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Enhanced replication of avian-origin H3N2 canine influenza virus in eggs, cell cultures and mice by a two-amino acid insertion in neuraminidase stalk.

Lin Y, Xie X, Zhao Y, Kalhoro DH, Lu C, Liu Y - Vet. Res. (2016)

Bottom Line: Compared to the short stalk strain (without 2-aa insertion), the long stalk strain (with 2-aa insertion) exhibited higher peak titers and greater yields in Madin-Darby canine kidney (MDCK) cells, chicken embryo fibroblasts and canine bronchiolar epithelial cells, as well as much larger plaques in MDCK cell monolayers.However, in chickens, the two viral strains showed no significant difference with nearly the same proportion of detectable viral RNA loads in tissues.These observations suggest that the 2-aa insertion in the NA stalk acquired by avian-origin H3N2 CIV helps to enhance viral replication and is likely a result of adaptive evolution in canine hosts.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Canine influenza virus (CIV) is a newly identified, highly contagious respiratory pathogen in dogs. Recent studies indicate that avian-origin H3N2 CIV are circulating in Chinese dogs. To investigate the effects of a two-amino acid (2-aa) insertion naturally occurring at the distal end of the neuraminidase (NA) stalk found in Chinese isolates since 2010 on virus replication and virulence, we rescued the CIV strain, A/canine/Jiangsu/06/2011(H3N2) and its NA mutant without the 2-aa insertion using reverse genetics. The NA stalk length affected virus growth in cell culture. Compared to the short stalk strain (without 2-aa insertion), the long stalk strain (with 2-aa insertion) exhibited higher peak titers and greater yields in Madin-Darby canine kidney (MDCK) cells, chicken embryo fibroblasts and canine bronchiolar epithelial cells, as well as much larger plaques in MDCK cell monolayers. Furthermore, mice inoculated with the long stalk strain showed more severe pathologic damage in lung and higher proportion of detectable viral RNA in tissues. The long stalk strain induced local IFN-γ production with faster kinetics and higher levels in mice. However, in chickens, the two viral strains showed no significant difference with nearly the same proportion of detectable viral RNA loads in tissues. These observations suggest that the 2-aa insertion in the NA stalk acquired by avian-origin H3N2 CIV helps to enhance viral replication and is likely a result of adaptive evolution in canine hosts.

No MeSH data available.


Related in: MedlinePlus

Replication of r-06 and r-06/NA2 strains in MDCK, CEF and CBE cell cultures. MDCK, CEF and CBE cells were infected with the two viruses at an MOI of 0.001, 0.01 and 0.01, respectively, and the viral NS1 expression was determined at 24 h after infection by immunofluorescence microscopy using an anti-NS1 antibody. A Indirect immunofluorescence staining of infected cells. B FACS analysis of infected cells. Red bar indicates the positive region. The infected cells in which fluorescence intensities (FIs) were larger than those in mock infected cells were confirmed as positive (right part of the mock histogram, red bar region). The area of red bar region represents the count of NS1+cells. C Quantification of the NS1+ proportion of infected cells in FACS analysis. NS1+ ratio was calculated by the proportion of cells of red bar region in all cells by CFlow plus software. D Quantification of NS1 expression by mean fluorescence intensities (MFIs) in FACS analysis. NS1 expression is depicted as fold MFI of mock-infected cells. MFIs of NS1+ cells (in red bar region) were calculated by CFlow plus software. *P < 0.05, **P < 0.01.
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Fig4: Replication of r-06 and r-06/NA2 strains in MDCK, CEF and CBE cell cultures. MDCK, CEF and CBE cells were infected with the two viruses at an MOI of 0.001, 0.01 and 0.01, respectively, and the viral NS1 expression was determined at 24 h after infection by immunofluorescence microscopy using an anti-NS1 antibody. A Indirect immunofluorescence staining of infected cells. B FACS analysis of infected cells. Red bar indicates the positive region. The infected cells in which fluorescence intensities (FIs) were larger than those in mock infected cells were confirmed as positive (right part of the mock histogram, red bar region). The area of red bar region represents the count of NS1+cells. C Quantification of the NS1+ proportion of infected cells in FACS analysis. NS1+ ratio was calculated by the proportion of cells of red bar region in all cells by CFlow plus software. D Quantification of NS1 expression by mean fluorescence intensities (MFIs) in FACS analysis. NS1 expression is depicted as fold MFI of mock-infected cells. MFIs of NS1+ cells (in red bar region) were calculated by CFlow plus software. *P < 0.05, **P < 0.01.

Mentions: To evaluate the virus infectivity efficiency, viral NS1 expression was determined at 24 h after infection by immunofluorescence microscopy using an anti-NS1 antibody in all three cell lines. We inoculated confluent cell monolayers with r-06 or r-06/NA2 viruses, and positive cells exhibited green staining. From Figure 4A, we can see that in all three cells, stronger fluorescence was observed in r-06-infected cells than r-06/NA2-infected cells. Especially in MDCK cells, r-06 infection resulted in a noticeably greater number of stained cells than r-06/NA2 infection.Figure 4


Enhanced replication of avian-origin H3N2 canine influenza virus in eggs, cell cultures and mice by a two-amino acid insertion in neuraminidase stalk.

