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Department of Virology, Erasmus Medical Centre, PO Box 2040, 3000 CA Rotterdam, The Netherlands
Correspondence
Ron A. M. Fouchier
r.fouchier{at}erasmusmc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Falsey et al., 2003; van den Hoogen et al., 2001, 2003; Williams et al., 2006). Clinical symptoms range from mild respiratory problems to severe cough, bronchiolitis and pneumonia (van den Hoogen et al., 2003; Williams et al., 2006). AMPV causes respiratory tract illness in domestic poultry (Cook, 2000). AMPV infections frequently become complicated with secondary bacterial infections resulting in air sacculitis and pneumonia (Jirjis et al., 2004), whereas AMPV infections without any secondary infections are mainly confined to the nasal turbinate (NT) and trachea (Jirjis et al., 2002; Velayudhan et al., 2005). AMPVs have been classified into four subgroups, A–D (Bayon-Auboyer et al., 1999; Eterradossi et al., 1995; Juhasz & Easton, 1994; Seal, 1998), and AMPV-C is more closely related to HMPV than the other AMPV subgroups (Govindarajan & Samal, 2004, 2005; Govindarajan et al., 2004). It has been demonstrated that HMPV probably originated from birds and that a common ancestor of AMPV-C and HMPV existed around 200 years ago (de Graaf et al., 2008b).
The metapneumovirus genome is non-segmented and of antisense polarity. Genome replication requires the viral genomic RNA (vRNA) to be encapsidated in a ribonucleoprotein complex, comprising the nucleoprotein (N), which encapsidates the viral genome, the phosphoprotein (P), the large polymerase protein (L) and the putative transcriptional elongation factor (M2.1). These polymerase complex proteins can be functionally exchanged between HMPV and AMPV-C in both mini-replicon systems and chimeric viruses (de Graaf et al., 2008a; Govindarajan et al., 2006; Pham et al., 2005). Metapneumoviruses encode three membrane glycoproteins, the attachment (G), the small hydrophobic (SH) and the fusion (F) proteins. The HMPV and AMPV-C SH and G proteins are type II membrane proteins that are not essential for virus replication in vitro (Biacchesi et al., 2004; Naylor et al., 2004). Moreover, recombinant HMPV from which the SH gene has been deleted replicates with normal kinetics in hamsters and monkeys (Biacchesi et al., 2004, 2005). In contrast, HMPV from which the G gene has been deleted is viable but attenuated (Biacchesi et al., 2004, 2005). Thus, it appears that the F protein by itself can promote attachment and fusion. The F protein is a type I membrane protein and is synthesized as an inactive precursor F0 that is cleaved by host proteases into the functional subunits F1 and F2 (Lamb et al., 2006; Russell et al., 2001). The F protein mediates fusion of the virus envelope with the cell membrane during viral entry and induces syncytium formation in infected cells (Herfst et al., 2008; Schowalter et al., 2006). For bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV), the F protein determines the cellular host range (Schlender et al., 2003).
It was suggested previously that HMPV can infect turkey poults, despite the inability to isolate viable virus from these animals (Velayudhan et al., 2006). Here, we have shown that AMPV, but not HMPV, can infect and replicate in turkey poults. To determine which genes contribute to this difference in host range, chimeric HMPVs were created that contained individual genes of AMPV-C. These chimeric viruses were tested in quail fibroblast (QT6) cells, which are permissive for replication of AMPV-C, but relatively non-permissive for HMPV. The F protein was found to be primarily responsible for this difference in host tropism. Furthermore, all chimeric viruses in which polymerase component proteins were exchanged replicated to lower titres compared with HMPV. This phenomenon was investigated by exchanging polymerase components between HMPV and AMPV-C mini-replicon systems, revealing that chimeric HMPV/AMPV-C polymerase components are not functional in QT6 cells. This study provides insight into the determinants of metapneumovirus host tropism, specificity and functional interaction of the polymerase proteins and F protein-promoted fusion.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was cultured in Iscove's modified Dulbecco's medium (IMDM; BioWhittaker) supplemented with 10 % fetal calf serum (FCS), 100 IU penicillin ml–1, 100 µg streptomycin ml–1 and 2 mM glutamine as described previously (Herfst et al., 2004). Baby hamster kidney cells stably expressing T7 RNA polymerase (BSR-T7), a kind gift of Dr K. Conzelmann (Buchholz et al., 1999), were grown in Dulbecco's modified Eagle's medium (DMEM; BioWhittaker) supplemented with 10 % FCS, non-essential amino acids, 100 IU penicillin ml–1, 100 µg streptomycin ml–1, 2 mM glutamine and 0.5 mg G418 ml–1 (Invitrogen). For HMPV rescue, Vero-118 and BSR-T7 cells were co-cultured in DMEM supplemented with 3 % FCS, 100 IU penicillin ml–1, 100 µg streptomycin ml–1, 2 mM glutamine and 0.25 mg trypsin ml–1. For virus propagation and titration of HMPV, all chimeric viruses and AMPV-C (Colorado strain; Intervet), Vero-118 cells were grown in IMDM supplemented with 4 % bovine serum albumin fraction V (Invitrogen), 100 IU penicillin, 2 mM glutamine and 3.75 µg trypsin ml–1. QT6 cells were cultured in Medium 199 (Cambrex Bio Science) supplemented with 5 % FCS, 1 % chicken serum, 5 % tryptose phosphate broth, 100 IU penicillin ml–1, 100 µg streptomycin ml–1, 2 mM glutamine, 0.75 mg sodium bicarbonate ml–1 and non-essential amino acids. For virus propagation, 0.25 mg trypsin ml–1 was added. For purification and concentration, viruses were subjected to one freeze–thaw cycle after which cell-free supernatants were purified and concentrated using a 30–60 % (w/w) sucrose gradient.
