Comparison of influenza viruses isolated from man and from whales

Bulktin of the World Health Organlvatlon, 56 (6): 923-930 (1978) Comparison of influenza viruses isolated from man and from whales D. K. Lvov,' V. M. ZDANOV,' A. A. SAZONOV,1 N. A. BRAUDE,' E. A. VLADIMRTCEVA,1 L. V. AGAFONOVA,1 E. I. SKLJANSKA lA,' N. V. KAVERTN,1 V. I. REzNIK,4 T. V. PYSINA,2 A. M. OkERovI6,6 A. A. BmERzN,3 I. A. MJASNIKOVA,' R. Y. PODCERNJAEVA,1 S. M. KL KO,1 V. P. ANDREPEv,1 & M. A. YAKHNO1 Four isolates of influenza virus strains from Moscow and Habarovsk that caused outbreaks of influenza in November and December 1977 in several cities of the USSR were studied and their haemagglutinins and neuraminidases were compared with those of other human and animal influenza viruses including A/whale/Pacific Ocean/76. In HI tests these isolates, designated A/USSR/77, reacted with immune serum against A/FM/1/47 (HIN1) to the homologous titre, and with antiserum against A/whale/PO/19/76 virus to 1/8 of the homologous titre. In neuraminidase inhibition tests all A/USSR/77 isolates showed the presence of human Ni type neuraminidase, more closely related to A/sw/New Jersey/76 (HswlN1) than to A/FM/1/47 (HINI) virus. The haemagglutinin of A/whale/Pacific Ocean/19/76 virus occupies an intermediate position betweenHO andHI, but its neuramini- dase is close to Nav2. The virus from whales multiplies better at low (280C) and at high (40°C) temperatures than do the viruses ofhuman origin that were tested. An epidemic caused by a virus characterized antigenically as HlNl was first detected in the far east region of the USSR in early November 1977. The initial outbreak and the subsequent nationwide epidemic has been described previously (1). During 1975-76 lung and liver specimens were collected from the striped whale (Balaenopteridae) in the South Pacific and influen A viruses were isolated from them that were shown also to have H antigens related to the HO, Hi group. In this paper we describe the antigenic and some biological properties of current HINI viruses isolated from man and compare them with those of the viruses isolated from the whales. 1D. I. Ivanovskii Institute of Virology, Academy of Medical Sciences, Moscow, USSR. ' Scientific Research Institute of Epidemiology and Microbiology, Vladivostok, Ministry of Health, RSFSR, USSR. ' Pacific Institute of Fishery and Oceanography, Vla- divostok, Academy of Sciences, USSR. ' Habarovsk Sanitary-Epidemiological Station, Habarovsk, USSR. 'Moscow Sanitary-Epidemiological Station, Moscow, USSR. MATERIALS AND METHODS Viruses Strains A/Moscow/0778/77, A/Moscow/0782/77, and A/Moscow/0897/77 were isolated on 21-24 November 1977, from young adult patients in Moscow by A. M. OWerovid. Strain A/Habarovsk/ 03466/77 was isolated on 24 November 1977, from a sick child in Habarovsk by V. I. Reznik. These strains are designated A/USSR/77. After isolation in chick embryos, the strains were passaged only twice. Specimens from striped whales (Balaenopteridae) were collected in 1975-76 by scientists with a whaler flotilla in the South Pacific. Seventy-two specimens from lungs and 65 from livers were taken from 72 whales. The specimens were stored at -20°C for 4 months in 50% glycerol prepared in phosphate buffer (pH 7.2) with antibiotics (10 000 IU of penicillin and 1500 IU of streptomycin per ml). After inoculation into chick embryos in this labora- tory, 13 strains of influenza virus were isolated from the lung specimens and 1 strain from a liver specimen. The viruses were isolated at second passages. 