B)
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Correlation in virus titer results by two methods was determined by linear regression. The best-fit curve was plotted as a dashed line. (C) Microscopic morphology of LLCMK2 cells infected by TN/94-49/A2 at the indicated time post-infection (10x phase contrast, scale bar 400 µm).
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Fig.6 Direct plaque assay can be applied in animal samples. (A) C57BL/6 mice were infected with 1.0 x 106 PFU TN/94-49/A2 via intra-nasal (I.N.) or intra-tracheal (I.T.) route with 2 mice per group. The lung and nasal turbinate (NT) were harvested on day 4 post-infection. Serially-diluted lung and NT homogenates were inoculated onto LLC-MK2 cells, and direct plaque assay was performed as described in Materials and
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Methods. One representative image is shown for lung and NT samples. (B) Virus titer of lung and NT homogenate in (A) by either indirect immunostaining or agarose plaque assay. Mean ± SD from two replicates are shown.
Fig.7 Other HMPV strains form plaques in direct plaque assay. (A) Morphology of
plaques formed by HMPV isolates TN/1501/A1, TN/94-49/A2, OH/202/B1, and TN/91-
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320/B2 in a direct plaque assay. The overlay medium contained 1.5 µg/ml trypsin-
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TPCK. Plaques were visualized on day 6 by crystal violet. (B) Morphology of plaques formed by recombinant HMPV strains as in (A) (C-D) Microscopic images (10x field) of
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plaques formed by HMPV clinical isolates (C) or recombinant viruses (D).
Fig.8 Some other closely-related viruses also formed plaques in direct plaque
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assay. (A) Plaque morphology of HMPV (TN/94-49/A2), recombinant respiratory
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syncytial virus (rRSV-A2-K-Line19F), human parainfluenza virus type 3 (HPIV3), and human parainfluenza virus type 5 (HPIV5) under agarose overlay. The direct plaque assay was performed with 1.5 µg/ml trypsin-TPCK for 6 days. (B) Microscopic images of
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plaques (10x field) formed by the four viruses in (A).
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Figures:
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Table:
Table 1. Recipe for HMPV direct plaque assay overlay medium Final conc.
Stock
Amount for 100 ml
MEM
1x
2x
50 ml
Glutamine
2 mM
200 mM
Gentamicin
50 µg/ml
50 mg/ml
Amphotericin B
2.5 µg/ml
250 µg/ml
HEPES
25 mM
1 M (pH7.4)
2.5 ml
NaHCO3
0.12%
7.5%
1.6 ml
BSA
0.05%
5%
Agarose
0.25%
1%
diH2O
-
-
Trypsin-TPCK
1.5 µg/ml
2 mg/ml
75 µl
Actinomycin-D (opt.)
0.1 µg/ml
1 mg/ml
10 µl
1 ml
100 µl 1 ml
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1 ml
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‡Abbreviation
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Ingredients‡
25 ml 17.8 ml
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for ingredients: minimum essential media (MEM), 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), sodium bicarbonate (NaHCO3), bovine serum albumin (BSA), deionized water (diH2O), and L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)treated trypsin (Trypsin-TPCK).
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S0166-0934(18)30116-2 https://doi.org/10.1016/j.jviromet.2018.05.012 VIRMET 13471
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
4-3-2018 24-5-2018 24-5-2018
Please cite this article as: Xu J, Zhang Y, Williams JV, Development and optimization of a direct plaque assay for trypsin-dependent human metapneumovirus strains, Journal of Virological Methods (2018), https://doi.org/10.1016/j.jviromet.2018.05.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
HMPV plaque assay
Xu et al.
Development and optimization of a direct plaque assay for trypsin-dependent human metapneumovirus strains
a
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Jiuyang Xua,b, Yu Zhanga, and John V. Williamsa,‡
Department of Pediatrics, University of Pittsburgh School of Medicine, Children’s
Hospital of Pittsburgh of UPMC, Pittsburgh, PA 15224, USA b
Department of Basic Medical Sciences, Tsinghua University School of Medicine, Beijing
Corresponding Author:
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‡
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100084, China
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John V. Williams, Children’s Hospital of Pittsburgh, Division of Pediatric Infectious
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Diseases, 4401 Penn Ave, Rangos 9122, Pittsburgh, PA 15224, Phone: (412) 692-8298
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Fax: (412) 692-7636, Email: [email protected]
Highlights
A direct plaque assay for HMPV trypsin-dependent strains was developed.
This assay yields similar sensitivity and results with immuno-staining assay.
This assay is economical, timing-saving, and does not require use of antibodies.
This assay is applicable to HMPV from all subgroups and animal tissue samples.
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Summary
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Human metapneumovirus (HMPV) is a non-segmented, negative strand RNA virus belonging to the family Pneumoviridae, previously a subfamily of Paramyxoviridae. It is a leading cause of lower respiratory tract infection in infants, children, and adults with
underlying medical conditions. HMPV grows poorly in cell culture and requires trypsin to cleave and mature the virus particles, which adds to the challenge of HMPV research.
Currently, an indirect immuno-staining assay is commonly used to titrate HMPV, which is time-consuming and costly. In order to simplify virus quantification for HMPV, a direct
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plaque assay was developed. By optimizing trypsin concentration and other
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supplements in the agarose overlay, it was found that HMPV strains from all four
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subgroups formed clear and countable plaques 5-7 days post-infection. Animal tissue homogenate can also be directly titrated with this assay. Compared with the traditional
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assay, the direct plaque assay yields similar titer result, but saves time and eliminates the use of antibodies. Potentially, it can also be applied to plaque purification for HMPV
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clinical isolates. The direct plaque assay will be a valuable tool in HMPV research.
