Isolation of swine-like influenza A(H1N1) viruses from man in Switzerland and The Netherlands

Ann. Inst. Pasteur/Virol. 1988, 139, 429-437

Q ELSEVIER

Paris

1988

ISOLATION OF SWINE-LIKE INFLUENZA A(HIN1) VIRUSES FROM M A N IN SWITZERLAND A N D THE NETHERLANDS J.C. de Jong (1), M.F. Paccaud (2), F.M. de Ronde-Verloop (1), N.H. Huffels (I), C. Verwei (1), T.F. Weijers (i), p . j . Bangma (3), E. van Kregten (3), J.A.M. Kerckhaert (4), F. Wicki (5) and W. Wunderli (6)

(1) Rijksinstituut voor Volksgezondheid en Milieuhygi~ne, Laboratory o f Virology, 3720 BA Bilthoven (The Netherlands), (2) Institute o f Hygiene, Virus Laboratory and National Influenza Centre, Geneva (Switzerland), (3) St. Elisabeth Hospital, Amersfoort (The Netherlands), (4) Hospital de Liehtenberg, Laboratory o f Immunology, Amersfoort (The Netherlands), (5) Willisau (Switzerland), (6) Institute of Immunology and Virology, Ziirich (Switzerland)

SUMMARY Swine influenza A (H1N1) viruses were isolated from two people in Switzerland and one in the Netherlands in early 1986. In haemagglutinationinhibition and neuraminidase-inhibition assays, the three viruses were closely related to one another and to the A / N e w Jersey/8/76 strain. The Swiss patients showed only mild symptoms, whereas the Dutch patient suffered from severe pneumonia. Two of the patients had been in close contact with diseased pigs. No such contact could be established for the third patient. None of the three individuals was known to suffer from immunodeficiency. No manto-man transmission of the virus has been detected. KEY-WORDS: Swine, Influenza, Haemagglutination, Neuraminidase; Inhibition, Europe, Humans, H1N1 viruses.

Submitted June 20, 1988, accepted July 9, 1988.

430

J.C. D E J O N G A N D COLL.

INTRODUCTION Swine influenza A (H1N1) virus, designated as sent paper, was first isolated from swine in 1930 perienced a high incidence o f infection with this E u r o p e , its presence in these animals had not been then, outbreaks of S w H I N 1 virus disease in swine creased in severity and frequency [12].

SwH1N1 virus in the pre[18]. Pigs in the U S A exvirus for m a n y years. In reported until 1976. Since on this continent have in-

Transmission of SwH1N1 virus to humans was first demonstrated in 1961, when the Czechoslovakia Influenza Centre isolated the virus from five sporadic infections in the winter o f 1959/60 [9]. The sources o f infection were unclear in those cases. In 1966, serological evidence of infection with the virus was f o u n d among humans in Illinois, USA, w h o had had regular exposure to pigs [17]. In spite o f increased interest and isolation facilities, the swine virus has been isolated only rarely from man even in regions where the virus is endemic in pigs. This is p r o b a b l y due to its low pathogenicity for humans. In 1976, a small outbreak o f SwH1N1 virus among military recruits in Fort Dix, New Jersey, USA, drew world-wide attention [10]. To the authors' knowledge only nine other, sporadic, cases of isolation of the virus from man have been recorded in the literature, including six cases in the U S A f r o m 1974 to 1982 and three in the Asian part o f the U S S R in 1983 [19, 3, 4, 15, 5]. To date, there have been no such reports from Europe. In the present paper, we give detailed epidemiological, clinical and virological information on three cases o f S w H I N 1 virus infection in man. T w o o f the subjects lived in Switzerland and one in the Netherlands. A short account of these events has been published [7].