3760 - 923- D. K. LVOV ET AL. Table 1. Reference sera used Antiserum Source Obtained from Virus of sera A/PR/8/34 A/Bel/42 A/WSN-A/duck/Engl/56 A/whale/PO/1 9/76 A/FM/1/47 A/Moscow/0897/77 A/Sw/lowa/1 3/30 A/Sw/Wisc/67 A/NJ/76 A/NJ/76-A/PR/34 (x-53) A/Sing/i /57 A/H K/1 /68 A/P.Ch/1/73 A/HK/1/68-A/PR/34 (R-2069) A/eq/Prague/56 A/eq/Miami/63 A/chick FPV/Rostock/34 A/turk/England/63 A/duck/England/56 A/duck/Czechoslovakia/56 A/tem/SA/61 A/turkey/Canada/63 A/shearwater/Australia/71 A/duck/Ukraine/63 A/tern/Turkmenia/73-A/Bel/42 (R-6a) A/turkey/Ontario/67 A/turkey/Wisc/66-A/tern/SA/61 goat goat rat rabbit goat rabbit rat goat rat rabbit goat goat rat rabbit rat rat goat rat rat rat rat rat rat rat rabbit rat rabbit R. Webster R. Webster Institute of Virology Institute of Virology R. Webster Institute of Virology Institute of Virology R. Webster Institute of Virology E. Kilbourne R. Webster R. Webster Institute of Virology E. Kilbourne Institute of Virology Institute of Virology R. Webster Institute of Virology Institute of Virology Institute of Virology Institute of Virology Institute of Virology Institute of Virology Institute of Virology Institute of Virology Institute of Virology E. Kilbourne Immune sera Immune sera to the strain A/whale/Pacific Ocean/19/76 was prepared by immunization of white rats. All 14 isolates were neutralized with this serum diluted 1:2560 in HI tests and 1:30 in NI tests, so were considered identical. Therefore, only the prototype strain A/whale/Pacific Ocean/19/76 was used in further studies. The reference sera used are listed in Table 1. Serological reactions Haemagglutination inhibition (HI) tests were performed by a method described previously (2). The sera were first treated with standard preparations of receptor-destroying enzyme. Neuraminidase inhi- bition (NI) tests were carried out by the method described by Webster & Pereira (3). Electron microscopy Purified and concentrated virus preparations were used. The preparations were layered and contrasted with a 1 % uranyl acetate solution in methanol. Biological properties The methods used, such as cultivation at various temperatures, elution characteristics, plaque-forming capacity, etc., have been described previously (4). Antigenic formula HONI HONavl HOHI Nav2 HINl Hswl Nl Hswi Ni H2N2 H3N2 H3NI Heqi Neq1 Heq2Neq2 Hav1Nl Hav2Nav2 Hav3Navl Hav4Navl Hav5Nav2 Hav6Neq2 Hav6Nav5 Hav7Neq2 Hav7N1 Hav8Nav4 Hav9Nav2 924 COMPARISON OF INFLUENZA VIRUSES All the strains studied in polyacrylamide gel were cultivated in chick embryos. Virus was concentrated by differential centrifugation and purified in linear 15-60% sucrose gradients and centrifuged at 2400 g for 16 h at + 50C. The purified virus was stored at -700C. Electrophoresis in polyacrylamide gel Polypeptides were analysed in sodium dodecyl- sulfate (SDS) containing 10% polyacrylamide gel, using a tris-glycerol system. The samples were treated for 2 min at 100°C in lysis buffer containg 0.1 mol/litre dithiothreitol, 2% SDS, 10% glycerol, and 0.2% bromophenol blue. Samples containing lOO,pg of virus protein in 20 ml were layered on gel and electrophoresis was performed at 40 V for 17-18 h. After electrophoresis, the gel was stained with 0.2% amido black and washed with a mixture of ethanol, acetic acid, and water (5:1:5 respectively). Polyacrylamide gel analysis of virus-specific proteins synthesized in the infected cells Dog kidney (MDCK) cells in monolayer culture were infected at the rate of 50 TCID50/cell. After labelling, the cells were washed with buffered saline (0.15 mol/litre NaCl, 0.01 mol/litre