Detection of influenza C virus by a real-time RT-PCR assay

Abstract

Background: Influenza C virus can cause both upper and lower respiratory tract infections and has been reported to be prevalent in children. However, these infections have been under-diagnosed, and epidemiological data available are limited due to the lack of convenient detection assays.

Objective: Design and validate a real-time reverse-transcriptase PCR (rt RT-PCR) assay for the detection of influenza C.

Study design: Respiratory samples from two primary settings, namely, children who were hospitalized or seen in the emergency department, and respiratory outbreaks for which no other viral etiology was found were used for the detection of influenza C.

Results and conclusions: The assay was sensitive and specific for the detection of influenza C. Eleven of 474 (2·32%) patients, all less than 10 years of age, were positive for influenza C. The strains clustered into two lineages, namely C/Kanagawa and C/Sao Paulo, based upon sequencing of the hemagglutinin-esterase gene. Epidemiological data showed that a higher proportion of influenza C infections occur in younger children and during the winter months. This is the first report of the detection of influenza C in Alberta, Canada, and suggests that the detection of this virus should be included in respiratory virus testing panels.

Keywords: Influenza C; real-time PCR; respiratory virus.

Figure 1

Age range of patients positive for influenza C virus. The number of samples tested and positives detected in each group is indicated. Also indicated is the percentage of positive samples detected in each age group.

Figure 2

Seasonality of influenza C virus. The number of samples tested, positives detected, and percentage of positive samples in each month is indicated.

Figure 3

Phylogenetic tree showing the relationship between influenza C viruses based on partial hemagglutinin‐esterase (HE) gene sequence. The phylogenetic tree includes the six classically different lineages of influenza C (representatives in bold), positive samples from this study, representative sequences from different parts of the world and one sequence from a pig isolate for comparison. The Genbank numbers for sequences used in the alignment are as follows: Taylor_47 =  C/Taylor/1233/47 (M11637.1), Aichi_81 = Aichi/1/81 (D28970), Sao Paulo_82 = C/Sao Paulo/378/82 (AB035364.1), Kanagawa_76 = C/Kanagawa/1/76 (D63470), and Yamagata_81 = Yamagata/26/81 (D28971.1), Aichi_99 = C/Aichi/1/99 (AB182357), Catalonia_2009 = C/Catalonia/1457/2009 (HM748633.1), England_83 = C/England/892/83 (M11642.1), Fukuoka_2004 = C/Fukuoka/2/2004 (AB252164.1), Georgia_69 = C/Georgia/1/69 (AB035359.1), Greece_79 = C/Greece/1/79 (AB035363), Johannesburg_66 = C/Johannesburg/66 (M17868), Kyoto_79 = C/Kyoto/1/79 (D63472), Miyagi_92 = C/Miyagi/3/92 (AB219076), Mississippi_80 = C/Mississippi/80 (M11640), Paris_67 = C/Paris/1/67 (AB035357), Kansas_79 = C/Kansas/2/79 (AB035361.1), Yamagata_93 = C/Yamagata/1/93 (AB035365.1), Singapore_2006 = C/Singapore/DSO‐050530/2006 (GQ853455.1), Yamagata_2004 = C/Yamagata/3/2004 (AB252153.1), Yamagata_98 = C/Yamagata/6/98 (AB064402.1), pigBeijing_81 = C/pig/Beijing/115/81 (M11644.1), Yamagata_88 = C/Yamagata/3/88 (D63473.1), New Jersey_76 = C/New Jersey/1/76 (AB035362.1), Sapporo_71 = C/Sapporo/71 (D63468.1).

Figure 4

Phylogenetic tree showing the relationship between influenza C viruses based on partial M gene sequence. The phylogenetic tree compares the matrix gene from the lineages defined based on the M and hemagglutinin‐esterase (HE) genes (representatives in bold), representative sequences from different parts of the world and one sequence from a pig isolate for comparison. The Genbank numbers for sequences used in the alignment are as follows: Pig_Beijing_81 = C/pig/Beijing/115/81(AB000722.1); Mississippi_80 = C/Mississippi/80 (AB000720.1); Kyoto_79 = C/Kyoto/1/79 (AB000609.1); England_83 = C/England/83 (AB000725.1); Yamagata_81 = C/Yamagata/26/81(AB000721.1); Kanagawa_76 = C/Kanagawa/1/76 (AB000606.1); Johannesburg_66 = C/Johannesburg/1/66 (AM410041.1); Greece_79 = C/Greece/79 (AB099602.1); NewJersey_76 =  C/NewJersey/76 (AB099600.1); Taylor_47 = C/Taylor/1233/47 (D26546.1); Yamagata_88 = C/Yamagata/1/88 (D16261.1); Miyagi_92 = C/Miyagi/2/92 (D87384.1); Sao Paulo_82 = C/Sao Paulo/378/82 (AB035372.1); Yamagata_93 = C/Yamagata/1/93 (AB035373.1); Aichi_81 = C/Aichi/1/81 (D16260.1); Aichi_99 = C/Aichi/1/99 (D16260.1); Kansas/1/79 = C/Kansas/1/79 (AB099601.1); Aomori_74 = C/Aomori/74 (D16259.1).