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Keywords: Human metapneumovirus; Plaque assay; Quantification
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1. Introduction
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Human metapneumovirus (HMPV) belongs to the family Pneumoviridae, previously a member of Paramyxoviridae (Adams et al., 2016; Kuhn et al., 2015). Although HMPV was discovered in the year 2001, antibodies against HMPV can be detected in human
serum samples collected more than 60 years ago (van den Hoogen et al., 2001). HMPV is a leading cause of lower respiratory tract infection in children (Williams et al., 2004).
Almost all children have been exposed to HMPV by the age of five (van den Hoogen et al., 2001). HMPV is also among the most commonly detected pathogens in pneumonia-
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associated hospitalization for both adults and children (Jain et al., 2015a; Jain et al.,
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2015b). Despite the burden of HMPV, no antiviral treatment or vaccine is commercially
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available.
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HMPV grows poorly in cell culture, posing a challenge to basic research. Trypsin is usually supplemented in cell culture medium to facilitate virus growth (Tollefson, Cox,
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and Williams, 2010; van den Hoogen et al., 2001). The quantification assays to measure virus titer for HMPV are limited. Most groups use an indirect immuno-staining-
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based plaque assay to titrate infectious virus particles in virus stocks, in which either HMPV anti-sera (Biacchesi et al., 2004; Guerrero-Plata et al., 2006; Williams et al.,
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2005) or monoclonal antibodies targeting F or N proteins (Deffrasnes et al., 2008; Zhang et al., 2012) are used to detect viral antigens. Plaques (virus foci) are visualized
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by staining with conjugated secondary antibodies and substrates. However, the immuno-staining procedure is time-consuming and expensive. Alternatively, viral genomic RNA copy number determined by RT-qPCR is a sensitive method for viral
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RNA detection, but it cannot provide information on the viability of virus. Indeed, it has been reported that HMPV RNA can be detected in mice lungs months after infectious virus is cleared (Erickson et al., 2012). Other quantification methods, including cytopathogenic-effect (CPE)-based TCID50 assays (Hamelin et al., 2005; Zhang et al., 2012) and recombinant viruses encoding reporting genes (de Graaf et al., 2007; Zhou et
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al., 2013), are either less sensitive or limited to specific recombinant strains. An easier, more cost-effective, and universal quantification assay to titrate HMPV is needed. Previously, Zhang et al (Zhang et al., 2012) developed a direct agarose plaque assay for avian metapneumovirus (AMPV), the other member of subfamily
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Metapneumovirinae. The assay can be applied to certain HMPV strains that have selfcleavage sites in F protein and are independent of trypsin. However, most HMPV strains require trypsin to grow, and therefore cannot be titrated with this assay.
In this study, we developed and optimized a direct agarose plaque assay for HMPV trypsin-dependent strains. This assay yields comparable titer results with the
conventional indirect immuno-staining assay. Viral plaques can be visualized and
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counted 5-7 days post-infection by staining with crystal violet. Actinomycin-D, an
inhibitor of cellular but not viral RNA transcription (Reich et al., 1961; Reich et al., 1962),
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can be supplemented in the overlay medium to increase plaque size. We have tested
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2. Materials and methods
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this assay in titrating animal tissue homogenates and various clinical isolates of HMPV.
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2.1 Cells
Rhesus monkey kidney cells LLC-MK2 (ATCC CCL-7) were maintained in OPTIMEM reduced serum medium (Invitrogen) containing 2% heat-inactivated fetal bovine
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serum (FBS). African green monkey kidney cells Vero E6 (ATCC CRL-1586), human bronchial epithelial cells BEAS-2B (ATCC CRL-9609), and human lung epithelial cell
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A549 (ATCC CCL-185) were maintained in DMEM (Corning) containing 10% FBS. The cell culture medium was also supplemented with 2 mM glutamine, 2.5 µg/ml
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amphotericin B, and 50 µg/ml gentamicin. All cells were routinely tested for mycoplasma contamination with a PCR-based detection kit according to manufacturer’s directions (Venor GeM mycoplasma detection kit, Sigma). 2.2 Viruses The HMPV clinical isolates TN/1501/A1, TN/94-49/A2, OH/202/B1, and TN/91320/B2 were originally obtained from nasopharyngeal specimens collected from children 4
with acute respiratory illness in Nashville, TN, and Cincinnati, OH. All viruses were grown in LLC-MK2 cells in serum-free OPTI-MEM supplemented with 1 µg/ml TPCKtreated trypsin (Invitrogen) at 37℃. All clinical isolates were used at <10 passages in cell culture. Purified virus working stocks (around passage 10) were generated by ultra-
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centrifuge on a 20% sucrose cushion as described previously (Williams et al., 2005). The infectious cDNA clone and supporting plasmids of HMPV NL/1/00 and NL/1/99 were kindly provided by Dr. Bernadette G. van den Hoogen (Erasmus Medical Center). Recombinant viruses (rNL/1/00 and rNL/1/99) were generated as previously described (Herfst et al., 2004; van den Hoogen and Fouchier, 2017). Successful virus recovery was confirmed by immunostaining and were propagated in LLC-MK2 cells.
