Frequent Reassortment among Influenza C Viruses

Abstract

In a 9-year survey from December 1990 to December 1999 in Sendai City, Japan, we succeeded in isolating a total of 45 strains of influenza C virus. These 45 strains were isolated in clusters within 4 months in a year, especially from winter to early summer. Previous studies of the hemagglutinin-esterase genes of various influenza C virus isolates revealed the existence of five distinct virus lineages (Aichi/1/81-, Yamagata/26/81-, Mississippi/80-, Sao Paulo/82-, and Kanagawa/1/76-related lineage) in Japan between 1970 and the early 1990s (Y. Matsuzaki, K. Mizuta, H. Kimura, K. Sugawara, E. Tsuchiya, H. Suzuki, S. Hongo, and K. Nakamura, J. Gen. Virol. 81:1447-1452, 2000). Antigenic and genetic analyses of the 45 strains showed that they could be divided into these five virus lineages and a few antigenic groups were cocirculating in Sendai City. In 1990 and 1991 the dominant antigenic group was the Aichi/1/81 virus group, and in 1992 it was Yamagata/26/81 virus group. The Mississippi/80 virus group was isolated from 1993 to 1996, and the Yamagata/26/81 virus group reemerged in 1996 and continued to circulate until 1999. This finding led us to a speculation that the replacement of the dominant antigenic groups had occurred by immune selection within the human population in the restricted area. Phylogenetic analysis of seven RNA segments showed that 44 viruses among the 45 strains isolated in our surveillance work were reassortant viruses that have various genome compositions distinguishable from those of the reference strains of the each lineage. This observation suggests that the reassortment between two different influenza C virus strains occurs frequently in nature and the genome composition of influenza C viruses may influence their ability to spread in humans.

Influenza C virus usually causes a mild upper respiratory illness (9) but can also cause lower respiratory infections such as bronchitis and pneumonia (20). Although influenza C virus is isolated only infrequently, the majority of humans acquire antibodies to the virus early in life (7, 23), indicating that this virus is widely distributed throughout the world (6, 8). Recurrent infection with this virus occurs frequently in children as well as in adults (7, 9, 10, 14). The genome of influenza C virus consists of seven RNA segments which encode three polymerase proteins (PB2, PB1, and P3), hemagglutinin-esterase (HE) glycoprotein, nucleoprotein (NP), matrix (M1) protein, CM2 protein, and two nonstructural proteins (NS1 and NS2) (reviewed in reference 13). Antigenic variation exists among influenza C virus isolates, as demonstrated clearly by antigenic analysis with anti-HE monoclonal antibodies (MAbs) (1, 12, 16, 18, 29). Nevertheless, analysis with polyclonal immune sera showed a high degree of cross-reaction among all the isolates examined so far (6, 11, 16, 18, 21), indicating that influenza C virus is antigenically much more stable than human influenza A virus. The significance of antigenic variation in influenza C virus epidemiology remains to be clarified.

Early studies on the RNA genomes of various isolates suggested that influenza C virus epidemiology might be characterized by the presence of many cocirculating variants (4, 5). To obtain more information about influenza C virus epidemiology, we initiated surveillance for influenza C virus infections in Yamagata City, Japan, in 1988 and in the adjacent city of Sendai, Japan, in 1990 (20). We compared the HE gene sequence among the 42 strains isolated between 1947 and 1993 and showed the existence of six discrete lineages represented by Taylor/47, Kanagawa/1/76 (KA176), Yamagata/26/81 (YA2681), Aichi/1/81 (AI181), Sao Paulo/378/82 (SP82), and Mississippi/80 (MS80), four of which (YA2681-, AI181-, SP82-, and MS80-related lineages) circulated in Japan in the 1980s and the early 1990s (18). We obtained direct evidence of cocirculation that two strains belonging to different lineages were isolated only 1 day apart from two children living within walking distance of each other(16). Thus, mixed infection with influenza C virus belonging to different lineages is likely to occur in humans, resulting in the emergence of reassortment virus characterized by exchange of genome segments between two different strains (2, 18, 25, 26, 32). Recently, we succeeded in detecting influenza C virus outbreaks for the first time in Yamagata City, which were caused by a reassortant virus (19). This led us to a speculation that the genome composition of influenza C viruses influences their ability to spread in humans.

