A chi es o Vi ology 53, 1--7 ( 977)
A chi es o Vi ology
© by Sp inge -Ve lag 977
Isola ion o an In luenza Vi us, Simila o A/Po Chalme s/l/73
(H3N2) om a Common Mu e a Sakhalin Island in U.S.S.R
(S ain A/eommonMu e/Sakhalin/l/74)
By
A. A. SAzo~-o 1, D. K. L o , 1%. G. W~ST~Ra, T. V. SO~OLOVA 2,
N'. A. BRAUDE 1, and N. V. PORTYANKO 1
1 I ano sky Ins i u e o Vi ology, Moscow, U.S.S.R.,
2 Sakhalin Sani a y--Epidemiologieal S a ion, U.S.S.R.,
3 S . Jude Child en's Resea ch Hospi al, Memphis, Tennessee, U.S.A.
Wi h 1 Figu e
Accep ed July 12, 1976
Summa y
An in luenza A i us isola ed om he cloaca o a common mu e was cha -
ac e ized an igenically as H3N2 and was mos closely ela ed o he A/Po
Chalme s/1/73 s Mn. Se ological s udies o se a collec ed om common mu es
in he a ea o i us isola ion showed ha 21 pe cen o he bi ds had an ibodies
o Hong Kong in luenza i us.
In oduc ion
The impo ance o in luenza A i uses ~n he animal and bi d popula ions o
he wo ld ha e no been ully elucida ed. The numbe o in luenza i uses in he
lowe o de s is no known, no is hei impo ance in he o igin o new pandemic
s ains o human in luenza i uses. The numbe o in luenza i uses isola ed om
bi ds has inc eased apidly du ing he pas ew yea s wi h an awa eness o he
impo ance in unde s anding he ecology o in luenza i uses (2, 4, 5, 8--18, 22).
I he p esen s udy, an in luenza A i us simila o Po , Chalme s/1/73 which
was isola ed om a common mu e in he Fa Eas o he U.S.S.R. is epo ed.
Ma e ials and Me hods
S udy A ea
Samples o se ological and i ological s udies we e collec ed om eo nmoJ1 min e
(U ia aalge)
nes ing on Tuleni Island in Augus , 1974. Tuleni Island is loca ed 20 km
om ~he eas e n coas line o Sakhalin Island in Te penia Bay, U.S.S.R. The island
(600 In long and 100 m wide) is a ocky pla eau; he beaches a e occupied by u seMs
(Callo hinchus u sinus L.)
and sea lions
(Eume hiasjuba u8 Sch)
om Ap il o No em-
be . The op o he pla eau is occupied om May--Augus by common mu es and by
A ch. Vi ol. 53/1--2 1
2 A.A. SAzo~o
e al. :
ki ewakes
(Rissa idac yla).
The common mu es and u seals mix, p o iding
bioceno ie connec ions be ween hese species.
Fo i ologic examina ions acheal and eloaeal swabs we e collec ed om 100 com-
mon mu es. Swabs we e aken in o plas ic ubes con aining issue cul u e media 199
and 1 pe cen bo ine se um albumin wi h 2000 uni s o penicillin and 500 ~g o
s ep omycin. Samples we e ozen in liquid ni ogen o anspo o he labo a o y.
Fo i us isola ion s udies 10-day-old chick emb yos we e inocula ed by he
amnio ie and allan oic ou e and incuba ed a 35 ° C o 48 hou s.
To minimize he possibili y o labo a o y con amina ion, he ini ial isola ion and
he ollowing 3 passages we e done in he i ology labo a o y a Sakhalinsk Sani a y-
:Epidemiologie S a ion, la e passages we e done a he Depa men o Ecology- o Vi uses
o he I ano sky Ins i u e o Vi ology. All samples we e passaged 3 imes be o e hey
we e conside ed nega i e.
Vi us isola es we e concen a ed and pa ially pu i ied by adso p ion and elu ion
om chick e y h oey es and inally sedimen ed h ough a suc ose g adien (10--60 pe
cen suc ose in phospha e-bu e ed saline, pH 7.2).
An ise a
Immune an ise a o he isola ed haemagglu inin and neu aminidase subuni s o
he e e ence s ains o in luenza A i uses we e made wi h ax~ igen emulsi ied in
F eund's comple e adju an (20).