MATERIALS AND METHODS Case 1, Switzerland. On January 23, 1986, a 50-year old employee of a mill in Central Switzerland, who was prone to serious crises of asthmatiform bronchitis, consulted his physician for malaise and afebrile rhinitis. These symptoms developed about three days after he had observed, as the supervisor of the mill-owned piggery, the first cases of a relatively severe influenza-like illness which rapidly spread among the pigs. Clinical examination of the patient revealed no sign other than slight rhinopharyngitis.

HI = haemagglutination inhibition. NI = neuraminidase inhibition,

SwH1N1 = swineinfluenza A(H1N1). tMK = tertiarycynomolgusmonkeykidney.

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Recovery was complete in three days. Nasopharyngeal specimens for virus isolation and blood specimens were collected on January 23 and February 18, 1986. Nasal swabs and paired sera were also collected from four pigs with influenza symptoms. Case 2, Switzerland. A 3-year old patient who lived in a small village in western Switzerland consulted for a febrile pharyngitis and a slight myringitis on January 31, 1986. As part of a sentinel and rapid diagnosis programme, a nasopharyngeal aspirate to be tested by enzyme-linked immunosorbent assay (ELISA) was collected, but no blood specimens were taken. The child had had no known contact with pigs.

Case 3, The Netherlands. On March 12, 1986, a 29-year old farmer living in the centre of the Netherlands presented with clinical influenza. He rapidly developed pneumonia, resembling staphylococcal pneumonia, which necessitated artificial respiration for twelve days. Administration of various antibiotics did not improve his condition. Sputum samples were taken on March 15 and 19, and serum samples on March 19 and April 1, 1986. Eventually, he recovered and was discharged from the hospital on April 18, 1986. At about the same time as the farmer, a number of pigs at his farm had developed respiratory symptoms. The patient had had regular contact with these animals over this period. On May 23, 1986, sera were taken from 5 of these pigs. On May 7, 1986, serum specimens were obtained from 7 individuals living or working on the farm in the beginning of March. On April 28, 1986, serum samples were collected from 20 nurses who had tended the patient in the hospital.

Reference strains. Influenza A(H1N1) and A(H3N2) virus reference strains were obtained from the World Influenza Centre at Mill Hill (Director Dr J.J. Skehel). The strain A/swine/Netherlands/3/80(SwH1N1) was kindly supplied by Dr W.E.P. Beyer from the National Influenza Centre in Rotterdam. The latter strain has been described by Masurel et al. [13].

Laboratory procedures. At Geneva, the nasopharyngeal specimens collected from case 1 were inoculated into the amniotic cavity of embryonated fowl eggs and onto trypsin-treated MDCK, human embryonic fibroblast, HeLa OHIO, HCV1, RC37 and LLC-MK2 cell cultures for virus isolation. The paired sera were examined in complement-fixation and haemagglutination-inhibition (HI) tests for antibody titre rises against various respiratory agents. Four-fold or greater rises were considered significant. The nasopharyngeal aspirate from case 2 was inoculated into embryonated fowl eggs. In the Netherlands, the sputum sample from case 3 taken on March 15 was examined for pathogenic bacteria, and that taken on March 19 for respiratory viruses in tertiary cynomolgus monkey kidney (tMK) cells, human embryonic fibroblast, HEp-2 and rhinovirus-sensitive HeLa cell cultures. The paired sera from the patient were assayed in HI tests [8] and in neuraminidase-inhibition (NI) tests as described by Aymard-Henry et al. [2], modified according to the suggestion of Russ et al. [16]. On May 7, 1986, blood was taken from the patient and his lymphocytes were examined in a transformation assay to evaluate his cellular immunocompetence.

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RESULTS Isolation of S w H 1 N 1 viruses. C a s e 1. ~ F r o m the nasopharyngeal material of the 50-year old miller, SwH1N1 virus was isolated in embryonated fowl eggs and in MDCK cell cultures after one blind passage. The isolate was labelled influenza A / G e n e v a / 5200/86(H1N1). In identification experiments, the egg-isolated virus was used. C a s e 2. ~ In the nasopharyngeal aspirate from the 3-year old child, influenza A virus antigens were detected by ELISA. After inoculation and one blind passage in eggs, this specimen yielded an SwH1N1 virus. This strain was called influenza A/Geneva/5521/86(H1N1).

