- Email: [email protected]
Pathogenicity of highly pathogenic avian influenza viruses of H5N1 subtype isolated in Thailand for different poultry species
Available online at www.sciencedirect.com
Veterinary Microbiology 133 (2009) 65–74 www.elsevier.com/locate/vetmic
Pathogenicity of highly pathogenic avian influenza viruses of H5N1 subtype isolated in Thailand for different poultry species Takehiko Saito a,b,*, Chiaki Watanabe a,b, Nobuhiro Takemae a,b, Arunee Chaisingh c, Yuko Uchida a,b, Chantanee Buranathai d, Hirofumi Suzuki a, Masatoshi Okamatsu a, Tadao Imada e, Sujira Parchariyanon c, Nimit Traiwanatam c, Shigeo Yamaguchi a a
Research Team for Zoonotic Diseases, National Institute of Animal Health, National Agriculture and Food Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan b Thailand-Japan Zoonotic Diseases Collaboration Center (ZDCC), Bangkok, Thailand c National Institute of Animal Health, Bangkok, Thailand d Department of Livestock and Development, Ministry of Agriculture and Cooperatives, Bangkok, Thailand e Kyushu Branch, National Institute of Animal Health, National Agriculture and Food Organization (NARO), 2702 Nakayamacho, Kagoshima 891-0105, Japan Received 16 April 2008; received in revised form 13 June 2008; accepted 26 June 2008
Abstract Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused several rounds of outbreaks in Thailand. In this study, we used 3 HPAI viruses isolated in Thailand in January 2004 from chicken, quail, and duck for genetic and pathogenetic studies. Sequence analysis of the entire genomes of these isolates revealed that they were genetically similar to each other. Chickens, quails, domestic ducks, and cross-bred ducks were inoculated with these isolates to evaluate their pathogenicity to different host species. A/chicken/Yamaguchi/7/04 (H5N1), an HPAI virus isolated in Japan, was also used in the chicken and quail studies for comparison. All four isolates were shown to be highly pathogenic to chickens and quails, with 100% mortality by 106 EID50 inoculants of the viruses. They caused sudden death in chickens and quails within 2–4 days after inoculation. The mean death times (MDT) of quails infected with the Thai isolates were shorter than those of chickens infected with the same isolates. Mortality against domestic and cross-bred ducks ranged from 50 to 75% by intranasal inoculation with the 106 EID50 viruses. Neurological symptoms were observed in most of the inoculated domestic ducks and appeared less severe in the cross-bred ducks. The MDTs of the ducks infected with the Thai isolates were 4.8–6 days post-inoculation. Most of the surviving ducks infected with the Thai isolates had sero-converted until 14 dpi. Our study illustrated the pathobiology of the Thai isolates against different poultry species and would provide useful information for improving control strategies against HPAI. # 2008 Elsevier B.V. All rights reserved. Keywords: HPAI; Poultry; Influenza virus; Pathogenicity
* Corresponding author at: Research Team for Zoonotic Diseases, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Tel.: +81 29 838 7802; fax: +81 29 838 7802. E-mail address: [email protected] (T. Saito). 0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.06.020
66
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
1. Introduction Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have been proven widespread in Southeast Asia since 2004. Thailand is one of the countries heavily affected by the HPAI outbreaks. Since HPAI was first recognized in that country in January 2004, nearly 2000 outbreaks have been recognized there. Several species of poultry, including chickens, quails, and domestic ducks, as well as humans, tigers, and wild birds, were involved in those outbreaks. Involvement of many species of poultry, as well as of mammalians and wildlife, is a unique property of the recent HPAI incidents in the region. In 14 countries worldwide, 372 human cases were confirmed between 2004 and March 2008, among which 235 were fatal. In Thailand alone, 25 human cases have been confirmed. In the early period of the HPAI outbreaks, outbreaks in tigers that had been fed infected chicken carcasses were reported in Thailand (Amonsin et al., 2006; Keawcharoen et al., 2004). Infection of dogs and cats in Thailand was also reported (Songserm et al., 2006; Songsermn et al., 2006). Several species of wild birds were also confirmed to be infected by H5N1 HPAI viruses in Thailand (Melville and Shortridge, 2006). It has been apparent that the pathogenicity of the currently circulating HPAI viruses of the H5N1 subtype, which are descendants of A/goose/Guangdong/1/96, against avian and mammalian species has been changing as the viruses have become prevalent in Eurasia and Africa. Type A influenza viruses, except H7N7 equine influenza A viruses, had been considered to require adaptation before causing lethal infection in a mouse model (Kawaoka, 1991). However, HPAI viruses that were isolated from patients suffering from HPAI infection in Hong Kong in 1997 caused lethal and systematic infection in a mouse experimental model without adaptation (Lu et al., 1999). It had also been thought that H5 HPAI virus infection did not result in severe diseases in waterfowl. HPAI viruses of the H5 subtype had never been isolated from wild birds, except from terns in South Africa in 1961. Such observations were also shown to be no longer valid after a die-off of wild birds by the H5N1 viruses was recognized in Hong Kong in 2002. Some of the viruses isolated from the outbreak were shown to cause lethal infection in mallards (Anas platyrhynchos) (Sturm-
Ramirez et al., 2004). During the 2004–2005 HPAI outbreaks, nearly 50 species of wild birds were shown to be affected by the H5N1 viruses in East and Southeast Asia (Melville and Shortridge, 2006). Furthermore, more than 6000 migratory birds died from infection with HPAI viruses of the H5N1 subtype at Qinghai Lake, China, in May 2005 (Chen et al., 2005; Liu et al., 2005). Following the Qinghai outbreak, related strains, so-called ‘‘Qinghai strains’’, spread worldwide, reaching Europe and Africa (Alexander, 2007). Similar strains also re-emerged at Qinghai Lake in May 2006 (Wang et al., 2008) Because of the changing pathogenicity of the currently circulating H5N1 viruses against different hosts, it is crucial to understand the pathobiology of the viruses in different hosts in order to establish efficacious eradication strategies. In this study, we characterized three HPAI viruses isolated in Thailand in January 2004 genetically and pathobiologically. Experimental infection studies using four different poultry species demonstrated a divergence in their pathogenicity against poultry, although genetic characterization revealed extensive similarities among those isolates. The results obtained in this study would provide useful information for improving surveillance and eradication strategies and would provide a clue to understanding the epidemiology of the HPAI viruses.
2. Materials and methods 2.1. Viruses Three isolates from Thailand (Table 1) were obtained from the National Institute of Animal Health, Thailand. They, as well as A/chicken/Yamaguchi/7/ 2004, were isolated and propagated with 10- to 11day-old embryonated hen’s eggs at 37 8C. All of the experiments using intact viruses, including animal experiments, were carried out in BSL-3 facilities at the National Institute of Animal Health, Japan. 2.2. Sequence analyses Viral RNA was extracted from virus-containing allantoic fluid by using a commercial kit (RNeasy, Qiagen, Hilden, Germany). After reverse transcription with Superscript III (Invitrogen, Carlsbad, CA, USA),
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
67
Table 1 Thailand HPAI viruses used in this study Strains
Date of collection
Province/city
EID50/mla
ELD50/mlb
EID50/ELD50
TCID50/mlc
Ck/Suphanburi/1/04
2004/1/21
9.1
7.4
47
6.5 (6.3)
512
Qa/Angthong/71/04 Dk/Angthong/72/04 Ck/Yamaguchi/7/04
2004/1/23 2004/1/23 2004/1/9
Suphanburi/Bang Pla Mok Angthong/Pa Mok Angthong/Pa Mok Yamaguchi/Ato
9.0 8.1 8.7
9.0 6.4 8.7
1 47 1
6.5 (7.5) 7.0 (6.7) 7.6 (7.6)
1024 64 64
a b c
HA titer
Virus titer expressed as log10. Lethality was determined at 48 h after inoculation and titers were expressed as log10. Virus titer was measured by infecting MDCK cells with and without (in parentheses) trypsin.
cDNAs were amplified by polymerase chain reaction (PCR) with ExTaq (Takara, Shiga, Japan) with segment-specific primers (the primer sequences are available upon request). PCR products were purified by the QIAquick PCR purification kit (Qiagen) and subjected to sequencing with the Big Dye Terminator sequencing kit, version 3.1 (Applied Biosystems, Foster City, CA, USA). Reactions were analyzed on an ABI Prism 3100 genetic analyzer (Applied Biosystems). The nucleotide sequences obtained were assembled by using Sequencher version 4.6 (Hitachi Software Engineering, Tokyo, Japan) and were translated into deduced amino acid sequences by GENETYX-WIN (GENETYX, Tokyo, Japan). The nucleotide sequences obtained in this study are available from GenBank under accession numbers AB440324–AB440347. 2.3. Serological tests A hemagglutination inhibition (HI) test was performed by a conventional microplate method with chicken red blood cells. Serum samples drawn from surviving animals at the end of a 14-day observation period after inoculation were treated with RDE (II) (Denka Seiken, Niigata, Japan) to remove non-specific inhibitors in sera. 2.4. Experimental infection of poultry Specific pathogen-free (SPF) chickens (Gallus gallus domesticus) and Japanese quails (Coturnix japonica) were purchased from Nisseiken (Yamanashi, Japan). Conventional domestic ducks (Cherry Valley) and cross-bred ducks were purchased from farms in Japan. The cross-bred ducks used in this study were F1
or F2 between mallards and domestic ducks. All of the birds were 4 weeks old at the time of inoculation. Groups of 8 birds of respective species were inoculated intranasally with 106 50% egg infective dose (EID50) of each virus. In the chicken experiment, 104 and 102 EID50 virus doses were also used for A/chicken/ Suphanburi/1/2004 and A/quail/Angthong/71/2004. Inoculated birds were observed for clinical symptoms for 14 days. Oropharyngeal and cloacal swabs were taken from birds that had died during the observation period. Swabs were suspended in Dulbecco’s Modified Eagle’s Medium containing antibiotics and bovine serum albumin. After brief centrifugation, the supernatants were stored at 80 8C until used. Swab samples were titrated in embryonated hen’s eggs. The antibody responses of the surviving birds against the inoculated virus were examined by means of HI assay. The experimental procedures and care of animals were approved by the Animal Experiment Committee of National Institute of Animal Health.
