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Genome and infection characteristics of human parechovirus type 1: the interplay between viral infection and type I interferon antiviral system.

Chang JT, Yang CS, Chen YS, Chen BC, Chiang AJ, Chang YH, Tsai WL, Lin YS, Chao D, Chang TH - PLoS ONE (2015)

Bottom Line: Human parechoviruses (HPeVs), members of the family Picornaviridae, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis.A facilitated cytopathic effect and increased viral titers were reached after serial viral passages in Vero cells, with viral genome mutation found in later passages.The characterization of the complete genome and infection features of HPeV1 provide comprehensive information about this newly isolated HPeV1 for further diagnosis, prevention or treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan; Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan; Department of Pediatrics; Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.

ABSTRACT
Human parechoviruses (HPeVs), members of the family Picornaviridae, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis. A new contemporary strain of HPeV1, KVP6 (accession no. KC769584), was isolated from a clinical specimen. Full-genome alignment revealed that HPeV1 KVP6 shares high genome homology with the German strain of HPeV1, 7555312 (accession no. FM178558) and could be classified in the clade 1B group. An intertypic recombination was shown within the P2-P3 genome regions of HPeV1. Cell-type tropism test showed that T84 cells (colon carcinoma cells), A549 cells (lung carcinoma cells) and DBTRG-5MG cells (glioblastoma cells) were susceptible to HPeV1 infection, which might be relevant clinically. A facilitated cytopathic effect and increased viral titers were reached after serial viral passages in Vero cells, with viral genome mutation found in later passages. HPeV1 is sensitive to elevated temperature because 39C incubation impaired virion production. HPeV1 induced innate immunity with phosphorylation of interferon (IFN) regulatory transcription factor 3 and production of type I IFN in A549 but not T84 cells. Furthermore, type I IFN inhibited HPeV1 production in A549 cells but not T84 cells; T84 cells may be less responsive to type I IFN stimulation. Moreover, HPeV1-infected cells showed downregulated type I IFN activation, which indicated a type I IFN evasion mechanism. The characterization of the complete genome and infection features of HPeV1 provide comprehensive information about this newly isolated HPeV1 for further diagnosis, prevention or treatment strategies.

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HPeV1 propagation and replication analysis.(A) Left panel, plaque-forming assay of HPeV1 titration ofP5-P10 progeny. Right panel, cytopathic effect (CPE) formation, HPeV1viral titer, and the results of VP1 sequence for each progeny. P5, ND:not determined; P6-P8, sequence no change; P9-P10, the point nucleotideand amino acid mutation are indicated. (B) A549 cells and(C) T84 cells were infected with each progeny at MOI =1; plaque-forming assay of virus production kinetics in culture mediumharvested at indicated times. (D) RT-qPCR analysis of mRNAlevel of HPeV1 structure-protein (VP1) positive sense (+) and negativesense (-) in A549 cells (2×105, upper panels) and T84cells (2×105, lower panels) infected with HPeV1 (MOI =1). Data are mean±SD. (E) Immunoblotting of VP0viral protein and loading control, β-actin, in A549 cells(2×105, upper panels) and T84 cells(2×105, lower panels) infected with HPeV1 (MOI =1) as indicated. (F) A549 cells (2×105,upper panel) and T84 cells (2×105, lower panel) wereinfected with HPeV1 (MOI = 1); plaque-forming assay of virus productionin culture supernatant at the indicated times. Data aremean±SD.
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pone.0116158.g003: HPeV1 propagation and replication analysis.(A) Left panel, plaque-forming assay of HPeV1 titration ofP5-P10 progeny. Right panel, cytopathic effect (CPE) formation, HPeV1viral titer, and the results of VP1 sequence for each progeny. P5, ND:not determined; P6-P8, sequence no change; P9-P10, the point nucleotideand amino acid mutation are indicated. (B) A549 cells and(C) T84 cells were infected with each progeny at MOI =1; plaque-forming assay of virus production kinetics in culture mediumharvested at indicated times. (D) RT-qPCR analysis of mRNAlevel of HPeV1 structure-protein (VP1) positive sense (+) and negativesense (-) in A549 cells (2×105, upper panels) and T84cells (2×105, lower panels) infected with HPeV1 (MOI =1). Data are mean±SD. (E) Immunoblotting of VP0viral protein and loading control, β-actin, in A549 cells(2×105, upper panels) and T84 cells(2×105, lower panels) infected with HPeV1 (MOI =1) as indicated. (F) A549 cells (2×105,upper panel) and T84 cells (2×105, lower panel) wereinfected with HPeV1 (MOI = 1); plaque-forming assay of virus productionin culture supernatant at the indicated times. Data aremean±SD.