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The recombinant virus rNL/1/00-F bearing a trypsin-independent cleavage site in the fusion (F) protein and rNL/1/00-F-GFP, which contains a green fluorescent protein
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(GFP) insertion at the MluI site in the leader of NL/1/00 strain, were kindly provided by
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Dr. Jianrong Li and generated as described previously (Zhang et al., 2012). The
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recombinant virus rCAN97-83-GFP was purchased from ViraTree (MPV-GFP1) and amplified in LLC-MK2 cells.
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HPIV5 (ATCC-VR288) was propagated in LLC-MK2 cells in OPTI-MEM containing
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2% FBS. HPIV3 (obtained from a nasopharyngeal specimen collected from a child with acute respiratory illness in Nashville, TN) was grown in LLC-MK2 cells in OPTI-MEM supplemented with 1 µg/ml TPCK-treated trypsin for 5 passages before generating the
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sucrose-purified working stock. Recombinant RSV with red fluorescent protein rRSVA2-K-Line19F was kindly provided by Dr. Martin L. Moore (Hotard et al., 2012) and was
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amplified in HEp-2 cells in DMEM containing 10% FBS. 2.4 Indirect immuno-staining assay for HMPV
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The immuno-staining assay was described previously (Williams et al., 2005). In brief,
confluent LLC-MK2 cells in 24-well plates were infected in triplicate with 10-fold serially diluted virus. After 1h incubation at room temperature on a rocking platform, the cells were overlaid with 1 ml overlay medium containing 0.75% methylcellulose in OPTIMEM, supplemented with 5 µg/ml trypsin-EDTA (Invitrogen). After 4-5 days’ incubation
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at 37℃, the monolayers were formalin fixed, rinsed, and stained with guinea pig antiHMPV serum prepared by infecting guinea pigs with sucrose-purified HMPV TN/9449/A2 (Williams et al., 2005) (1:1000 dilution), followed by peroxidase-labeled goat antiguinea pig immunoglobulin (KPL) (1:1000 dilution). Plaques were developed using
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TrueBlue substrate (KPL). 2.5 Direct plaque assay for HMPV
LLC-MK2 cells were seeded in 6-well plates at a density of 1.2x106 cells per well. After overnight incubation at 37℃, the monolayer was washed three times with PBS containing 1mM CaCl2 and inoculated with 10-fold serially diluted virus. After 1h
incubation at room temperature with constant shaking, the monolayer was overlaid with
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2 ml overlay medium containing 0.25% low-melting-point (LMP) agarose (GenePure),
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1.5 µg/ml TPCK-treated trypsin, and other ingredients listed in Table 1. Where indicated, the overlay medium was supplemented with 0.1 µg/ml Actinomycin-D
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(Sigma). All ingredients in Table 1 were prepared in stock solution stored at -20℃ and
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mixed before each assay. The plates were incubated at 4 ℃ for 30 min to allow the agarose to solidify. After incubation at 37℃ for 6 days, the cells were fixed with 10%
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formalin (v/v) for 1h. The agarose overlay was discarded, and the plaques were
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visualized by staining the monolayer with 0.1% crystal violet (w/v) in 20% ethanol (v/v). Alternatively, the monolayer was topped and stained with 2 ml agarose containing
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0.03% neutral red at 37℃ 16h before fixation. Plaque images were taken by FUJIFILM LAS-3000 and quantified by ImageJ FIJI software (Schindelin et al., 2012).
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2.6 Growth kinetics of HMPV LLC-MK2 cells were inoculated with HMPV strain TN/94-49/A2 at a multiplicity of
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infection (MOI) of 0.01 in T25 flasks. The inoculum was removed after adsorption at 37℃ for 1 h. Cells were cultured at 37℃ with OPTI-MEM containing 0.5 µg/ml TrypsinTPCK. Every day, 400 µl supernatant fluid was collected, snap-frozen and stored at 80℃. Fresh medium with trypsin was replenished. Virus titer was determined using either indirect immunostaining assay or direct agarose plaque assay as described above.
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2.7 Animal experiments C57BL/6 mice were purchased from The Jackson Laboratory, and were maintained under specific-pathogen-free environment at the University of Pittsburgh under the approved IACUC protocol number 15025585. Seven-week old female mice were
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anesthetized with 0.5% isoflurane and infected with HMPV strain TN/94-49/A2 at 1.0x106 PFU per animal via intra-tracheal (I.T.) or intra-nasal (I.N.) route. Four days
post-infection, the lung and nasal turbinate (NT) were harvested, homogenized in OPTIMEM, and clarified by centrifuging at 1,200 rpm for 5 min before titration. 2.8 Statistical analysis
Statistical analysis was performed by student’s t-test or one-way ANOVA followed by
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multiple comparison test using GraphPad Prism 6 software. A value of P<0.05 was
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3. Results
3.1 HMPV formed plaques in an agarose overlay plaque assay in LLC-MK2 cells
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First, we wanted to determine whether HMPV can form plaques under agarose
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overlay. Previously Zhang et al (Zhang et al., 2012) reported a direct plaque assay for a recombinant HMPV strain with self-cleavable F protein. However, they did not observe
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plaque formation by a wild-type HMPV strain with trypsin-dependent F cleavage site. Since higher trypsin concentration has been reported to enhance HMPV growth in cell culture (Tollefson et al., 2010), we hypothesized that higher concentration of trypsin
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would also facilitate HMPV plaque formation. To test this, we performed a direct HMPV plaque assay in LLC-MK2 cells with trypsin-TPCK concentration ranging from 0.2 µg/ml
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to 5.0 µg/ml in overlay medium. BSA and CaCl2 were supplemented in the overlay medium to stabilize the cell monolayer (Table 1). An HMPV A2 strain TN/94-49/A2 was used in this assay. As Fig.1A shows, although viral plaques were barely visible with low trypsin concentration (< 0.4 µg/ml), clear countable plaques were observed with 1.0-2.0 µg/ml trypsin after 7 days incubation. Further increase of trypsin concentration (5.0 µg/ml) caused disruption of the cell monolayer and made it difficult to count the plaques
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on day 7. Similarly, 5.0 µg/ml trypsin also reduced the monolayer integrity at earlier time points (Fig.2A), and thus did not facilitate earlier enumeration although plaque sizes were larger under this condition. We also tested BEAS-2B, A549, and VeroE6 cells for HMPV plaque formation. However, the cell monolayers were all disrupted with 1.0 µg/ml trypsin in overlay medium and no plaques were observed in these cell lines (data not
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shown).