3. Results 3.1. Virus properties Three strains of HPAI viruses of the H5N1 subtype—A/chicken/Suphanburi/1/2004 (CkSup), A/ duck/Angthong/72/2004 (DkAng), and A/quail/ Angthong/71/2004 (QaAng)—were isolated in the early period of HPAI outbreaks in Thailand in January 2004 (Table 1). CkSup was isolated from a dead chicken, whereas DkAng and QaAng were isolated from live domestic duck and quail, respectively. Suphanburi and Angthong are provinces in central Thailand.
68
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
Table 2 Amino acid differences among the Thai isolates used in this studya Strain
Gene segment and amino acid number PA
Ck/Suphanburi/1/04 Qa/Angthong/71/04 Dk/Angthon g/72/04 a
PB1
PB2
HA
NP
NA
NS1
NS2
122
76
192
386
195
250
140 (HA1)
456
43
57
73
115
V
N D D
R K
R K K
D
A V V
E K K
V
H Q
A
S
A
S
T
T
E
N
A
Differences from Ck/Suphanburi/1/04 are shown.
Sequence analysis revealed a high rate of identity among those isolates, suggesting a rapid dissemination of the HPAI viruses within the country in the early phase of the outbreak. The sequences of the entire genomes of three isolates were determined, and more than 99% identity was observed in both the nucleotide and deduced amino acid sequences of all 11 coding regions among the isolates. In particular, 100% identity in the deduced amino acid sequence was observed with M1, M2, and PB1-F2 proteins among all three isolates. In NP, NS1, and NS2 proteins, CkSup and QaAng showed 100% identity and CkSup and DkAng showed 100% identity in NA protein. The amino acid differences among the Thai isolates used in this study are listed in Table 2. All of the viruses replicated to higher titers in embryonated eggs than in MDCK cells (Table 1). Lethality to eggs, judged at 48 h after inoculation, differed among the isolates. The 50% egg infectious dose (EID50) and the 50% egg lethal dose (ELD50) were almost the same for QaAng; on the other hand, the ratios between them were approximately 50 for CkSup and DkAng, indicating that QaAng was the
most virulent to embryonated eggs among the isolates used with a given dose. QaAng gave 10-times higher titer in MDCK cells when it was cultured without exogenous trypisn, whereas the other two isolates showed comparable titers with and without trypsin in MDCK cells. The highly pathogenic nature of the three Thai isolates, along with Ck/Yamaguchi/07/2004 (CkYama), was demonstrated by intranasal administration against two species of gallinaceous birds: chickens and Japanese quail (Table 3). In the eight birds tested for each species, 100% mortality was observed with an intranasally administered virus of 106 EID50. Both species died without showing apparent symptoms; the rather sudden death appears to be a characteristic of infection with those viruses. For chickens, mean death time (MDT) was less than 3 days postinoculation with the Thai isolates as well as with CkYama (Table 3). The shortest MDT was observed for chickens inoculated with DkAng (1.4 days postinoculation, DPI), which was significantly shorter than MDTs obtained for CkSup ( p = 0.001) and CkYama ( p = 0.014). For quails, all of the birds
Table 3 Mortality and sero-conversion of experimentally inoculated poultry Strain
Ck/Suphanburi/1/04 Qa/Angthong/71/04 Dk/Angthong/72/04 Ck/Yamaguchi/7/04 a b c d
Chicken
Quail
Duck
Cross-bred duck
Mortality (%)
MDTa
Mortality (%)
MDT
Mortality (%)
MDT
Ab (+)/ survivors
Mortality (%)
MDT
Ab (+)/ survivors
100 100 100 100
2.3 1.9 1.4 c 2.0
100 100 100 100
1.4 1.1 1.0 3.4 d
62.5 62.5 50 NT
6.0 6.0 6.0 NT
2/3 (128)b 3/3 (102) 0/4
50 50 75
6.3 5.3 4.8
4/4 (45) 2/4 (16) 0/2
Mean death time (days postinoculation) among dead animals. Geometric mean of HI antibody titers among sero-positive animals at 14 DPI is shown in parentheses. Significant to Ck/Suphanburi ( p = 0.005) and Ck/Yamaguchi ( p = 0.014) by ANOVA, followed by Tukey analysis. Significant to the rest of the group by ANOVA ( p < 0.001).
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
inoculated with DkAng died within 1 DPI (MDT = 1.0), and within 2 DPI with CkSup (MDT = 1.4) and QaAng (MDT = 1.1). It appears that quails died earlier than chickens when they were inoculated with either CkSup ( p = 0.005) or QaAng ( p = 0.025) (Table 3). In contrast, the MDT was significantly longer for quails inoculated with CkYam than for chickens ( p < 0.001). 3.2. Pathogenicity in gallinaceous poultry The lethal dose against chickens was shown to be different between CkSup and QaAng when 102 and 104 EID50 of CkSup or QaAng were inoculated intranasally to 8 chickens in each group and observation for mortality was for two weeks (Fig. 1a and b). Only 3 out of the 8 chickens inoculated with 104 EID of CkSup died. On the other hand, 6 out of 8 died after inoculation with the same dose of QaAng. As described above, all chickens inoculated with 106 EID50 of either CkSup or QaAng died within 4 DPI or 2 DPI, respectively. None of the chickens inoculated with 102 EID50 of any of these viruses died during the observation period. These results indicated that the 50% chicken lethal dose (CLD50) of CkSup could be more than 104 EID50 and that of QaAng could be less than 104 EID50. This suggests that there was an approximately 100-fold
69
difference between CLD50 of CkSup and QaAng. In addition, none of the surviving chickens had seroconverted at 14 DPI against any of the inoculated strains. Virus titers in cloacal and oropharyngeal swabs of dead quails were shown to be different from CkSup and CkYam (Fig. 2). Postmortem swabs from 3 out of the 5 quails that died at 1 DPI and of all 3 that died at 2 DPI inoculated with CkSup, and from 3 out of the 5 quails that died at 3 DPI and of all 3 that died at 4 DPI inoculated with CkYam, were subjected to virus titration in embryonated eggs. None of the cloacal swabs from the quails that died at 1 DPI yielded any virus above the detection level (<101.2 EID50/ml). Virus titers in the oropharyngeal swabs obtained from quails that died at 1 DPI showed lower titers (2101.3, 2101.3, and 102.9 EID50/ml) than those obtained from quails that died at 2 DPI. No significant difference was observed in virus titers of swabs collected from quails inoculated with CkYam at different time points. Mean virus titers in postmortem cloacal and oropharyngeal swabs from quails inoculated with CkSup were significantly lower than those inoculated with CkYam (Fig. 2). 3.3. Pathogenicity in anseriform poultry Pathogenicity against anseriformes birds was evaluated by intranasal inoculation of the viruses to
Fig. 1. Survival rates of chickens infected with various doses of Ck/Suphanburi/1/04 or Qa/Angthong/71/04. (^) 1 106 EID50/animal intranasally, (&) 1 104 EID50/animal intranasally, (~) 1 102 EID50/animal intranasally.