Mentions: To understand whether viral features of the cytopathic effect (CPE) and viralgenome are changed during viral amplification, we propagated the HPeV1 KVP6virus from the 4th progeny (P4) in Vero cells with serialsub-passaging. We found no CPE at post-infection day 10 in P5 infected cells,which might due to the extremely low virion production. Nevertheless, P6 progenyshowed CPE and plaque formation; CPE was accelerated from day 9 to 3 in thelater progenies, and viral titer was increased at P9 and P10 (Fig. 3A, left panels). Genomemutation during virus passage was reported in hepatitis A and E virus infectionand severe acute respiratory syndrome (SARS-CoV) infection [38,39,40]. We analyzed the VP1sequence from different progenies, because in P9 of HPeV1 KVP6, the nucleotideposition 2984 adenosine was replaced by thymine and resulted in a mutation inamino acid position 768 from Asparagine to Isoleucine (Fig. 3A, right panel). Becausethe amino acid position 768 is close to the RGD motif (position 763~765),whether this mutation relates to receptor binding might be of interest.Mutations other than the 2984 positive one may exist in other regions of thegenome. These data suggest that HPeV1 KVP6 virulence and viral titer could beamplified by serial passage in vitro; however, the genomeinstability would appear during the passages.


Genome and infection characteristics of human parechovirus type 1: the interplay between viral infection and type I interferon antiviral system.

Chang JT, Yang CS, Chen YS, Chen BC, Chiang AJ, Chang YH, Tsai WL, Lin YS, Chao D, Chang TH - PLoS ONE (2015)

HPeV1 propagation and replication analysis.(A) Left panel, plaque-forming assay of HPeV1 titration ofP5-P10 progeny. Right panel, cytopathic effect (CPE) formation, HPeV1viral titer, and the results of VP1 sequence for each progeny. P5, ND:not determined; P6-P8, sequence no change; P9-P10, the point nucleotideand amino acid mutation are indicated. (B) A549 cells and(C) T84 cells were infected with each progeny at MOI =1; plaque-forming assay of virus production kinetics in culture mediumharvested at indicated times. (D) RT-qPCR analysis of mRNAlevel of HPeV1 structure-protein (VP1) positive sense (+) and negativesense (-) in A549 cells (2×105, upper panels) and T84cells (2×105, lower panels) infected with HPeV1 (MOI =1). Data are mean±SD. (E) Immunoblotting of VP0viral protein and loading control, β-actin, in A549 cells(2×105, upper panels) and T84 cells(2×105, lower panels) infected with HPeV1 (MOI =1) as indicated. (F) A549 cells (2×105,upper panel) and T84 cells (2×105, lower panel) wereinfected with HPeV1 (MOI = 1); plaque-forming assay of virus productionin culture supernatant at the indicated times. Data aremean±SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380134&req=5

