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Genetic relatedness of hemagglutinins of the H1 subtype of influenza a viruses isolated from swine and birds
VIROLOGY
129,521-523 (1983)
Genetic Relatedness of Hemagglutinins of the Hl Subtype of Influenza A Viruses Isolated from Swine and Birds C. SCHOLTISSEK,* *Institut
H. B~JRGER,* P. A. BACHMANN,?
AND C. HANNOUN*
Virolcgti, Justus-Liebig-Universitiit, Giessen, West Gemany; ~Institut fiir Medizinische M~ikrobiologie, Iqfektims- urd Seuchemnedizin, LudwigM~milians-University, Munich, West Germany; and $.Institut Pasteur, Paris, France
fiir
Received April 20,
1983;
accepted June 20,
1983
The hemagglutinin (HA) gene of the influenza virus subtype HlNl isolated from pigs and birds has been analyzed by the hybridization technique. According to the RNase protection data the HA genes of recent isolates from pigs in Northern Europe are genetically more closely related to those of isolates from birds in Europe and North America than to those of isolates from pigs in the United States, Taiwan, and Italy. Thus, two different HlNl subtypes are circulating in the pig population. The results are consistent with the view that HlNl viruses can be transmitted from birds to pigs and/or vice versa.
It has been suggested that human pandemic influenza A strains might be created in nature by reassortment during double infection of a common host with a prevailing human virus and a strain infecting animals, especially birds. In this context pigs have been mentioned as possible candidates to be such a common host. It is of interest, therefore, to compare influenza virus strains isolated from pigs with those found in humans and birds. Recently, influenza viruses related to swine influenza viruses (HlNl) were isolated from ducks in North America (I), Hong Kong (Z), and Europe (3, 4). On the other hand, in 1979 influenza viruses of the HlNl subtype were isolated from diseased pigs in Germany (Westfalia), which were not recognized in this area before (5). Later on corresponding swine influenza viruses were also found in other parts of Germany (6), France (7), and Belgium (8). In Germany the virus was isolated only from clinically affected pigs. The clinical symptoms were typical for swine influenza. The virus had apparently not been introduced before 1979, since HI antibodies against swine influenza virus were not present in 745 sera collected from pigs on 161 farms in this area for diagnostic purpose during and prior to 1978 (5). In France, the virus seemed to be enzootic
and in the majority of cases, infection was asymptomatic although contagion rates were high and diffusion was rapid in contaminated farms. There was no evidence for infection of man, even in persons in close contact with infected animals (7’). There is serological evidence that these newly recognized strains isolated from pigs are more closely related serologically to HlNl bird isolates than to the classical swine influenza virus strains (8). In addition, swine influenza-like HlNl strains from birds readily infect pigs and spread among them by contact (3). Therefore, we have started an analysis of recent European HlNl isolates by the RNA hybridization technique and compared them with those obtained in other geographic regions. The virus strains listed in Table 1 were investigated. The A/swine/Arnsberg/6554/79 strain was kindly provided by Dr. K. H. Witte (5). The A/duck/Alberta/35/76 strain has been provided by Drs. V. Hinshaw and R. G. Webster. The strains A/swine/France/MAR/80, A/swine/France/STE/80, and A/swine/ France/OLI/80 were isolated in January or February 1980 when first evidence of the introduction of HlNl (SW) virus in France was obtained. The former was isolated from sick pigs during an outbreak of 521
0042-6822/83 $3.00 Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
SHORT
522
COMMUNICATIONS TABLE
1
RNase PROTECTION OF HYBRIDIZATION OF %P-LABELED vRNA WITH NONLABELED
CRNA OF VARIOUS
Percentage
cRNA of
Country of isolation
A/duck/Bavaria/2/77 A/swine/l976/31 A/swine/France/STE&O A/duck/Alberta/35176 A/swine/Arnsberg/6554/79 A/swine/France/MAR/80 A/swine/France/OLI/80 A/swine/Germany/Z/81 A/swine/Belgium/2179 A/duck/Schleswig/21/79 A/swine/Taiwan/l/76 A/coot/Schleswig/4/79 A/New Jersey/8176 A/swine/Germany/l/82 A/swine/Italy/81 A/swine/Iowa/30 A/PR/8/34 A/FPV/Rostoek/34 (H7Nl)
Germany United States France Canada Germany France France Germany Belgium Germany Taiwan Germany United States Germany Italy United States United States Germany
“All strains listed belong to the HlNl *nd = not done.
