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Effect of in vitro syncytium formation on the severity of human metapneumovirus disease in a murine model.

Aerts L, Cavanagh MH, Dubois J, Carbonneau J, Rhéaume C, Lavigne S, Couture C, Hamelin MÈ, Boivin G - PLoS ONE (2015)

Bottom Line: Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infections (ARTI) in children, elderly individuals and immunocompromised patients.In vitro, different HMPV strains can induce variable cytopathic effects ranging from large multinucleated syncytia to focal cell rounding.In vivo, however, the virulence and replicative capacity of the different HMPV strains did not appear to be solely dependent on the F gene but also on the viral background, with the strains containing the C-85473 background inducing more weight loss as well as increased lung viral titers, pro-inflammatory cytokines and inflammation than strains containing the CAN98-75 background.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Infectiologie of the Centre Hospitalier Universitaire de Québec and Université Laval, Quebec, Canada.

ABSTRACT
Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infections (ARTI) in children, elderly individuals and immunocompromised patients. In vitro, different HMPV strains can induce variable cytopathic effects ranging from large multinucleated syncytia to focal cell rounding. In this study, we investigated the impact of different in vitro phenotypes of two HMPV strains on viral replication and disease severity in a BALB/c mouse model. We first generated two recombinant GFP-expressing HMPV viruses: C-85473, a syncytium-inducing strain (rC-85473) belonging to the A1 subtype and CAN98-75, a focal cell rounding-inducing strain (rCAN98-75) of the B2 subtype. We subsequently exchanged the F genes of both strains to create the chimeric viruses rC-85473_F and rCAN98-75_F. We demonstrated that the F protein was the sole protein responsible for the syncytium phenotype and that viruses carrying a syncytium-inducing F protein replicated to significantly higher titers in vitro. In vivo, however, the virulence and replicative capacity of the different HMPV strains did not appear to be solely dependent on the F gene but also on the viral background, with the strains containing the C-85473 background inducing more weight loss as well as increased lung viral titers, pro-inflammatory cytokines and inflammation than strains containing the CAN98-75 background. In conclusion, the F protein is the main determinant of syncytium formation and replication kinetics in vitro, although it is not the only factor implicated in HMPV disease severity in mice.

No MeSH data available.


Related in: MedlinePlus

Real-time cell analysis of recombinant HMPV strains.LLC-MK2 monolayers in 96 well-plates were infected with rHMPV at an MOI of 0.01 (a) Output of one real-time cell analysis (RTCA) experiment; data was normalized using mock-infected wells and normalized cell index is plotted. (b) Mean time until cell index is reduced by 50% from 4 independent experiments is plotted. *, p < 0.05 using unpaired, two-tailed Student t-test.
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pone.0120283.g004: Real-time cell analysis of recombinant HMPV strains.LLC-MK2 monolayers in 96 well-plates were infected with rHMPV at an MOI of 0.01 (a) Output of one real-time cell analysis (RTCA) experiment; data was normalized using mock-infected wells and normalized cell index is plotted. (b) Mean time until cell index is reduced by 50% from 4 independent experiments is plotted. *, p < 0.05 using unpaired, two-tailed Student t-test.

Mentions: We also investigated the effect of HMPV infection on the state of LLC-MK2 monolayers using RTCA. This method measures the change in electrical impedance across a cell monolayer in real-time. A parameter called cell index (CI) is used to quantify cell status based on the detected cell-electrode impedance; an elevated CI means that cells have fully adhered to the well and have proliferated, whereas a low CI indicates changes in morphology and viability of the cell monolayer. The changes in CI during an infection experiment using RTCA is shown in Fig. 4a and mean time until the normalized CI was reduced by 50% is reported in Fig. 4b. On average, it took rCAN98–75 28 h longer to reduce the CI by 50% than rC-85473 ((118 ± 9 h compared to 91 ± 8 h). Exchanging the F protein resulted in reverse phenotypes with 50% reduction of CI obtained by 98 ± 4 h and 115 ± 2 h for rCAN98–75_F and rC-85473_F, respectively. These data show that the HMPV strains carrying the syncytium-inducing F protein from C-85473 alter the cell state of infected cells faster than viruses carrying the F protein from CAN98–75.