It is worthwhile noting in connection with this fortuitously diagnosed case of SwH1N1 virus infection that a seroepidemiological survey indicated that the virus in swine had spread epizootically to the region where the patient was living during the same m o n t h in which the child was infected. C a s e 3. ~ From the sputum taken on March 19, 1986, from the 29-year old farmer, an SwH1N1 virus was isolated in tMK cells. The virus will be referred to as influenza A / N e t h e r l a n d s / 3 8 6 / 8 6 ( H 1 N 1 ) . The cellular immunocompetence of the patient, as tested in a lymphocyte transformation assay, seemed to be normal. There was no evidence for any underlying disease.

In none of the three cases was any other virus or pathogenic bacterium isolated from the patients. Pigs. ~ From nasal swabs collected from 2 pigs from the piggery of case 1, two SwH1N1 viruses were isolated in eggs. One of these viruses, influenza A/swine/Geneva/5175/86(H1N1) was used in the present study. Three other SwHIN1 viruses isolated from pigs elsewhere in Switzerland were also studied, namely influenza A/swine/Geneva/5186, 5257 and 5258/86(H1N1). These isolates were obtained from specimens collected in St Gallen on January 17, 1986, in Lucerne on December 26, 1985, and in Thurgau on December 27, 1985, respectively. As a reference virus for SwH1N1 viruses from the Netherlands, influenza A/swine/Netherlands/3/80(H1N1) was used [13].

HI and NI tests.

In HI and NI assays, the SwH1N1 viruses in this study resembled the SwH1N1 viruses recovered from pigs between 1930 and 1976 (tables I and II). The strains are closely related to one another as well as to A / N e w Jersey/8/76 and to A / s w i n e / G e r m a n y / 2 / 8 1 virus (not shown) and to other SwH1N1 viruses isolated from swine in Europe since 1982 (Dr J.J. Skehel, London, personal communication). No relationship was found with the H1N1 virus strains isolated from humans after 1933.

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IN MAN

TABLE I. - - H a e m a g g l u t i n a t i o n - i n h i b i t i o n (HI) tests w i t h S w H 1 N 1 viruses. HI titres of post-infection ferret antisera to: Virus strains, grown in tMK cells A/Netherlands/386/86(SwH1N1) (3) A/Geneva/5200/86(SwH1N1) (1) A/Geneva/5521/86(SwH1N1) (2) A/swine/Netherlands/3/80(SwH1N1) A/swine/Geneva/5175/86(SwH1N1) (1) A/swine/Geneva/5186/86(SwH1N1) A/swine/Geneva/5257/86(SwI-llN1) A/swine/Geneva/5258/86(SwH1N1) A/swine/S15/30 Du(SwH1N1) A/swine/Cambridge/39(SwH1N1) A/swine/H0ng Kong/1/74(SwH1N1) A/swine/Italy/1443/76(SwH1N1) A/New Jersey/8/76(SwH1Nl) A/Puerto Rico/8/34(H1N1) A/Fort Monmouth/1/47(H1N1)

N'86 N'80 S15 Cam HK Ital NJ PR8 FM

480 320 320 40 160 480 160 80 20 160 40 80 160 -

640 640 320 320 320 480 160 160 30 480 120 60 240 -

A/fhile/1/83(H1N1)

-

-

A/Philippines/2/82(H3N2)

-

-

60 40 80 40 20 60 40 40 60 40 15 40 15 30 40 30 80 40 40 80 40 20 40 160 10 80 640 120 640 160 160 160 40 240 -

240 160 160 40 160 160 160 80 40 160 320 320 160

160 160 160 30 80 160 160 80 40 120 160 60 160

. . . . . . . . . . . . . 1280

. . . . . . . . . . . . .

. . . . . . . . . . . . .