Next, we wanted to characterize the plaque-forming kinetics of HMPV in LLC-MK2 cells. LLC-MK2 cells were infected with serially diluted TN/94-49/A2 and incubated in overlay medium containing 2.0 µg/ml trypsin-TPCK at 37℃ for 3 to 7 days. Visible
HMPV plaques were detected as early as on day 3 post-infection, and clear countable
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plaques began to form on day 5 (Fig. 1B). Increased plaque size and decreased
background were observed over time (Fig.1B-E). The plaque formation was examined
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by light microscopy and multinucleated giant cells were observed around the edge of
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plaques (Fig.1C), suggesting that these plaques were formed by HMPV-induced cell
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fusion. We also counter-stained the plaques by immuno-staining assay and confirmed that cells around the plaques were positive for HMPV antigen (data not shown).
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Although varying incubation time changed plaque sizes greatly, it was found not to alter virus titers calculated on day 5, 6, or 7 post-infection (Fig.1F). This suggests that
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the plaque numbers are consistent after clear countable plaques are formed. Therefore, it is possible to optimize assay time for better plaque morphology without affecting
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titration results.
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3.2 Optimization of agarose plaque assay The conditions of the plaque assay were then optimized to obtain clear plaques in
the shortest time. LLC-MK2 cells were infected by the same amount of virus in each
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well, overlaid with medium containing trypsin-TPCK from 0 µg/ml to 5.0 µg/ml, and incubated for 3 to 7 days (Fig. 2A). Although trypsin supports HMPV plaque formation, high concentrations of trypsin-TPCK (3.0-5.0 µg/ml) disrupted the cell monolayer and increased background. Trypsin-TPCK within 1.0-2.0 µg/ml was found to be optimal because the plaques were clear and the cell monolayer was intact. In addition, there was no significant difference among titers observed within this trypsin concentration
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range (Fig.2B). Therefore, we incubated cells with 1.5 µg/ml trypsin for 6 days, or 2.0 µg/ml trypsin for 7 days in subsequent experiments. Previously, Actinomycin-D (AcD) has been reported to enhance plaque formation for AMPV and trypsin-independent HMPV strains (Zhang et al., 2012). We wanted to test
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whether AcD also accelerates plaque development of trypsin-dependent HMPV strains. AcD at a final concentration of 0, 0.1, or 0.2 µg/ml were included in overlay medium and plaques were fixed and developed under the previously optimized trypsin conditions
(Fig. 3A). Although AcD significantly increased plaque size at 0.1 µg/ml, it disrupted the cell monolayer at 0.2 µg/ml (Fig.3C-D). In addition, this enhancement by AcD did not affect plaque number (Fig.3B). We conclude that AcD at low concentration (e.g. 0.1 µg/ml) can be used to further increase plaque sizes or potentially reduce the
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development time.
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Methylcellulose has also been tested for direct HMPV plaque assay. Previously,
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0.75% of methylcellulose in overlay medium has been used in immuno-staining, and the
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ready-to-use medium is easy to handle. However, 0.75% methylcellulose did not support HMPV plaque formation at conditions optimized for the agarose plaque assay
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(Fig.4A). CPE was present with 5.0 µg/ml trypsin-TPCK, but no plaques were observed.
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As an alternative to low-melting-point (LMP) agarose, non-LMP agarose also facilitated clear HMPV plaque formation at the optimized concentration for LMP agarose (0.1%-0.5%) (Fig.4B). However, non-LMP agarose solidified very quickly at room
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temperature and therefore was more difficult to handle than LMP agarose, especially if multiple plates were processed at the same time. The recipe of 0.25% LMP agarose
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was adopted because it supported clear plaque formation and had a reasonable solidification time (about 30 min at 4℃), compared with more than 1h for 0.1% LMP.
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We also tested neutral red dye as an alternative to crystal violet staining. To stain
the plaques with neutral red, another layer of agarose containing 0.03% neutral red was topped on the overlay agarose plug one day before fixation. After incubation at 37℃ overnight (about 16 h), cells were fixed with 10% formalin and plaques were directly counted without removing the agarose. Neutral red staining was also able to produce
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good contrast between HMPV plaques and background (Fig.4C), thus serving as a good alternative to crystal violet in HMPV plaque assay. 3.3 Comparing direct plaque assay with indirect immuno-staining assay To evaluate its accuracy and reliability, we applied the direct plaque assay to
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measuring the growth kinetics of TN/94-49/A2 in LLC-MK2 cells in parallel with the conventional indirect immuno-staining assay. As shown in Fig.5A, TN/94-49/A2 had an exponential growth phase from day 1 to day 3, and the virus titer dropped on day 4
when massive CPE appeared and cells began to detach (Fig.5C). The growth curve generated by the direct plaque assay was consistent with that by the conventional
indirect assay (Fig.5A). Virus titers from three independent experiments were combined
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to evaluate if the two methods produced equivalent results. The linear regression fit
curve showed a strong correlation between the two assays (slope =0.96±0.03, R2=0.98)
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with the indirect immuno-staining assay.