In this study, we characterize influenza C viruses antigenically and genetically isolated in a 9-year survey (December 1990 to December 1999) performed in Sendai City and demonstrate the two findings that influenza C virus strains belonging to different antigenic groups cocirculate and the replacement of the dominant group occurs in this restricted area and that reassortment between two different influenza C virus strains occurs frequently, resulting in epidemics of influenza C virus by the reassortant viruses.

MATERIALS AND METHODS

Viruses.

A total of 45 strains of influenza C virus were obtained from throat swabs of pediatric patients (<15 years of age) with acute respiratory illness who visited three hospitals (Sendai National Hospital, Tohoku Koseinenkin Hospital, and Nakajima Hospital) and two clinics (Nagai Children's Clinic and Shoji Clinic) in Sendai, Japan, between December 1990 and December 1999. For this study, all these viruses were reisolated from throat swabs and passaged and propagated by inoculating them into the amniotic cavity of 9-day-old embryonated hen's eggs, and these amniotic fluids were used for the following experiments.

Ten older strains (Sapporo/71 [SA71], Aomori/74 [AO74], KA176, Kyoto/1/79 [KY179], Shizuoka/79 [SHI79], MS80, YA2681, AI181, SP82, and Yamagata/1/86 [YA186]) were also used for comparison.

HI test.

Four anti-HE MAbs characterized previously (30, 31) were used. Antisera against five different isolates (AI181, Yamagata/7/81 [YA781], MS80, Yamagata/1/92[YA192] and AO74) were prepared in chickens (11). The hemagglutination inhibition (HI) test was done in microtiter plates with 0.5% chicken erythrocytes. Briefly, 50 μl of virus suspension (16 hemagglutinin U/ml) was added to each well containing 50 μl of twofold-diluted MAbs or chicken antisera. After incubation for 30 min at room temperature, 100 μl of 0.5% chicken erythrocytes was added to all wells and plates were stored for 60 min at 4°C. The HI titer was expressed as the reciprocal of the highest antibody dilution which completely inhibited hemagglutination. Precautions over inhibitors were not necessary because little or no inhibitor activity was detected in mouse ascitic fluid and chicken antisera.

Nucleotide sequencing and phylogenetic analysis.

Viral RNA was extracted from 200 μl of the virus-containing amniotic fluid by using an RNeasy mini kit (Qiagen) and eluted in 30 μl of RNase-free distilled water. The cDNA was synthesized in a final reaction volume of 20 μl, containing 10 μl of RNA, 2 μl of 10× reaction buffer, 500 μM (each) deoxynucleoside triphosphate, 40 U of RNase inhibitor (Promega), 1 μg of universal primer complementary to positions 1 to 12 at the 3′ end of RNA, and 20 U of avian myeloblastosis virus reverse transcriptase XL (Life Science). The reaction mixture was incubated at 42°C for 60 min and heated to 95°C for 5 min to inactivate the enzyme activity. By using the resulting cDNA as a template, the individual RNA segments were amplified by PCR through 35 cycles of the thermocycler program described previously (12). The PCR products were purified by a rapid gel filtration with a Chroma spin column (Clontech) and then sequenced by using a BigDye Terminator cycle sequencing FS ready reaction kit on an ABI Prism 310 (Applied Biosystems) automatic sequencer. Nucleotide sequences of the oligonucleotide primers used for PCR amplification and sequencing are available from authors upon request. Sequence data were analyzed with the PHYLIP program (version 3.573c), and phylogenetic trees were constructed by the neighbor-joining method (28) using the same software.

Nucleotide sequence accession numbers.

The nucleotide sequences determined in this study have been submitted to the DDBJ/EMBL/GenBank databases and assigned the accession numbers AB086656 to AB086811.

RESULTS

Isolation of influenza C virus in Sendai City.

During a 9-year survey from December 1990 to December 1999, a total of 35,859 throat swab specimens were collected from children with acute respiratory illness and examined for the presence of influenza C virus. The results obtained are shown in Fig. 1. In 1990 and 1991, 10 strains of influenza C viruses were isolated between December 1990 and March 1991. In 1992, three strains were isolated in July and September, seven strains were isolated between February and May 1993, three strains were isolated in June 1994, nine strains were isolated between April and July 1996, nine strains were isolated between December 1997 and March 1998, and four strains were isolated in June and July 1999. In 1995, influenza C virus was not isolated. The average isolation rate for every year was 0.12%, nearly equal to the result presented our previous report (0.16%) (20). Clusters of isolates were observed mainly in December and January and between May and July, and the period from beginning to end in a cluster was within 4 months. Moreover, the duration between two clusters of isolates varies, ranging from 4 months (from 27 September 1992 to 2 February 1993) to 22 months (from 30 June 1994 to 30 April 1996).