70
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
Fig. 2. Whiskered box plots of virus titers in cloacal or oropharyngeal swabs from quails inoculated with Ck/Suphanburi/1/04 or Ck/Yamaguchi/7/04.
domestic ducks and cross-bred ducks. Domestic ducks inoculated with the Thai isolates died at a mean death time of 6 DPI, and mortality rates were 50–63% (Table 3 and Fig. 3a and b). Hemorrhages of beak, depression, and torticollis were commonly observed symptoms among the dying ducks. Some of the survivors recovered from torticollis and general behavioral aberrations during the observation period. The mortality rate and MDT of cross-bred ducks infected with the Thai isolates were also similar to those obtained for domestic ducks: 50–75% mortality
Fig. 4. Virus titers in postmortem cloacal or oropharyngeal swabs taken from dead ducks inoculated with Ck/Suphanburi/1/04. Marks under dotted lines mean virus titers under the detection level, which was 1.19 log10 EID50/ml.
and 4.8–6.3 DPI for MDT. In contrast to domestic ducks, cross-bred ducks showed no apparent symptoms, except depression, before death. Virus titers in postmortem cloacal and oropharyngeal swabs from the domestic ducks infected with CkSup were measured (Fig. 4). Both cloacal and oropharyngeal swabs obtained from the ducks that died at 4 DPI showed high virus titers. Although virus replication was confirmed in one oropharyngeal swab obtained from a duck that died at 7 DPI, other swabs,
Fig. 3. Survival rates of (a) domestic ducks or (b) cross-bred ducks inoculated intranasally by 1 106 EID50/animal of the Thai HPAI isolates ((*) Ck/Suphanburi/1/04, (&) Dk/Angthong/72/04, (~) Qa/Angthong/71/04).
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
one oropharyngeal and two cloacal, did not yield viruses above detectable level (101.2 EID50/ml). Likewise, neither cloacal nor oropharyngeal swabs taken at 8 DPI yielded a virus. These observations suggested that ducks infected with the Thai isolates did not always die at the peak of virus replication in the organs. Sero-conversion was confirmed in most of the surviving domestic and cross-bred ducks, which were inoculated with either CkSup or QaAng. On the other hand, it was also noteworthy that none of the survivors infected with DkAng sero-converted in those experiments. These results clearly demonstrated that the Thai strains used in this study were infectious and significantly lethal to domestic and cross-bred ducks at a given dose.
4. Discussion Our study demonstrated that the HPAI viruses isolated in Thailand during the early period of the outbreaks were lethal not only in gallinaceous birds, chicken and Japanese quail, but also in anseriformes birds. Although the strains are genetically highly similar to each other, their pathobiological features varied among the strains. It is noteworthy that the Thai strains used in this study killed quails faster than chickens, while Ck/Yamaguchi killed chickens faster. In previous reports (Perkins and Swayne, 2001; Isoda et al., 2006), several H5N1 viruses killed chickens faster than quail, as we observed for Ck/Yamaguchi. It has been suggested that the maximal pathogenicity of HPAI viruses was achieved in their host of origin because of species adaptation (Perkins and Swayne, 2001). However, this was not the case in our experiment; there were no significant differences among the three Thai strains isolated from three distinct hosts—chicken, quail, and duck—in their ability to kill quails. The Thai HPAI viruses used in this study might have adapted to quail before being transmitted to chickens, thus showing higher virulence in the former. Quail has been suggested to serve as an intermediate host that provides an environment in which influenza viruses can generate variants that can be transmitted to chickens (Perez et al., 2003; Xu et al., 2007). It was also shown that quails were more susceptible to Gs/HK/437-4/99 (H5N1) infection than
71
chickens (Webster et al., 2002). Different genetic constellations may also explain the differences in pathobiology between the Thai isolates and CkYam. The genotype of the Thai isolates was designated genotype Z (Puthavathana et al., 2005), whereas, that of CkYam was designated genotype V (Mase et al., 2005). There are several differences in deduced amino acids throughout their genome, suggesting that some of the different amino acids and/or combinations of them cause the differences in the pathobiology of the viruses. Another intriguing observation obtained with the experimental infection study in quail was that virus titers in postmortem oropharyngeal and cloacal swabs were significantly higher in quails infected with CkYam than in those infected with CkSup, although CkSup killed quails much more rapidly than CkYam. Level of virus replication in the respiratory tract or intestine may not necessarily correlate with virulence. A small plaque clone of a human isolate, A/Vietnam/ 1203/04, has low pathogenicity to mallards, while a large plaque clone of the same isolate is highly pathogenic to mallards (Hulse-Post et al., 2007). It was previously shown that both clones replicated comparably to each other in the trachea and cloaca of intravenously inoculated ducks. Virus replication in a particular target organ other than respiratory or intestinal organs may contribute to the virulence of the Thai isolates in quails. An MDCK-passaged strain of A/Hong Kong/156/97 (H5N1), which was isolated from an index case of a Hong Kong H5N1 incident in 1997, replicated well in mouse brain after intranasal inoculation and was highly virulent in mouse, whereas an egg-passaged strain of the same virus did not replicate in mouse brain and had low virulence in mouse (Hiromoto et al., 2000). Both strains replicated to similar levels in the lungs of intranasally inoculated mice. Differences in tissue tropism and/or in virus replication in a particular tissue of the H5N1 viruses in quails need to be examined to elucidate the variability of virulence in quails. Since late 2002, H5N1 influenza viruses with high pathogenicity to ducks have been recognized. The three Thai isolates used in this study were mild to highly pathogenic to both domestic and cross-bred ducks, in contrast with previous studies with Thai H5N1 isolates, which demonstrated that the isolates were relatively mildly pathogenic to older ducks (Hulse-Post et al., 2005; Pantin-Jackwood et al.,
72
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
2007). Pathogenicity to ducks varies considerably from strain to strain among the H5N1 HPAI isolated after late 2002, and even genetically similar strains could differ in their virulence in ducks (SturmRamirez et al., 2004; Swayne and Pantin-Jackwood, 2006). Although mortality rates of the Thai isolates were comparable between domestic and cross-bred ducks, neurological symptoms were less apparent in cross-bred ducks. The cross-bred ducks used in this study were F1 or F2 between mallards and domestic ducks. Age, which influences the immunological status of an animal, also affects the outcome of infection with the H5N1 HPAI viruses in ducks (Swayne and Pantin-Jackwood, 2006; Pantin-Jackwood et al., 2007). As-yet unidentified differences in genetic background as well as in the immunological status of the ducks appeared to moderate the manifestation of symptoms. A high incidence of sero-conversion of the ducks that survived infection with the Thai isolates was evident. This sharply contrasted with the chicken experimental study, in which none of the chickens surviving from 104 EID50 virus infection seroconverted. Ducks that survived HPAI infection could be protected or experience asymptomatic infections by a second exposure to another H5N1 virus, because of serum antibody against the viruses. In the asymptomatic infection of ducks, it is possible that the serum antibody may select an antigenic variant in vivo, causing antigenic drift of the viruses, as suggested in vaccinated chickens (Lee et al., 2004). Another study suggested that long-lasting virus shedding alone in an individual duck could provide an opportunity to generate an antigenic variant (Hulse-Post et al., 2005). Since duck farming is a significant livestock industry in Southeast Asia, the involvement of ducks in such antigenic drift mechanisms raise a concern on poultry vaccination strategies. Antigenic variants could silently circulate and be amplified within duck populations. Emerging antigenic variants would hamper vaccine efficacy in poultry in countries where a vaccination strategy is a part of an HPAI eradication program. Thus, control measures in duck populations in Southeast Asia should be given a high priority in order to eradicate HPAI in the region. Although all of the Thai isolates examined were 100% lethal to chickens with 106 EID50 viruses, the lethal dose of QaAng to chickens was demonstrated to
be higher than that of CkSup. QaAng may replicate more efficiently in an infected host. This would give it an advantage in overcoming a host defense system such as innate immunity and subsequently allow it to lethally infect chickens. Supporting this speculation is the fact that none of the survivors sero-converted in experimental infection with 104 and 102 EID50 of QaAng and difference between EID50 and ELD50. It has been suggested that high virus replication in a target organ was one of the factors determining the severity of the disease in a bird host (Swayne, 2007). Among the six amino acid differences across the whole genome between CkSup and QaAng, four were concentrated in the polymerase genes: 3 (N76D, R192K, and R386K) in the PB1 gene and 1 (A250V) in the PB2 gene. Molecular changes in the polymerase genes were shown to associate with the virulence of an H5N1 virus, A/Vietnam/1203/04, in mallards (HulsePost et al., 2007). T515A in the PA gene or Y436H in the PB1 gene was shown to abolish the virulence of A/ Vietnam/1203/04 to mallards. Although none of the differences observed between CkSup and QaAng corresponded to those substitutions, it could be speculated that some of the differences, and/or a combination of differences, may confer the strains with replication ability in chickens, resulting in a difference in virulence between the two strains. The other differences, one in HA1 (E140K) and the other in NA (H43Q), may not be involved in replication ability, although the possibility of their involvement cannot be completely ruled out. E140K resides on the globular head region of the HA1, but is not involved in receptor binding (Ha et al., 2001), and H43Q resides within the stalk of the NA protein (Miki et al., 1983), where substitution is less likely to affect the enzymatic activity of the NA. Our current study clearly demonstrated that the Thai isolates have a unique pathobiology in gallinaceous birds compared to observations previously reported. This indicates that the HPAI viruses currently circulating in the world have been changing their characteristics not only against ducks (SturmRamirez et al., 2004) but also against other avian species. Another important finding of this study is that postmortem swabs from H5N1 HPAIV infected dead ducks may not always contain a detectable level of virus with the virus isolation in eggs. This phenomenon would make field surveillance of the HPAI virus
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
in the duck population more difficult and would result in underestimation of the incidence of infection. Ducks infected with the HPAI virus could tolerate infection asymptomatically or with only slight manifestations. Even if the ducks die from infection, postmortem diagnosis may not reveal replication of the virus if the death occurred more than a certain period after infection. Control of HPAI viruses in the duck population is crucial for the effective control of HPAI in poultry in Southeastern Asia. Our study emphasizes the importance of understanding the pathobiology of currently circulating H5N1 viruses in ducks and poultry other than chickens in order to improve eradication strategies against those viruses.