Plasmid construction.
The full-length cDNA plasmids for HMPV (lineage B1, strain NL/1/99) and the full-length chimeric HMPV cDNA plasmids containing the N, P or L gene of AMPV-C have been described previously (de Graaf et al., 2008a; Herfst et al., 2004). For construction of the full-length chimeric HMPV cDNA plasmids containing the F, G or SH gene of AMPV-C, fragments of HMPV were amplified by PCR and cloned into pBluescript SK+ or pCR4TOPO (Invitrogen). All fragments were cloned such that type II restriction sites replaced the F, G or SH open reading frame (ORF). The F, G and SH ORFs of AMPV-C were amplified by PCR using primers flanked by type II restriction sites and cloned into pBluescript SK+ or pCR4TOPO (Invitrogen) containing the HMPV fragments. Using unique restriction sites, the fragments containing the desired ORF were swapped back into the full-length HMPV cDNA plasmids. All plasmid inserts were sequenced to ensure the absence of undesired mutations. All primer sequences are available upon request.
A pCAGGS plasmid expressing HMPV F (lineage B1, strain NL/1/99) has been described previously (here renamed pCAGGS-Fh) (Herfst et al., 2008). For construction of the AMPV-C F expression plasmid, the AMPV-C F ORF was amplified by PCR and cloned into the multiple cloning site of pCAGGS, using the EcoRI and XhoI restriction sites, to yield pCAGGS-Fa. Next, chimeric F expression vectors were constructed in which the F1 or F2 region or the C-terminal region of F1 of HMPV and AMPV-C F were exchanged. For the chimeric F expression vectors in which F1 and F2 were exchanged, the F1 and F2 regions were amplified separately by PCR using primers flanked by type II restriction sites for regions within the F gene, and EcoRI and XhoI at the start of the F2 region and the end of the F1 ORF, respectively. These fragments were combined and cloned into pCAGGS, resulting in pCAGGS-F2a–F1h and pCAGGS-F2h–F1a. The F2 region included the cleavage site. For the chimeric F expression vectors in which the C-terminal region of F1 was exchanged, nt 1–1308 (aa 1–436) and nt 1309–1614/1620 (aa 437–538/540; the F gene of HMPV and AMPV differ by 2 aa in length) of the HMPV and AMPV F ORFs were amplified separately by PCR using primers flanked by type II restriction sites for regions within the F gene, and EcoRI and XhoI at the start of the F2 region and the end of the F1 ORF, respectively. These fragments were combined and cloned into pCAGGS, resulting in pCAGGS-F2h–F1ha and pCAGGS-F2a–F1ah. All plasmid inserts were sequenced to ensure the absence of undesired mutations. All primer sequences are available upon request.
Animal experiments.
Nicholas strain turkey poults (Meleagris gallopavo) were obtained from AMPV antibody-free parents and raised in isolation facilities. As a precaution, the turkey poults were given 10 % enrofloxacin (5 ml in 10 l water) until they were 11 days of age. At 2 weeks of age, serum samples were collected and tested for AMPV antibodies using an IDEXX Flockchek ELISA kit (IDEXX Laboratories). At 3 weeks of age, the poults (n=6) were inoculated with 200 µl containing 105 or 107 50 % tissue culture infectious dose (TCID50) purified AMPV-C or HMPV (lineage B1, strain NL/1/99) ml–1, diluted in PBS. The poults were inoculated with a 50 µl volume in each conjunctival space and nostril. During the first experiment, swabs were collected from the choanal clefts and nostrils of turkey poults for a period of 10 days, using sterile cotton swabs (Greiner), placed in 2 ml transport medium (Hanks' balanced salt solution containing 10 % v/v glycerol, 200 U penicillin ml–1, 200 µg streptomycin ml–1, 100 U polymyxin B sulfate ml–1 and 250 µl gentamicin ml–1; MP Biomedicals) and stored at –80 °C. At 10 days post-inoculation (p.i.), serum samples were collected and tested for AMPV antibodies using an IDEXX Flockchek ELISA kit. For the second experiment, at 3 weeks of age, the poults (n=6) were inoculated with 200 µl containing 105 TCID50 purified AMPV-C or HMPV ml–1, diluted in PBS (50 µl in each conjunctival space and nostril). At 3, 5 and 7 days p.i., NTs, trachea and lungs were collected, snap-frozen immediately and stored at –80 °C until further processing. All animal studies were approved by an independent Animal Ethics Committee and the Dutch authority for working with genetically modified organisms and were carried out in accordance with animal experimentation guidelines.