FIG. 1.

Monthly distribution and antigenicity of influenza C virus strains isolated in Sendai City between December 1990 and December 1999.

Antigenic analysis of influenza C viruses isolated in Sendai City between 1990 and 1999.

The 45 influenza C virus strains isolated in Sendai City between 1990 and 1999 were examined in HI tests for reactivity with four different anti-HE MAbs characterized previously (30, 31). Antigenicity of all the isolates and HI titers of the representative strains were shown in Fig. 1 and Table 1, respectively. The reactivity patterns of all the isolates except Miyagi/9/96 (MI996) were similar to one of the four known antigenic groups represented by AI181, YA2681, MS80 and SP82. MI996 displayed reactivity patterns similar to that of KA176, which was clearly distinguishable from those of the four antigenic groups. There was no virus with an HE antigenicity similar to that of KA176 among >70 influenza C virus isolates obtained in Japan from 1978 to 1995. This might suggest that the KA176-like virus emerged for the first time in 20 years in Japan. The representative isolates were further examined by HI tests for reactivity with chicken antisera to five different antigenic strains. Viruses belonging to the same group could not be distinguished from each other and had similar antigenicity to their reference strains, whereas antigenic differences between viruses belonging to different groups could be detected clearly.

TABLE 1.

Antigenic analysis of representative influenza C virus isolates by HI tests with anti-HE MAbs and chicken antiviral sera

Virus strain	HI titer (HI units/ml) of anti-HE MAbs				HI titer (HI units/ml) of antiserum to:
	Q5	U4	U1	MS2	AI181	YA781b	MS80	YA192c	AO74d
Reference
AI181	160	5,120	1,600	<	5,120	320	1,280	640	640
YA2681	12,800	80	3,200	<	1,280	2,560	2,560	640	640
MS80	<a	<	128,000	25,600	640	80	20,480	320	320
SP82	128,000	12,800	512,000	<	1,280	320	2,560	2,560	640
KA176	2,560	40	<	<	2,560	640	1,280	1,280	2,560
Aichi/1/81 group
MI190	160	2,560	640	<	5,120	320	1,280	640	1,280
MI591	320	2,560	640	<	5,120	320	1,280	640	1,280
Yamagata/26/81 group
MI391	25,600	<	640	<	1,280	5,120	1,280	640	640
MI192	16,000	80	32,000	<	640	2,560	1,280	320	1,280
MI696	25,600	40	6,400	<	640	2,560	640	320	320
MI597	51,200	40	1,600	<	1,280	5,120	1,280	640	640
MI499	51,200	40	1,600	<	1,280	5,120	1,280	640	640
Mississippi/80 group
MI493	<	<	64,000	256,000	1,280	160	10,240	640	640
MI394	<	<	32,000	64,000	1,280	160	20,480	640	640
MI496	<	<	128,000	64,000	1,280	160	20,480	640	640
SaoPaulo/82 group (MI593)	32,000	6,400	32,000	<	1,280	320	2,560	2,560	1,280
Kanagawa/1/76 group (MI996)	640	40	<	<	2,560	640	1,280	640	2,560

^a<, Titer below 40.

^bThe HE antigenicity of YA781 was identical to that of Yamagata/26/81 (11, 16).

^cThe HE antigenicity of YA192 was identical to that of SaoPulo/378/82 (18).

^dThe HE antigenicity of AO74 was identical to that of Kanagawa/1/76 (21).

Nucleotide sequence and phylogenetic analysis of the HE genes of influenza C virus strains isolated in Sendai City between 1990 and 1999.

In order to confirm the results of the antigenic analysis, the sequence of the HE gene (nucleotide 64 to 1,989) was determined for 12 strains (Table 2) and a phylogenetic tree was constructed by using them in addition to the 62 published sequences (1, 3, 4, 12, 16, 17, 18, 19, 21, 22, 24, 25, 26, 27, 33) as well as the sequences of two old strains (YA186 and SHI79) determined here.

TABLE 2.