The an igen mix u e was injec ed in o goa s ---in o he ail as well as in amuscula -
ly. The animals ecei ed a second dose o an igen in adju an plus an in a enous
injec ion o sMine 40 days la e . Blood samples we e collec ed 7 days a e he second
injec ion and se um s o ed a --20 ° C. An ise a o an igenic hyb id i uses we e p e-
pa ed in abbi s as p e iously desc ibed (20).
Se ological Tes s
Ha~magglu ina ion i a ions (HA) and haemagglu ina ion inhibi ion (HI) es s
we e pe o med in plas ic ays wi h ecep o -des oying enzyme (I~DE)- ea ed se a
as p e iously desc ibed (6). Neu aminidase i a ions (NA) we e done by he me hod
o WA~RE~- (19) and A~*~INOFF (3). Neu aminidase inhibi ion (NI) es s we e pe o med
as p e iously desc ibed (21) using p eincuba ion o i us-an ibody and subs a e
( e uin) a 37 ° C o 30 minu es p io o assay o ee sialic acid. In ac in luenza i uses
we e used in all es s.
Immunodi usion es s we e pe o med in 1.5 pe cen aga ose (A37) dissol ed :in
phospha e-bu e ed saline (PBS) (pH 7.2) con aining 0.1 pe cen sa kosyl NL 97 and
0.1 pe cen sodium azide (20). Pu i ied i us (HA 6.0 log10 uni s/m ) was dis up ed
wi h 0.1 pe cen sa kosyl NL 97 and he s one concen a ion was added o he an ise a
be o e addi ion o he pla es o p e en non-speci ic p ecipi a ion bands. Single adial
immunodi usion pla es (SRID) (1) con aining dis up ed A/chicken/Ge many/N/49
(I-Ia 2 Neq 1) in luenza i us we e used o de ec an ibodies o in luenza A ibonueleo-
p o ein; o he pla es con aining in~ae A/Hong Kong/68 (X-31) we e used o de ec
an ibodies o he su ace an igens o A/ Iong Kong/68 in luenza i us. 5 ~zl o un ea ed
se a we e added o SRID pla es. Posi i e and nega i e con ol se a we e added o each
pla e.
Resul s
Vi us Isola ion
One haemagglu ina ing agen was isola ed om he eloacal sample o a
common mu e (Table 1). The agen was i s de ec ed in he second passage in he
allan oie ca i y and subsequen ly g ew o a high i e , bo h in he allan oie and
amnio ie ca i ies. Vi uses we e no isola ed om he acheal swabs.
Ini ial s udies in HI es s using speci ic an ise a o he isola ed haemagg u inin
o all o he e e ence s ains o in luenza A i uses showed ha he agen isola ed
Isola ion o an In luenza Vi us (I- 3N2) om a Common Mu e in U.S.S.I~. 3
om he common mu e was ei he o he H3 (Hong Kong) o Ha 7 (Duck/
Uk aine/63) sub ypes. In o de o u he cha ac e ize his i us, i was s udied
in HI es s wi h an ise a o he a ian s o Hong Kong in luenza i us.
Table 1. Isola ion el a hemagglu ina ing agen / ein he cloaca o/ a common mu e in
chick emb yos
Passage numbe s
Rou e o
inocula ion Ini ial
I
II III
IV V VI
Amnio ie 0 0 0 @ ~ - - ~"
1:16
b 1:128 1:512
Allan oic 0 0 -}- ~ -}- NT~ NT
:32
a Shows de ec able hemagglu ina ion
b e:magglu inin i e
o No es ed
Cha ac e iza ion o/ he Haemagglu inin An igen
In HI es s he A/common mu e/74 in luenza i us is mos closely ela ed
o A/Po Chalme s/1/73 in luenza i us (Table 2) al hough i also eac s wi h
an ise a o Hong Kong/68 and England/42/72 o lowe i e s. In gel di usion es s,
he A/common mu e/74 in luenza i us shows a line o pa ial iden i y wi h Hong
Kong/68 (Fig. i a) and a line o iden i y wi h Po Chalme s/73 (Fig. I b). The
mul iple lines o p ecipi a ion wi h speci ic an ise a o he haemagglu inin o
in luenza i uses has been epo ed p e iously (7) and may e lec he mul iple
an igenic de e minan s on he haemagglu inin subuni s.