Virus titrations.
Tissues from the inoculated poults were homogenized using a Polytron homogenizer (Kinematica AG) in infection medium. After removal of tissue debris by centrifugation, the supernatants were used for virus titration in Vero-118 cells. Titres were calculated per gram of tissue, with a detection limit of 101.8, 101.1 and 100.7 TCID50 g–1 for NT, trachea and lung samples, respectively. Choanal and nasal swab samples were centrifuged to remove debris, and the supernatants were used for virus titration in Vero-118 cells.
Virus titres were determined as described previously (Herfst et al., 2004). Confluent monolayers of Vero-118 cells in 96-well plates were spin-inoculated (15 min, 2000 g) with 100 µl of tenfold serial dilutions of each sample and incubated at 37 °C. After 2 h and again after 3–4 days, the inoculum was replaced with fresh infection medium. Seven days after inoculation, infected wells were identified by immunofluorescence assays with HMPV-specific polyclonal antiserum raised in guinea pigs, as described previously (van den Hoogen et al., 2001). Titres were expressed as TCID50 values, calculated as described by Reed & Muench (1938).
Real-time RT-PCR.
Total RNA was isolated from 200 µl choanal or nasal swab material, using a MagNA Pure LC system with a MagNA Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics), with an elution volume of 50 µl. HMPV genome copies were detected by TaqMan real-time RT-PCR as described previously (Maertzdorf et al., 2004; van den Hoogen et al., 2007).
Replication curves.
Replication curves were generated as described previously (Herfst et al., 2004). Flasks (25 cm2) containing confluent Vero-118 or QT6 cells were inoculated for 2 h at 37 °C with HMPV, AMPV-C or one of the chimeric viruses at an m.o.i. of 0.1. After adsorption of virus to the cells, the inoculum was removed and cells were washed twice with medium before addition of 7 ml fresh medium and incubation at 37 °C. Each day, 0.5 ml supernatant was collected and replaced by fresh medium. Plaque assays were performed in Vero-118 cells to determine virus titres as described previously (de Graaf et al., 2008a).
Mini-genome assays.
QT6 cells grown in six-well plates were transfected with 1 µg vector expressing the vRNA-like molecule, 0.8 µg pCITE-N, 0.4 µg pCITE-P, 0.4 µg pCITE-L, 0.4 µg pCITE-M2.1, 0.4 µg pTS27 (a constitutive β-galactosidase expression vector, kindly provided by Dr M. H. Malim, King's College London School of Medicine, UK) and 1.5 µg pAR3126, a plasmid expressing T7 RNA polymerase (a kind gift of Dr J. Dunn, Brookhaven National Laboratory, NY, USA) using Lipofectamine 2000 (Invitrogen). Cells were analysed 3 days after transfection by ELISA for chloramphenicol acetyl transferase (CAT) and β-galactosidase (Roche Diagnostics) according to the instructions of the manufacturer. All transfections were carried out in triplicate and CAT values were standardized to 10 ng β-galactosidase to control for transfection efficiency and sample processing.
Cell-content mixing assays.
Cell-content mixing assays in Vero-118 cells were performed as described previously (Herfst et al., 2008). Two wells of a six-well plate containing Vero-118 cells were each transfected with 2 µg pCAGGS-F and 0.4 µg pTS27, using Lipofectamine 2000. One well was co-transfected with 2 µg pLTR-CAT [containing the CAT gene under the control of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)] and the other well with 2 µg pTat (expressing the HIV-1 transactivator of transcription) (a kind gift of Dr R. Gruters; Gruters et al., 1991). On day 1 post-transfection, both cell populations were harvested using trypsin/EDTA, mixed and plated in three wells of a six-well plate. Trypsin was added to the medium the following day. On day 3 post-transfection, cell lysates were harvested and CAT and β-galactosidase expression was quantified by ELISA (Bioconnect). In this assay, cell-content mixing, as a result of F-mediated fusion, resulted in Tat-mediated transactivation of the HIV-1 LTR and hence induction of CAT expression. CAT expression was normalized based on β-galactosidase expression.