Acknowledgement This work was supported in part by the program of Founding Research Center for Emerging and Reemerging Infectious Diseases launched by a project commissioned by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan.
References Alexander, D.J., 2007. Summary of avian influenza activity in Europe, Asia, Africa, and Australasia, 2002–2006. Avian Dis. 51, 161–166. Amonsin, A., Payungporn, S., Theamboonlers, A., Thanawongnuwech, R., Suradhat, S., Pariyothorn, N., Tantilertcharoen, R., Damrongwantanapokin, S., Buranathai, C., Chaisingh, A., Songserm, T., Poovorawan, Y., 2006. Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand. Virology 344, 480–491. Chen, H., Smith, G.J., Zhang, S.Y., Qin, K., Wang, J., Li, K.S., Webster, R.G., Peiris, J.S., Guan, Y., 2005. Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature 436, 191–192. Ha, Y., Stevens, D.J., Skehel, J.J., Wiley, D.C., 2001. X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc. Natl. Acad. Sci. U.S.A. 98, 11181–11186. Hiromoto, Y., Saito, T., Lindstrom, S., Nerome, K., 2000. Characterization of low virulent strains of highly pathogenic A/Hong Kong/156/97 (H5N1) virus in mice after passage in embryonated hens’ eggs. Virology 272, 429–437. Hulse-Post, D.J., Franks, J., Boyd, K., Salomon, R., Hoffmann, E., Yen, H.L., Webby, R.J., Walker, D., Nguyen, T.D., Webster, R.G., 2007. Molecular changes in the polymerase genes (PA and PB1) associated with high pathogenicity of H5N1 influenza virus in Mallard ducks. J. Virol. 81, 8515–8524.
73
Hulse-Post, D.J., Sturm-Ramirez, K.M., Humberd, J., Seiler, P., Govorkova, E.A., Krauss, S., Scholtissek, C., Puthavathana, P., Buranathai, C., Nguyen, T.D., Long, H.T., Naipospos, T.S., Chen, H., Ellis, T.M., Guan, Y., Peiris, J.S., Webster, R.G., 2005. Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia. Proc. Natl. Acad. Sci. U.S.A. 102, 10682–10687. Isoda, N., Sakoda, Y., Kishida, N., Bai, G.R., Matsuda, K., Umemura, T., Kida, H., 2006. Pathogenicity of a highly pathogenic avian influenza virus, A/chicken/Yamaguchi/7/04 (H5N1) in different species of birds and mammals. Arch. Virol. 151, 1267–1279. Kawaoka, Y., 1991. Equine H7N7 influenza A viruses are highly pathogenic in mice without adaptation: potential use as an animal model. J. Virol. 65, 3891–3894. Keawcharoen, J., Oraveerakul, K., Kuiken, T., Fouchier, R.A., Amonsin, A., Payungporn, S., Noppornpanth, S., Wattanodorn, S., Theambooniers, A., Tantilertcharoen, R., Pattanarangsan, R., Arya, N., Ratanakorn, P., Osterhaus, D.M., Poovorawan, Y., 2004. Avian influenza H5N1 in tigers and leopards. Emerg. Infect. Dis. 10, 2189–2191. Lee, C.W., Senne, D.A., Suarez, D.L., 2004. Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. J. Virol. 78, 8372–8381. Liu, J., Xiao, H., Lei, F., Zhu, Q., Qin, K., Zhang, X.W., Zhang, X.L., Zhao, D., Wang, G., Feng, Y., Ma, J., Liu, W., Wang, J., Gao, G.F., 2005. Highly pathogenic H5N1 influenza virus infection in migratory birds. Science 309, 1206. Lu, X., Tumpey, T.M., Morken, T., Zaki, S.R., Cox, N.J., Katz, J.M., 1999. A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans. J. Virol. 73, 5903–5911. Mase, M., Kim, J.H., Lee, Y.J., Tsukamoto, K., Imada, T., Imai, K., Yamaguchi, S., 2005. Genetic comparison of H5N1 influenza A viruses isolated from chickens in Japan and Korea. Microbiol. Immunol. 49, 871–874. Melville, D.S., Shortridge, K.F., 2006. Migratory waterbirds and avian influenza in the East Asian-Australasian Flyway with particular reference to the 2003–2004 H5N1 outbreak. In: Boere, G.C., Galbraith, C.A., Stroud, D.A. (Eds.), Waterbirds Around the World. The Stationery Office, Edinburgh, UK, pp. 432–438. Miki, T., Nishida, Y., Hisajima, H., Miyata, T., Kumahara, Y., Nerome, K., Oya, A., Fukui, T., Ohtsuka, E., Ikehara, M., et al., 1983. The complete nucleotide sequence of the influenza virus neuraminidase gene of A/NJ/8/76 strain and its evolution by segmental duplication and deletion. Mol. Biol. Med. 1, 401– 413. Pantin-Jackwood, M.J., Suarez, D.L., Spackman, E., Swayne, D.E., 2007. Age at infection affects the pathogenicity of Asian highly pathogenic avian influenza H5N1 viruses in ducks. Virus Res. 130, 151–161. Perez, D.R., Lim, W., Seiler, J.P., Yi, G., Peiris, M., Shortridge, K.F., Webster, R.G., 2003. Role of quail in the interspecies transmission of H9 influenza A viruses: molecular changes on HA that correspond to adaptation from ducks to chickens. J. Virol. 77, 3148–3156.
74
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
Perkins, L.E., Swayne, D.E., 2001. Pathobiology of A/chicken/Hong Kong/220/97 (H5N1) avian influenza virus in seven gallinaceous species. Vet. Pathol. 38, 149–164. Puthavathana, P., Auewarakul, P., Charoenying, P.C., Sangsiriwut, K., Pooruk, P., Boonnak, K., Khanyok, R., Thawachsupa, P., Kijphati, R., Sawanpanyalert, P., 2005. Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand. J. Gen. Virol. 86, 423–433. Songserm, T., Amonsin, A., Jam-on, R., Sae-Heng, N., Pariyothorn, N., Payungporn, S., Theamboonlers, A., Chutinimitkul, S., Thanawongnuwech, R., Poovorawan, Y., 2006. Fatal avian influenza A H5N1 in a dog. Emerg. Infect. Dis. 12, 1744–1747. Songsermn, T., Amonsin, A., Jam-on, R., Sae-Heng, N., Meemak, N., Pariyothorn, N., Payungporn, S., Theamboonlers, A., Poovorawan, Y., 2006. Avian influenza H5N1 in naturally infected domestic cat. Emerg. Infect. Dis. 12, 681–683. Sturm-Ramirez, K.M., Ellis, T., Bousfield, B., Bissett, L., Dyrting, K., Rehg, J.E., Poon, L., Guan, Y., Peiris, M., Webster, R.G., 2004. Reemerging H5N1 influenza viruses in Hong Kong in 2002 are highly pathogenic to ducks. J. Virol. 78, 4892–4901.
Swayne, D.E., 2007. Understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds. Avian Dis. 51, 242–249. Swayne, D.E., Pantin-Jackwood, M., 2006. Pathogenicity of avian influenza viruses in poultry. Dev. Biol. (Basel) 124, 61–67. Wang, G., Zhan, D., Li, L., Lei, F., Liu, B., Liu, D., Xiao, H., Feng, Y., Li, J., Yang, B., Yin, Z., Song, X., Zhu, X., Cong, Y., Pu, J., Wang, J., Liu, J., Gao, G.F., Zhu, Q., 2008. H5N1 avian influenza re-emergence of Lake Qinghai: phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation. J. Gen. Virol. 89, 697–702. Webster, R.G., Guan, Y., Peiris, M., Walker, D., Krauss, S., Zhou, N.N., Govorkova, E.A., Ellis, T.M., Dyrting, K.C., Sit, T., Perez, D.R., Shortridge, K.F., 2002. Characterization of H5N1 influenza viruses that continue to circulate in geese in southeastern China. J. Virol. 76, 118–126. Xu, K.M., Li, K.S., Smith, G.J., Li, J.W., Tai, H., Zhang, J.X., Webster, R.G., Peiris, J.S., Chen, H., Guan, Y., 2007. Evolution and molecular epidemiology of H9N2 influenza A viruses from quail in southern China, 2000–2005. J. Virol. 81, 2635–2645.
