perpetuation of influenza a viruses in alaskan waterfowl reservoirs.pdf

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Arch Virol(1995) 140:1163-1172

_ A r c h i v e s

ro logy

© Spr inger Ver lag 1995

P r i n t e d i n

ust r ia

P e r p e t u a t i o n o f i n f l u e n z a A v i ru s e s in A l a s k a n w a t e r f o w l r e se r vo i rs

T. Ito 1 K. OkazakP Y. Kawaokaz A . Takada 1 R.G . W ebster2 and H. Kidax

~Department of V eterinaryHygieneand Microbiology,Schoolof V eterinary Medicine,

Hok kaido University,Sapporo, Japan

2Department of Virologyand Molecular Biology,St. Jude Children sResearch H ospital,

Mem phis, Tennessee, U.S.A.

Accepted Ma rch 31, 1995

S u m m a r y . T o p r o v i d e i n f o r m a t i o n o n t h e m e c h a n i s m o f p e r p e t u a t i o n o f i n fl u e n z a

viruses amo ng w ater fow l reservoir s in na ture , v i ro logica l surve i l lance was car r ied

ou t in A la s ka d u r ing the i r b r e e d ing s e a s on in s um m e r f r om 1991 to 1994. I n f lue nz a

v i r u se s we r e i s o la te d m a in ly f r om f e c al s a m ple s o f da bb l ing duc ks in the i r ne s t ing

p la c e s in c e n t r a l Ala s ka . The num be r s o f s ub type s o f 108 in f lue nz a v i ru s i s o la te s

w e r e 1 H 2 N 3 , 3 7 H 3 N 8 , 5 5 H 4 N 6 , 1 H 7 N 3 , 1 H S N 2 , 1 H 1 0 N 2 , 11 H 1 0 N 7 , a n d

H 10 N9. Inf luenza v iruses were a lso iso la ted f rom w ater sam ples of the lakes where

the y ne s t. Eve n in Se p te m be r o f 1994 wh e n the m os t duc ks ha d l e f t f o r m ig r a t io n

to s ou th , v i r u se s we r e s t il l i s o la te d f r om the l a ke w a te r. Phy loge n e t i c a na lys i s o f

the NP ge ne s o f the r e p r e s e n ta t ive i s o la te s s howe d tha t the y be long to the

N or t h Am e r ic a n l ine a ge o f a v ia n in f lue nz a v i ru s e s, s ugge s ting tha t the m a jo r i ty o f

t h e w a t e rf o w l s b r e ed i n g i n ce n t ra l A l a s k a m i g r a te t o N o r t h A m e r i c a a n d n o t t o

As ia . The p r e s e n t r e s u l t s s uppor t the no t ion tha t in f lue nz a v i r u s e s ha ve be e n

m a i n t a i n e d i n w a t er f o w l p o p u l a t i o n b y w a t e r - b o rn e t r a n sm i s s i o n a n d r e ve a le d t h e

me chan ism of year -by-y ear perpe tu a t ion of the v iruses in the lakes where they breed .

In tr od u c t i on

Since 1956 num erous inf luenza A viruses have been isolated from a var iety of

avian species [ 1]. All o f the different subtypes o f inf luenza A viruses (H 1 to H 14 an d N1

to N9) are circulating in aquatic birds , par ticular ly in migratory waterfowls [11] .

W aterfow ls are, therefore, ra ted as a majo r reservoir o f influenza A viruses in natu re [34].

I n f lue nz a A v i r u s e s o f a v ia n o r ig in ha ve be e n im p l ic a te d in ou tb r e a ks o f

inf lu enz a in ma m m als , such as seals [12, 32, 33] , wh ales [14] , pigs [25] , m ink s [20],

and horses [9] as wel l as in dom es t ic po ul t r ies [3 , 10]. These ev idences indica te tha t a

va s t in f lue nz a v i r u s ge ne poo l f o r f u tu r e m a m m a l ia n in f lue nz a e x i s t s in a v ia n

s ou rc e s . S inc e e a c h pa nde m ic o f hum a n in f lue nz a f i rs t a ppe a r e d in C h ina [ 34 ], the

pos s ib i l i ty ha s be e n r a i s e d tha t s ou the r n Ch ina i s a n in f lue nz a e p ic e n te r [27] . Ne w

hum a n pa nde m ic s t r a in s a r e be l i e ve d to a r i s e by ge ne t i c r e a s s o r tm e n t be twe e n