pone.0116158.g003: HPeV1 propagation and replication analysis.(A) Left panel, plaque-forming assay of HPeV1 titration ofP5-P10 progeny. Right panel, cytopathic effect (CPE) formation, HPeV1viral titer, and the results of VP1 sequence for each progeny. P5, ND:not determined; P6-P8, sequence no change; P9-P10, the point nucleotideand amino acid mutation are indicated. (B) A549 cells and(C) T84 cells were infected with each progeny at MOI =1; plaque-forming assay of virus production kinetics in culture mediumharvested at indicated times. (D) RT-qPCR analysis of mRNAlevel of HPeV1 structure-protein (VP1) positive sense (+) and negativesense (-) in A549 cells (2×105, upper panels) and T84cells (2×105, lower panels) infected with HPeV1 (MOI =1). Data are mean±SD. (E) Immunoblotting of VP0viral protein and loading control, β-actin, in A549 cells(2×105, upper panels) and T84 cells(2×105, lower panels) infected with HPeV1 (MOI =1) as indicated. (F) A549 cells (2×105,upper panel) and T84 cells (2×105, lower panel) wereinfected with HPeV1 (MOI = 1); plaque-forming assay of virus productionin culture supernatant at the indicated times. Data aremean±SD.
Mentions: To understand whether viral features of the cytopathic effect (CPE) and viralgenome are changed during viral amplification, we propagated the HPeV1 KVP6virus from the 4th progeny (P4) in Vero cells with serialsub-passaging. We found no CPE at post-infection day 10 in P5 infected cells,which might due to the extremely low virion production. Nevertheless, P6 progenyshowed CPE and plaque formation; CPE was accelerated from day 9 to 3 in thelater progenies, and viral titer was increased at P9 and P10 (Fig. 3A, left panels). Genomemutation during virus passage was reported in hepatitis A and E virus infectionand severe acute respiratory syndrome (SARS-CoV) infection [38,39,40]. We analyzed the VP1sequence from different progenies, because in P9 of HPeV1 KVP6, the nucleotideposition 2984 adenosine was replaced by thymine and resulted in a mutation inamino acid position 768 from Asparagine to Isoleucine (Fig. 3A, right panel). Becausethe amino acid position 768 is close to the RGD motif (position 763~765),whether this mutation relates to receptor binding might be of interest.Mutations other than the 2984 positive one may exist in other regions of thegenome. These data suggest that HPeV1 KVP6 virulence and viral titer could beamplified by serial passage in vitro; however, the genomeinstability would appear during the passages.

Bottom Line: Human parechoviruses (HPeVs), members of the family Picornaviridae, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis.A facilitated cytopathic effect and increased viral titers were reached after serial viral passages in Vero cells, with viral genome mutation found in later passages.The characterization of the complete genome and infection features of HPeV1 provide comprehensive information about this newly isolated HPeV1 for further diagnosis, prevention or treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan; Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan; Department of Pediatrics; Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.

ABSTRACT
Human parechoviruses (HPeVs), members of the family Picornaviridae, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis. A new contemporary strain of HPeV1, KVP6 (accession no. KC769584), was isolated from a clinical specimen. Full-genome alignment revealed that HPeV1 KVP6 shares high genome homology with the German strain of HPeV1, 7555312 (accession no. FM178558) and could be classified in the clade 1B group. An intertypic recombination was shown within the P2-P3 genome regions of HPeV1. Cell-type tropism test showed that T84 cells (colon carcinoma cells), A549 cells (lung carcinoma cells) and DBTRG-5MG cells (glioblastoma cells) were susceptible to HPeV1 infection, which might be relevant clinically. A facilitated cytopathic effect and increased viral titers were reached after serial viral passages in Vero cells, with viral genome mutation found in later passages. HPeV1 is sensitive to elevated temperature because 39C incubation impaired virion production. HPeV1 induced innate immunity with phosphorylation of interferon (IFN) regulatory transcription factor 3 and production of type I IFN in A549 but not T84 cells. Furthermore, type I IFN inhibited HPeV1 production in A549 cells but not T84 cells; T84 cells may be less responsive to type I IFN stimulation. Moreover, HPeV1-infected cells showed downregulated type I IFN activation, which indicated a type I IFN evasion mechanism. The characterization of the complete genome and infection features of HPeV1 provide comprehensive information about this newly isolated HPeV1 for further diagnosis, prevention or treatment strategies.

Show MeSH
Related in: MedlinePlus