Veterinary Microbiology 133 (2009) 65–74 www.elsevier.com/locate/vetmic
Pathogenicity of highly pathogenic avian influenza viruses of H5N1 subtype isolated in Thailand for different poultry species Takehiko Saito a,b,*, Chiaki Watanabe a,b, Nobuhiro Takemae a,b, Arunee Chaisingh c, Yuko Uchida a,b, Chantanee Buranathai d, Hirofumi Suzuki a, Masatoshi Okamatsu a, Tadao Imada e, Sujira Parchariyanon c, Nimit Traiwanatam c, Shigeo Yamaguchi a a
Research Team for Zoonotic Diseases, National Institute of Animal Health, National Agriculture and Food Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan b Thailand-Japan Zoonotic Diseases Collaboration Center (ZDCC), Bangkok, Thailand c National Institute of Animal Health, Bangkok, Thailand d Department of Livestock and Development, Ministry of Agriculture and Cooperatives, Bangkok, Thailand e Kyushu Branch, National Institute of Animal Health, National Agriculture and Food Organization (NARO), 2702 Nakayamacho, Kagoshima 891-0105, Japan Received 16 April 2008; received in revised form 13 June 2008; accepted 26 June 2008
Abstract Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused several rounds of outbreaks in Thailand. In this study, we used 3 HPAI viruses isolated in Thailand in January 2004 from chicken, quail, and duck for genetic and pathogenetic studies. Sequence analysis of the entire genomes of these isolates revealed that they were genetically similar to each other. Chickens, quails, domestic ducks, and cross-bred ducks were inoculated with these isolates to evaluate their pathogenicity to different host species. A/chicken/Yamaguchi/7/04 (H5N1), an HPAI virus isolated in Japan, was also used in the chicken and quail studies for comparison. All four isolates were shown to be highly pathogenic to chickens and quails, with 100% mortality by 106 EID50 inoculants of the viruses. They caused sudden death in chickens and quails within 2–4 days after inoculation. The mean death times (MDT) of quails infected with the Thai isolates were shorter than those of chickens infected with the same isolates. Mortality against domestic and cross-bred ducks ranged from 50 to 75% by intranasal inoculation with the 106 EID50 viruses. Neurological symptoms were observed in most of the inoculated domestic ducks and appeared less severe in the cross-bred ducks. The MDTs of the ducks infected with the Thai isolates were 4.8–6 days post-inoculation. Most of the surviving ducks infected with the Thai isolates had sero-converted until 14 dpi. Our study illustrated the pathobiology of the Thai isolates against different poultry species and would provide useful information for improving control strategies against HPAI. # 2008 Elsevier B.V. All rights reserved. Keywords: HPAI; Poultry; Influenza virus; Pathogenicity
* Corresponding author at: Research Team for Zoonotic Diseases, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Tel.: +81 29 838 7802; fax: +81 29 838 7802. E-mail address: [email protected] (T. Saito). 0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.06.020
66
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
1. Introduction Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have been proven widespread in Southeast Asia since 2004. Thailand is one of the countries heavily affected by the HPAI outbreaks. Since HPAI was first recognized in that country in January 2004, nearly 2000 outbreaks have been recognized there. Several species of poultry, including chickens, quails, and domestic ducks, as well as humans, tigers, and wild birds, were involved in those outbreaks. Involvement of many species of poultry, as well as of mammalians and wildlife, is a unique property of the recent HPAI incidents in the region. In 14 countries worldwide, 372 human cases were confirmed between 2004 and March 2008, among which 235 were fatal. In Thailand alone, 25 human cases have been confirmed. In the early period of the HPAI outbreaks, outbreaks in tigers that had been fed infected chicken carcasses were reported in Thailand (Amonsin et al., 2006; Keawcharoen et al., 2004). Infection of dogs and cats in Thailand was also reported (Songserm et al., 2006; Songsermn et al., 2006). Several species of wild birds were also confirmed to be infected by H5N1 HPAI viruses in Thailand (Melville and Shortridge, 2006). It has been apparent that the pathogenicity of the currently circulating HPAI viruses of the H5N1 subtype, which are descendants of A/goose/Guangdong/1/96, against avian and mammalian species has been changing as the viruses have become prevalent in Eurasia and Africa. Type A influenza viruses, except H7N7 equine influenza A viruses, had been considered to require adaptation before causing lethal infection in a mouse model (Kawaoka, 1991). However, HPAI viruses that were isolated from patients suffering from HPAI infection in Hong Kong in 1997 caused lethal and systematic infection in a mouse experimental model without adaptation (Lu et al., 1999). It had also been thought that H5 HPAI virus infection did not result in severe diseases in waterfowl. HPAI viruses of the H5 subtype had never been isolated from wild birds, except from terns in South Africa in 1961. Such observations were also shown to be no longer valid after a die-off of wild birds by the H5N1 viruses was recognized in Hong Kong in 2002. Some of the viruses isolated from the outbreak were shown to cause lethal infection in mallards (Anas platyrhynchos) (Sturm-
Ramirez et al., 2004). During the 2004–2005 HPAI outbreaks, nearly 50 species of wild birds were shown to be affected by the H5N1 viruses in East and Southeast Asia (Melville and Shortridge, 2006). Furthermore, more than 6000 migratory birds died from infection with HPAI viruses of the H5N1 subtype at Qinghai Lake, China, in May 2005 (Chen et al., 2005; Liu et al., 2005). Following the Qinghai outbreak, related strains, so-called ‘‘Qinghai strains’’, spread worldwide, reaching Europe and Africa (Alexander, 2007). Similar strains also re-emerged at Qinghai Lake in May 2006 (Wang et al., 2008) Because of the changing pathogenicity of the currently circulating H5N1 viruses against different hosts, it is crucial to understand the pathobiology of the viruses in different hosts in order to establish efficacious eradication strategies. In this study, we characterized three HPAI viruses isolated in Thailand in January 2004 genetically and pathobiologically. Experimental infection studies using four different poultry species demonstrated a divergence in their pathogenicity against poultry, although genetic characterization revealed extensive similarities among those isolates. The results obtained in this study would provide useful information for improving surveillance and eradication strategies and would provide a clue to understanding the epidemiology of the HPAI viruses.
2. Materials and methods 2.1. Viruses Three isolates from Thailand (Table 1) were obtained from the National Institute of Animal Health, Thailand. They, as well as A/chicken/Yamaguchi/7/ 2004, were isolated and propagated with 10- to 11day-old embryonated hen’s eggs at 37 8C. All of the experiments using intact viruses, including animal experiments, were carried out in BSL-3 facilities at the National Institute of Animal Health, Japan. 2.2. Sequence analyses Viral RNA was extracted from virus-containing allantoic fluid by using a commercial kit (RNeasy, Qiagen, Hilden, Germany). After reverse transcription with Superscript III (Invitrogen, Carlsbad, CA, USA),
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
67
Table 1 Thailand HPAI viruses used in this study Strains
Date of collection
Province/city
EID50/mla
ELD50/mlb
EID50/ELD50
TCID50/mlc
Ck/Suphanburi/1/04
2004/1/21
9.1
7.4
47
6.5 (6.3)
512
Qa/Angthong/71/04 Dk/Angthong/72/04 Ck/Yamaguchi/7/04
2004/1/23 2004/1/23 2004/1/9
Suphanburi/Bang Pla Mok Angthong/Pa Mok Angthong/Pa Mok Yamaguchi/Ato
9.0 8.1 8.7
9.0 6.4 8.7
1 47 1
6.5 (7.5) 7.0 (6.7) 7.6 (7.6)
1024 64 64
a b c
HA titer
Virus titer expressed as log10. Lethality was determined at 48 h after inoculation and titers were expressed as log10. Virus titer was measured by infecting MDCK cells with and without (in parentheses) trypsin.
cDNAs were amplified by polymerase chain reaction (PCR) with ExTaq (Takara, Shiga, Japan) with segment-specific primers (the primer sequences are available upon request). PCR products were purified by the QIAquick PCR purification kit (Qiagen) and subjected to sequencing with the Big Dye Terminator sequencing kit, version 3.1 (Applied Biosystems, Foster City, CA, USA). Reactions were analyzed on an ABI Prism 3100 genetic analyzer (Applied Biosystems). The nucleotide sequences obtained were assembled by using Sequencher version 4.6 (Hitachi Software Engineering, Tokyo, Japan) and were translated into deduced amino acid sequences by GENETYX-WIN (GENETYX, Tokyo, Japan). The nucleotide sequences obtained in this study are available from GenBank under accession numbers AB440324–AB440347. 2.3. Serological tests A hemagglutination inhibition (HI) test was performed by a conventional microplate method with chicken red blood cells. Serum samples drawn from surviving animals at the end of a 14-day observation period after inoculation were treated with RDE (II) (Denka Seiken, Niigata, Japan) to remove non-specific inhibitors in sera. 2.4. Experimental infection of poultry Specific pathogen-free (SPF) chickens (Gallus gallus domesticus) and Japanese quails (Coturnix japonica) were purchased from Nisseiken (Yamanashi, Japan). Conventional domestic ducks (Cherry Valley) and cross-bred ducks were purchased from farms in Japan. The cross-bred ducks used in this study were F1
or F2 between mallards and domestic ducks. All of the birds were 4 weeks old at the time of inoculation. Groups of 8 birds of respective species were inoculated intranasally with 106 50% egg infective dose (EID50) of each virus. In the chicken experiment, 104 and 102 EID50 virus doses were also used for A/chicken/ Suphanburi/1/2004 and A/quail/Angthong/71/2004. Inoculated birds were observed for clinical symptoms for 14 days. Oropharyngeal and cloacal swabs were taken from birds that had died during the observation period. Swabs were suspended in Dulbecco’s Modified Eagle’s Medium containing antibiotics and bovine serum albumin. After brief centrifugation, the supernatants were stored at 80 8C until used. Swab samples were titrated in embryonated hen’s eggs. The antibody responses of the surviving birds against the inoculated virus were examined by means of HI assay. The experimental procedures and care of animals were approved by the Animal Experiment Committee of National Institute of Animal Health.