Cell-content mixing assays in QT6 cells were performed as described previously (Rawling et al., 2008), with minor adjustments. Two wells of a six-well plate containing QT6 cells were each transfected with 2 µg pCAGGS-F and 0.4 µg pTS27, using Lipofectamine 2000. One well was co-transfected with 2 µg pTM1-GFP [containing the green fluorescent protein (GFP) gene under control of the T7 RNA polymerase promoter; a kind gift of Dr J. Melero, Centro Nacional de Biologia Fundamental, Instituto de Salud Carlos III, Madrid, Spain] and the other well with 2 µg pAR3126. On day 1 post-transfection, both cell populations were harvested using trypsin/EDTA, mixed and plated in three wells of a six-well plate. The following day, trypsin was added to the medium. On day 3 post-transfection, cell lysates were harvested, and GFP and β-galactosidase expression was quantified by ELISA. In this assay, cell-content mixing, as a result of F-mediated fusion, resulted in T7 RNA polymerase-mediated expression of GFP. GFP expression was normalized based on β-galactosidase expression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). For the animals inoculated with 2x106 TCID50 AMPV-C, a clear peak of virus replication could be observed at day 2 p.i., followed by a rapid decrease in virus titres. No virus could be isolated from the nasal swabs. For the animals inoculated with 2x104 TCID50 AMPV-C, the increase and subsequent decrease in virus titres was more gradual compared with animals inoculated with 2x106 TCID50 AMPV-C. Moreover, the duration of viral shedding was longer, as virus was still isolated from most animals at 7 days p.i, whereas for the animals inoculated with 2x106 TCID50 AMPV-C, only one animal shed virus at day 6 and no virus could be detected at day 7 p.i. At 10 days p.i., serum samples were collected from all animals and tested by ELISA. Seroconversion was observed for all AMPV-C-inoculated animals, but not for HMPV-inoculated animals (data not shown).
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Next, two groups of 18 turkeys were inoculated with 2x104 TCID50 HMPV or AMPV-C. Six animals from each group were euthanized at 3, 5 and 7 days p.i. and NT, trachea and lung samples were collected (Fig. 2). Virus titres in the NTs of AMPV-C-inoculated animals were highest at day 5 p.i. and virus could be isolated from all six animals. Virus titres observed at days 3 and 7 p.i. were at least 100-fold lower. In the trachea of AMPV-C-inoculated animals, virus titres were higher at day 5 p.i. compared with days 3 and 7 p.i. No virus could be isolated from the lungs at days 3, 5 and 7 p.i. of the AMPV-C inoculated animals. No virus could be isolated from the NT, trachea or lungs at day 3, 5 or 7 p.i. of the HMPV-inoculated animals. Based on these results, we concluded that HMPV does not replicate in turkey poults.
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and data not shown). In contrast to the avian cell lines, Vero-118 cells supported efficient replication of both HMPV and AMPV-C (Fig. 3) (de Graaf et al., 2008a).
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First, multi-step replication curves were generated to compare the replication efficiency of the chimeric viruses with those of the parental viruses HMPV and AMPV-C in Vero-118 cells. AMPV-C replicated faster in Vero-118 cells than HMPV. Furthermore, HMPV/NAMPV-C, HMPV/FAMPV-C, HMPV/SHAMPV-C, HMPV/GAMPV-C and HMPV/LAMPV-C replicated to similar titres as wild-type HMPV. HMPV/PAMPV-C replicated to slightly higher titres than the wild-type HMPV, as described previously (de Graaf et al., 2008a; Pham et al., 2005).
Next, multi-step replication curves were generated to compare the replication efficiency of the chimeric viruses with those of the parental viruses HMPV and AMPV-C in QT6 cells. AMPV-C replicated efficiently in QT6 cells in contrast to HMPV, which replicated to at least 1000-fold lower titres. HMPV/NAMPV-C, HMPV/PAMPV-C, HMPV/GAMPV-C and HMPV/LAMPV-C did not replicate in QT6 cells. In contrast, HMPV/SHAMPV-C replicated to similar titres as the wild-type HMPV. HMPV/FAMPV-C replicated at least tenfold more efficiently than HMPV, but less efficiently than AMPV-C. These results indicated that metapneumovirus F protein is an important determinant of host tropism.
