Download Continuing Evolution of H3, H4, H6 and H9 Influenza A Viruses in

Continuing Evolution of H3, H4, H6 and H9 Influenza A Viruses
in Live Bird Markets in Korea with Potential for Expanding Host Range
HyunHyun-Jeong Lee1, YuYu-Na Lee1, DongDong-Hun Lee1, HaHa-Na Youn1, JiJi-Sun Kwon2, YounYoun-Jeong Lee2, MinMin-Chul Kim2, OkOk-Mi Jeong2,
HyunJun-Hun Kwon2, JoongJoong-Bok Lee1, SeungSeung-Young Park1, InIn-Soo Choi1 and ChangChang-Seon Song1*
Hyun-Mi Kang2, Jun1College of Veterinary Medicine, Konkuk University, Seoul, Korea
2Avian Disease Division, National Veterinary Research & Quarantine
Quarantine Service, Anyang, Korea
INTRODUCTION
MATERIALS & METHODS
• In Korea, there have been extensive gene pools for influenza viruses to generate multiple
reassortants.
Sampling and virus isolation
• Live bird markets (LBMs) are highly productive sources of avian influenza viruses (AIVs) because
they provide an ideal environment for viral reassortment and interspecies transmission.
Phylogenetic and molecular analysis
• However, only limited reports provided information about ecology of AIV circulating in Korean LBM.
Animal experiment
• In this study, we performed nationwide surveillance on AIV from LBM in Korea from 2006 to 2008
• To understand the epidemiology of the AIVs in Korean LBM
• To understand the role of LBMs as sources of AIV evolution
• To determine the animal species that possess the potentials of expanding host range of AIVs
Table 1. Summary of avian influenza viruses isolated from LBMs by host species
H9N2 (chicken farm)
• From September 2006 through March 2008, a total of 644 tissue specimens were collected from
different species in 13 different LBMs of South Korea and used for virus isolation.
• Eight gene segments of representative isolates were compared with those of influenza viruses
isolated from domestic chickens, wild birds, human, swine and equine.
• Twelve representative viral isolates were inoculated with 105.0~6.0 EID50 of each virus.
• Re-isolate inoculated virus from each animals.
Chicken & Quail (2~4wks old)
In trachea & CT
on 3 dpi.
Mouse (5~6wks old)
In lung
on 5 dpi.
Dog (10wks old)
In nose
daily for 12 days p.i.
H9N2 (LBM)
CK/Korea/96-like
Viral subtype
No of
Total
samples H3N2 H3N8 H4N2 H4N6 H6N2 H9N2 UnidentifiedA (%)
Order
Species
Anseriformes
Pekin duck
153
5
2
Mallard duck
116
3
1
Galliformes
Geese
8
Muscovy duck
8
Korean native chicken
1
6
2
16
1
6
3
16
133
17
17
Silky fowl
78
11
11
Pheasant
34
1
1
Turkey
54
Quail
25
Layer
12
Bantam
5
Guinea fowl
4
Columbiformes Pigeon
Carnivora
1
1
CK/Korea/96
Total
CK/Korea/Q19/04
Chicken
PB2
PB1
PA
HA
NP
NA
M
NS
2
1
7
1
1
644
9
1
Pekin Duck
3
1
1
2
44
5
(b) H3
American avian-like
Eurasian avian-like
Eurasian poultry-like
NS B allele
LBM1354/06
2
CK/Korea/99-like
Korean LBM-like
H3N2 (LBM)
1
LBM76/07
CK/Korea/Q19/04-like
6
Dog
Cat
CK/Korea/99
65
H3N8 (LBM)
LBM78/07
LBM448/07
LBM182/07
H4N6 (LBM)
LBM187/08
H6N2 (LBM)
LBM399/09
PB2
PB1
PA
HA
NP
NA
M
NS
AHA
and NA subtype of four isolates were partially identified; one of H3, one of H4, one of H6, and one of N2.