3. Results 3.1. Virus properties Three strains of HPAI viruses of the H5N1 subtype—A/chicken/Suphanburi/1/2004 (CkSup), A/ duck/Angthong/72/2004 (DkAng), and A/quail/ Angthong/71/2004 (QaAng)—were isolated in the early period of HPAI outbreaks in Thailand in January 2004 (Table 1). CkSup was isolated from a dead chicken, whereas DkAng and QaAng were isolated from live domestic duck and quail, respectively. Suphanburi and Angthong are provinces in central Thailand.
68
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
Table 2 Amino acid differences among the Thai isolates used in this studya Strain
Gene segment and amino acid number PA
Ck/Suphanburi/1/04 Qa/Angthong/71/04 Dk/Angthon g/72/04 a
PB1
PB2
HA
NP
NA
NS1
NS2
122
76
192
386
195
250
140 (HA1)
456
43
57
73
115
V
N D D
R K
R K K
D
A V V
E K K
V
H Q
A
S
A
S
T
T
E
N
A
Differences from Ck/Suphanburi/1/04 are shown.
Sequence analysis revealed a high rate of identity among those isolates, suggesting a rapid dissemination of the HPAI viruses within the country in the early phase of the outbreak. The sequences of the entire genomes of three isolates were determined, and more than 99% identity was observed in both the nucleotide and deduced amino acid sequences of all 11 coding regions among the isolates. In particular, 100% identity in the deduced amino acid sequence was observed with M1, M2, and PB1-F2 proteins among all three isolates. In NP, NS1, and NS2 proteins, CkSup and QaAng showed 100% identity and CkSup and DkAng showed 100% identity in NA protein. The amino acid differences among the Thai isolates used in this study are listed in Table 2. All of the viruses replicated to higher titers in embryonated eggs than in MDCK cells (Table 1). Lethality to eggs, judged at 48 h after inoculation, differed among the isolates. The 50% egg infectious dose (EID50) and the 50% egg lethal dose (ELD50) were almost the same for QaAng; on the other hand, the ratios between them were approximately 50 for CkSup and DkAng, indicating that QaAng was the
most virulent to embryonated eggs among the isolates used with a given dose. QaAng gave 10-times higher titer in MDCK cells when it was cultured without exogenous trypisn, whereas the other two isolates showed comparable titers with and without trypsin in MDCK cells. The highly pathogenic nature of the three Thai isolates, along with Ck/Yamaguchi/07/2004 (CkYama), was demonstrated by intranasal administration against two species of gallinaceous birds: chickens and Japanese quail (Table 3). In the eight birds tested for each species, 100% mortality was observed with an intranasally administered virus of 106 EID50. Both species died without showing apparent symptoms; the rather sudden death appears to be a characteristic of infection with those viruses. For chickens, mean death time (MDT) was less than 3 days postinoculation with the Thai isolates as well as with CkYama (Table 3). The shortest MDT was observed for chickens inoculated with DkAng (1.4 days postinoculation, DPI), which was significantly shorter than MDTs obtained for CkSup ( p = 0.001) and CkYama ( p = 0.014). For quails, all of the birds
Table 3 Mortality and sero-conversion of experimentally inoculated poultry Strain
Ck/Suphanburi/1/04 Qa/Angthong/71/04 Dk/Angthong/72/04 Ck/Yamaguchi/7/04 a b c d
Chicken
Quail
Duck
Cross-bred duck
Mortality (%)
MDTa
Mortality (%)
MDT
Mortality (%)
MDT
Ab (+)/ survivors
Mortality (%)
MDT
Ab (+)/ survivors
100 100 100 100
2.3 1.9 1.4 c 2.0
100 100 100 100
1.4 1.1 1.0 3.4 d
62.5 62.5 50 NT
6.0 6.0 6.0 NT
2/3 (128)b 3/3 (102) 0/4
50 50 75
6.3 5.3 4.8
4/4 (45) 2/4 (16) 0/2
Mean death time (days postinoculation) among dead animals. Geometric mean of HI antibody titers among sero-positive animals at 14 DPI is shown in parentheses. Significant to Ck/Suphanburi ( p = 0.005) and Ck/Yamaguchi ( p = 0.014) by ANOVA, followed by Tukey analysis. Significant to the rest of the group by ANOVA ( p < 0.001).
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
inoculated with DkAng died within 1 DPI (MDT = 1.0), and within 2 DPI with CkSup (MDT = 1.4) and QaAng (MDT = 1.1). It appears that quails died earlier than chickens when they were inoculated with either CkSup ( p = 0.005) or QaAng ( p = 0.025) (Table 3). In contrast, the MDT was significantly longer for quails inoculated with CkYam than for chickens ( p < 0.001). 3.2. Pathogenicity in gallinaceous poultry The lethal dose against chickens was shown to be different between CkSup and QaAng when 102 and 104 EID50 of CkSup or QaAng were inoculated intranasally to 8 chickens in each group and observation for mortality was for two weeks (Fig. 1a and b). Only 3 out of the 8 chickens inoculated with 104 EID of CkSup died. On the other hand, 6 out of 8 died after inoculation with the same dose of QaAng. As described above, all chickens inoculated with 106 EID50 of either CkSup or QaAng died within 4 DPI or 2 DPI, respectively. None of the chickens inoculated with 102 EID50 of any of these viruses died during the observation period. These results indicated that the 50% chicken lethal dose (CLD50) of CkSup could be more than 104 EID50 and that of QaAng could be less than 104 EID50. This suggests that there was an approximately 100-fold
69
difference between CLD50 of CkSup and QaAng. In addition, none of the surviving chickens had seroconverted at 14 DPI against any of the inoculated strains. Virus titers in cloacal and oropharyngeal swabs of dead quails were shown to be different from CkSup and CkYam (Fig. 2). Postmortem swabs from 3 out of the 5 quails that died at 1 DPI and of all 3 that died at 2 DPI inoculated with CkSup, and from 3 out of the 5 quails that died at 3 DPI and of all 3 that died at 4 DPI inoculated with CkYam, were subjected to virus titration in embryonated eggs. None of the cloacal swabs from the quails that died at 1 DPI yielded any virus above the detection level (<101.2 EID50/ml). Virus titers in the oropharyngeal swabs obtained from quails that died at 1 DPI showed lower titers (2101.3, 2101.3, and 102.9 EID50/ml) than those obtained from quails that died at 2 DPI. No significant difference was observed in virus titers of swabs collected from quails inoculated with CkYam at different time points. Mean virus titers in postmortem cloacal and oropharyngeal swabs from quails inoculated with CkSup were significantly lower than those inoculated with CkYam (Fig. 2). 3.3. Pathogenicity in anseriform poultry Pathogenicity against anseriformes birds was evaluated by intranasal inoculation of the viruses to
Fig. 1. Survival rates of chickens infected with various doses of Ck/Suphanburi/1/04 or Qa/Angthong/71/04. (^) 1 106 EID50/animal intranasally, (&) 1 104 EID50/animal intranasally, (~) 1 102 EID50/animal intranasally.