Influenza C virus strains sequenced in this study among the isolates in Sendai City, Japan

Virus strain	Abbreviation	Date of sample collection (day-mo-yr)	HE gene lineagea
Miyagi/1/90	MI190	21-Dec-90	AI
Miyagi/5/91	MI591	25-Feb-91	AI
Miyagi/7/91	MI791	21-Mar-91	AI
Miyagi/9/91	MI991	27-Mar-91	YAb
Miyagi/2/92	MI292	27-Sep-92	YAb
Miyagi/1/93	MI193	02-Feb-93	MSb
Miyagi/2/93	MI293	03-Feb-93	MSb
Miyagi/3/93	MI393	13-Apr-93	MSb
Miyagi/4/93	MI493	20-Apr-93	MSb
Miyagi/5/93	MI593	06-May-93	SP
Miyagi/6/93	MI693	17-May-93	MSb
Miyagi/7/93	MI793	19-May-93	MSb
Miyagi/1/94	MI194	22-Jun-94	MSb
Miyagi/2/94	MI294	24-Jun-94	MSb
Miyagi/3/94	MI394	30-Jun-94	MSb
Miyagi/2/96	MI296	16-May-96	YA
Miyagi/8/96	MI896	23-May-96	MSb
Miyagi/9/96	MI996	03-Jun-96	KA
Miyagi/4/96	MI496	19-Jun-96	MSb
Miyagi/6/96	MI696	03-Jul-96	YA
Miyagi/7/96	MI796	15-Jul-96	MSb
Miyagi/1/97	MI197	01-Dec-97	YA
Miyagi/2/98	MI298	07-Jan-98	YA
Miyagi/4/98	MI498	27-Mar-98	YA
Miyagi/1/99	MI199	13-Jun-99	YA
Miyagi/3/99	MI399	21-Jul-99	YA

^aAbbreviations: AI, AI181-related lineage; YA, YA2681-related lineage; MS, MS80-related lineage; SP, SP82-related lineage; KA, KA176-related lineage.

^bHE gene sequence was determined previously (3, 12, 19).

As shown in Fig. 2, the HE genes of influenza C viruses analyzed were divided into six discrete lineages, represented by Taylor/47, AI181, SP82, KA176, YA2681, and MS80. As expected from the results of antigenic analysis, MI190, MI591, and MI791 were within the AI181-related lineage, MI593 was within the SP82-related lineage, and MI996 was within the KA176-related lineage. We reported previously that the HE genes of the YA2681-related lineage can be divided into two distinct subgroups, represented by YA2681 and Pig/Beijing/115/81 [PB11581], and the HE genes of MI391, MI991, and MI292 were closely similar to those of PB11581, which is a swine isolate obtained in China (12). The seven strains isolated in Sendai City between 1996 and 1999, which have antigenicity similar to that of YA2681, were within the PB11581-related sublineage. Furthermore, it was revealed that the nucleotide sequences of their HE genes were highly homologous (99.6%) to those of the 1996 and 1998 Yamagata strains which were isolated in an epidemic of influenza C virus in Yamagata City. The 12 strains isolated from 1993 to 1996 that had antigenicity similar to that of MS80 were within the MS80-related lineage, as reported previously (19).

FIG. 2.

Phylogenetic tree of influenza C virus HE genes. The region from nucleotides 64 to 1989 was used for analysis. The Sendai isolates (those listed in Table 2 and MI391) with HE antigenicities identical to those of AI181, YA2681, MS80, SP82, and KA176 are marked (∗, †, ‡, §, and ¶, respectively). Horizontal distances are proportional to the minimum number of nucleotide differences needed to join the gene sequences. Numbers below or above the branches are the bootstrap probabilities (percentages) of each branch as determined by the PHYLIP program (version 3.573c).

Phylogenetic analyses of individual RNA segments of influenza C virus strains.

To determine the genome composition of the influenza C viruses isolated by our surveillance work, partial nucleotide sequences of PB2 (position 52 to 520), PB1 (position 50 to 425), P3 (position 49 to 420), and NP (position 71 to 670) genes as well as complete coding region sequences of the M (position 26 to 1,147) and NS (position 26 to 889) genes were determined for the 26 isolates in Sendai City (Table 2), as well as for five old strains (SA71, AO74, KY179, SHI79, and YA186). The phylogenetic trees of individual genes were constructed by using the sequences of these 31 strains in addition to the 24 previously reported sequences (2, 12, 18, 19, 25, 26, 32).