A/duck/ Bavaria/2/77 100 73 94 96 nd* 90 94 94 95 nd 73 96 72 94 71 71 nd 33
SEGMENT 4 (HA)
INFLUENZA
STRAINS
RNase protection after hybridization with vRNA segment 4 of A/swine/ 1976/31 68 100 72 64 67 nd 69 nd 71 69 95 nd nd 65 nd 97 93 34
subtype except A/FPV/Rostock/34,
severe swine influenza in the south of France, the two latter from apparently healthy pigs in Normandy and Brittany (Q, and they were kindly provided by Dr. Gourreau. The hybridization technique has been described in detail elsewhere (9). It was difficult to incorporate sufficient 32P into the virion RNA (vRNA) segment 4 (hemagglutinin gene) of the influenza virus strains investigated, and to obtain sufficiently concentrated complementary RNA (cRNA) probes. Therefore, the following controls were included for the calculation of the percentage RNase protection. (i) In order to control the background radioactivity of the gel, a band of the same size as the HA band was cut out in the immediate neighbourhood of the HA band. The RNA extracted from this band was hybridized under identical conditions as
OF FOUR HlNl
A STRAINS
A/swine/ France/STE&O 94 71 100 91 96 94 94 96 100 96 71 91 65 97 62 70 nd 32
A/duck/ Alberta/35176 92 67 93 100 92 92 94 91 92 91 65 nd 64 92 nd 67 nd 30
which is a H7N1 strain.
the HA-RNA samples. These control values never exceeded 20% of those of the HA hybridization. They were subtracted before calculation of the percentage RNase protection of the HA genes. (ii) In order to be sure that the concentration of cRNA was sufficient to work under saturation conditions, hybridization was also done against RNA segment 7 (M protein gene), which is the most conserved gene of influenza A viruses (9). If the hybridization with RNA segment 7 was less than 90%, the corresponding batch of cRNA was either discarded or the amount of cRNA was drastically increased. The error width of the method is about 5%. Thus, if the RNase protection is about 95% the two strains under investigation are expected to be genetically highly related in their HA genes differing from each other presumably only by a few point mutations.
SHORT
523
COMMUNICATIONS
The radioactivity in the various aliquots of the vRNA of the four strains was between 600 and 2000 cpm. The RNase protection after homologous and heterologous hybridization is a relative measure of the genetic relatedness between various influenza virus strains. In Table 1 data are presented on the RNase protection after cross-wise hybridization of vRNA segment 4 of various HlNl strains isolated from birds and pigs. It can be seen that HA genes of the recent bird isolates from the United States and Germany are closely related to each other and to HlNl isolates from pigs in Northern Europe. These HAS can be clearly differentiated genetically from the HA genes of HlNl strains isolated about 50 years ago in the United States or more recent isolates from Taiwan, Italy, or the United States, which also are closely related to each other. Furthermore, the HA gene of the A/swine/1976/ 31 strain is closely related to that of the PR8 strain (Table 1). Since the HA gene of PR8 exhibits almost 100% RNase protection after hybridization with cRNA of the FM1 strain (IO), and the FMl-HA differs from the HA genes of recent HlNl strains isolated from humans only by a few point mutations (11), the recent HlNl isolates from birds and pigs of Northern Europe are less closely related genetically to the human HlNl hemagglutinins when the latter were compared with those obtained from swine strains isolated in the United States. These data indicate that there are two different HlNl influenza virus subtypes circulating in the pig population; it is suggested that viruses carrying the HA gene of such strains might be transferred from birds to pigs and/or vice versa. Very recently an HlNl influenza A strain has ‘been isolated from turkeys in Colorado. However, this strain is more
closely related to the swine influenza strains isolated in the United States than to the A/duck/Alberta/35/76 virus (I.%‘), which again suggests that bird influenza virus genes might cross the species barrier going to pigs or vice versa. ACKNOWLEDGMENTS The skillful technical assistance of Mrs. K. Miiller is gratefully acknowledged. The work was supported by the Sonderforschungshereich 47 of the Deutsche Forschungsgemeinschaft. REFERENCES 1. HINSHAW, J Gen