70
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
Fig. 2. Whiskered box plots of virus titers in cloacal or oropharyngeal swabs from quails inoculated with Ck/Suphanburi/1/04 or Ck/Yamaguchi/7/04.
domestic ducks and cross-bred ducks. Domestic ducks inoculated with the Thai isolates died at a mean death time of 6 DPI, and mortality rates were 50–63% (Table 3 and Fig. 3a and b). Hemorrhages of beak, depression, and torticollis were commonly observed symptoms among the dying ducks. Some of the survivors recovered from torticollis and general behavioral aberrations during the observation period. The mortality rate and MDT of cross-bred ducks infected with the Thai isolates were also similar to those obtained for domestic ducks: 50–75% mortality
Fig. 4. Virus titers in postmortem cloacal or oropharyngeal swabs taken from dead ducks inoculated with Ck/Suphanburi/1/04. Marks under dotted lines mean virus titers under the detection level, which was 1.19 log10 EID50/ml.
and 4.8–6.3 DPI for MDT. In contrast to domestic ducks, cross-bred ducks showed no apparent symptoms, except depression, before death. Virus titers in postmortem cloacal and oropharyngeal swabs from the domestic ducks infected with CkSup were measured (Fig. 4). Both cloacal and oropharyngeal swabs obtained from the ducks that died at 4 DPI showed high virus titers. Although virus replication was confirmed in one oropharyngeal swab obtained from a duck that died at 7 DPI, other swabs,
Fig. 3. Survival rates of (a) domestic ducks or (b) cross-bred ducks inoculated intranasally by 1 106 EID50/animal of the Thai HPAI isolates ((*) Ck/Suphanburi/1/04, (&) Dk/Angthong/72/04, (~) Qa/Angthong/71/04).
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
one oropharyngeal and two cloacal, did not yield viruses above detectable level (101.2 EID50/ml). Likewise, neither cloacal nor oropharyngeal swabs taken at 8 DPI yielded a virus. These observations suggested that ducks infected with the Thai isolates did not always die at the peak of virus replication in the organs. Sero-conversion was confirmed in most of the surviving domestic and cross-bred ducks, which were inoculated with either CkSup or QaAng. On the other hand, it was also noteworthy that none of the survivors infected with DkAng sero-converted in those experiments. These results clearly demonstrated that the Thai strains used in this study were infectious and significantly lethal to domestic and cross-bred ducks at a given dose.
4. Discussion Our study demonstrated that the HPAI viruses isolated in Thailand during the early period of the outbreaks were lethal not only in gallinaceous birds, chicken and Japanese quail, but also in anseriformes birds. Although the strains are genetically highly similar to each other, their pathobiological features varied among the strains. It is noteworthy that the Thai strains used in this study killed quails faster than chickens, while Ck/Yamaguchi killed chickens faster. In previous reports (Perkins and Swayne, 2001; Isoda et al., 2006), several H5N1 viruses killed chickens faster than quail, as we observed for Ck/Yamaguchi. It has been suggested that the maximal pathogenicity of HPAI viruses was achieved in their host of origin because of species adaptation (Perkins and Swayne, 2001). However, this was not the case in our experiment; there were no significant differences among the three Thai strains isolated from three distinct hosts—chicken, quail, and duck—in their ability to kill quails. The Thai HPAI viruses used in this study might have adapted to quail before being transmitted to chickens, thus showing higher virulence in the former. Quail has been suggested to serve as an intermediate host that provides an environment in which influenza viruses can generate variants that can be transmitted to chickens (Perez et al., 2003; Xu et al., 2007). It was also shown that quails were more susceptible to Gs/HK/437-4/99 (H5N1) infection than
71
chickens (Webster et al., 2002). Different genetic constellations may also explain the differences in pathobiology between the Thai isolates and CkYam. The genotype of the Thai isolates was designated genotype Z (Puthavathana et al., 2005), whereas, that of CkYam was designated genotype V (Mase et al., 2005). There are several differences in deduced amino acids throughout their genome, suggesting that some of the different amino acids and/or combinations of them cause the differences in the pathobiology of the viruses. Another intriguing observation obtained with the experimental infection study in quail was that virus titers in postmortem oropharyngeal and cloacal swabs were significantly higher in quails infected with CkYam than in those infected with CkSup, although CkSup killed quails much more rapidly than CkYam. Level of virus replication in the respiratory tract or intestine may not necessarily correlate with virulence. A small plaque clone of a human isolate, A/Vietnam/ 1203/04, has low pathogenicity to mallards, while a large plaque clone of the same isolate is highly pathogenic to mallards (Hulse-Post et al., 2007). It was previously shown that both clones replicated comparably to each other in the trachea and cloaca of intravenously inoculated ducks. Virus replication in a particular target organ other than respiratory or intestinal organs may contribute to the virulence of the Thai isolates in quails. An MDCK-passaged strain of A/Hong Kong/156/97 (H5N1), which was isolated from an index case of a Hong Kong H5N1 incident in 1997, replicated well in mouse brain after intranasal inoculation and was highly virulent in mouse, whereas an egg-passaged strain of the same virus did not replicate in mouse brain and had low virulence in mouse (Hiromoto et al., 2000). Both strains replicated to similar levels in the lungs of intranasally inoculated mice. Differences in tissue tropism and/or in virus replication in a particular tissue of the H5N1 viruses in quails need to be examined to elucidate the variability of virulence in quails. Since late 2002, H5N1 influenza viruses with high pathogenicity to ducks have been recognized. The three Thai isolates used in this study were mild to highly pathogenic to both domestic and cross-bred ducks, in contrast with previous studies with Thai H5N1 isolates, which demonstrated that the isolates were relatively mildly pathogenic to older ducks (Hulse-Post et al., 2005; Pantin-Jackwood et al.,
72
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
2007). Pathogenicity to ducks varies considerably from strain to strain among the H5N1 HPAI isolated after late 2002, and even genetically similar strains could differ in their virulence in ducks (SturmRamirez et al., 2004; Swayne and Pantin-Jackwood, 2006). Although mortality rates of the Thai isolates were comparable between domestic and cross-bred ducks, neurological symptoms were less apparent in cross-bred ducks. The cross-bred ducks used in this study were F1 or F2 between mallards and domestic ducks. Age, which influences the immunological status of an animal, also affects the outcome of infection with the H5N1 HPAI viruses in ducks (Swayne and Pantin-Jackwood, 2006; Pantin-Jackwood et al., 2007). As-yet unidentified differences in genetic background as well as in the immunological status of the ducks appeared to moderate the manifestation of symptoms. A high incidence of sero-conversion of the ducks that survived infection with the Thai isolates was evident. This sharply contrasted with the chicken experimental study, in which none of the chickens surviving from 104 EID50 virus infection seroconverted. Ducks that survived HPAI infection could be protected or experience asymptomatic infections by a second exposure to another H5N1 virus, because of serum antibody against the viruses. In the asymptomatic infection of ducks, it is possible that the serum antibody may select an antigenic variant in vivo, causing antigenic drift of the viruses, as suggested in vaccinated chickens (Lee et al., 2004). Another study suggested that long-lasting virus shedding alone in an individual duck could provide an opportunity to generate an antigenic variant (Hulse-Post et al., 2005). Since duck farming is a significant livestock industry in Southeast Asia, the involvement of ducks in such antigenic drift mechanisms raise a concern on poultry vaccination strategies. Antigenic variants could silently circulate and be amplified within duck populations. Emerging antigenic variants would hamper vaccine efficacy in poultry in countries where a vaccination strategy is a part of an HPAI eradication program. Thus, control measures in duck populations in Southeast Asia should be given a high priority in order to eradicate HPAI in the region. Although all of the Thai isolates examined were 100% lethal to chickens with 106 EID50 viruses, the lethal dose of QaAng to chickens was demonstrated to
be higher than that of CkSup. QaAng may replicate more efficiently in an infected host. This would give it an advantage in overcoming a host defense system such as innate immunity and subsequently allow it to lethally infect chickens. Supporting this speculation is the fact that none of the survivors sero-converted in experimental infection with 104 and 102 EID50 of QaAng and difference between EID50 and ELD50. It has been suggested that high virus replication in a target organ was one of the factors determining the severity of the disease in a bird host (Swayne, 2007). Among the six amino acid differences across the whole genome between CkSup and QaAng, four were concentrated in the polymerase genes: 3 (N76D, R192K, and R386K) in the PB1 gene and 1 (A250V) in the PB2 gene. Molecular changes in the polymerase genes were shown to associate with the virulence of an H5N1 virus, A/Vietnam/1203/04, in mallards (HulsePost et al., 2007). T515A in the PA gene or Y436H in the PB1 gene was shown to abolish the virulence of A/ Vietnam/1203/04 to mallards. Although none of the differences observed between CkSup and QaAng corresponded to those substitutions, it could be speculated that some of the differences, and/or a combination of differences, may confer the strains with replication ability in chickens, resulting in a difference in virulence between the two strains. The other differences, one in HA1 (E140K) and the other in NA (H43Q), may not be involved in replication ability, although the possibility of their involvement cannot be completely ruled out. E140K resides on the globular head region of the HA1, but is not involved in receptor binding (Ha et al., 2001), and H43Q resides within the stalk of the NA protein (Miki et al., 1983), where substitution is less likely to affect the enzymatic activity of the NA. Our current study clearly demonstrated that the Thai isolates have a unique pathobiology in gallinaceous birds compared to observations previously reported. This indicates that the HPAI viruses currently circulating in the world have been changing their characteristics not only against ducks (SturmRamirez et al., 2004) but also against other avian species. Another important finding of this study is that postmortem swabs from H5N1 HPAIV infected dead ducks may not always contain a detectable level of virus with the virus isolation in eggs. This phenomenon would make field surveillance of the HPAI virus
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
in the duck population more difficult and would result in underestimation of the incidence of infection. Ducks infected with the HPAI virus could tolerate infection asymptomatically or with only slight manifestations. Even if the ducks die from infection, postmortem diagnosis may not reveal replication of the virus if the death occurred more than a certain period after infection. Control of HPAI viruses in the duck population is crucial for the effective control of HPAI in poultry in Southeastern Asia. Our study emphasizes the importance of understanding the pathobiology of currently circulating H5N1 viruses in ducks and poultry other than chickens in order to improve eradication strategies against those viruses.