1 1 6 4 T I t o e t a l

h u m a n a n d n o n - h u m a n v i r u se s [6, 2 1 ,3 1 ]. I t h as b e e n s h o w n t h a t H 2 N 2 A s i a n a n d

H 3 N 2 H o n g K o n g p a n d e m i c in f l u e n za v i ru s e s a r e g en e t ic r e a s s o r ta n t s b e t w e e n

s t r a in s o f h u m a n a n d a v i a n o r i g i n [1 6, 24 , 30 ]. A n t i g e n i c a n d g e n e t i c a n a l y s e s o f H 3

i n f l u e n z a v i r u s e s i s o l a t e d f r o m w i l d d u c k s c a p t u r e d o n t h e A s i a n P a c i f i c f l y w a y i n

J a p a n a n d d o m e s t i c d u c k s i n s o u t h e r n C h i n a h a v e s h o w n t h a t t h e h e m a g g l u t i n i n s

( H A s ) o f t h e s e v ir u s e s w e r e c l o s e l y r e l a t e d t o t h o s e o f t h e e a r l ie s t h u m a n H 3 v i r u se s

[1 8, 3 5]. T h e s e fi n d i ng s i n d i c a t e d t h a t t h e H A g e n e o f t h e h u m a n p a n d e m i c s t r ai n , A /

H o n g K o n g / 6 8 ( H 3 N 2 ) w a s d e r i v e d f r o m s u c h a n a v i a n v i r u s [ 1 9 ] . T h e r e f o r e ,

e p i d e m i o l o g i c a l su r v e il l an c e o f a v i a n i n f lu e n z a w o u l d p r o v i d e i n f o r m a t i o n o n t h e

n e x t e p i d e m i c s fo r o t h e r a n i m a l s p ec ie s, i n c l u d in g h u m a n p a n d e m i c s .

W a t e r f o w l s s u c h a s d u c k s a n d g e es e i n th e n o r t h e r n h e m i s p h e r e m i g r a t e t o

s o u t h i n a u t u m n , b a c k t o n o r t h i n s pr in g , a n d b r e e d i n s u m m e r i n th e n e s ti n g p la c es

i n n o r t h e r n t e r r i t o r i e s s u c h as A l a s k a , C a n a d a , o r S i b e r ia . E x t e n s i v e s u rv e i l l a n c e o f

i n f lu e n z a in m i g r a t o r y w a t e r f o w l s w a s c a r r i e d o u t i n m a r s h a l i n g a r e a s , w h e r e t h e y

w e r e g a t h e r i n g in p r e p a r a t i o n o f m i g r a t i o n t o s o u t h , i n C a n a d a f r o m 19 78 t o 1 98 3

[1 3] . H o w e v e r , l i t tl e is k n o w n a b o u t h o w i n f l u e n z a v i ru s e s a r e m a i n t a i n e d i n d u c k

p o p u l a t i o n i n t h e i r b r e e d i n g p la c e s .

I t w a s s u g g e s t e d t h a t s o m e d u c k s ( e .g . N o r t h e r n p i n ta i l) s h o u l d m i g r a t e f r o m

A l a s k a t h r o u g h A l e u t i a n I s la n d s t o A s i a, a l t h o u g h t h e m a j o r i t y o f m i g r a t o r y d u c k s

i n A s i a a r e f r o m S i b e r i a [ 36 ]. D i s t i n c t l i n e a g e s o f t h e g e n e s o f i n f l u e n z a v i r u s e s

i s o l a t e d f r o m a v i a n s p e c ie s i n d i f fe r e n t r e g i o n s o f t h e w o r l d ( e .g . N o r t h A m e r i c a a n d

Eu r a s i a ) w e r e d e m o n s t r a t e d [ 4 , 5 , 7 , 8 , 15 , 2 2] . P h y l o g e n e t i c a n a l y s i s o f t h e g e n e s o f

i n f l u e n z a v ir u s i s o la t e s, t h e r e f o r e , w o u l d m a k e i t p o s s i b l e to p r e s u m e t h e f l y w a y s o f

t h e h o s t b i r d s .