V. S., WEBSTER, R. G., and TURNER, viral! 41, 115-127 (1978).
B.,
2. BU’ITERFIELD, W. K., CAMPBELL, C. H., WEBSTER, R. G., and SHORTRIDGE, K. F., J. In&t. D&I 138, 686-689 (1978). 3. Orr~s, K., and BACHMANN, P. A., Arch Viral 63, 185-190 (1980). 4 OTTIS, K., and BACHMANN, P. A., Zbl Vet. Med B30, 22-35 (1983). 5. WILE, K. H., NIENHOFF, U., and PRAGER, D., 606 (1981).
H., ERNST, H., SCHMIDT,
Tierhztl.
Umsch 36,591-
6. OTTIS, K., BOLLWAHN, W., BACHMANN, P. A., and HEINRITZI, K., Tier&ztL UWLSC~36, 608-612 (1981). 7. GOURREAU,
J. M., HANNOUN,
Ann Viral (Inst. Pasteur)
C., and KAISER, C., 132E, 287-294 (1981).
8. PENSAERT, M., OITIS, K., VANDEP~, M. M., and BACHMANN, P. A., Bull 75-78 (1981).
J., KAPLAN, ?KH;O. 59,
9. SCHOLTISSEK, C., HARMS, E., ROHDE, W., ORLICH, M., and Rorr, R., Virology 74, 332-344 (1976). 10. SCHOLTISSEK, C., ROHDE, W., HARMS, R., Virology 79, 330-336 (1977). 11. SCHOLTISSEK, C., VON HOYNINGEN, R., Virology 69, 613-617 (1978).
E., and ROTI!, V., and Roar,
12. HINSHAW, V. S., WEBSTER, R. G., and BEAN, W. S. In “The Origin of Pandemic Influenza Viruses” (W. G. Laver, ed.), pp. 181-189. Elsevier/North-Holland, New York/Amsterdam/ Oxford, 1983.
129,521-523 (1983)
Genetic Relatedness of Hemagglutinins of the Hl Subtype of Influenza A Viruses Isolated from Swine and Birds C. SCHOLTISSEK,* *Institut
H. B~JRGER,* P. A. BACHMANN,?
AND C. HANNOUN*
Virolcgti, Justus-Liebig-Universitiit, Giessen, West Gemany; ~Institut fiir Medizinische M~ikrobiologie, Iqfektims- urd Seuchemnedizin, LudwigM~milians-University, Munich, West Germany; and $.Institut Pasteur, Paris, France
fiir
Received April 20,
1983;
accepted June 20,
1983
The hemagglutinin (HA) gene of the influenza virus subtype HlNl isolated from pigs and birds has been analyzed by the hybridization technique. According to the RNase protection data the HA genes of recent isolates from pigs in Northern Europe are genetically more closely related to those of isolates from birds in Europe and North America than to those of isolates from pigs in the United States, Taiwan, and Italy. Thus, two different HlNl subtypes are circulating in the pig population. The results are consistent with the view that HlNl viruses can be transmitted from birds to pigs and/or vice versa.