As shown in Fig. 3, the PB2 genes were split into six lineages, represented by strains MS80, KA176, AI181, YA2681, SP82, and PB11581, but one isolate in 1971 (SA71) did not belong to any lineage. In the PB1 gene tree, four lineages, represented by AI181, MS80, KA176, and YA2681, were identified. The P3 genes were divided into five different lineages, represented by MS80, KA176, AI181, SP82, and YA2681. The NP genes were split into six distinct lineages, represented by MS80, YA2681, KA176, AI181, PB11581, and MI193, but the phylogenetic positions of SA71 and KY179 were discrete from these lineages. In the tree of the M gene sequences, there were five lineages represented by AO74 (previously designated lineage III by Tada et al. [32]), AI181 (lineage II), MS80 (lineage II), and SP82 and YA2681 (lineage I). The NS gene sequences were split into five discrete lineages, represented by AI181, KA176, MS80, SP82, and YA2681. The branch clusters containing AI181, KA176, and MS80 were previously designated lineage A and that containing YA2681 was designated lineage B by Alamgir et al. (2).

FIG. 3.

Phylogenetic trees for the PB2 (A), PB1 (B), P3 (C), NP (D), M (E,) and NS (F) genes of influenza C virus isolates. The nucleotide sequences of the following regions were used for analysis: nucleotides 52 to 520 for the PB2 gene, nucleotides 50 to 425 for the PB1 gene, nucleotides 49 to 420 for P3 genes, nucleotides 71 to 670 for the NP gene, nucleotides 26 to 1,147 for the M gene, and nucleotides 28 to 889 for the NS gene. The Sendai isolates listed in Table 2, having the HE genes of the AI181-, YA2681-, MS80-, SP82-, and KA176-related lineages, are marked (∗, †, ‡, §, and ¶, respectively). Horizontal distances are proportional to the minimum number of nucleotide differences needed to join the gene sequences. Numbers below or above the branches are the bootstrap probabilities (percentages) of each branch as determined by the PHYLIP program (version 3.573c).

MI190, MI591, and MI791 with HE genes on the AI181-related lineage were located on the AI181 virus lineage in PB2, PB1, NP, and M trees but had P3 and NS genes closely related to those of the YA2681-like virus. The phylogenetic position of MI593 having HE gene on SP82-related lineage was identical to Yamagata/1/93 that had all of the six RNA segments closely related to SP82 as previously described (18). In the trees of six gene segments, MI991 and MI292 with HE genes on the YA2681-related lineage were located on the similar lineage with PB11581. PB11581-like virus forms a distinct lineage different from that of YA2681-like virus in the PB2 and NP gene trees (12). The remaining seven strains with HE genes on the YA2681-related lineage in 1996 to 1999 (MI296, MI696, MI197, MI298, MI498, MI199, and MI399) like MI991 and MI292 had PB2, PB1, M, and NS genes closely similar to those of the PB11581-like virus. However, the seven strains were located on the MS80 virus lineage in the trees of the P3 and NP gene, separated from MI991 and MI292. This genome composition was indistinguishable from that of the influenza C virus strains that caused outbreaks in Yamagata City in 1996 and 1998. MI996 with the HE gene on the KA176-related lineage were located on a different virus lineage from KA176 in PB2, PB1, P3, NP, M, and NS gene trees. Interestingly, all of these six internal protein genes of this virus were closely related to those of the 1996 to 1999 isolates with HE genes on the YA2681-related lineage as described above.

The 12 strains having a HE gene belonging to the MS80-related lineage were isolated in Sendai City between 1993 and 1996. Analysis of the sequence of the six gene segment revealed the 12 isolates could be divided into four groups based on their genome compositions that were represented by MI393, MI193, MI793, and MI896 (Table 3). The P3, NP, and M genes of MI393 and MI493 were on the MS80 virus lineage, but the PB2 was on the PB11581 virus lineage, PB1 and NS genes were on the YA2681 virus lineage. This genome composition was identical to that of Yamagata/5/92 (YA592), which was antigenically indistinguishable from MS80 isolated for the first time in Tohoku district (which includes Yamagata and Miyagi prefectures) in 1992. This result confirms our previous suggestion based on oligonucleotide map comparison that MI393 and MI493 as well as YA592 are reassortant viruses between MS80-like virus and PB11581-like virus (26). Six additional isolates from 1993 and 1994 with HE genes belonging to the MS80-related lineage (MI193, MI293, MI693, MI194, MI294, and MI394) were also reassortants. The PB2, P3, M, and NS genes of the six isolates were within the YA2681 virus lineage, the PB1 gene was within the KA176 virus lineage, and the NP gene was within the MI193 virus lineage. MI793 and 2 isolates in 1996 (MI496 and MI796) possessed the HE, PB2, and NS genes belonging to the MS80 virus lineage, the PB1 gene belonging to the KA176 virus lineage, and the NP gene belonging to the PB11581 virus lineage. MI896 was a reassortant which had received the PB2 and NP genes from a PB11581-like virus, the PB1 gene from a KA176-like virus, and the P3, M, and NS genes from a YA2681-like virus. These observations indicate that the three or four groups of reassortant viruses, as summarized in Table 3, with the antigenicity of an MS80-like virus cocirculate in a geographically restricted area.