Acknowledgement This work was supported in part by the program of Founding Research Center for Emerging and Reemerging Infectious Diseases launched by a project commissioned by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan.
References Alexander, D.J., 2007. Summary of avian influenza activity in Europe, Asia, Africa, and Australasia, 2002–2006. Avian Dis. 51, 161–166. Amonsin, A., Payungporn, S., Theamboonlers, A., Thanawongnuwech, R., Suradhat, S., Pariyothorn, N., Tantilertcharoen, R., Damrongwantanapokin, S., Buranathai, C., Chaisingh, A., Songserm, T., Poovorawan, Y., 2006. Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand. Virology 344, 480–491. Chen, H., Smith, G.J., Zhang, S.Y., Qin, K., Wang, J., Li, K.S., Webster, R.G., Peiris, J.S., Guan, Y., 2005. Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature 436, 191–192. Ha, Y., Stevens, D.J., Skehel, J.J., Wiley, D.C., 2001. X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc. Natl. Acad. Sci. U.S.A. 98, 11181–11186. Hiromoto, Y., Saito, T., Lindstrom, S., Nerome, K., 2000. Characterization of low virulent strains of highly pathogenic A/Hong Kong/156/97 (H5N1) virus in mice after passage in embryonated hens’ eggs. Virology 272, 429–437. Hulse-Post, D.J., Franks, J., Boyd, K., Salomon, R., Hoffmann, E., Yen, H.L., Webby, R.J., Walker, D., Nguyen, T.D., Webster, R.G., 2007. Molecular changes in the polymerase genes (PA and PB1) associated with high pathogenicity of H5N1 influenza virus in Mallard ducks. J. Virol. 81, 8515–8524.
73
Hulse-Post, D.J., Sturm-Ramirez, K.M., Humberd, J., Seiler, P., Govorkova, E.A., Krauss, S., Scholtissek, C., Puthavathana, P., Buranathai, C., Nguyen, T.D., Long, H.T., Naipospos, T.S., Chen, H., Ellis, T.M., Guan, Y., Peiris, J.S., Webster, R.G., 2005. Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia. Proc. Natl. Acad. Sci. U.S.A. 102, 10682–10687. Isoda, N., Sakoda, Y., Kishida, N., Bai, G.R., Matsuda, K., Umemura, T., Kida, H., 2006. Pathogenicity of a highly pathogenic avian influenza virus, A/chicken/Yamaguchi/7/04 (H5N1) in different species of birds and mammals. Arch. Virol. 151, 1267–1279. Kawaoka, Y., 1991. Equine H7N7 influenza A viruses are highly pathogenic in mice without adaptation: potential use as an animal model. J. Virol. 65, 3891–3894. Keawcharoen, J., Oraveerakul, K., Kuiken, T., Fouchier, R.A., Amonsin, A., Payungporn, S., Noppornpanth, S., Wattanodorn, S., Theambooniers, A., Tantilertcharoen, R., Pattanarangsan, R., Arya, N., Ratanakorn, P., Osterhaus, D.M., Poovorawan, Y., 2004. Avian influenza H5N1 in tigers and leopards. Emerg. Infect. Dis. 10, 2189–2191. Lee, C.W., Senne, D.A., Suarez, D.L., 2004. Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. J. Virol. 78, 8372–8381. Liu, J., Xiao, H., Lei, F., Zhu, Q., Qin, K., Zhang, X.W., Zhang, X.L., Zhao, D., Wang, G., Feng, Y., Ma, J., Liu, W., Wang, J., Gao, G.F., 2005. Highly pathogenic H5N1 influenza virus infection in migratory birds. Science 309, 1206. Lu, X., Tumpey, T.M., Morken, T., Zaki, S.R., Cox, N.J., Katz, J.M., 1999. A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans. J. Virol. 73, 5903–5911. Mase, M., Kim, J.H., Lee, Y.J., Tsukamoto, K., Imada, T., Imai, K., Yamaguchi, S., 2005. Genetic comparison of H5N1 influenza A viruses isolated from chickens in Japan and Korea. Microbiol. Immunol. 49, 871–874. Melville, D.S., Shortridge, K.F., 2006. Migratory waterbirds and avian influenza in the East Asian-Australasian Flyway with particular reference to the 2003–2004 H5N1 outbreak. In: Boere, G.C., Galbraith, C.A., Stroud, D.A. (Eds.), Waterbirds Around the World. The Stationery Office, Edinburgh, UK, pp. 432–438. Miki, T., Nishida, Y., Hisajima, H., Miyata, T., Kumahara, Y., Nerome, K., Oya, A., Fukui, T., Ohtsuka, E., Ikehara, M., et al., 1983. The complete nucleotide sequence of the influenza virus neuraminidase gene of A/NJ/8/76 strain and its evolution by segmental duplication and deletion. Mol. Biol. Med. 1, 401– 413. Pantin-Jackwood, M.J., Suarez, D.L., Spackman, E., Swayne, D.E., 2007. Age at infection affects the pathogenicity of Asian highly pathogenic avian influenza H5N1 viruses in ducks. Virus Res. 130, 151–161. Perez, D.R., Lim, W., Seiler, J.P., Yi, G., Peiris, M., Shortridge, K.F., Webster, R.G., 2003. Role of quail in the interspecies transmission of H9 influenza A viruses: molecular changes on HA that correspond to adaptation from ducks to chickens. J. Virol. 77, 3148–3156.
74
T. Saito et al. / Veterinary Microbiology 133 (2009) 65–74
Perkins, L.E., Swayne, D.E., 2001. Pathobiology of A/chicken/Hong Kong/220/97 (H5N1) avian influenza virus in seven gallinaceous species. Vet. Pathol. 38, 149–164. Puthavathana, P., Auewarakul, P., Charoenying, P.C., Sangsiriwut, K., Pooruk, P., Boonnak, K., Khanyok, R., Thawachsupa, P., Kijphati, R., Sawanpanyalert, P., 2005. Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand. J. Gen. Virol. 86, 423–433. Songserm, T., Amonsin, A., Jam-on, R., Sae-Heng, N., Pariyothorn, N., Payungporn, S., Theamboonlers, A., Chutinimitkul, S., Thanawongnuwech, R., Poovorawan, Y., 2006. Fatal avian influenza A H5N1 in a dog. Emerg. Infect. Dis. 12, 1744–1747. Songsermn, T., Amonsin, A., Jam-on, R., Sae-Heng, N., Meemak, N., Pariyothorn, N., Payungporn, S., Theamboonlers, A., Poovorawan, Y., 2006. Avian influenza H5N1 in naturally infected domestic cat. Emerg. Infect. Dis. 12, 681–683. Sturm-Ramirez, K.M., Ellis, T., Bousfield, B., Bissett, L., Dyrting, K., Rehg, J.E., Poon, L., Guan, Y., Peiris, M., Webster, R.G., 2004. Reemerging H5N1 influenza viruses in Hong Kong in 2002 are highly pathogenic to ducks. J. Virol. 78, 4892–4901.
Swayne, D.E., 2007. Understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds. Avian Dis. 51, 242–249. Swayne, D.E., Pantin-Jackwood, M., 2006. Pathogenicity of avian influenza viruses in poultry. Dev. Biol. (Basel) 124, 61–67. Wang, G., Zhan, D., Li, L., Lei, F., Liu, B., Liu, D., Xiao, H., Feng, Y., Li, J., Yang, B., Yin, Z., Song, X., Zhu, X., Cong, Y., Pu, J., Wang, J., Liu, J., Gao, G.F., Zhu, Q., 2008. H5N1 avian influenza re-emergence of Lake Qinghai: phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation. J. Gen. Virol. 89, 697–702. Webster, R.G., Guan, Y., Peiris, M., Walker, D., Krauss, S., Zhou, N.N., Govorkova, E.A., Ellis, T.M., Dyrting, K.C., Sit, T., Perez, D.R., Shortridge, K.F., 2002. Characterization of H5N1 influenza viruses that continue to circulate in geese in southeastern China. J. Virol. 76, 118–126. Xu, K.M., Li, K.S., Smith, G.J., Li, J.W., Tai, H., Zhang, J.X., Webster, R.G., Peiris, J.S., Chen, H., Guan, Y., 2007. Evolution and molecular epidemiology of H9N2 influenza A viruses from quail in southern China, 2000–2005. J. Virol. 81, 2635–2645.