One AIV was not identified subtype.
H3N2 (LBM)
Table 2. Replication of representative influenza viruses in chickens, quails, and mice
QuailsA
ChickensA
Subtype
Isolate
Tra
CT
Tra
CT
Lung
H3N2
DK/LBM1354/06
2/4
0/4
0/4
0/4
4/4
H3N8
DK/LBM347/07
1/4
0/4
0/4
0/4
0/4
H3N8
DK/LBM182/07
0/4
0/4
0/4
0/4
0/4
H4N6
DK/LBM187/08
1/4
0/4
0/4
0/4
2/4
H4N2
3/4
MAL/LBM188/08
3/4
0/4
0/4
4/4
H6N2
DK/LBM399/07
1/4
1/4
0/4
0/4
4/4
H6N2
DK/LBM1674/07
1/4
0/4
2/4
0/4
4/4
H9N2
CK/LBM341/07
2/4
0/4
H9N2
DK/LBM186/07
0/4
0/4
3/4
1/4
0/4
H9N2
CK/LBM76/07
3/4
0/4
3/4
0/4
0/4
H9N2
DK/LBM446/07
4/4
0/4
4/4
1/4
0/4
H9N2
CK/Kor/006/96
4/4
3/4
4/4
0/4
0/4
0/4
0/4
LBM397/07
LBM1621/07
0/4
Mallard
(a) H9
Table 3. Replication of H3 influenza viruses in dogs
Clinical signs
H3N2
Canine/LBM412/08 Dog
FeverA
3/3
Cough Sneeze
3/3
3/3
canine/412/08
Virus
SeroconversionC
replicationB
3/3
3/3
H3N2
DK/LBM1354/06
Duck
0/3
3/3
3/3
0/3
3/3
H3N8
DK/LBM347/07
Duck
0/3
3/3
3/3
3/3
3/3
AFever
is defined by rectal temperature over 39.5°C.
BNumber of virus positive dogs/number of inoculated dogs. The infectious dose was 105EID .
50
For 12 days, nasal swabs were examined for virus shedding by RRT-PCR.
CNumber of virus positive dogs/number of inoculated dogs.
At 17 days post-infection, ELISA antibody titers were regarded as positive if percent inhibition (PI) was >50.
Korean
LBM
WB
Mammals
0.02
(c) N2
Canine
Figure 1. Gene constellation of AIVs isolated from LBM in Korea. Genetically similar genes
are shown in the same color. B allele; B allele in the tree of NS gene, Gs/Gd;
Goose/Guangdong/1/96(H5N1)
of virus positive birds/number of inoculated birds. The infectious dose was 105~106 EID50.
At 3 days post-infection, Tra and CTs were tested to detect inoculated virus. Tra, trachea; CT, cecal tonsil.
BNumber of virus positive mice/number of inoculated mice. The infectious dose was 105EID .
50
At 5 days post-infection, lungs were tested to detect inoculated virus.