Table 2. Cha ac e iza ion o~ he haemagglu inin on he A/common mu e/1/74 in luenza
i us
HI i e s o he ollowing in luenza i uses
An ise a o Hong Kong/68 England/72 Po Chalme s/73 Common mu e/74
Aiehi/2/68
1800 ~
600 200 280
England/42/72
1200 3000 1600 1800
Po Chalme s/1/73 400 2200 3000 4000
Figu es gi e he ecip ocal o he dilu ion inhibi ing h ee ou o ou haemagglu ina -
ing doses o i us. AJ1 ise a we e p epa ed in chickens o he in ac in luenza i uses
and we e bled 14 days la e
Cha ac e iza ion o/ he iVeu aminidase An igens
Neu aminidase inhibi ion es s wi h speci ic an ise a o all o he p o o ype
in luenza A i us neu aminidases showed ha he only an ise a ha neu Mized
he enzyme o he i us isola ed om a common mu e was he N2 )qoe ( esul s
no shown). The neu aminidase an igen was u he cha ac e ized using an ise a
o di e en a ian s o Iong Kong in luenza i us (Table 3). The neu aminidase
on he A/Common mu e/Sakhalin/1/7¢ in luenza i us appea s o be mos closely
ela ed o Po Chalme s/1/73 in luenza i us.
1"
4 A.A. SAzoNo
e al. :
Isola ion o an In luenza Vi us (H3N2) om a Common Mu e in U.S.S.R. 5
Se ological S udies on Se a / om Common Mu es
Se a collec ed om 142 common mu es on Tuleni ]island in Augus 1974 we e
examined o an ibodies o in luenza i uses in I I es s. 21 pe cen o he se a
con ained an ibodies o A/Hong Kong/1/68 in luenza i us. The majo i y o he
se a (92) we e also examined in SRID es s o an ibodies o Hong Kong/68
i luenza i us and o an ibodies o he ibonucleop o ein o in luenza A i uses;
21 pe cen o he se a eac ed o A/Hong Ken /68 and 23 pe cen eac ed wi h
he RNP o in luenza A i uses,
Table 3.
Iden i]ica ion o] he neu aminidase an igen on an in luenza A i us isola ed
/ ein a common mu~ e in U.S.S.R.
An ise a
A/Japan/57 I-Iong Ken /68 England/42/72 Po Chalme s/i/73
[Equinel (n)- [Equine 1 (~)- [Equine 1 (R)-
(isola ed Hong Kong/68 England/72 Po Chalme s/73
Vi uses N 2) (N)] (~)] (N)]
A/Japa~n/305/57
200
80 < 10 < 10
A/Hong Kong/1/68 30
200
< 10 < 10
A/England/42/72 30 30
600 O0
A/Po Chalme s/73 < 10 < 10 50
100
A/common mu e/74 20 20 50 100
An ise a o A/Japan/57 we e p epa ed wi h isola ed neu aminidase
Aaa ise a o Hong Kong/1/68, England/42/72 and Po Chalme s/1/73 neu aminidase
we e p epa ed agains an igenic hyb ids possessing equine/P ague/i/56 hemagglu inin
and he app op ia e neu aminidase. Values ep esen he ecip ocal o he dilu ion
causing 50 pe cen inhibi ion o in~s neu aminidase gi ing an app oxima e O.D.
alue o 0.50
Discussion
An in luenza A i us isola ed om he cloaca o a common mu e caugh on
Tuleni Island o ile eas coas o U.S.S.R. was shown o possess H 3N2 an igens.
The haemagglu ini ~ and neu aminidase subuni s we e mos closely ela ed o
he A/Po Chalme s/1/73 a ian o Hong Kong in luenza i us. Se ological
s udies o se a om common mu es collec ed a Tuleni Island showed ha 21 pe
cen o he bi ds had an ibodies o Hong Kong in luenza i us.