T o p r o v i d e i n f o r m a t i o n h o w i n f l u e n z a v i r u s e s a r e m a i n t a i n e d i n d u c k

p o p u l a t i o n i n th e i r b r e e d i n g a r e a s a n d t h e m e c h a n i s m o f p e r p e t u a t i o n o f t h e v ir u s es ,

v i r o l o g ic a l s ur v e i ll a n c e o f a v i a n i n f l u e n z a w a s m a d e i n A l a s k a f r o m 1 99 1 t o 1 99 4 a n d

p h y l o g e n e t i c a n a l y s e s o f th e g e n e s o f th e i s o l a t e s w e r e c a r r i e d o u t .

M a t e r i a l s a n d m e t h o d s

Sample collection

In ea ch July fro m 1991 to 1994, 3 120 fecal materials of birds, comprising 2 345 ducks, m ainly

mallards An as platyrhynchos) an d pintails Anas ac uta), 663 Can ada geese B ranta canadensis) 6

Tun dra swans

Cygnus columb ianus),

7 dunlins

Calidris alpina),

and 10 M ew gulls

Larus can us),

were collected at 15 different points in A laska o f the Un ited States (Fig. 1 and Table 1). In

1992-1994, 81 water sam ples were also collected from 11 different ponds and lakes were ducks an d

geese were nesting. In Septemb er 1994, after almost du cks migrated to south, 21 water samples of

Big Minto Lak e where the viruses were isolated from ducks in the sum mer w ere collected. Samples

were put into cryotubes, the lids sealed, and transp orted w ith dry ice to the D epartm ent of

Virology and Molecular Biology,St. Jude Children s R esearch Hospital, Mem phis, Tennessee and

then stored at- 80 °C until assayed.

Virus isolation

Ea ch fecal sample was pu t in to phosphate-buffered saline (PBS; pH 7.2) containing antibiotics

(2 000 units of penicillin,400 ~ g o f streptomycin, 40 ~g o f gentamycin and 200 units ofpolymyxin B



D u c k i n f l u e n z a v i r u s e s i n A l a s k a 1 1 6 5

~ CORVILLE DELTA

~ PRUDHOE BAY

MALLARD LAKE HEART LAKE

CANVASBACK

BIG M~NTO LAKE

~ '% LAKE

'o o

FAIRBANKS

NENANA o

o DELTA ]CT

DENALI NAT'L PARK o

ANCHORAGE

J

I

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. i

I

LAKE HOOD/SPENARD

LAKE CHENEY

POTTERMARSH

WESTCHESTER LAGOON

F i g . 1 . L o c a t i o n o f 1 5 d i f f er e n t p o i n t s w h e r e f e c a l m a t e r i a l s o f w a t e r f o w l s w e r e c o l l e c t e d i n

A las ka . - - - - A r c t i c c i rc l e

p e r m l ) t o g i v e 1 0 - 2 0 s u s p e n s i o n . T h e s u s p e n s i o n w a s c e n t ri f u g e d a t 2 5 0 0 r p m f o r 2 0 m i n u t e s .

O n e t e n t h m l o f t h e s u p e r n a t a n t w a s i n o c u l a t e d i n t o a l l a n t o i c c a v i t ie s o f t w o 9 - 1 1 - d a y - o l d f e r ti l e

h e n ' s e g g s . T h e e g g s w e r e i n c u b a te d a t 3 5 ° C f o r 2 t o 3 d a y s u n l e s s d e a t h o f t h e e m b r y o w a s

d e t e c t e d . A t t h e e n d o f t h e i n c u b a t i o n p e r i o d o r u p o n e m b r y o d e a t h , t h e a l l a n t o i c f lu i d s w e r e

te s te d for h e ma gg lut ina t ing ac tiv ity .

W a t e r s a m p l e s w e r e c o n c e n t r a t e d u s i n g f o r m a l i n - tr e a t e d c h i c k e n e r y t h r o cy t e s a s f o ll o w s : t w o

m l o f 5 f o r m a l i n -t r e a te d c h i c k e n er y t h r o cy t e s in 3 0 x P B S w a s a d d e d t o e a c h 5 0 m l o f w a t e r

s a m p l e a n d m i x e d . A f t e r i n c u b a t i o n f o r 1 h a t 4 ° C , s a m p l e s w e r e ce n t r if u g e d a t 1 5 0 0 rp m f o r 5

m i n u t e s . A f t e r r e m o v i n g t h e s u p e r n a ta n t , 0 . 2 m l o f P B S c o n t a i n i n g a n t i b i o t i cs w e r e a d d e d a n d t h e

e r y t h ro c s ~ e s w e r e r es u s p e n d e d . O n e t e n t h m l o f t h e r e s u s p e n d e d e r y t h r o cy t e s w a s i n je c t ed i n t o

a l l a n t o i c c a v i ty o f 2 e g g s p e r s a m p l e . E a c h u n c o n c e n t r a t e d w a t e r s a m p l e w a s a l s o i n o c u l a t e d .