Replication of metapneumovirus vRNA-like molecules by chimeric polymerase complexes
All chimeric HMPVs in which polymerase complex proteins were exchanged with those of AMPV-C replicated to similar or higher titres as the wild-type virus in Vero-118, but were attenuated in QT6 cells. To investigate the functional interaction between the polymerase complex proteins of human and avian metapneumoviruses in QT6 cells as the cause of this attenuation, a mini-replicon assay was performed. The HMPV and AMPV-C mini-replicon systems were both tested and the HMPV N, P, L, and M2.1 expression plasmids were exchanged individually with those of AMPV-C to mimic the gene configuration of the chimeric viruses (Fig. 4). The HMPV and AMPV-C mini-replicon systems were both functional in QT6 cells. However, the HMPV vRNA-like molecules were not replicated at all by the chimeric HMPV/AMPV-C polymerase complexes. This reduction in replication or transcription efficiency observed for the chimeric polymerase complexes could thus be responsible for the impaired replication of the chimeric viruses in QT6 cells.
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). HMPV/NAMPV-C, HMPV/PAMPV-C, HMPV/GAMPV-C, HMPV/SHAMPV-C and HMPV/LAMPV-C replicated in Vero-118 cells to high titres with minimal CPE, in contrast to AMPV-C and HMPV/FAMPV-C, both of which resulted in pronounced CPE (Fig. 5, left panels). Profound CPE was also observed in QT6 cells inoculated with AMPV-C and HMPV/FAMPV-C, whereas HMPV and HMPV/SHAMPV-C, which only replicated to low titres in QT6 cells, displayed minimal CPE (Fig. 5, right panels), suggesting that the F protein was responsible for this difference.
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). The chimeric F expression plasmids were tested in a cell-content mixing assay in both Vero-118 (Fig. 6b) and QT6 (Fig. 6c) cells using an LTR–CAT or pT7–GFP-based system, respectively. The LTR–CAT-based system yielded higher signals compared with the pT7–GFP-based system in Vero-118 cells, but the HIV-1 LTR was less functional in the avian cell line (data not shown).
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In QT6 cells, higher levels of CAT expression were observed for the AMPV-C F protein than for the HMPV F. Thus, AMPV F was more fusogenic than HMPV F in both Vero-118 and QT6 cells. Furthermore, the chimeric F proteins resulted in similar trends in fusion activity in Vero-118 and QT6 cells. Although in Vero-118 cells F2a–F1ah yielded the highest levels of CAT, F2a–F1h resulted in the highest levels of GFP expression in QT6 cells. In conclusion, the F2 region of AMPV-C F was responsible for the increase in fusogenic properties in both QT6 and Vero-118 cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This conclusion was based on clinical signs and immunohistochemical findings only, as no seroconversion or recovery of infectious HMPV could be demonstrated (Velayudhan et al., 2006). Inoculation with HMPV thus resulted in an abortive infection, whilst AMPV-C replicated efficiently. Here, we have shown clear differences in the host range of HMPV and AMPV-C. An inoculum dose of 2x104 or 2x106 TCID50 AMPV-C resulted in robust infection of the respiratory tract of turkeys and seroconversion after 10 days. After inoculation with 2x104 TCID50, AMPV-C was detected in the NT and trachea. No virus was isolated from the lungs, in agreement with previous experiments that demonstrated replication in the upper respiratory tract only (Jirjis et al., 2002). In sharp contrast, no infectious virus could be recovered from tracheal swabs of turkeys inoculated with an inoculum dose of 2x104 or 2x106 TCID50 HMPV. These results were confirmed by RT-PCR, using tests that are more sensitive than virus isolation (Maertzdorf et al., 2004). In addition, after inoculation with 2x104 TCID50 HMPV, no virus was detected in the NT, trachea or lungs at day 3, 5 or 7 p.i. Therefore, we concluded that HMPV does not replicate in turkeys, in contrast to AMPV-C.
To investigate this difference in host range, an in vitro cell-culture model was set up. Mammalian Vero-118 cells supported the replication of both HMPV and AMPV-C. In contrast, avian QT6 cells were highly permissive for AMPV-C replication, but did not result in efficient replication of HMPV. Replication curves in Vero-118 cells of chimeric HMPVs in which the N, P, SH, F, G and L genes were replaced with those of AMPV-C revealed similar replication kinetics to the wild-type HMPV with the exception of HMPV/PAMPV-C, which gave slightly higher virus titres. However, in QT6 cells, large differences in replication kinetics were observed. QT6 cells supported efficient replication of AMPV-C, but supported replication to a much lower extent for HMPV. Recombinant HMPVs in which the N, P, G and L genes were replaced with those of AMPV-C replicated to lower titres than the wild-type virus. In contrast, the chimeric virus containing the F protein of AMPV-C replicated more efficiently than HMPV. Similar results were found in QT35 cells (data not shown). This indicated that the F protein is the main determinant of host tropism of metapneumoviruses in vitro. Interestingly, for HRSV and BRSV, similar differences in host range have been described in vivo and in vitro; using primary cell lines it was found that the F protein is the main determinant of cellular host range (Schlender et al., 2003).