tris-hydro- chloride, pH 7.5), dissolved in lysis buffer heated for 2 min at 100°C, layered on top of the gradient (6-12%) polyacrylamide gel slab, and incubated under Eagle's minimal essential medium (MEM) for 5 h at 37°C. Then the culture fluid was discarded and the cells were washed three times with Hanks' balanced salt solution (BSS) and incubated further in the presence of"4C-Chlorella hydrolysate (740 kBq/ ml in Hanks' BSS) for 90 min. RESULTS The object of the first experiments was to determine the antigenic properties of the influenza viruses isolated from whales, of which the prototype was strain A/whale/PO/19/76. The results of the HI tests are shown in Table 2. It can be seen that the haemagglutinin of A/whale/ PO/19/76 virus cross-reacts with immune sera to the antigenic complex HO-H1-Hswl-Hav5 and that it is more closely related to HO of the strain A/Bel/42 (1/16 of the homologous titre) than to HO of the earlier strain A/PR8/34 (1/32) or to Hi ofA/FM/1/47 (1/32). Thus, the haemagglutinin of this virus may be said to occupy an intermediate position between HO and HI. Antiserum to virus A/whale/PO/19/76 Table 2. HI tests with A/whale/Pacific Ocean/i 9/76 virus a Reciprocal ire of immune sera with: Immune sera to haemagglutinins homologous A/whale/PO/ virus 19/76 HO A/PR8/34 640 20 HO A/Bel/42 1280 80 HI A/FM/1/47 640 20 Hswl A/sw/lowa/30 1280 10 Hav5 A/tern/SA/61 80 <10 A/whale/PO/19/76 b 2560 2560 A/Moscow/0782/77 c 2560 <10 a Negative results were obtained with immune sera to haemag- glutinins H2, H3, Heql, Heq2Z Havl, Hav2, Hav3, Hav4, Hav5, Hav6, Hav7, Hav8, Hav9. b Titres with antigen A/USSR/77: 320 A/PR8/34: 320 A/FM/1/47: 160 A/Bel/42: 320 A/WSN/33: 320 c Titres with antigens A/PR/8/34, A/Bel/47, A/WSN/33: <10 A/FM/1 /47: 640 Table 3. NI tests with A/whale/Pacific Ocean/i 9/76 virus a Reciprocal titres of immune sera with: Immune sera to neuraminidase homologous A/whale/PO/ virus 19/76 Nav2 A/Tern/SA/61 30 30 Nav2 A/tern/Turkm/1 8/73 30 20 Nav2 R-136 (Hav9Nav2) 60 30 A/whale/PO/1 9/76 30 30 a Negative results were obtained with immune sera to neura- minidases Ni, N2, Neql, Neq2, Navl, Nav3, Nav4, Nav5, Nav6. reacts with virus A/USSR/77 to 1/8 of the homo- logous titre (Table 5). Table 3 presents results of neuraminidase inhibi- tion tests with the whale virus. It can be seen that the neuraminidase of A/whale/PO/19/76 is closely related or identical to neuraminidase Nav2. Thus A/whale/PO/19/76 possesses an antigenic formula (HO-Hi Nav2), not known before for human or animal influenza viruses. 925 D. K LVOV ET AL. Fig. 1. Electron micrograph of virions of A/whale/Pacific Ocean/I 9/76 virus. Negative contrasting. Electron-micrographs (Fig. 1) show that the morphology of the virus does not differ from that of other influenza A viruses. Both round and thread- like or irregular virions are observed. Typical structures of ribonucleoprotein are also observed in some preparations as well as spikes of haemaggu- tinin. Some of the biological properties of A/whale/ PO/19/76 virus and A/WSN/33 (HON1) and A/FM/1/47 (H1N1) viruses were also compared. Table 4 shows: (a) the whale virus multiplies better at low (280C) and at high (400C) temperatures than do the two other viruses, (b) it is not sensitive to inhibitors in normal horse serum, (c) it produces turbid plaques in chick embryo fibroblast mono- layers, (d) its haemagglutinin is moderately thermo- stable, and (e) its elution speed is intermediate. The heat resistance of the hgglutinin of the whale virus is intermediate between that of A/WSN/33 and that of A/FM/1/47 and