den t i f i ca t i on o f so l a t es

A l l h e m a g g I u t i n a t i n g a g e n t s w e r e id e n t i f ie d i n h 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 a n d n e u r a m i n i d a s e -

i n h i b i t i o n t e s t s u s i n g s p e c i f ic a n t i s e r a t o t h e r e f e r e n c e s t r a in s o f i n f l u e n z a v i r u s e s [ 2 , 2 6 ] . T h e

a n t i s e r a w e r e u s e d a s p r e v i o u s l y p r e p a r e d a n d d e s c r i b e d [ 17 ].

Ph y t o g e n e t i c a n a l ysi s o f h e N P ge n es

P h y l o g e n e t i c a n a l y s is w a s b a s e d o n p a r t ia l n u c l e o t id e s e q u e n c e s o f th e N P g e n e s ( p o s i t i o n s

1 2 9 1 - 1 4 3 0 ) o f i n f l u e n z a v ir u s e s , d e t e r m i n e d b y r e v er s e t r a n s c r i p t io n a n d P C R d i r e c t s e q u e n c i n g

u s i n g v i ra l R N A a s a t e m p l a t e a n d o l i g o n u c t e o t i d e p r i m e r s . S e q u e n c e d a t a o f e a c h g e n e , t o g e th e r

w i t h s e q u e n c e s f r o m G e n B a n k , w e r e a n a l y ze d b y N e i g h b o r - j o in i n g m e t h o d [ 23 ] u s i n g a c o m p u t e r

s o f t w a r e , O D E N v e r s i o n 1 . 1 .1 ( Y a s u o I n a , N a t i o n a l I n s t i t u te o f G e n e t i c s, M i s h i m a , J a p an ).



1 1 6 6 T I t o e t a l

R e s u l t s

Isolation o f influenza A viruses r om fec al samp les o f waterfowls

To assess if waterfowls harbor influenza A viruses at their breeding areas, we

collected 3 120 fresh fecal samples of birds in different areas in Alaska, USA during

their late breeding season from 1991 to 1994. From these samples, 205 hemag-

glutinating agents were isolated. Of these, 108 were serologically identified as

influenza viruses and the remainings as paramyxoviruses. Most of the influenza

virus isolates were obtained from fecal samples of ducks, while only 4 were isolated

from those of geese Table 1). These viruses were isolated mainly from the samples

collected in the central Alaska i.e., pond in Nenana, Big Minto Lake, and Mallard

Lake on the Yukon fiat), while the frequencies of influenza virus isolation were very

low in north and south Alaska. These results indicate that influenza viruses have

been maintained in duck population in the major breeding areas of central Alaska.

Antigen ic classi fication o f the influenza A virus isolates

As shown in Table 2, subtypes of the influenza virus isolates from ducks were H3N8,

H8N2, H10N2, H10N7, and H10N9 in 1991, H4N6 and H10N7 in 1992, H3NS,

H4N6, and H7N3 in 1993, and H2N3, H3NS, and H4N6 in 1994. In each of Big

Minto Lake and Heart Lake, influenza viruses of different subtypes were isolated at

the same time see footnote of Table 1). Additionally, different subtypes ofinfiuenza

viruses were isolated in different years in the same lake Big Minto Lake and

Mallard Lake). These results indicate that different subtypes of influenza A viruses

co-circulate in duck populat ion in their breeding areas.

Isolation of influenza A viruses rom take water

To investigate the mechanism of perpetuation of the viruses from year to year, virus

isolation was carried out from water samples of the lakes where ducks were nesting.