The HRSV G protein has an important role in the attachment of viral particles to the host cell and it has been suggested that cellular glycosaminoglycans or heparin-like molecules are involved in this binding (Hallak et al., 2000a, b; Krusat & Streckert, 1997). The HRSV G protein is not essential for viral viability in vitro, but HRSV from which the G gene has been deleted is highly attenuated in vivo, as it does not replicate in the upper respiratory tract and replicates at a very low level in the lower respiratory tract of mice (Teng et al., 2001). Similar to HRSV, HMPV and AMPV-C G proteins are not essential for virus replication in vitro (Biacchesi et al., 2004; Naylor et al., 2004). Moreover, HMPV from which the G gene has been deleted is attenuated but viable in hamsters and non-human primates (Biacchesi et al., 2004, 2005). Thus, it appears that the F protein by itself can promote both attachment and fusion. The fact that chimeric HMPV containing the F protein of AMPV-C replicated to lower titres than AMPV-C could be due to a functional restriction of one of the other HMPV proteins in QT6 cells because of host incompatibility.
Chimeric HMPVs containing polymerase complex proteins of AMPV-C did not replicate in QT6 cells. To test whether this difference was due to differences in replication and transcription efficiency of the chimeric polymerase complexes, mini-replicon assays were performed using similar chimeric polymerase complexes. Indeed, chimeric polymerase complexes were dysfunctional with respect to replicating and transcribing the vRNA-like molecules. In contrast, in mammalian cell lines, gene exchange between HMPV and AMPV-C resulted in only modest differences in polymerase complex activity both in mini-replicon assays and in replication curves using chimeric viruses (de Graaf et al., 2008a).
It has been proposed that HMPV originated from a cross-species transmission event of an AMPV-C-like virus from birds to humans around 200 years ago. As there is a wider variety of AMPV subgroups compared with HMPV, it has been hypothesized that the direction of transmission was from birds to humans (de Graaf et al., 2008b). Therefore, it is of interest that HMPV did not replicate efficiently in QT6, QT35 (data not shown) and CEF cells (van den Hoogen et al., 2001). In contrast, AMPV-C was more promiscuous in its cellular host range. Despite its avian origin, AMPV-C replicates to high titres in mammalian cell lines such as Vero-118, baby hamster kidney cells (BHK-21), baby grivet monkey kidney (BGM) and African green monkey kidney cells (MA-104) (Tiwari et al., 2006). Moreover, AMPV-C can infect and replicate in hamsters (de Graaf et al., 2008a).
HMPV is a poor inducer of syncytium formation in infected cultures, in contrast to AMPV-C. By comparing chimeric AMPV-C/HMPV viruses in cell cultures, it was found that the F protein caused this difference. To investigate this phenomenon, cell-content mixing assays were performed. It was found that the F protein of AMPV-C was more fusogenic in Vero-118 and Q6 cells than HMPV. The region responsible for the difference in fusion was further mapped using chimeric AMPV-C/HMPV F expression plasmids. This analysis revealed that the F2 region of the F protein was responsible for the differences in fusion capacity. Surprisingly, F2a–F1ah gave higher fusion activity in Vero cells and F2a–F1h in QT6 cells compared with Fa. This increase in fusion activity could not be explained and requires further research.
From these studies, we conclude that AMPV-C and HMPV display clear differences in their ability to replicate in turkeys, and that the F protein is responsible for the differences in host tropism of HMPV and AMPV-C. The AMPV-C F protein was more fusogenic than the F protein of HMPV, in both avian and mammalian cells. Although the F protein was the main determinant of host tropism, host tropism is not necessarily related to fusion efficiency. The difference in host tropism and fusion efficiency may be caused by a differential capacity to bind to cellular receptors. Future work should be directed towards identifying the virus receptors on the host cell that are recognized by the F proteins of HMPV and AMPV-C.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Biacchesi, S., Skiadopoulos, M. H., Yang, L., Lamirande, E. W., Tran, K. C., Murphy, B. R., Collins, P. L. & Buchholz, U. J. (2004). Recombinant human metapneumovirus lacking the small hydrophobic SH and/or attachment G glycoprotein: deletion of G yields a promising vaccine candidate. J Virol 78, 12877–12887.
Biacchesi, S., Pham, Q. N., Skiadopoulos, M. H., Murphy, B. R., Collins, P. L. & Buchholz, U. J. (2005). Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates. J Virol 79, 12608–12613.
Buchholz, U. J., Finke, S. & Conzelmann, K. K. (1999). Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73, 251–259.