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(Fig.5B). This shows that the direct plaque assay is accurate, reliable, and equivalent
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3.4 Testing viral burden in mice tissue homogenates To further explore application of the plaque assay, we titrated HMPV in animal
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samples. Seven-week old C57BL/6 mice were infected with 1.0 x 106 PFU TN/94-49/A2
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via intra-nasal (I.N.) or intra-tracheal (I.T.) route. Lung and nasal turbinate (NT) were harvested on day 4 post-infection, and the tissue homogenate was titrated with direct
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plaque assay and indirect immunostaining assay in parallel. As shown in Fig.6A, HMPV strain TN/94-49/A2 was still able to form plaques under agarose overlay despite the presence of inflammatory cytokines in animal samples, although plaques in the lowest
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dilution were smaller in size. Virus titers determined by the direct plaque assay was comparable to those by the indirect immunostaining assay in samples from all four
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groups (Fig.6B). Therefore, the direct plaque assay can be used to titrate animal tissue homogenates. 3.5 Direct plaque assay is applicable to HMPV common laboratory strains and clinical isolates
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Next, we wanted to study whether plaque formation under agarose overlay was strain-specific or universal for HMPV. We chose clinical isolate strains TN/1501/A1, OH/202/B1, and TN/91-320/B2 apart from the reference strain TN/94-49/A2, together representing all four HMPV subgroups. All clinical isolates tested could form countable plaques under the agarose overlay (Fig.7A). Light microscopic images of the plaques
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showed the presence of syncytia around the edge of plaques, suggesting that the
plaques were formed by viral infection (Fig.7C). Recombinant viruses based on the
genomic sequence of several commonly-used HMPV strains were also generated. All recombinant viruses, rNL/1/99, rNL/1/00-F with trypsin-independent cleavage site, rNL/1/00-F-GFP, and rCAN97-83-GFP, formed clear plaques under the agarose
overlay, and syncytia were observed around the plaques (Fig.7B, D). We concluded that
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this direct plaque assay can be applied to multiple strains of HMPV.
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3.6 HPIV3 and HPIV5 but not HRSV interferes with the HMPV direct plaque assay
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Some closely related viruses in the pneumovirus and paramyxovirus families were
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also tested for plaque formation under this assay. We found that human parainfluenza virus type 3 and type 5 (HPIV3 and HPIV5), but not a recombinant strain of human
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respiratory syncytial virus (rRSV-A2-K-Line19F), were able to form clear plaques under the agarose overlay optimized for HMPV (Fig.8A-B). This suggests that this plaque
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assay cannot differentiate HMPV from other closely related pathogens if co-infection
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occurs.
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4. Discussion
Here we reported a direct plaque assay for trypsin-dependent HMPV strains.
Previously, a direct plaque assay was developed for AMPV (Zhang et al., 2012) and
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was applicable to trypsin-independent strains of HMPV. However, most HMPV laboratory strains and clinical isolates have trypsin-dependent F protein cleavage sequences (Yang et al., 2009). In the current study, by increasing trypsin concentration and modifying ingredients in overlay medium, we have developed and optimized a direct agarose plaque assay for HMPV trypsin-dependent strains.
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The incubation time (5-7 days) in direct plaque assay is similar to that of traditional indirect immuno-staining (4-5 days), but the staining time is much shorter; staining the cell monolayer with crystal violet can be finished within one minute, compared with hours of blocking and antibody staining in the indirect assay. Moreover, the direct
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plaque assay conserves resources for antibody/animal anti-sera. The direct plaque assay has wide applications to titrate HMPV in both cell culture and animal tissue homogenates. The assay can detect recombinant viruses derived from several commonly used lab strains, as well as clinical isolates from all four
subgroups. Since the virus is not inactivated in this assay, the direct agarose plaque
assay can also be applied for traditional plaque purification of virus. The main limitation of the direct plaque assay is that it cannot differentiate HMPV from other closely-related
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viruses, thus the indirect antibody-based method is still necessary in cases of co-
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infection.