TABLE 3.

Genome compositions of influenza C virus isolates, as determined by phylogenetic analysis

Virus strain	RNA segmenta
	PB2	PB1	P3	HE	NP	M	NS
AI181-related lineage
AI181	A	A	A	A	A	A	A
YA186	A	A	Y	A	A	A	Y
MI190	A	A	Y	A	A	A	Y
MI591	A	A	Y	A	A	A	Y
MI791	A	A	Y	A	A	A	Y
YA2681-related lineage
SA71	SA	K	Y	Y	SA	Y	K
KY179	Y	K	Y	Y	SA	Y	K
SHI79	Y	Y	Y	Y	Y	Y	Y
YA2681	Y	Y	Y	Y	Y	Y	Y
YA788	Y	Y	Y	Y	Y	Y	Y
MI991	P	Y	Y	Y	P	Y	Y
MI292	P	Y	Y	Y	P	Y	Y
MI296	P	Y	M	Y	M	Y	Y
MI696	P	Y	M	Y	M	Y	Y
MI197	P	Y	M	Y	M	Y	Y
MI298	P	Y	M	Y	M	Y	Y
MI498	P	Y	M	Y	M	Y	Y
MI199	P	Y	M	Y	M	Y	Y
MI399	P	Y	M	Y	M	Y	Y
MS80-related lineage
MS80	M	M	M	M	M	M	M
KY4182	M	M	M	M	M	M	M
MI393	P	Y	M	M	M	M	Y
MI493	P	Y	M	M	M	M	Y
MI193	Y	K	Y	M	MI	Y	Y
MI293	Y	K	Y	M	MI	Y	Y
MI693	Y	K	Y	M	MI	Y	Y
MI194	Y	K	Y	M	MI	Y	Y
MI294	Y	K	Y	M	MI	Y	Y
MI394	Y	K	Y	M	MI	Y	Y
MI793	M	K	Y	M	P	Y	M
MI496	M	K	Y	M	P	Y	M
MI796	M	K	Y	M	P	Y	M
MI896	P	K	Y	M	P	Y	Y
SP82-related lineage
SP82	S	Y	S	S	Y	S	S
MI593	S	Y	S	S	Y	S	S
RA176-related lineage
AO74	K	A	K	K	K	AO	M
KA176	K	K	K	K	K	Y	K
MI177	K	K	K	K	K	Y	K
MI996	P	Y	M	K	M	Y	Y

^aAbbreviations: A, AI181-like strain; Y, YA2681-like strain; SA, SA71-like strain; K, KA176-like strain; P, PB11581-like strain; M, MS80-like strain; MI, MI193-like strain; S, SP82-like strain; AO, AO74-like strain.

Origin of influenza C virus strains having HE gene belonging to the MS80-related lineage.