Cook, J. K. (2000). Avian rhinotracheitis. Rev Sci Tech 19, 602–613.[Medline]
Crowe, J. E., Jr (2004). Human metapneumovirus as a major cause of human respiratory tract disease. Pediatr Infect Dis J 23, S215–S221.[Medline]
de Graaf, M., Herfst, S., Schrauwen, E. J., Choi, Y., van den Hoogen, B. G., Osterhaus, A. D. & Fouchier, R. A. (2008a). Specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses. J Gen Virol 89, 975–983.
de Graaf, M., Osterhaus, A. D. M. E., Fouchier, R. A. M. & Holmes, E. C. (2008b). Evolutionary dynamics of human and avian metapneumoviruses. J Gen Virol 89, 2933–2942.
Eterradossi, N., Toquin, D., Guittet, M. & Bennejean, G. (1995). Evaluation of different turkey rhinotracheitis viruses used as antigens for serological testing following live vaccination and challenge. Zentralbl Veterinarmed B 42, 175–186.[Medline]
Falsey, A. R., Erdman, D., Anderson, L. J. & Walsh, E. E. (2003). Human metapneumovirus infections in young and elderly adults. J Infect Dis 187, 785–790.[CrossRef][Medline]
Govindarajan, D. & Samal, S. K. (2004). Sequence analysis of the large polymerase (L) protein of the US strain of avian metapneumovirus indicates a close resemblance to that of the human metapneumovirus. Virus Res 105, 59–66.[CrossRef][Medline]
Govindarajan, D. & Samal, S. K. (2005). Analysis of the complete genome sequence of avian metapneumovirus subgroup C indicates that it possesses the longest genome among metapneumoviruses. Virus Genes 30, 331–333.[CrossRef][Medline]
Govindarajan, D., Yunus, A. S. & Samal, S. K. (2004). Complete sequence of the G glycoprotein gene of avian metapneumovirus subgroup C and identification of a divergent domain in the predicted protein. J Gen Virol 85, 3671–3675.
Govindarajan, D., Buchholz, U. J. & Samal, S. K. (2006). Recovery of avian metapneumovirus subgroup C from cDNA: cross-recognition of avian and human metapneumovirus support proteins. J Virol 80, 5790–5797.
Gruters, R. A., Otto, S. A., Al, B. J., Verhoeven, A. J., Verweij, C. L., Van Lier, R. A. & Miedema, F. (1991). Non-mitogenic T cell activation signals are sufficient for induction of human immunodeficiency virus transcription. Eur J Immunol 21, 167–172.[Medline]
Hallak, L. K., Collins, P. L., Knudson, W. & Peeples, M. E. (2000a). Iduronic acid-containing glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection. Virology 271, 264–275.[CrossRef][Medline]
Hallak, L. K., Spillmann, D., Collins, P. L. & Peeples, M. E. (2000b). Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection. J Virol 74, 10508–10513.
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. & other authors (2004). Recovery of human metapneumovirus genetic lineages A and B from cloned cDNA. J Virol 78, 8264–8270.
Herfst, S., Mas, V., Ver, L. S., Wierda, R. J., Osterhaus, A. D., Fouchier, R. A. & Melero, J. A. (2008). Low pH induced membrane fusion mediated by human metapneumoviruses F protein is a rare, strain dependent phenomenon. J Virol 82, 8891–8895.
Jirjis, F. F., Noll, S. L., Halvorson, D. A., Nagaraja, K. V. & Shaw, D. P. (2002). Pathogenesis of avian pneumovirus infection in turkeys. Vet Pathol 39, 300–310.
Jirjis, F. F., Noll, S. L., Halvorson, D. A., Nagaraja, K. V., Martin, F. & Shaw, D. P. (2004). Effects of bacterial coinfection on the pathogenesis of avian pneumovirus infection in turkeys. Avian Dis 48, 34–49.[CrossRef][Medline]
Juhasz, K. & Easton, A. J. (1994). Extensive sequence variation in the attachment (G) protein gene of avian pneumovirus: evidence for two distinct subgroups. J Gen Virol 75, 2873–2880.
Krusat, T. & Streckert, H. J. (1997). Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells. Arch Virol 142, 1247–1254.[CrossRef][Medline]
Kuiken, T., Van Den Hoogen, B. G., Van Riel, D. A., Laman, J. D., Van Amerongen, G., Sprong, L., Fouchier, R. A. & Osterhaus, A. D. (2004). Experimental human metapneumovirus infection of cynomolgus macaques (Macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract. Am J Pathol 164, 1893–1900.
Lamb, R. A., Paterson, R. G. & Jardetzky, T. S. (2006). Paramyxovirus membrane fusion: lessons from the F and HN atomic structures. Virology 344, 30–37.[CrossRef][Medline]
Maertzdorf, J., Wang, C. K., Brown, J. B., Quinto, J. D., Chu, M., De Graaf, M., Van Den Hoogen, B. G., Spaete, R., Osterhaus, A. D. & Fouchier, R. A. (2004). Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages. J Clin Microbiol 42, 981–986.