It has been suggested that human pandemic influenza A strains might be created in nature by reassortment during double infection of a common host with a prevailing human virus and a strain infecting animals, especially birds. In this context pigs have been mentioned as possible candidates to be such a common host. It is of interest, therefore, to compare influenza virus strains isolated from pigs with those found in humans and birds. Recently, influenza viruses related to swine influenza viruses (HlNl) were isolated from ducks in North America (I), Hong Kong (Z), and Europe (3, 4). On the other hand, in 1979 influenza viruses of the HlNl subtype were isolated from diseased pigs in Germany (Westfalia), which were not recognized in this area before (5). Later on corresponding swine influenza viruses were also found in other parts of Germany (6), France (7), and Belgium (8). In Germany the virus was isolated only from clinically affected pigs. The clinical symptoms were typical for swine influenza. The virus had apparently not been introduced before 1979, since HI antibodies against swine influenza virus were not present in 745 sera collected from pigs on 161 farms in this area for diagnostic purpose during and prior to 1978 (5). In France, the virus seemed to be enzootic
and in the majority of cases, infection was asymptomatic although contagion rates were high and diffusion was rapid in contaminated farms. There was no evidence for infection of man, even in persons in close contact with infected animals (7’). There is serological evidence that these newly recognized strains isolated from pigs are more closely related serologically to HlNl bird isolates than to the classical swine influenza virus strains (8). In addition, swine influenza-like HlNl strains from birds readily infect pigs and spread among them by contact (3). Therefore, we have started an analysis of recent European HlNl isolates by the RNA hybridization technique and compared them with those obtained in other geographic regions. The virus strains listed in Table 1 were investigated. The A/swine/Arnsberg/6554/79 strain was kindly provided by Dr. K. H. Witte (5). The A/duck/Alberta/35/76 strain has been provided by Drs. V. Hinshaw and R. G. Webster. The strains A/swine/France/MAR/80, A/swine/France/STE/80, and A/swine/ France/OLI/80 were isolated in January or February 1980 when first evidence of the introduction of HlNl (SW) virus in France was obtained. The former was isolated from sick pigs during an outbreak of 521
0042-6822/83 $3.00 Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
SHORT
522
COMMUNICATIONS TABLE
1
RNase PROTECTION OF HYBRIDIZATION OF %P-LABELED vRNA WITH NONLABELED
CRNA OF VARIOUS
Percentage
cRNA of
Country of isolation
A/duck/Bavaria/2/77 A/swine/l976/31 A/swine/France/STE&O A/duck/Alberta/35176 A/swine/Arnsberg/6554/79 A/swine/France/MAR/80 A/swine/France/OLI/80 A/swine/Germany/Z/81 A/swine/Belgium/2179 A/duck/Schleswig/21/79 A/swine/Taiwan/l/76 A/coot/Schleswig/4/79 A/New Jersey/8176 A/swine/Germany/l/82 A/swine/Italy/81 A/swine/Iowa/30 A/PR/8/34 A/FPV/Rostoek/34 (H7Nl)
Germany United States France Canada Germany France France Germany Belgium Germany Taiwan Germany United States Germany Italy United States United States Germany
“All strains listed belong to the HlNl *nd = not done.
A/duck/ Bavaria/2/77 100 73 94 96 nd* 90 94 94 95 nd 73 96 72 94 71 71 nd 33
SEGMENT 4 (HA)
INFLUENZA
STRAINS
RNase protection after hybridization with vRNA segment 4 of A/swine/ 1976/31 68 100 72 64 67 nd 69 nd 71 69 95 nd nd 65 nd 97 93 34
subtype except A/FPV/Rostock/34,
severe swine influenza in the south of France, the two latter from apparently healthy pigs in Normandy and Brittany (Q, and they were kindly provided by Dr. Gourreau. The hybridization technique has been described in detail elsewhere (9). It was difficult to incorporate sufficient 32P into the virion RNA (vRNA) segment 4 (hemagglutinin gene) of the influenza virus strains investigated, and to obtain sufficiently concentrated complementary RNA (cRNA) probes. Therefore, the following controls were included for the calculation of the percentage RNase protection. (i) In order to control the background radioactivity of the gel, a band of the same size as the HA band was cut out in the immediate neighbourhood of the HA band. The RNA extracted from this band was hybridized under identical conditions as
OF FOUR HlNl
A STRAINS
A/swine/ France/STE&O 94 71 100 91 96 94 94 96 100 96 71 91 65 97 62 70 nd 32
A/duck/ Alberta/35176 92 67 93 100 92 92 94 91 92 91 65 nd 64 92 nd 67 nd 30
which is a H7N1 strain.
the HA-RNA samples. These control values never exceeded 20% of those of the HA hybridization. They were subtracted before calculation of the percentage RNase protection of the HA genes. (ii) In order to be sure that the concentration of cRNA was sufficient to work under saturation conditions, hybridization was also done against RNA segment 7 (M protein gene), which is the most conserved gene of influenza A viruses (9). If the hybridization with RNA segment 7 was less than 90%, the corresponding batch of cRNA was either discarded or the amount of cRNA was drastically increased. The error width of the method is about 5%. Thus, if the RNase protection is about 95% the two strains under investigation are expected to be genetically highly related in their HA genes differing from each other presumably only by a few point mutations.