A/USSR/77. Virus A/USSR/77 is as sensitive to inhibitors in normal horse serum as is A/FM/1/47, but it does not form plaques on chick embryo fibroblast cultures. The results of the study of haemagglutinins of viruses isolated during the outbreaks in November 1977 are shown in Table 5. It can be seen that the haemagglutinin of the newly isolated viruses is closely related or identical to HI of the prototype virus A/FM/1/47 and partly (1/8) to the haemagglu- tinin of the whale virus. Negative results were obtained with other haemagglutinins of human and animal inluenza viruses (except for some crossing with A/Bal/42). Table 6 shows the results of the study of the neuraminidase of the recently isolated inflz viruses. It can be seen that their neuraminidase is morerelatedtothatofA/sw/NewJersey/76 (HswlNl) virus than to that of A/FM/1/47 (HINI) virus. In the polyacrylamide gel pattern of virus. specific proteins synthesized in MDCK cells, the most striking difference between A/FM/1/47 and both Port Chalmers and the newly isolated strain A/Habarovsk/77 was a much greater mobility ofNS protein (Fig. 2). Any differences in mobility of the HA were barely detectable. No differences could be revealed in the mobilities of NP and M proteins. 926 COMPARISON OF INFLUENZA VIRUSES Table 4. Some biological properties a of A/whale/Pacific Ocean/I 9/76 virus as compared with some influenza A viruses of human origin Sensitivity rt(oi Do Titre Of to rC(olls) Tie of hae- ErVirus haemagglutinin inhibitors Plaques magglutinin (hours) serum 28tC 37TC 40C A/whale/PO/1 9/76 +(HO-HI Nav2) 640 - turbid 8.0 8.5 8.0 60 2 A/WSN/33 +(HONI) 640 - clear 5.2 7.4 6.5 10 4 A/FM1 /47 +(HI NI) 320 + clear 6.5 7.5 6.0 120 1 A/USSR/77 not not(Hl NI) 320 + - 5.0 7.2 tested 80 tested arct -reproduction in chick embryos at various temperatures; Tie = thermoresistance of haemag- glutinin at 56T; Erc = 100 % elution from chicken erythrocytes. Neither neuraminidase (NA) nor P-proteins could be identified in the gels against the background of cellular proteins. Thus, the newly isolated epidemic strains have the antigenic formula HINI. The haemagglutinin therefore corresponds to that of the early HI variant, although there are some crosses with the late HO variant and HO-HI of the whale virus, while the neuraminidase is more similar to genealogical precursors of HINI virus. The polypeptides ofthe epidemic strainA/Moscow/ 0897/77 and of the virus A/whale/PO/19/76 were compared with those of the human viruses A/PR/8/ 34, A/FM/l/47, and A/WSN/33 by electrophoresis Table 5. Identification of haemagglutinins of newly isolated epidemic influenza viruses (A/USSR/77) a Reciprocal titres of sera with viruses: Immune sera to haemagglutinins A/Moscow A/Moscow A/Moscow 0778/77 0782/77 0897/77 Homologous HOA/PR8/34 < 10 <10 20 640 HO R-3a (HONav2) <10 <10 < 10 2560 HO R-5a (HONav2) <10 <10 <10 1280 HO A/Bel/42 80 40 80 1280 HO-HI A/whale/PO/19/76 320 320 320 2560 HI A/FM/1/47 640 640 640 640 H2 A/Sing/57 <10 <10 <10 1280 H3 A/HK/68 <10 <10 <10 5120 H3A/P.Ch./73 <10 <10 <10 1280 H3A/Tokyo/75 <10 <10 <10 160 H3 A/Vict/75 <10 <10 <10 160 a Negative results were obtained with immune sera to haemagglutinins Hswl, Heql, Heq2, Havl, Hav2, Hav3, Hav4, Hav5, Hav6, Hav7, Hav8, Hav9. 