Chil Phil

. . . . . . . . . . . . .

2560 1920 640

Figure in parentheses denotes the associated case. - indicates < 10. Homologous titres are underlined.

TABLE II. - - N e u r a m i n i d a s e - i n h i b i t i o n V i r u s s t r a i n s , g r o w n in t M K cells A/Netherlands/386/86(SwH 1N1) (3) A / G e n e v a / 5 2 0 0 / 8 6 ( S w H 1N1) (1) A / G e n e v a / 5 5 2 1 / 8 6 ( S w H 1N1) (2) A / s w i n e ~ N e t h e r l a n d s ~ 3 / 8 0 ( S w H 1N 1) A / s w i n e / G e n e v a / 5 1 7 5 / 8 6 ( S W H 1N1) (1) A/swine/S15/30 Du(SwH1N1) A/New Jersey/8/76(SwH1N1) X - 5 3 a ( S w H 1 N 1 ) (*) A / N e t h e r l a n d s / 3 4 5 / 8 5 ( H 1N 1) A/Philippines/2/82(H3N2)

( N I ) tests w i t h S w H 1 N 1 v i r u s e s . N I titres o f a n t i s e r a t o : S 15/30

X-53a

360 100 230 150 150 1070 610 600 -

280 300 280 110 140 330 1040 1000 14 -

Figure in parentheses denotes the associated case. - indicates < 1 ; n d = not done. Homologous titres are underlined. (*) Recombinant strain derived from A / N e w Jersey/8/76(SwH1N1).

Phi/82

m

k

m

R

nd 43

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J.C. D E J O N G A N D C O L L .

Serological tests.

H I assays with the paired sera f r o m cases 1 and 3 demonstrated a significant rise in antibody titres against several SwH1N1 viruses (table III), indicating recent infection with the isolated viruses. The farmer in case 3 had also m o u n t e d rises in a n t i b o d y titres against H1N1 and H3N2 viruses currently circulating among humans. No rise in antibody titres against other respiratory agents was f o u n d in complement-fixation and HI tests with the paired sera in cases 1 and 3. The five pigs from the f a r m in case 3, four of which had fallen ill a few days earlier than the farmer, showed serological evidence of previous infection with SwH1N1 virus 2 months later (table IV). Seven persons f r o m the case 3 f a r m and 20 persons who had nursed the patient in the

TABLE III. - - H a e m a g g l u t i n a t i o n - i n h i b i t i o n (HI) tests with paired sera f r o m h u m a n cases i and 3.

HI titres (*) of sera from: Case 1 Case 3 ac con ac con

Virus strains, grown in tMK cells

< 6 < 6 < 6 8 24 12 nd

A/Netherlands/386/86(SwH 1N1) (3) A/Geneva/5200/86(SwH 1N 1) (1) A/Geneva/5521/86(SwH 1N 1) (2) A/swine/Hong Kong/1/74(SwH1N1) A/Chile/1 / 83(H 1N 1) A/Philippines/2/82(H3N2) A/Netherlands/331/85(H3N2)

24 32 12 24 24 12 nd

6 nd nd 6 24 192 32

192 nd nd 128 128 384 128

Figure in brackets denotes the associatedcase. (*) HI titres of acute (ac) and convalescent(con) sera, respectively. nd = not done.

TABLZ IV. - - H a e m a g g l u t i n a t i o n - i n h i b i t i o n (HI) tests with sera f r o m five pigs f r o m the f a r m o f h u m a n case 3.

HI titres of serum taken from pig number:

Virus strains, grown in tMK cells

A/Netherlands/386/86(SwH 1N 1) A/swine/Hong Kong/1/74(SwH1N1) A/Chile/1/83(H1N1) A/Singapore/I/57(H2N2) A/Netherlands/331/85(H3N2)

1

2

3

4

5

48 12 < 6 < 6 < 6

192 96 < 6 < 6 < 6

96 48 <6 <6 <6

96 48 <6 <6 <6

/> 768 768 <6 <6 <6

Pig number 2 was not ill during the epidemicamongthe other pigs.