Source
H3N2 (LBM)
LBM188/08
PB2
PB1
PA
HA
NP
NA
M
NS
ANumber
Subtype Isolate
H4N2 (LBM)
MiceB
DK/Kor/S8/03(H3N2)
DK/LBM1258/06(H3N2)
DK/LBM182/07(H3N8)
DK/LBM377/07(H3N8)
DK/LBM1621/07(H3N2)
DK/LBM448/07(H3N2)
DK/LBM78/07(H3N2)
DK/LPM86/06(H3N2)
DK/LPM18/04(H3N2)
DK/LPM23/05(H3N2)
AB/Korea/JN-2/06(H3N2)
canine/korea/01/07(H3N2)
canine/korea/09/08(H3N2)
canine/LBM412/08(H3N2)
DK/LBM176/08(H3N2)
MAL/LBM397/07 (H3N2)
DK/LBM1354/06(H3N2)
MAL/LBM347/07(H3N8)
AB/Korea/KN-2/05(H3N2)
SW/Iowa/533/99(H3N2)
SW/Korea/JNS06/04(H3N2)
Panama/2007/99(H3N2)
Wisconsin/67/2005(H3N2)
Equine/Kentucky/5/02(H3N8)
Equine/Ohio/1/03(H3N8)
CK/LBM341/07(H9N2)
CK/LBM453/07(H9N2)
sCK/LBM1632/07(H9N2)
CK/Kor/Q19/04(H9N2)
CK/LBM76/07(H9N2)
DK/LBM186/07(H9N2)
CK/Kor/188/03(H9N2)
CK/Kor/Q30/04(H9N2)
CK/Kor/311/02(H9N2)
CK/Kor/029/99(H9N2)
CK/Kor/006/96(H9N2)
DK/HK/Y439/97(H9N2)
Teal/Primorie/3628/02(H9N2)
HK/1073/99(H9N2)
Qa/HK/G1/97(H9N2)
DK/HK/Y280/97(H9N2)
CK/Beijing/1/94(H9N2)
TY/MN/38391/6/95(H9N2)
Qa/Arkansas/29209
-1/93(H9N2)
0.02
CK/Korea/96
-like
G1-like
Y280-like
American
-like
0.02
DK/LBM448/07(H3N2)
sCK/LBM1632/07(H9N2)
DK/LBM78/07(H3N2)
CK/Korea/LPM23/05(H3N2)
CK/Korea/LPM18/04(H3N2)
CK/Korea/LPM86/06(H3N2)
MAL/LBM1674/07(H6N2)
DK/LBM176/08(H3N2)
MAL/LBM397/07(H3N2)
DK/LBM1354/06(H3N2)
DK/LBM399/07(H6N2)
DK/LBM1258/06(H3N2)
CK/LBM453/08(H9N2)
MAL/LBM1621/07(H3N2)
DK/Korea/S8/03(H3N2)
AB/Korea/JN-2/06(H3N2)
canine/korea/01/07(H3N2)
canine/korea/09/08(H3N2)
canine/LBM412/08(H3N2)
AB/Korea/KN-2/05(H3N2)
CK/Kor/029/99(H9N2)
CK/Kor/Q19/04(H9N2)
CK/LBM341/07(H9N2)
DK/LBM186/07(H9N2)
MAL/LBM188/08(H4N2)
CK/LBM76/07(H9N2)
sCK/LBM446/07(H9N2)
Panama/2007/99(H3N2)
Wisconsin/67/05(H3N2)
SW/Iowa/533/99(H3N2)
SW/Korea/JNS06/04(H3N2)
Korean
LBM
CK/Korea/96
-like
Mammals
Figure 2. Phylogenetic analysis of avian influenza viruses isolated from LBMs.(a) H9 HA gene; (b) H3 HA gene; (c) N2 NA gene. Bold type indicates the viruses examined in this study.
CK, chicken; DK, duck; MAL, mallard; sCK, silky chicken; AB, aquatic bird; SW, swine; TY, turkey; Qa, quail; HK, Hong Kong; Kor, Korea; MN, Minnesota.
RESULTS
DISCUSSIONS
Virus isolation and distribution of subtypes (Table 1)
• Sixty-five AIVs (H3, H4, H6 and H9) were isolated from 644 tissue samples collected in LBMs.
• Prevalence of subtypes: H9 (44) > H3 (13) > H4 & H6 (3)
• Most H9 subtypes were isolated from Galliformes and other subtypes were isolated from
Anseriformes.
• A single H3N2 virus isolated from nasal swabs of farmed dogs sold in LBMs.
Phylogenetic study & molecular analysis
• H9N2 viruses circulating in chickens have continued to maintain its lineage since the first
description, and internal genes of them were introduced into H3, H4, and H6 viruses in ducks.
• There were genetic diversity of AIV circulating in duck population.