Hong Kong in luenza i us and i s a ian s a e ubiqui ous h oughou , he
wo ld in hei abili y o in ec many domes ic and e al species. These i uses
ha e been isola ed om many species, including pigs, eM es, chickens, dogs,
ca s, monkeys, gibbons, baboons, and e al sea bi ds [ o e iew see EASTERDAY
Fig. l a (le ). Double Immunodi usion. Cen e : An ise um o he isola ed hem-
agglu inin o A/I-long Ken /I/68 in luenza i us
Fig. i b ( igh ). Cen e : An ise um o he isola ed hemagglu inin o A/Po Chalme s/
1/73. Ou e :
Chick/Ge m/N A/ehick/Ge many/"N'/49,
Common mu e A/common
mu e/Sakhalin/i/74, !4ong Kong A/Hong Kong/I/68, Wag ail A/wag ail/Uk aine/
107/74, Ilong I~2ong A/Hong Kong/I/68, Pigeon A/pigeon/Uk aine/lll/74, Po
ChMme s A/Po Chalme s/I/73, Blank Saline
Gel di usion es s we e done as desc ibed in Ma e ials and Me hods
6 A.A. SAzo~-o
e al. :
(5)]. The isola ion o a human in luenza i us (A/Po Chalme s/73) om common
mu es aises he ques ion o whe he common mu es con ac his i us om
man o whe he i uses o he A/Po Chalme s/73 ype ha e been in bi d popula-
ions o a long pe iod o ime. The la e possibili y seems unlikely, so we mus
assume ha common mu es con ac hese i uses ei he di ec ly o indi ec ly
om man. I is no known whe he he A/common mu e/74 in luenza i us will
pe sis in he common mu e popula ion; se ological s udies sugges ha 21 pe
cen o he popula ion had expe ienced his i us be o e Augus 1974. S udies
on Hong Kong in luenza i uses in chickens (8, 22) ha e shown ha his i us
is capable o ci cula ing in an a ian popula ion wi h pe iodic epizoo ics. O he
s udies on gong Kong in luenza i uses in pigs (Sc~ILD, G. C., pe sonal communi-
ca ion) sugges ha his i us has no es ablished i sel in he po cine popula ion
bu is ein oduced a in e als om he human popula ion.
The impo ance o pelagic bi ds o he a ious domes ic and e al animals om
which gong Kong in luenza i uses ha e been isola ed and hei po en ial as
ese oi s o gong Kong in luenza i uses emains o be es ablished. To da e,
ansmission s udies wi hin o be ween e al species, ha e no been done. The
possibili y emains, howe e , ha . a wide ange o animals and bi ds could se e
as po en ial ese oi s o gong Kong in luenza i uses.
Aeknowledgmen s
This s udy was made possible by he So ie -Ameiican P og am o Scien i ic
Collabo a ion on In luenza, P oblem A ea 3. "Ecology o in luenza i uses", and was
suppo ed in pa by con ac A152524 om Na ional Ins i u e o Alle gy and In ec-
ious Diseases.
Re~e enees
I. Ad anced Labo a o y Techniques o In luenza Diagnosis, U.S. Dep . o Heal h,
Educa ion and V~%] a e, Public Heal h Se ice, A lan a, Ga. (1975).
2. ALEXANDER, D. J., ALLAN, W. H., HAI~KNESS, J. W., HALL, S. A.: Isola ion o
in luenza i us om psi acines. ]~es. e . Sci. 17, 125--127 (1974).
3. AMINOFF, D. : Me hods o he quan i a i e es ima ion o N-ace yl-neu aminie acid
and hei applica ion o hyd olysa es o sialamucolids. Biochem. J. 81, 384~392
(1961).
4. BUTTE!aFIELD, W. K., VEDLOUTSeHNIG, I~. I., D~DIRI, A. H. : Isola ion and iden i i-
ca ion o mixo i uses om domes ic and impo ed a ian species. A ian Dis. 17,
155--159 (1973).
5. EASTEI%DAY, B. C. : Anima~ In luenza. In: KILBOURNE, E.D. (ed.), The In luenza
Vi uses and In luenza, p. 449--481. New Yo k: Academic P ess i975.
6. FAZEI4AS DE ST. G OT , S., WEBSTEE, ~. G. : Disquisi ions on o iginal an igenic
sin. I. E idence in man. J. exp. Med. 124, 331--345 (1966).
7. LAVER, W. G., DOW~IE, J. C., WEBSTE:a, R. G. : S udies on an igenic a ia ion in
in luenza i us. E idence o mul iple an igenic de e minan s on he hemagglu inin
subuni s o A/Hong Kong/68 (H 3N 2) i us and /be A/England/72 s ains. Vi ology
59,
230--244 (1974).