In 1992, two viruses were isolated from the samples from different lakes in Alaska,

where the same subtypes of influenza virus were isolated from ducks Table 3). In

the summer of 1994, 7 viruses were isolated from lake water and out of these, 2

samples contained 1018 and 10 2 8EIDs0 per ml of water samples. In the autumn of

1994, after most ducks left for migration to south, virus isolation was carried out

from the water samples of the Big Minto Lake where the viruses were isolated in the

summer. Three viruses were isolated from the water samples. These results suppor t

the notion that influenza viruses have been maintained in waterfowls by water-borne

transmission.

Phylogenetic a nalyses o f influenza virus isolates

To investigate the flyways of waterfowls carrying influenza viruses, phylogenetic

analyses of the NP genes of 18 representative strains which were isolated in different

years in the different lakes in Alaska was performed. Figure 2 shows an evolutionary

tree constructed on the basis of partial sequences of the NP genes together with



Duck influenza viruses in Alaska

Table 1. Isolation of influenzaviruses from fecal samples ofwaterfowls

in Alaska in 1991-1994

1167

Location Species No. of samples with virus/

total no. of samples tested

t991 1992 1993

1994

Lake Hood/Spenard Duck 0/302 2d/317 0/26 0/15

Goose 4~/197 0/39 0/2

Gull 0/3

Lake Cheney Duck 0/293 0/133 0134

Goose 0/185 0/8 0/3

Potter Marsh Duck 0/1 0/18

Goose 0/130

Gull 0/4

Westchester Lagoon Duck 0/134

Goose 0/2

Lake Hanger Duck 0144

Fairbanks Duck 01156 0/116 0/54

Dunlin 0/7

Gull 0/3

Nenana Duck 14b/65 0/41 2h/2

Delta Junction Duck 0/27

Denali Nat'l Park Duck 0/6

BigMinto lake Duck 9~/149 14f1205 4i128

Goose 0/9

Mallard Lake Duck 46e/103 7g/53 0/24

Goose 013

Heart Lake Duck 0/29 6J/39

Canvasback Lake Duck 0/6

Corville Delta Duck 0/14

Goose 0/16

Swan 0/6

Prudhoe Bay Goose 0/69

Total 27/1585 481994 21/376 12/165

No. of each antigenic subtype of he isolates is as follows: 3 H10N7 and 1 H8N2, u 14 H3N8,

7 H 10 N7, 1 H 10N2, and 1 H 10Ng, a 1 H4 N6 and 1 H10N7, e46 H4N6, f7 H3N8, 6 H4N6, and

~ j

1 H7N3,g7H3N8, 2H3N8,4H3N8,and3H3N8,2H4N6, 1 H2N3

those from GenBank. Alth ough the sequence data o f the recent isolates in old world

was not available, the present approach is valid because distinct lineages between the

genes of isolates from avian species in North America and Eurasia have been

demon st ra ted previous ly in several reports [4, 5, 7, 8, 15, 22]. The results show ed

that the NP genes of the viruses isolated from ducks in Alaska belong to the Nor th

Ame ric an lineage of avian influenza viruses. These results suggest that waterfowls

carrying these viruses migrate thro ugh the Conti nent of Nort h America to south

and not through Aleutian Islands to Asia.



1168 T. Ito et al.

Table 2. Antigenic characterization ofinfluenzaA virus isolates from fecal samples

of waterfowls in Alaska from 1991 to 1994

Antigenic

subtype

Number of influenza A viruses

isolated in the following year

1~1 1~2 1~3 1~4

o t a l

H2N3 1 1

H3N8 14 14 9 37

H4N6 47 6 2 55

HTN3 1 1

H8N2 1 1

H10N2 1 1

H10N7 10 1 11

H10N9 1 1

Total 27 48 21 12 108

Table 3 Isolation ofinfluenzaviruses from water samples of lakes in Alaska

in 1992-1994

Location

No. of samples with virus/

total no. of samples tested

1992 1993 1994 1994

summer summer summer antumn

Lake Hood/Spenard 1~/4 0/2

Lake Cheney 0/3 0/3

Potter Marsh 0/4

Westchester Lagoon 0/1

Lake Hanger 0/4

Fairbanks 0/5

Big Minto Lake 0/10

Mallard Lake 1b/2 0/5

Heart Lake 0/5

Canvasback Lake 0/3

Corville Delta 0/t

7o/13 3d/21

0/17

Total 2/23 0/28 7/30 3/21

No. of each antigenic subtype of the isolates is as follows: ~1 H4N6 b 1 H4N6 c4 H3N8 and 3

H4N6 2 H3N8 and 1 H7N3

D i s c u s s i o n

Hins haw et al . [13] demon stra ted high incidence of infection of ducks in the

marsh aling areas for their migra tory flyway in Cana da at the end of breeding season.