• Seven genes of CIV was closely related to those of at least one isolate of H3 and H4 viruses
from ducks in LBMs (Fig 1).
• Therefore, we suggest that ducks in LBM play a role in mixing vessels for AIVs to generate novel
reassortants including CIV.
Phylogenetic analysis (Figure 1 & 2)
• In H9 HA tree, all H9 viruses belonged to the Korea lineage (CK/Korea/96-like lineage) that has
been prevalent in chicken farms in Korea and genetically far from other Asian H9 lineage (Fig 2-a).
• In H3 HA tree, all H3 viruses formed Korea LBM lineage within Eurasian avian lineage (Fig 2-b).
• N2 genes of the AIVs in LBMs were divided into two clusters, LBM lineage and Korea lineage
(Fig 2-c).
• Six internal genes of H9N2 viruses of Korea lineage were widely dispersed in AIV gene pool
together with those of AIVs in Eurasian aquatic birds.
• There were genetic diversity of AIV in LBMs leading to generate numerous reassortants including
CIV (Figure 1).
Animal experiments (Table 2 & 3)
• Quail (Table 2)
Animal challenge study
• Chicken: H3 viruses from ducks have not adapted to chickens despite introduction of internal
genes from H9N2 viruses circulating chickens. In contrast, H4 and H6 viruses from ducks seems
to have potentials to infect chickens.
• Mice: Unlike H9N2 virus, replication of H3, H4, and H6 viruses in mice suggest that the AIV from
ducks possess potentials to expand the host range to mammalians.
• Quail: Quails may play a role in the ecology of AIVs based on the susceptibility to multiple
subtypes of AIVs.
• Dog: AIVs circulating in ducks, particularly the H3 subtype, in LBMs have the potential of
crossing species barriers.
¾ Most isolates replicated in the respiratory tracts.
• Chicken (Table 2)
¾ All the H9N2 viruses from chickens replicated well in trachea of chickens, as well as single H4N2 and
H6N2 virus from ducks. However, none of H3 viruses replicated in chickens.
• Mice (Table 2)
¾ H3, H4, and H6 viruses from ducks replicated well without pre-adaptation.
¾ In contrast, none of H9 viruses from chickens were reisolated in the lung of inoculated mice.
• Dog (Table 3)
¾ CIV and two AIV examined replicate in dogs.
¾Clinical sign : fever (>39.5℃), sneezing, nasal discharge, and coughing.
¾CIV induced severe clinical signs, but 2 H3 viruses from ducks induced mild clinical signs without
fever.
¾Virus shedding: detected both in dogs inoculated with CIV and one AIV.
¾Seroconversion: observed in all dogs inoculated with CIV or AIV.
¾Gross lesions: multifocal to coalescing reddish consolidations observed in lung of all infected dogs.
Molecular characterization
• Amino acid alteration observed in several AIVs showing ability to replicate in mice
¾ M1 protein: V15I (associated with pathogenecity in mice).
• Amino acid alteration observed in CIV
¾ HA protein
¾Lys-to-Arg substitution at position -4 from the cleavage site (RQTR*GLF)
¾Q226L and G288S in receptor binding site (preferentially recognize SAα2,6Gal)
¾ PB1-F2 protein: N66S (correlated with pathogenicity in mouse)
CONCLUSIONS
• Korean LBM played an important role in extending genetic diversity of influenza
viruses in Korea.
• The newly evolved AIVs have been continuously generated by reassortment
events in ducks in LBMs with the potential of expanding the host range to
mammalians.
• Continued monitoring of poultry population, in particular quails and ducks, in
LBM need to better understand the influenza ecology and interspecies
transmission, as a component of pandemic preparedness.
ACKNOWLEDGEMENTS
This study was financially supported by Grant no. M-AD15-2006-07-01 from National Veterinary
Research and Quarantine Service (NVRQS) of Korea.
Corresponding Author Contact Information
E-mail: [email protected]
Phone: +82-2-450-3712