SWINE-LIKE I N F L U E N Z A VIRUSES I N M A N

435

hospital were examined for HI antibodies against SwH1N1 virus. Only the 65-year old father of the patient, who also lived on the farm, was positive in that test.

DISCUSSION

Influenza viruses circulating among pigs are type A, subtypes H3N2 [20] and H1N1 [12]. The H3N2 viruses are antigenically similar to H3N2 virus strains recently found to be prevalent in the h u m a n population [14]. In contrast, the SwH1N1 viruses are not cross-reactive in HI tests with the H1N1 viruses which have been circulating in m a n since 1933 [1]. Since the great epidemics of 1918-1919, influenza virus with SwH1N1 surface antigens seems to have disappeared from the h u m a n population, apart from a few incidental cases presumably following contact with infected pigs. In recent years, SwH1N1 virus has become widespread in European pigs [12] and sporadic infections in m a n from this source are bound to occur. To the authors' knowledge, the infections reported here are the first evidence that this expectation is valid. In conformity with earlier cases in the USA, the three infections described in the present report appear not to have been transmitted to h u m a n contacts. The observation of anti-SwH1N1 antibodies in the serum of the father of patient 3 can readily be explained either by his age (65), or by direct infection from the diseased pigs which he had cared for daily during their illness. Generally, the virus is considered only weakly pathogenic for imm u n o c o m p e t e n t humans. Indeed, the illnesses in two of the three cases were mild. The cause o f the serious disease in case 3 is unknown. Serum specimens from this patient showed a rise in HI antibody titres against SwHIN1 virus as well as against the h u m a n viruses A/Chile/1/83(H1N1) and A/Netherlands/331/85(H3N2). The latter could have been anamnestic responses. Such heterologous responses have been described. Among 146 respiratory patients with an HI titre increase to influenza A(H3N2) virus in 1976 or 1977, six demonstrated a rise against the SwH1N1 virus as well [6]. Still, it cannot be excluded that the farmer's disease was aggravated by a concomitant infection with an influenza A(H1N1) or an H3N2 virus, both of which were circulating among the h u m a n population at the time. The pathogenic potential of SwH1N1 virus infections remains to be established.

ACKNOWLEDGEMENTS

The authors wish to thank patient 3, his family and the staff of the farm for their much appreciated cooperation in this study.

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J.C. D E JONG A N D COLL.

RI~SUME INFECTIONS HUMAINES PAR LE VIRUS DE LA GRIPPE PORCINE A EN SUISSE ET AUX PAYS-BAS

(H1N1)

Trois souches de virus de la grippe porcine <>A(H1N1) ont 6t6 isol6es chez l'homme (deux cas en Suisse, un cas aux Pays-Bas), au d6but de l'ann6e 1986. Ces souches sont, sur le plan antig6nique, 6troitement apparent6es entre elles et proches de la souche Influenza A / N e w Jersey/8/76(SwH1N1). Les sujets suisses n'ont pr6sent6 qu'une affection b6nigne alors que le malade hollandais a souffert d'une pneumonie grave. Dans deux de ces trois cas, il y avait eu un contact &roit avec des porcs atteints de grippe porcine. Aucun des trois sujets ne pr6sentait de d6ficience immunologique. On n ' a pas pu mettre en 6vidence de transmission interhumaine du virus. MOTS-CLI~S" Grippe, Porc, H6magglutination, Neuraminidase; Inhibition, Europe, Homme, Virus H1N1.