927 D. K. LVOV ET AL. Table 6. Identification of neuraminidase of newly isolated epidemic influenza viruses (A/USSR/77) a Reciprocal titres of sera with viruses: Immune sera to neuraminidases A/Moscow A/Moscow A/Moscow A/Habarovsk Homolo- 0778/77 0782/77 0897/77 034581/77 gous Ni A/FM/1/47 60 30 20 30 90 Ni A/NJ/78 60 20 60 30 30 N1 x-53 20 30 60 30 120 Ni A/FPV/Rostok 20 30 60 <10 not tested N1 R-6a 20 20 30 <10 30 NI R-2069 30 20 20 20 60 N2 A/Sing/57 <10 <10 <10 n.t. 60 N2 A/HK/68 <10 <10 <10 n.t. 60 N2A/P.Ch./73 <10 <10 <10 n.t. 100 N2 A/Tokyo/75 <10 <10 <10 n.t. 40 N2 A/Vict/75 <10 <10 <10 n.t. 20 a Negative results were obtained with immune sera to neuraminidases Neql, Neq2, Navl, Nav2, Nav3, Nav4, Nav5, Nav6. Fig. 2. Polyacrylamide gel analysis of virus-infected MDCK cells. The cells were infected at the rate of 50 TCID50/cell and labelled with 14C-ChlorelI hydro- lysate (740 kBq/ml) from 5 h to 6.5 h postinfection. Electrophoresis in gradient gel with 6-12% concen- tration of acrylamide for 4 h at 24 mA. U = uninfec- ted cells; Fm = A/FM/1/47(HINI); P.Ch. = A/Port Chalmers/1/73 (H3N2); H = A/Habarovsk/77. 928 COMPARISON OF IFUENZA VIRUSES Fig. 3. Autoradiogram of electropherogram of polypeptides of human influenza viruses and A/whale/Pacific Ocean/19/76 virus. Human influenza strains: A/WSN/33-strips 1 and 10; A/PR/8/34-strips 2 and 6; A/ Moscow/0897/77-strips 3 and 7; A/FM/i/47-strips 5 and 9. Influenza virus isolated from whales, A/whale/ PO/19/76-strips 4 and 8. The arrows show the possible positions of neuraminidase in A/WSN/33 and A/whalef PO/1 9/76. and the results are shown in Fig. 3. As can be seen, the epidemic strain A/Moscow/0987/77 has a poly- peptide composition similar to that of A/FM/1/47. The composition of virus A/whale/PO/19/76, except for one polypeptide which is evidently neuraminidase, is similar to that ofA/PR/8/34 virus. The polypeptide of A/whale/PO/19/76 that apparently represents neurainidase possesses lower mobility in gel, that is, higher molecular weight, than the corresponding polypeptide of the human virus A/PR/8/34 and is evidently similar to the neuraminidase ofA/WSN/33 virus. As shown in Fig. 2, both subunits of haemag- glutinin (HA2 and HAI) of A/Moscow/0987/77 and A/FM/1/47 strains, have lower mobility in the gel, that is, higher molecular weight, in comparison with the polypeptides of A/PR/8/34 and A/whale/PO/19/ 76 viruses. DISCUSSION From wild animals in the Pacific basin we isolated in 1974-76 influenza viruses that appear to be anti- genically related to known human epidemic viruses. A virus of the Hong Kong complex (H3N2) was isolated from a common murre (5), a virus of the Asian influenza complex (H2N2) was isolated from pintail ducks (6) and a virus with antigens HO- HlNav2 was isolated from whales. Analysis ofthe epidemic strain A/Moscow/0897/77 in polyacrylamide gel showed that its polypeptide composition is close to that of strain A/FM/1/47 (HlNl). The virus A/whale /PO/19/76 differs from these viruses in its polypeptide composition. How- ever, its polypeptides are similar to those of human influenza virus A/PR/8/34 (HONI) except for one peptide, which represents neuraminidase and whose electrophoretic mobility is close to that of the neuraminidase of A/WSN/33 (HONI) virus. The analysis of virus-specific peptides induced in MDCK cells showed that the mobility of the NS protein of A/FM/1/47 was greater than that of a newly isolated strain Habarovsk/77 and of Port Chalmers/ 1/73. These data are only preliminary. Thus, it has been demonstrated that in natural biocoenoses of the region where new epidemic human influenza viruses have often arisen, old viruses that disappeared 10-20 years ago still exist. It is probable that as a result of complex ecological processes, WSN PRB 08W? KHT Ftf HA DAG I A DIAGRAMMATIC REPRESENTATION 929 930 D. K. LVOV ET AL. including recombination, some