As stated above, the three groups represented by MI193, MI793, and MI896, which have HE genes belonging to the MS80-related lineage, are reassortants that obtained the PB1 gene from a KA176-like virus. But a virus having the HE gene belonging to the KA176-related lineage has not been isolated in Japan since 1977 (Miyagi/77 [MI77]). To obtain information about the parental origin of the MS80-like virus strains, the nucleotide sequences of the six internal genes were determined for the old strains from the 1970s (SA71, AO74, KY179, and SHI79) and then compared with those of the MS80-like strains. Phylogenetic trees are shown in Fig. 2 and 3, and the genome composition and the deduced pattern for gene reassortments are summarized in Table 3 and Fig. 4. The PB1, M, and NS genes of KA176 and MI77 were located on a different lineage from AO74, which has the HE gene of a KA176-related lineage, but on a similar lineage to SA71, which has the HE gene of a YA2681-related lineage. This indicates that KA176 and MI77 are reassortant viruses that inherit the HE, PB2, P3, and NP genes from an AO74-like virus and the PB1, M, and NS genes from an SA71-like virus. Among the three old strains having the HE gene belonging to the YA2681-related lineage (SA71, KY179, and SHI79), only SHI79 possessed a genome composition similar to that of YA2681. In the tree of the PB1 and NS genes, SA71 and KY179 were located not on the branch cluster of YA2681 but on that of KA176. The PB2 gene of SA71 and the NP genes of SA71 and KY179 seemed to be located on the independent position of any lineage recognized previously, shown as SA in Table 3. These results show that YA2681-like virus is a reassortant that has inherited PB2, P3, HE, and M genes from a KY179-like virus and PB1, NP, and NS genes from another parent not identified as yet. This reassortment event presumably occurred at the end of the 1970s. In the PB1 gene tree, among the four groups of MS80-related lineage mentioned above (Table 3), viruses in the three groups were located on the KA176 virus lineage together with SA71 and KY179. One group represented by MI193 was located in the same lineage as KY179 in the tree of PB2, P3, and M gene, and another group represented by MI793 was located at a phylogenetic position similar to that of KY179 in the tree of P3 and M gene. These observation suggest strongly that the influenza C virus strains with HE gene of MS80-related lineage, which possess PB1 genes on the KA176 virus lineage, have emerged by reassortment from two viruses closely related to KY179 and MS80 and thereafter were reassorted with a PB11581-like virus. We have previously reported that MS80-like viruses that had a genome composition identical to that of MS80 were newly introduced in the Kinki district in Japan between 1982 and 1983 (Kyoto/41/82 [KY4182], Nara/82, and Hyogo/1/83) (1). Therefore, it seems that the reassortment between KY179-like virus and MS80-like virus occurred in the early 1980s, although these viruses could not be identified, and then they reassorted with PB11581-like virus when it was isolated in Sendai City in 1991 and 1992.

FIG. 4.

Deduced pattern for gene reassortment among the Sendai isolates of influenza C virus having the HE genes of the AI181-, YA2681-, MS80-, and KA176-related lineage.

DISCUSSION

During a 9-year survey from December 1990 to December 1999 in Sendai City, we succeeded in isolating 45 strains of influenza C virus from more than 35,000 throat swab specimens taken from pediatric patients less than 15 years old with acute respiratory illness. In almost every year, the influenza C viruses were isolated in clusters within about 4 months in a year, especially in winter and spring to early summer.

We observed that the 45 strains could be divided into five antigenic groups, and there were no antigenic changes among the viruses belonging to the same antigenic groups during this period. For example, 24 strains of the viruses of YA2681 antigenic group were antigenically similar to one another by HI tests with not only anti HE MAbs but also polyclonal immune sera. Furthermore, these viruses had an antigenicity indistinguishable from those of YA2681 (Table 1) and SA71 (data not shown), which had been isolated 10 to 30 years before the surveillance. These observations suggest that the production of antigenic variants by immune selection has not occurred for the last 3 decades. This finding is well comparable with our previous indication that HE protein might no longer be able to evolve in response to antibody pressure because of a high degree of functional constraint on the change of its immunodominant region, which is near the receptor-binding site (15, 21). On the other hand, as shown in Fig. 1, it was also observed that a few antigenic groups were cocirculating in Sendai City and that there was a dominant antigenic group. In 1990 and 1991 the dominant antigenic group was the AI181 virus group, and in 1992 it was the YA2681 virus group. Viruses in the MS80 virus group were isolated from 1993 to 1996, and the YA2681 virus group reemerged in 1996 and continued to circulate until 1999. This observation led us to a speculation that the replacement of the dominant antigenic group has occurred by immune selection within the human population in the restricted area.

According to the previous report documenting influenza C virus outbreak in a children's home (9), the patients might have shed the viruses for a long period (more than 9 days). On the other hand, it was ascertained in the present study that influenza C viruses belonging to different lineages were cocirculating. Therefore, it seems that mixed infection with influenza C virus belonging to different lineages is likely to occur in humans, resulting in our finding that most of the circulating influenza C viruses are reassortants. The fact that all strains except MI593 isolated by our surveillance work are reassortant viruses genetically differentiated from the reference strains demonstrates that the influenza C virus strains reassort frequently in nature and acquire a survival advantage in the human population. Reassortment events may be a means of evolution for influenza C viruses, since the influenza C virus strain seems to have difficulty evolving by the emergence of antigenic variants because of the strict functional constraint on the HE protein.