SHORT
523
COMMUNICATIONS
The radioactivity in the various aliquots of the vRNA of the four strains was between 600 and 2000 cpm. The RNase protection after homologous and heterologous hybridization is a relative measure of the genetic relatedness between various influenza virus strains. In Table 1 data are presented on the RNase protection after cross-wise hybridization of vRNA segment 4 of various HlNl strains isolated from birds and pigs. It can be seen that HA genes of the recent bird isolates from the United States and Germany are closely related to each other and to HlNl isolates from pigs in Northern Europe. These HAS can be clearly differentiated genetically from the HA genes of HlNl strains isolated about 50 years ago in the United States or more recent isolates from Taiwan, Italy, or the United States, which also are closely related to each other. Furthermore, the HA gene of the A/swine/1976/ 31 strain is closely related to that of the PR8 strain (Table 1). Since the HA gene of PR8 exhibits almost 100% RNase protection after hybridization with cRNA of the FM1 strain (IO), and the FMl-HA differs from the HA genes of recent HlNl strains isolated from humans only by a few point mutations (11), the recent HlNl isolates from birds and pigs of Northern Europe are less closely related genetically to the human HlNl hemagglutinins when the latter were compared with those obtained from swine strains isolated in the United States. These data indicate that there are two different HlNl influenza virus subtypes circulating in the pig population; it is suggested that viruses carrying the HA gene of such strains might be transferred from birds to pigs and/or vice versa. Very recently an HlNl influenza A strain has ‘been isolated from turkeys in Colorado. However, this strain is more
closely related to the swine influenza strains isolated in the United States than to the A/duck/Alberta/35/76 virus (I.%‘), which again suggests that bird influenza virus genes might cross the species barrier going to pigs or vice versa. ACKNOWLEDGMENTS The skillful technical assistance of Mrs. K. Miiller is gratefully acknowledged. The work was supported by the Sonderforschungshereich 47 of the Deutsche Forschungsgemeinschaft. REFERENCES 1. HINSHAW, J Gen
V. S., WEBSTER, R. G., and TURNER, viral! 41, 115-127 (1978).
B.,
2. BU’ITERFIELD, W. K., CAMPBELL, C. H., WEBSTER, R. G., and SHORTRIDGE, K. F., J. In&t. D&I 138, 686-689 (1978). 3. Orr~s, K., and BACHMANN, P. A., Arch Viral 63, 185-190 (1980). 4 OTTIS, K., and BACHMANN, P. A., Zbl Vet. Med B30, 22-35 (1983). 5. WILE, K. H., NIENHOFF, U., and PRAGER, D., 606 (1981).
H., ERNST, H., SCHMIDT,
Tierhztl.
Umsch 36,591-
6. OTTIS, K., BOLLWAHN, W., BACHMANN, P. A., and HEINRITZI, K., Tier&ztL UWLSC~36, 608-612 (1981). 7. GOURREAU,
J. M., HANNOUN,
Ann Viral (Inst. Pasteur)
C., and KAISER, C., 132E, 287-294 (1981).
8. PENSAERT, M., OITIS, K., VANDEP~, M. M., and BACHMANN, P. A., Bull 75-78 (1981).
J., KAPLAN, ?KH;O. 59,
9. SCHOLTISSEK, C., HARMS, E., ROHDE, W., ORLICH, M., and Rorr, R., Virology 74, 332-344 (1976). 10. SCHOLTISSEK, C., ROHDE, W., HARMS, R., Virology 79, 330-336 (1977). 11. SCHOLTISSEK, C., VON HOYNINGEN, R., Virology 69, 613-617 (1978).
E., and ROTI!, V., and Roar,
12. HINSHAW, V. S., WEBSTER, R. G., and BEAN, W. S. In “The Origin of Pandemic Influenza Viruses” (W. G. Laver, ed.), pp. 181-189. Elsevier/North-Holland, New York/Amsterdam/ Oxford, 1983.