of the strains may have been selected and readapted to man from among the enormous and heterogenous population of animal influenza viruses. We wonder if this hap- pened with the new epidemic HlNl virus that reappreared in the human population after a 20-year absence and 1.5 years after an antigenically related virus had been isolated from whales. Influenza virus could spread to whales from birds through their virus-containing excrements, which are shed into the sea in large quantities around bird colonies. The possible intermediate role of plankton cannot be ruled out (7). Where birds nest, their faeces form an essential part of the food of zooplankton, in which influenza viruses can propably remain mechanically or biologically for a time. Whales of the family Balaenopteridae, most of whose diet consists of zooplankton, could be infected with influenza viruses during feeding. It may be signi- ficant that virus A/whale/PO/19/76 grows well both at a temperature of 280C (whale marker?) and also at a temperature of 40°C (bird marker?). Also the avian type of neuraminidase suggests that the whale virus originated from avian influenza viruses. R SUMmt ETUDE COMPARAE DE VIRUS GRIPPAUX ISOLtS CHEZ L'HOMM ET CHEZ DES BALENES Quatre isolats de virus grippaux responsables de flamb6es survenues en novembre et d6cembre 1977 dans plusieurs villes de l'URSS - notamment Moscou et Habarovsk d'oi proviennent les souches - ont fait l'objet de travaux visant a comparer leurs h6magglutinines et leurs neuraminidases avec celles d'autres virus de la grippe humaine et animale - A/whale/Pacific Ocean/ 19/76 en particulier. Dans les epreuves d'inhibition de 1'hemagglutination, ces isolats - A/USSR/77 - ont 6t6 neutralis6s par un imuns6rum contre A/FM/1/47 (HilNl) de titre homologue et ont reagi de fa9on partielle avec l'antis6rum contre A/whale/PO/19/76 (1/8 du titre homologue). Dans les epreuves portant sur les neura- minidases en revanche, les neuraminidases de type humain Ni mises en 6vidence dans tous les isolats Al USSR/77 se sont r6v6l6es plus proches de A/sw/New Jersey/76 (HswlNl) que deA/FM/1/47 (HlNl). L'h6mag- glutinine duvirusA/whale/PO/19/76occupe ainsi uneplace intermediaire entre HO et Hl, mais sa neuraminidase est voisine de Nav2. La souche provenant des baleines se mul- tiplie mieux a faible (28°C) et A haute (40°C) temp6ratures que les virus d'origine humaine cultiv6s dans les memes conditions. Dans les bioc6noses naturelles de la r6gion oii sont apparus les nouveaux virus i l'origine d'epid6mies de grippe humaine, on a pu dtablir la persistence d'anciens virus qui ne s'etaient pas manifest6s depuis 10-20 ans. Ainsi, il est possible qu'a la suite de processus 6cologiques complexes et notamment de recombinaison, la selection de certaines souches et leur r6adaptation a l'homme se soient operees au sein de l'enorme population h6t6ro- gene des virus de grippe animale. REFERENCES 1. ZDANOV, V. M. ET AL. Return of epidemic Al (HINI) influenza virus. Lancet, 1: 294-295 (1978). 2. Advanced laboratory techniquesfor influenza diagnosis. Atlanta, GA, US Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, 1975, 135 pp. (Immunology Series No. 6, Procedural Guide). 3. WEmsmR, R. G. & PEIRnmA, H. G. A common surface antigen in influenza viruses from human and avian sources. Journal ofgeneral virology, 3: 201-208 (1968). 4. PODCERNJAEVA, R. JA. Er AL. Forward investigations on interspecific hybridization of influenza A viruses. Voprosy virusologii, 2: 209-211 (1968). 5. SAZONOV, A. A. Er AL. 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