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Although this assay was developed for trypsin-dependent strains of HMPV, it can
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also be applied to the trypsin-independent strain rNL/1/00-F. The virus with selfcleavage site formed plaques in trypsin-free conditions (data not shown), but the
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addition of trypsin increased the plaque sizes and yielded clearer plaques. This suggests that trypsin should be a routine ingredient in the overlay recipe. We used
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trypsin-TPCK rather than trypsin-EDTA because EDTA chelates divalent cations required for integrin function, and RGD-binding integrins serve as receptors for HMPV
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(Cox et al., 2012; Cox et al., 2015; Cseke et al., 2009). We have noticed a few other factors for HMPV plaque formation. LLC-MK2 cells
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supported HMPV plaque formation in a wide range of initial seeding density (0.5 x 106 to 2.0 x 106 cells/well in a 6-well plate). Within this range, we observed that lower seeding
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density may increase plaque sizes, but it also causes higher background (data not shown). The 1.2 x 106 cells/well density was chosen in all experiments in balance of the plaque size and background signal. We also tested tissue culture treated 6-well plates from three suppliers (TPP, Greiner Bio-one, and Corning Costar). While all three plates supported HMPV plaque formation, we observed differences in plaque sizes and background signal noise among different brands -- HMPV plaques were slightly larger
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and clearer in TPP brand plates (data not shown). Therefore, TPP plates were used in all subsequent experiments. Viral factors also likely contribute to plaque formation, as different HMPV strains form plaques of different sizes under the same conditions. We hypothesize that the ability to form better plaques is correlated with better syncytium formation and in vitro replication. The viral factors that determine plaque size are yet to
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be identified. HMPV TN/94-49/A2 is the best plaque-forming strain tested in this assay, and also grows to higher titer and induces more CPE than other strains in cell culture (data not shown). Therefore, like other common HMPV strains, TN/94-49/A2 is likely partly cell-adapted but may serve as a good tool for HMPV research.
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Conflicts of interest statement
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The authors have no conflicts of interest to declare.
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Acknowledgement
This work was supported by NIH/NIAID grant R01 AI085062-06 to JVW. YZ received
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a Research Advisory Council (RAC) fellowship award from the Children’s Hospital of
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Pittsburgh of UPMC. JX received funding as visiting scholar to the University of Pittsburgh from China Scholarship Council ([2016]3097). We thank Dr. Bernadette G.
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van den Hoogen, Dr. Martin L. Moore, and Dr. Jianrong Li for the HMPV and RSV reverse genetics systems, and we thank Dr. Ursula Buchholz for BSR-T7/5 cells to
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generate recombinant viruses.
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Reference
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Biacchesi, S., Skiadopoulos, M.H., Tran, K.C., Murphy, B.R., Collins, P.L. and Buchholz, U.J., 2004. Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes. Virology 321, 247-59. Cox, R.G., Livesay, S.B., Johnson, M., Ohi, M.D. and Williams, J.V., 2012. The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins. J Virol 86, 12148-60. Cox, R.G., Mainou, B.A., Johnson, M., Hastings, A.K., Schuster, J.E., Dermody, T.S. and Williams, J.V., 2015. Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes. PLoS Pathog 11, e1005303. Cseke, G., Maginnis, M.S., Cox, R.G., Tollefson, S.J., Podsiad, A.B., Wright, D.W., Dermody, T.S. and Williams, J.V., 2009. Integrin alphavbeta1 promotes infection by human metapneumovirus. Proc Natl Acad Sci U S A 106, 1566-71. de Graaf, M., Herfst, S., Schrauwen, E.J.A., van den Hoogen, B.G., Osterhaus, A.D.M.E. and Fouchier, R.A.M., 2007. An improved plaque reduction virus neutralization assay for human metapneumovirus. Journal of Virological Methods 143, 169-174. Deffrasnes, C., Hamelin, M.E., Prince, G.A. and Boivin, G., 2008. Identification and evaluation of a highly effective fusion inhibitor for human metapneumovirus. Antimicrob Agents Chemother 52, 279-87. Erickson, J.J., Gilchuk, P., Hastings, A.K., Tollefson, S.J., Johnson, M., Downing, M.B., Boyd, K.L., Johnson, J.E., Kim, A.S., Joyce, S. and Williams, J.V., 2012. Viral acute lower respiratory infections impair CD8+ T cells through PD-1. J Clin Invest 122, 2967-82. Guerrero-Plata, A., Casola, A., Suarez, G., Yu, X., Spetch, L., Peeples, M.E. and Garofalo, R.P., 2006. Differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus. Am J Respir Cell Mol Biol 34, 320-9. Hamelin, M.E., Yim, K., Kuhn, K.H., Cragin, R.P., Boukhvalova, M., Blanco, J.C., Prince, G.A. and Boivin, G., 2005. Pathogenesis of human metapneumovirus lung infection in BALB/c mice and cotton rats. J Virol 79, 8894-903. Herfst, S., de Graaf, M., Schickli, J.H., Tang, R.S., Kaur, J., Yang, C.F., Spaete, R.R., Haller, A.A., van den Hoogen, B.G., Osterhaus, A.D. and Fouchier, R.A., 2004. Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA. J Virol 78, 8264-70. Hotard, A.L., Shaikh, F.Y., Lee, S., Yan, D., Teng, M.N., Plemper, R.K., Crowe, J.E., Jr. and Moore, M.L., 2012. A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis. Virology 434, 129-36. Jain, S., Self, W.H., Wunderink, R.G., Fakhran, S., Balk, R., Bramley, A.M., Reed, C., Grijalva, C.G., Anderson, E.J., Courtney, D.M., Chappell, J.D., Qi, C., Hart, E.M., Carroll, F., Trabue, C., Donnelly, H.K., Williams, D.J., Zhu, Y., Arnold, S.R., Ampofo, K., Waterer, G.W., Levine, M., Lindstrom, S., Winchell, J.M., Katz, J.M., Erdman, D., Schneider, E., Hicks, L.A., McCullers, J.A., Pavia, A.T., Edwards, K.M., Finelli, L. and Team, C.E.S., 2015a. Community-Acquired Pneumonia Requiring Hospitalization among U.S. Adults. N Engl J Med 373, 415-27. Jain, S., Williams, D.J., Arnold, S.R., Ampofo, K., Bramley, A.M., Reed, C., Stockmann, C., Anderson, E.J., Grijalva, C.G., Self, W.H., Zhu, Y., Patel, A., Hymas, W., Chappell, J.D., Kaufman, R.A., Kan, J.H., Dansie, D., Lenny, N., Hillyard, D.R., Haynes, L.M., Levine, M., Lindstrom, S., Winchell, J.M., Katz, J.M., Erdman, D., Schneider, E., Hicks, L.A., Wunderink, R.G., Edwards,
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K.M., Pavia, A.T., McCullers, J.A., Finelli, L. and Team, C.E.S., 2015b. Community-acquired pneumonia requiring hospitalization among U.S. children. N Engl J Med 372, 835-45. Kuhn, J.H., Dietzgen, R.G., Easton, A.J., Kurath, G., Nowotny, N., Rima, B., Rubbenstroth, D., Vasilakis, N., Walsh, J., Collins, P.L., Fouchier, R.A., Lamb, R., Maisner, A., Randall, R., Rota, P., Wang, L.-F. and Kondo, H., 2015. ICTV taxonomic proposal 2015.011a-gM.A.v2.Pneumoviridae. Elevate subfamily Pneumovirinae (family Paramyxoviridae) to the taxonomic rank of family, named Pneumoviridae, in the order Mononegavirales; change the name of genus Pneumovirus to Orthopneumovirus. Reich, E., Franklin, R.M., Shatkin, A.J. and Tatum, E.L., 1961. Effect of actinomycin D on cellular nucleic acid synthesis and virus production. Science 134, 556-7. Reich, E., Franklin, R.M., Shatkin, A.J. and Tatumel, 1962. Action of actinomycin D on animal cells and viruses. Proc Natl Acad Sci U S A 48, 1238-45. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A., 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-82. Tollefson, S.J., Cox, R.G. and Williams, J.V., 2010. Studies of culture conditions and environmental stability of human metapneumovirus. Virus Res 151, 54-59. van den Hoogen, B.G., de Jong, J.C., Groen, J., Kuiken, T., de Groot, R., Fouchier, R.A. and Osterhaus, A.D., 2001. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 7, 719-724. van den Hoogen, B.G. and Fouchier, R.A.M., 2017. Recovery of a Paramyxovirus, the Human Metapneumovirus, from Cloned cDNA. Methods Mol Biol 1602, 125-139. Williams, J.V., Harris, P.A., Tollefson, S.J., Halburnt-Rush, L.L., Pingsterhaus, J.M., Edwards, K.M., Wright, P.F. and Crowe, J.E., 2004. Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children. N Engl J Med 350, 443-450. Williams, J.V., Tollefson, S.J., Johnson, J.E. and Crowe, J.E., Jr., 2005. The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity. J Virol 79, 10944-51. Yang, C.F., Wang, C.K., Tollefson, S.J., Piyaratna, R., Lintao, L.D., Chu, M., Liem, A., Mark, M., Spaete, R.R., Crowe, J.E., Jr. and Williams, J.V., 2009. Genetic diversity and evolution of human metapneumovirus fusion protein over twenty years. Virol J 6, 138. Zhang, Y., Wei, Y., Li, J. and Li, J., 2012. Development and optimization of a direct plaque assay for human and avian metapneumoviruses. J Virol Methods 185, 61-8. Zhou, M., Kitagawa, Y., Yamaguchi, M., Uchiyama, C., Itoh, M. and Gotoh, B., 2013. Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase. J Clin Virol 56, 31-36.
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Fig.1 HMPV strain TN/94-49/A2 formed plaques in LLC-MK2 cells in an agarose overlay plaque assay. (A) Plaque morphology of HMPV strain TN/94-49/A2 with different trypsin concentrations. LLC-MK2 cells were infected with the same amount of virus (about 200 PFU per well) and overlaid with medium containing indicated amount of trypsin-TPCK. Plates were formalin fixed on day 7 and plaques were visualized by
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staining with 0.1% crystal violet. (B) Plaque morphology of TN/94-49/A2 in LLC-MK2
cells over time. Plaque assay was performed with 2.0 µg/ml trypsin-TPCK and plates
were fixed and stained at the indicated time post-infection. (C) Microscopic images of plaques formed by HMPV over time. Images taken under 2x stereoscope. (D-E)
Quantification of plaques. In each group, twenty single plaques were randomly selected and their sizes were quantified by ImageJ. Data were shown as mean ± SD (D) or
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median with range (E). (F) Quantification of plaque numbers over time. The same
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serially diluted virus samples were inoculated onto LLC-MK2 cells, and plaques were counted on the indicated days post-infection to determine the virus titer. Data shown as
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mean ± SD from three replicates (ns P>0.05).
Fig.2 Optimization of trypsin concentration in overlay medium. (A) LLC-MK2 cells
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were inoculated with the same amount of virus (about 30 PFU per well) on day 0 and
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were incubated with different doses of trypsin-TPCK (0-5.0 µg/ml) in overlay medium. Cells were fixed with 10% formalin at the indicated time (day 3 to day 7), and plaques were visualized by crystal violet staining. (B) Virus titer of TN/94-49/A2 was determined
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under agarose overlay with indicated amount of trypsin. Plaques were counted 6 days
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post-infection. Data shown as mean ± SD from three replicates (ns P>0.05).