Naylor, C. J., Brown, P. A., Edworthy, N., Ling, R., Jones, R. C., Savage, C. E. & Easton, A. J. (2004). Development of a reverse-genetics system for Avian pneumovirus demonstrates that the small hydrophobic (SH) and attachment (G) genes are not essential for virus viability. J Gen Virol 85, 3219–3227.
Pham, Q. N., Biacchesi, S., Skiadopoulos, M. H., Murphy, B. R., Collins, P. L. & Buchholz, U. J. (2005). Chimeric recombinant human metapneumoviruses with the nucleoprotein or phosphoprotein open reading frame replaced by that of avian metapneumovirus exhibit improved growth in vitro and attenuation in vivo. J Virol 79, 15114–15122.
Rawling, J., García-Barreno, B. & Melero, J. A. (2008). Insertion of the two cleavage sites of the respiratory syncytial virus fusion protein in Sendai virus fusion protein leads to enhanced cell–cell fusion and a decreased dependency on the HN attachment protein for activity. J Virol 82, 5986–5998.
Reed, L. J. & Muench, H. (1938). A simple method of estimating fifty percent end points. Am J Epidemiol 27, 493–497.
Russell, C. J., Jardetzky, T. S. & Lamb, R. A. (2001). Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion. EMBO J 20, 4024–4034.[CrossRef][Medline]
Schlender, J., Zimmer, G., Herrler, G. & Conzelmann, K. K. (2003). Respiratory syncytial virus (RSV) fusion protein subunit F2, not attachment protein G, determines the specificity of RSV infection. J Virol 77, 4609–4616.
Schowalter, R. M., Smith, S. E. & Dutch, R. E. (2006). Characterization of human metapneumovirus F protein-promoted membrane fusion: critical roles for proteolytic processing and low pH. J Virol 80, 10931–10941.
Seal, B. S. (1998). Matrix protein gene nucleotide and predicted amino acid sequence demonstrate that the first US avian pneumovirus isolate is distinct from European strains. Virus Res 58, 45–52.[CrossRef][Medline]
Teng, M. N., Whitehead, S. S. & Collins, P. L. (2001). Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo. Virology 289, 283–296.[CrossRef][Medline]
Tiwari, A., Patnayak, D. P., Chander, Y. & Goyal, S. M. (2006). Permissibility of different cell types for the growth of avian metapneumovirus. J Virol Methods 138, 80–84.[CrossRef][Medline]
van den Hoogen, B. G., de Jong, J. C., Groen, J., Kuiken, T., de Groot, R., Fouchier, R. A. & Osterhaus, A. D. (2001). A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 7, 719–724.[CrossRef][Medline]
van den Hoogen, B. G., van Doornum, G. J. J., Fockens, J. C., Cornelissen, J. J., Beyer, W. E. P., de Groot, R., Osterhaus, A. D. M. E. & Fouchier, R. A. M. (2003). Prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients. J Infect Dis 188, 1571–1577.[CrossRef][Medline]
van den Hoogen, B. G., Herfst, S., de Graaf, M., Sprong, L., van Lavieren, R., van Amerongen, G., Yuksel, S., Fouchier, R. A., Osterhaus, A. D. & de Swart, R. L. (2007). Experimental infection of macaques with human metapneumovirus induces transient protective immunity. J Gen Virol 88, 1251–1259.
Velayudhan, B. T., McComb, B., Bennett, R. S., Lopes, V. C., Shaw, D., Halvorson, D. A. & Nagaraja, K. V. (2005). Emergence of a virulent type C avian metapneumovirus in turkeys in Minnesota. Avian Dis 49, 520–526.[CrossRef][Medline]
Velayudhan, B. T., Nagaraja, K. V., Thachil, A. J., Shaw, D. P., Gray, G. C. & Halvorson, D. A. (2006). Human metapneumovirus in turkey poults. Emerg Infect Dis 12, 1853–1859.[Medline]
Williams, J. V., Wang, C. K., Yang, C. F., Tollefson, S. J., House, F. S., Heck, J. M., Chu, M., Brown, J. B., Lintao, L. D. & other authors (2006). The role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience. J Infect Dis 193, 387–395.[CrossRef][Medline]
Received 19 December 2008;
accepted 23 February 2009.
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), AMPV-C (
), HMPV/NAMPV-C (
), HMPV/PAMPV-C (
), HMPV/FAMPV-C (
), HMPV/SHAMPV-C (
), HMPV/GAMPV-C (
) or HMPV/LAMPV-C (
). Supernatants were collected daily and virus titres were determined by plaque assay. The results shown are representative of assays performed at least twice for each virus.