MI593 is a unique virus antigenically and genetically, together with two strains isolated in Yamagata City during 1992 and 1993 (YA192 and Yamagata/1/93). These strains are very similar to SP82 and may have been introduced into Japan shortly before 1992, but we have been unable to detect an SP82-like virus since then (18). AI181-like viruses had reassorted with a YA2681-like virus and spread in Sendai City between 1990 and 1991. This reassortant virus was already isolated in Yamagata City in 1986 (YA186) as well as in various areas of Japan from 1987 to 1990 (16, 24, 26), and disappeared after the last isolation in Yamagata City in 1992 (Y. Matsuzaki, unpublished results). We found that the strains with HE genes on a YA2681-related lineage continued to be isolated in Japan for the longest period. The YA2681-like virus used as a reference strain is also a reassortant that acquired HE, PB2, P3, and M genes from a KY179-like virus and the other genes from an unknown parent. The YA2681-like virus such as SHI79 and Yamagata/7/88 (YA788) had been circulating in Japan for about 10 years. The PB11581-like virus, which has an HE gene in the sublineage of the YA2681-related lineage, was introduced into Japan and isolated for the first time in Yamagata City in 1989 (YA1089), followed by isolation of six strains in Sendai City during 1991 to 1992, but was not isolated at all in either city thereafter (12, 16). The PB11581-like virus reemerged in 1996; it had been provided with P3 and NP genes from an MS80-like virus and caused outbreaks in Yamagata City detected for the first time in a large community (19). These reassortant viruses were also isolated in Sendai City from 1996 to 1999. It seems that a PB11581-like virus acquired the increased ability to prevail in humans through a reassortment event. The 12 isolates with HE genes in the MS80-related lineage isolated in Sendai City between 1993 and 1996 were all reassortants that inherited various genes of seven RNA segments from PB11581-like virus, presumably suggesting that this reassortment event seemed to occur around 1991 when PB11581-like virus was circulating in Sendai City. All 13 strains, including YA592, with HE genes in the MS80-related lineage isolated in Yamagata and Sendai cities were divided into four groups based on their genome composition, which were represented by MI193, MI393, MI793, and MI896. We documented previously that the three influenza C virus isolates of the MS80-like virus reference strain were introduced into the Kinki district of Japan in 1982 and 1983 (KY4182, Nara/82, and Hyogo/1/83) (1). Here we have obtained evidence that the three groups represented by MI193, MI793, and MI896 formerly received the reassortment of the genome between KY4182-like virus and KY179-like virus. Although these reassortants could not be isolated, they likely cocirculate elsewhere in Japan. Previously, we pointed out the possibility that KY4182-like virus also continues to circulate in Japan, because the epidemic strains in 1996 and 1998 in Yamagata City seemed to receive the P3 and NP genes from the Kinki strains described above rather than those of the Yamagata and Sendai strains represented by MI393 isolated in 1992 and 1993 (19). Moreover, our observation that the three groups of reassortants represented by MI193, MI393, and MI793 cocirculated in Sendai City at the same time in 1993 supports the idea that the viruses with HE genes in an MS80-related lineage reassort with other virus lineage strains in a complex fashion and some of them succeed in cocirculating. However, it is unknown why only the reassortants with the HE gene in the MS80-related lineage can coexist, although in the case of other virus lineages, one strain is selected and circulates over the parental viruses (Fig. 4). Surprisingly, a virus strain antigenically indistinguishable from KA176 was isolated in Sendai City in June 1996 (MI996). We had previously suggested that KA176-like virus might have died out (21), because none of the >70 influenza C virus isolates obtained from 1977 to 1995 had an HE antigenicity similar to that of KA176. Sequence analyses of seven RNA segments of MI996 showed that all of the six internal protein genes of this virus were closely related to those of the 1996 and 1998 epidemic strains of Yamagata and Sendai cities rather than those of KA176. This suggests that KA176-like virus barely circulated in some areas and reemerge by acquisition of selective advantage, receiving the same internal genes as those obtained by epidemic strains through a reassortment event.

The influenza C virus is antigenically stable, and some different antigenic groups cocirculate within the geographically restricted area. Thus, reassortment occurs frequently in nature, resulting in epidemics of influenza C virus that has acquired the increased ability to spread in humans over their parental viruses.