8. L o , D. IX2., SAZAI~OV, A. A., C~ER~ETSOV, IL . V. : Complex in es iga ion o
in luenza i us ecology a he Commodo e Islands. Vop . Vi usol. 18, 747--750
(1973).
9. L o , D. K., AIqDREEV, V. P., ISACHENKO, V. A., YAKE~O, M. A., ZAKSTELSKAYA,
L. YA., SOKOLOVA, ~. ~., ~ ASILIEV, V. N. : De ec ion o in luenza A i us ( e n)
Isola ion o an In luenza Vi us (II3N2) om a Common Mu e in U.S.S.R. 7
Tu kmenis an/18/73 (Hay 7 Nay 2) om S e na hi undo L. in hc eas e n pa o ile
Caspian sea ( he Tu kmen S.S.I%.). Ecology o Vi uses II, 88 -93 (1974).
10. MoFEI~RAN, J. B., CONDOR, T. J., COLloids, D. S., ALLA~, G. M.: Isola ion o an
a i ulen in luenza i us om a pa o . Ve . Ree. 95, 466-467 (1974).
11. ROSENBE~CE~, J. K., K~AUSS, ~¢V. C., SLE~O~S, R. D.: Isola ion o Newcas le
disease and ype A in luenza i uses om mig a o y wa e owl in A lan ic lyway.
A ian Dis. 18, 610--613 ( 974);
12. 1K0SLAYA, I. G., ROSLYAKOV, G. E., L o , D. Is2., ISACHE~KO, V. A., ZAKSTEL-
SKAYA, L. YA., T~oP, I. E. : Ci cula ion o a bo- mad myxo i uses in popula ions
o wa e owl and sho e bi ds o he lowe Amu . Ecology o Vi uses II, 148--156
(1974).
13. SAZANOV, A. A., L o , D. I ., ZAKSTELSKAYA, L. YA. : Se ological examina ion o
sea bi ds in he No h o he Fa Eas o he U.S.S.R. Ecology o Vi uses I, 41--46
(1973).
14. SAZANOV, A. A.,
L o ,
D. K., CHUM~KOV, V. M., SOKOLOVA, N. N.: Se ological
examina ion o popula ion and bi ds a , Commodo e Islands. Ecology o Vi uses II,
165- 169 (1974).
15. SLEMONS, R. D., COOPER, R. S., OSBO~', J. S. : Isola ion o ype A in luenza i uses
om impo ed exo ic bi ds. A ian Dis. 17, 746--751 (1973).
16. SLEMO~S, R. D., Jo~INSOS , D. C., MALONE, T. G. : In luenza ype A isola ed om
impo ed exo ic bi ds. A ian Dis. 17, 485--489 ( 973).
17. SLE3[ONN, ~. D., JORNSO~% D. C., OsBom% J. S., IIAVES, F. : Type A in luenza
i uses isola ed om wild ee- lying ducks in Cali o nia. A ian Dis. 18, 119--124
(1974).
18. TUlV OVA, B., EISENGARTEN, JcI. J., SIEB][~LIST-KEIgSTA_NTINOV, I., STU1VIpA, A.,
"WEBSTEI~, R. G. : A duck in luenza i us wi h haemagglu inin ela ed o ha o A/
Singapo e/57 (H2N2) i us. Ae a i ol. 19, 261 (1975).
9. W'A ~I~E?¢, L. : The hioba bi u ic acid assay o sialie acids. J. biol. Chem. 234,
1971--1975 (1959).
20. WEBSTEI% R. G., LAVEI~, W. G. : S udies on he o igin o pandemic in luenza.
I. An igenic analysis o Ae in luenza i uses isola ed be o e und a e he appea ance
o Hong Kong in luenza using an ise a o he isola ed hemagglu lnln subuni s.
Vi ology 48, 433--444 (1972).
21. WHO Repo : In luenza neu aminidase and neu aminidase-inhibi ion es p oce-
du es. Bull. Wld. Hl h. O g. 48, 199--202 (1973).
22. Z lEZ51ER, V. YU., ZAGIBAH~O, N. M. : Resul s o expe imen al in ec ion o chickens
wi h in luenza i uses o g oup A ( 3N2). Ecology o Vi uses II, 18--23 (1974).
Au ho s' add ess: D . A. A. SAzoz~-o , I ano sky Ins i u e o Vi ology, Moscow,
U.S.S.I~. Recei ed Ap il 27, 1976