REFERENCES [I] AYMARD,M., DOUGLAS)A.R., FONTAINE)M., GOURREAU)J.M., KAISER) C., MILLION) J. & SKEHEL, J.J.)Antigenic characterization of influenza A (HINI) viruses recently isolated from pigs and turkeys in France. Bull. WHO, 1985, 63, 537-542. [2] AYMARD-HENRY, M., COLEMAN, M.T., DOWDLE, W.R., LAVER, W.G., SCHILD, G.C. & WEBSTER, R.G., Influenza virus neuraminidase and neuraminidase-inhibition test procedures. Bull. WHO, 1973, 48, 199-202. [3] Centers for Disease Control, Influenza Surveillance Report No. 92 (p. 36). US Department of Health and Human Services, Public Health Service, Atlanta, GA, 1981. [4] Centers for Disease Control, Influenza Surveillance Report No. 93 (p. 23). US Department of Health and Human Services, Public Health Service, Atlanta, GA, 1983. [5] CHUVAI
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[9] DOWDLE,W.R. & HATTWICK,M.A.W., Swine influenza virus infections in humans. J. infect. Dis., 1977, 136, $386-$389. [10] GALASSO,G.J., TYERYAR, F.J. Jr, CATE, T.R., COUCH, R.B., GLEZEN, W.P., GROSS, P.A., KASEL, J.A., WEBSTER, R.G. & WmOHT, P.F., Clinical studies of influenza vaccines - - 1976. Conference held at the National Institutes of Health, Bethesda, Maryland. Session I. Early events. J. infect. Dis., 1977, 136, $347-396. [12] HINSHAW, V.S., ALEXANDER, D.J., AYMARD, M., BACHMANN, P.A., EASTERDAY, B.C., HANNOUN, C., KIDA, H., LIPKIND, M., MACI~NZm, J.S., NEROME, K., SCHILD, G.C., SCHOLTISSEK,C., SENNE, D.A., SHORTmDOE, K.F., SKEHEL, J.J. & WEBSTER, R.G., Antigenic comparisons of swine-influenza-like H1N1 isolates from pigs, birds and humans: an international collaborative study. Bull. WHO, 1984, 62, 871-878. [13] MASUREL,N., DE BOER, G.F., ANKER, W.J.J. &; HUFFELS,A.D.N.H.J., Prevalence of influenza viruses A-H1N1 and A-H3N2 in swine in the Netherlands. Comp. Immunol. Microbiol. infect. Dis., 1983, 6, 141-149. [14] NAKAJIMA,K., NAKAJIMA,S., SHORTRIDOE,K.F. & KENDAL,A.P., Further genetic evidence for maintenance of early Hong Kong-like influenza A(H3N2) strains in swine until 1976. Virology, 1982, 116, 562-572. [15] PATR1ARCA, P.A., KENDAL, A.P., ZAKOWSlO, Ph.C., COX, N.J., TRAUTMAN, M.S., CHERRY, J.D., AUERBACH, D.M., MCCUSKER, J., BELLIVEAtJ, R.R. & KAPPUS, K.D., Lack of significant person-to-person spread of swine influenza-like ~virus following fatal infection in an immunocompromised child. Am. J. EpidemioL, 1984, 119, 152-158. [16] Russ, G., VARECKOVA,E. t~ STYK,B., Steric effects in the reaction of influenza virus neuraminidases with antibodies. Acta Virol., 1974, 18, 299-306. [17] SCHNtJRRENBEROER,P.R., WOODS, G.T. & MARTIN, R.J., Serologic evidence of human infection with swine virus. Am. Rev. respir. Dis., 1970, 102, 356-361. [18] SHOeE, R.E., Swine influenza 3. Filtration experiments and etiology. J. exp. Med., 1931, 54, 373-380. [19] SMITH,T.F., BO~GERT,E.O. Jr, DOWDLE,W.R., NOBLE, G.R.; CAMPBELL,R.J. & VAN Sco~, R.E., Isolation of swine influenza virus from autopsy lung tissue of man. N. EngL J. Med., 1976, 294, 708-710. [20] WEBSTER,R. & LAVER,W.G., Antigenic variation of influenza viruses, in <> (E.D. Kilbourne) (pp. 269-314). Academic Press, New York, 1975.