Effect of in vitro syncytium formation on the severity of human metapneumovirus disease in a murine model.

Aerts L, Cavanagh MH, Dubois J, Carbonneau J, Rhéaume C, Lavigne S, Couture C, Hamelin MÈ, Boivin G - PLoS ONE (2015)

Real-time cell analysis of recombinant HMPV strains.LLC-MK2 monolayers in 96 well-plates were infected with rHMPV at an MOI of 0.01 (a) Output of one real-time cell analysis (RTCA) experiment; data was normalized using mock-infected wells and normalized cell index is plotted. (b) Mean time until cell index is reduced by 50% from 4 independent experiments is plotted. *, p < 0.05 using unpaired, two-tailed Student t-test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4372586&req=5

pone.0120283.g004: Real-time cell analysis of recombinant HMPV strains.LLC-MK2 monolayers in 96 well-plates were infected with rHMPV at an MOI of 0.01 (a) Output of one real-time cell analysis (RTCA) experiment; data was normalized using mock-infected wells and normalized cell index is plotted. (b) Mean time until cell index is reduced by 50% from 4 independent experiments is plotted. *, p < 0.05 using unpaired, two-tailed Student t-test.
Mentions: We also investigated the effect of HMPV infection on the state of LLC-MK2 monolayers using RTCA. This method measures the change in electrical impedance across a cell monolayer in real-time. A parameter called cell index (CI) is used to quantify cell status based on the detected cell-electrode impedance; an elevated CI means that cells have fully adhered to the well and have proliferated, whereas a low CI indicates changes in morphology and viability of the cell monolayer. The changes in CI during an infection experiment using RTCA is shown in Fig. 4a and mean time until the normalized CI was reduced by 50% is reported in Fig. 4b. On average, it took rCAN98–75 28 h longer to reduce the CI by 50% than rC-85473 ((118 ± 9 h compared to 91 ± 8 h). Exchanging the F protein resulted in reverse phenotypes with 50% reduction of CI obtained by 98 ± 4 h and 115 ± 2 h for rCAN98–75_F and rC-85473_F, respectively. These data show that the HMPV strains carrying the syncytium-inducing F protein from C-85473 alter the cell state of infected cells faster than viruses carrying the F protein from CAN98–75.

Bottom Line: Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infections (ARTI) in children, elderly individuals and immunocompromised patients.In vitro, different HMPV strains can induce variable cytopathic effects ranging from large multinucleated syncytia to focal cell rounding.In vivo, however, the virulence and replicative capacity of the different HMPV strains did not appear to be solely dependent on the F gene but also on the viral background, with the strains containing the C-85473 background inducing more weight loss as well as increased lung viral titers, pro-inflammatory cytokines and inflammation than strains containing the CAN98-75 background.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Infectiologie of the Centre Hospitalier Universitaire de Québec and Université Laval, Quebec, Canada.

ABSTRACT
Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infections (ARTI) in children, elderly individuals and immunocompromised patients. In vitro, different HMPV strains can induce variable cytopathic effects ranging from large multinucleated syncytia to focal cell rounding. In this study, we investigated the impact of different in vitro phenotypes of two HMPV strains on viral replication and disease severity in a BALB/c mouse model. We first generated two recombinant GFP-expressing HMPV viruses: C-85473, a syncytium-inducing strain (rC-85473) belonging to the A1 subtype and CAN98-75, a focal cell rounding-inducing strain (rCAN98-75) of the B2 subtype. We subsequently exchanged the F genes of both strains to create the chimeric viruses rC-85473_F and rCAN98-75_F. We demonstrated that the F protein was the sole protein responsible for the syncytium phenotype and that viruses carrying a syncytium-inducing F protein replicated to significantly higher titers in vitro. In vivo, however, the virulence and replicative capacity of the different HMPV strains did not appear to be solely dependent on the F gene but also on the viral background, with the strains containing the C-85473 background inducing more weight loss as well as increased lung viral titers, pro-inflammatory cytokines and inflammation than strains containing the CAN98-75 background. In conclusion, the F protein is the main determinant of syncytium formation and replication kinetics in vitro, although it is not the only factor implicated in HMPV disease severity in mice.

No MeSH data available.


Related in: MedlinePlus