They suggested that i t was probabl y due to large numbers of susceptible juvenile

birds in the areas. The present surveillance study of waterfowls in Alaska reveals



D u c k i n f l u e n z a v i r u s e s i n A la s k a 1 1 69

r D I d E g / 6 2 ( H 4 N 6 )

n / S A / 6 1 ( H S N 3 )

C k / G r / 4 9 ( H 1 0 N 7 )

I D I d E g / 5 6 ( H 1 1 N 6 )

= l i i ,,,,, D k / U / 6 3 ( H 3 N e )

I I ~ . . . . . . . F P V /D b /2 7 ( H 7 N 7 )

I I= I " F P V / R t / 3 4 ( H 7 N)

I l e D k / C z / f 6 1 , H 4 : )d / H q 7 7 ( H 4 N 6 )

W

' = D k / M r r v 7 4 ( H 3 N 8 )

I I D k , , A k / ~ O , , S l ( H 3 N e i ]

o n d i n N e n a n a

I D k / A k / 9 2 4 / 9 1 ( H 3 N S i t P o n d i n N e n a n a

I D W A k / 9 0 7 / g l (H 3 N e ) , ,] P o n d i n N e n a n a

M I / N Y / 7 8 ( H 2 N 2 )

D k / P n / 6 9 ( H S N 1 )

T y / O t / 6 8 ( H 8 N 4 )

T y / M n / 8 1 ( H 1 N 1 )

= " • I D k / A k / 2 6 9 5 / 9 2 ( H 4 N 6 ) , ,J M a l la r d L a k e

[ D k / A W 2 6 0 4 / 9 2 H 4 N 6 1 , ,, ,

M a l l a r d L a k e

T y / M n / 8 0 ( H 4 N 2 )

R t / N J / 8 5 ( H 4 N 6 )

t

o O O l / s i t e

~ , ~

' - " J A k / 3 1 0 7 / 9 3 ( H 3 N 8 ) [ B i g M i n to L a k e

I D k / A k / 3 1 1 1 / 9 3 ( H 7 N 3 ) '1 B i g M i n to L a k e

. I D I d A t d 3 2 4 2 " / 9 3 I H 3 N S i

B i g M i n t o

a k e

• I D k / A k / 3 2 4 5 '~ 3 ~ H 4 N 6 ) I B i g M i n to L a k e

" [ D k / A I d 3 2 1 0 / 9 3 ( H 4 N 6 ) ] B i g M i n to L a k e

• = , n • I D k / A k / 3 0 0 1 / 9 3 ( H 3 N e ) I M a l la r d L a k e

I D I d A I d 3 0 4 0 /9 3 1 H 3 N e ) I M a l la r d L a k e

I D I d A I d 2 3 3 3 / 9 2 /H 4 N 6 ) I L a k e S p e n a r d

I D k / A W 2 0 1 2 / 9 2 /H 1 0 N T ) I L a k e H o o d

i • [ D I d A k / 6 5 8 / 9 1 ( H 1 0 N 2 ) [ B i g M in t o L a k e

I D I d A I d S S 3 / 9 1 ( H 1 0 N 7 i , ]

B i g M i n to a k e

I D k / A k / 7 1 2 / 9 , , (H 1 0 N 9 ) ] B i g M in t o L a k e

I D k / A k / 7 0 2 / 9 1 ( H s N 2 ) I B i g a i n t o L a k e

C I d P n / 8 3 ( H 5 N 3 )