Lin Y, Xie X, Zhao Y, Kalhoro DH, Lu C, Liu Y - Vet. Res. (2016)

Replication of r-06 and r-06/NA2 strains in MDCK, CEF and CBE cell cultures. MDCK, CEF and CBE cells were infected with the two viruses at an MOI of 0.001, 0.01 and 0.01, respectively, and the viral NS1 expression was determined at 24 h after infection by immunofluorescence microscopy using an anti-NS1 antibody. A Indirect immunofluorescence staining of infected cells. B FACS analysis of infected cells. Red bar indicates the positive region. The infected cells in which fluorescence intensities (FIs) were larger than those in mock infected cells were confirmed as positive (right part of the mock histogram, red bar region). The area of red bar region represents the count of NS1+cells. C Quantification of the NS1+ proportion of infected cells in FACS analysis. NS1+ ratio was calculated by the proportion of cells of red bar region in all cells by CFlow plus software. D Quantification of NS1 expression by mean fluorescence intensities (MFIs) in FACS analysis. NS1 expression is depicted as fold MFI of mock-infected cells. MFIs of NS1+ cells (in red bar region) were calculated by CFlow plus software. *P < 0.05, **P < 0.01.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862097&req=5

Fig4: Replication of r-06 and r-06/NA2 strains in MDCK, CEF and CBE cell cultures. MDCK, CEF and CBE cells were infected with the two viruses at an MOI of 0.001, 0.01 and 0.01, respectively, and the viral NS1 expression was determined at 24 h after infection by immunofluorescence microscopy using an anti-NS1 antibody. A Indirect immunofluorescence staining of infected cells. B FACS analysis of infected cells. Red bar indicates the positive region. The infected cells in which fluorescence intensities (FIs) were larger than those in mock infected cells were confirmed as positive (right part of the mock histogram, red bar region). The area of red bar region represents the count of NS1+cells. C Quantification of the NS1+ proportion of infected cells in FACS analysis. NS1+ ratio was calculated by the proportion of cells of red bar region in all cells by CFlow plus software. D Quantification of NS1 expression by mean fluorescence intensities (MFIs) in FACS analysis. NS1 expression is depicted as fold MFI of mock-infected cells. MFIs of NS1+ cells (in red bar region) were calculated by CFlow plus software. *P < 0.05, **P < 0.01.
Mentions: To evaluate the virus infectivity efficiency, viral NS1 expression was determined at 24 h after infection by immunofluorescence microscopy using an anti-NS1 antibody in all three cell lines. We inoculated confluent cell monolayers with r-06 or r-06/NA2 viruses, and positive cells exhibited green staining. From Figure 4A, we can see that in all three cells, stronger fluorescence was observed in r-06-infected cells than r-06/NA2-infected cells. Especially in MDCK cells, r-06 infection resulted in a noticeably greater number of stained cells than r-06/NA2 infection.Figure 4

Bottom Line: Compared to the short stalk strain (without 2-aa insertion), the long stalk strain (with 2-aa insertion) exhibited higher peak titers and greater yields in Madin-Darby canine kidney (MDCK) cells, chicken embryo fibroblasts and canine bronchiolar epithelial cells, as well as much larger plaques in MDCK cell monolayers.However, in chickens, the two viral strains showed no significant difference with nearly the same proportion of detectable viral RNA loads in tissues.These observations suggest that the 2-aa insertion in the NA stalk acquired by avian-origin H3N2 CIV helps to enhance viral replication and is likely a result of adaptive evolution in canine hosts.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Canine influenza virus (CIV) is a newly identified, highly contagious respiratory pathogen in dogs. Recent studies indicate that avian-origin H3N2 CIV are circulating in Chinese dogs. To investigate the effects of a two-amino acid (2-aa) insertion naturally occurring at the distal end of the neuraminidase (NA) stalk found in Chinese isolates since 2010 on virus replication and virulence, we rescued the CIV strain, A/canine/Jiangsu/06/2011(H3N2) and its NA mutant without the 2-aa insertion using reverse genetics. The NA stalk length affected virus growth in cell culture. Compared to the short stalk strain (without 2-aa insertion), the long stalk strain (with 2-aa insertion) exhibited higher peak titers and greater yields in Madin-Darby canine kidney (MDCK) cells, chicken embryo fibroblasts and canine bronchiolar epithelial cells, as well as much larger plaques in MDCK cell monolayers. Furthermore, mice inoculated with the long stalk strain showed more severe pathologic damage in lung and higher proportion of detectable viral RNA in tissues. The long stalk strain induced local IFN-γ production with faster kinetics and higher levels in mice. However, in chickens, the two viral strains showed no significant difference with nearly the same proportion of detectable viral RNA loads in tissues. These observations suggest that the 2-aa insertion in the NA stalk acquired by avian-origin H3N2 CIV helps to enhance viral replication and is likely a result of adaptive evolution in canine hosts.

No MeSH data available.


Related in: MedlinePlus