Fig.3 Actinomycin-D increases plaque size without changing plaque counts. (A)
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LLC-MK2 cells were inoculated with the same amount of virus (about 30 PFU/well) and were incubated with the indicated amount of trypsin and actinomycin-D (AcD). Plaques were fixed and stained by crystal violet staining at the indicated time post-infection. (B) Virus titer of TN/94-49/A2 was determined by the direct plaque assay with or without AcD, in parallel with the indirect immuno-staining assay. Plaques were counted 6 days post-infection. Data were shown as mean ± SD from three replicates (ns P>0.05). (C-D)
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Quantification of plaques. Twenty single plaques were randomly selected and their sizes were quantified by ImageJ. Data were shown as mean ± SD (C) or median with range (D). Statistical analysis was performed only with mean ± SD (C) for simplicity (* P<0.05 by t-test). Plaques in AcD 0.2 µg/ml group were not quantified because the
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background was too light.
Fig.4 Effect of other overlay ingredients and dye. (A) Plaque morphology of TN/9449/A2 in overlay medium containing either 0.25% agarose or 0.75% methylcellulose.
The overlay medium also contained indicated concentration of trypsin-TPCK. Plaques were visualized on day 7. (B) Plaque formation under low-melting-point (LMP) or nonLMP agarose at indicated concentration. Serially diluted TN/94-49/A2 was inoculated
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onto LLC-MK2 cells and overlaid with media containing indicated amount of agarose.
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The plates were incubated at 4℃ until overlay medium completely solidified before
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being transferred to the 37℃ incubator. (C) Agarose plaque assay was performed as described in Materials and Methods. In crystal violet group, plaques were visualized on
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day 6 by staining with 0.1% crystal violet. In neutral red group, 2 ml 0.5% agarose containing 0.03% neutral red was topped on the overlay medium on day 5, and cells
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were fixed on day 6 to visualize the plaques.
Fig.5 Growth Curve of HMPV TN/94-49/A2 titrated by immunostaining and agarose
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plaque assay. (A) LLC-MK2 cells were infected with TN/94-49/A2 at MOI=0.01 in T25 flasks with 0.5 µg/ml trypsin-TPCK. Every day 400 µl supernatant fluid was collected
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and snap-frozen, and fresh medium containing trypsin was supplemented. Virus titer was determined by direct plaque assay and indirect immune-staining assay in parallel in three independent replicates. Dotted line shows the detection limit of the assay. (B)
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Correlation in virus titer results by two methods was determined by linear regression. The best-fit curve was plotted as a dashed line. (C) Microscopic morphology of LLCMK2 cells infected by TN/94-49/A2 at the indicated time post-infection (10x phase contrast, scale bar 400 µm).
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Fig.6 Direct plaque assay can be applied in animal samples. (A) C57BL/6 mice were infected with 1.0 x 106 PFU TN/94-49/A2 via intra-nasal (I.N.) or intra-tracheal (I.T.) route with 2 mice per group. The lung and nasal turbinate (NT) were harvested on day 4 post-infection. Serially-diluted lung and NT homogenates were inoculated onto LLC-MK2 cells, and direct plaque assay was performed as described in Materials and
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Methods. One representative image is shown for lung and NT samples. (B) Virus titer of lung and NT homogenate in (A) by either indirect immunostaining or agarose plaque assay. Mean ± SD from two replicates are shown.
Fig.7 Other HMPV strains form plaques in direct plaque assay. (A) Morphology of
plaques formed by HMPV isolates TN/1501/A1, TN/94-49/A2, OH/202/B1, and TN/91-
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320/B2 in a direct plaque assay. The overlay medium contained 1.5 µg/ml trypsin-
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TPCK. Plaques were visualized on day 6 by crystal violet. (B) Morphology of plaques formed by recombinant HMPV strains as in (A) (C-D) Microscopic images (10x field) of
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plaques formed by HMPV clinical isolates (C) or recombinant viruses (D).
Fig.8 Some other closely-related viruses also formed plaques in direct plaque
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assay. (A) Plaque morphology of HMPV (TN/94-49/A2), recombinant respiratory
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syncytial virus (rRSV-A2-K-Line19F), human parainfluenza virus type 3 (HPIV3), and human parainfluenza virus type 5 (HPIV5) under agarose overlay. The direct plaque assay was performed with 1.5 µg/ml trypsin-TPCK for 6 days. (B) Microscopic images of
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plaques (10x field) formed by the four viruses in (A).
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Figures:
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Table:
Table 1. Recipe for HMPV direct plaque assay overlay medium Final conc.
Stock
Amount for 100 ml
MEM
1x
2x
50 ml
Glutamine
2 mM
200 mM
Gentamicin
50 µg/ml
50 mg/ml
Amphotericin B
2.5 µg/ml
250 µg/ml
HEPES
25 mM
1 M (pH7.4)
2.5 ml
NaHCO3
0.12%
7.5%
1.6 ml
BSA
0.05%
5%
Agarose
0.25%
1%
diH2O
-
-
Trypsin-TPCK
1.5 µg/ml
2 mg/ml
75 µl
Actinomycin-D (opt.)
0.1 µg/ml
1 mg/ml
10 µl
1 ml
100 µl 1 ml
U
1 ml
N
A M
‡Abbreviation
SC RI PT
Ingredients‡
25 ml 17.8 ml
A
CC
EP
TE
D
for ingredients: minimum essential media (MEM), 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), sodium bicarbonate (NaHCO3), bovine serum albumin (BSA), deionized water (diH2O), and L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)treated trypsin (Trypsin-TPCK).
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