N

F ig . 2. P h y to g e n e t i c t r e e f o r a v i a n i n f l u e n z a A v i r u s N P g e n e s . H o r i z o n t a l d i s t a n c e s a r e

p r o p o r t i o n a l t o t h e m i n i m u m n u m b e r o f n u c l e o t i d e d i f fe r e nc e s r e q u i r e d t o j o i n n o d e s a n d

s e q u e n c e s . D o t s r e p r e s e n t t h a t t h e s e q u e n c e s a r e i d e n t i c a l w i th t h e n o d e s . V e r t i c a l li n e s a r e f o r

s p a c in g b r a n c h e s a n d l a b el s . V i r u s e s i s o l at e d f r o m w a te r f o w l s a t t h e i r n e s t i n g p o in t s i n A la s k a a r e

in b o x e s , t o g e th e r w i th t h e n a m e o f th e l a k e w h e r e t h e y w e r e i s o la t e d . A b b r e v i a t i o n s f o r v i ru s e s a r e

f o u n d in G o r m a n e t a l. [8 ]. E a c h t r e e w a s b r a n c h e d i n t o O l d W o r l d O W ) a n d N o r t h A m e r i c a n

N A ) s n b l i n e a g e s



1 1 7 0 T I t o e t a l

that high incidence of virus infection of ducks has already occurred in the breeding

places of ducks. Cont inuous surveillance of influenza viruses in migratory birds in

Canada and USA showed that the frequency of virus isolation from migratory

ducks coming from northern territories in autumn was high (more than 20 of

juveniles), whereas those from south in spring was extremely low (less than 0.25 )

[34], suggesting that viruses are mainly supplied when they migrate to south. The

present results suggest that first amplification of the virus by juvenile birds has

already occurred in the breeding areas of waterfowls and the viruses were brought to

southern area by their migration.

The mechanism of perpetuation of influenza viruses in avian species year by year

has no t been well understood. Influenza viruses were isolated from unconcentrated

lake water when migratory ducks are present [11]. In the present study, high titers of

the viruses were isolated from water samples of different lakes in the breeding areas

of ducks in northern high latitudes in summer (Lake Hood, Mallard lake and Big

Minto Lake). Furthermore , the viruses remained viable in the lake water after that

almost ducks left for migration to south. I t is possible that influenza viruses are

preserved in frozen lake water during winter when ducks are absent and that ducks

coming back from south are infected with these viruses in spring. Survival of

influenza viruses in water is dependent on the virus strain and the salinity, pH, and

temperature o f the water; at 17 °C some strains remain infectious for up to 207 days,

and at 4 °C they remain infectious for longer period [28, 29]. The major breeding

areas of ducks in Alaska located in high latitudes, 65-68 degrees north, near the

arctic regions. The take water in this area is cold enough for viruses to survive for

longer period in autumn when ducks leave for migration to south. Therefore,

influenza viruses could be preserved in the frozen lake water while ducks are absent.

The phylogenetic analysis of the NP genes of influenza virus isolates from ducks

in Alaska showed that all of those analyzed belonged to the North American lineage

(Fig. 2). Therefore, these viruses were not supposed to be the gene sources of Hong

Kong pandemic strains. Alternatively, Bean et al. [4] suggested that A/duck/

Hokkaido/21/82 (H3N8) which was isolated from a migratory duck captured on the

Asian Pacific flyway in Japan bore the North American type HA gene. There are

other major breeding areas of waterfowls in west and south Alaska such as Yuk on

Delta, Seward Peninsula, and St. Lawrence Island. The possibility that waterfowls

in these areas migrate to Asia through Aleutian Islands was retained. Phylogenetic

analysis of various virus isolates from waterfowls may make it possible to presume

the migratory flyways of host birds carrying influenza viruses.

A c k n o w l e d g e m e n t s

This studywas supportedby Grants-in-Aid or ScientificResearch03041013 and 05044117 from

the Ministry of Education, Scienceand Culture, Japan and in part by Public Health Service

Research Gran~s AI-29680 and AI-33898 from the National Institute of Allergyand Infectious

Disease, by Cancer Center Support (CORE) Grant CA-21765, and by the AmericanLebanese

Syrian AssociatedCharities. We are grateful o R. M. Oates, R. King, S. McLean, and T. Heuer

(theU.S. Fish and WildlifeService),J. M. Wright,and C. A. Smith theAlaskaDepartmentoffish

and Game) for their kind cooperation for the present surveillance study. We thank Hiroki

Takakuwa and AyakoShimizu or technicalassistance.



D uck inf luenza vi ruses in Alask a 1171

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Au thors address: D r . H. Kida , Depar tm ent o f Veter inary Hygiene and M icrobiology, School

of Veter inary Medic ine , Hok kaid o Un ivers ity , Sapporo 060, Japa n

Received Feb ruary 16, 1995

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