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Journal of General Virology (2010), 91, 2302–2306
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DOI 10.1099/vir.0.023267-0
Phenotypes influencing the transmissibility of highly
pathogenic avian influenza viruses in chickens
Koutaro Suzuki,1,2 Hironao Okada,2,3 Toshihiro Itoh,2,3 Tatsuya Tada1,2
and Kenji Tsukamoto1,2
Correspondence
Kenji Tsukamoto
[email protected]
1
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
2
Core Research for Evolutional Science and Technology, Japan Science and Technology
Corporation, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan
3
National Institute of Advanced Industrial Science and Technology, 1-2-1 Namiki, Tsukuba, Ibaraki
305-8564, Japan
Received 1 May 2010
Accepted 7 May 2010
To identify critical phenotypes that affect avian influenza virus transmission in chickens, we
compared the transmissibility of three H5N1 highly pathogenic viruses of different pathogenicity in
chickens by monitoring the exact time of death using wireless thermo-sensors. This study showed
that, despite quick deaths, the most virulent H5N1 A/chicken/Yamaguchi/7/2004 transmitted
quickly in chickens via contact and airborne routes. Intermediate virulent H5N1 A/chicken/
Miyazaki/K11/2007 spread moderately and less virulent H5N1 A/duck/Yokohama/aq10/2003,
causing severe clinical signs and a long period to death, spread slowly among the animals. The
transmissibility was correlated with virus titres of oropharyngeal and cloacal swabs, and the time
for swab virus titres to reach 50 % chicken infective dose affected the transmission speed. These
results demonstrate that peak virus titres excreted and the time required for virus titres to reach a
minimal chicken infectious dose may be the critical phenotypes influencing the transmissibility of
highly pathogenicity avian influenza viruses in chickens.
The incidence of outbreaks of low-pathogenicity (LP) and
high-pathogenicity (HP) avian influenza (AI) in domestic
poultry has increased worldwide (Alexander, 2007b; Senne,
2007; Swayne & Halvorso, 2003), resulting in great
economic losses in the poultry industry. The AI outbreaks
in poultry also raise concerns about food safety and public
health. From 2003 to March 2010, the H5N1 HPAI viruses
caused 291 human deaths in 15 countries.
Slemons, 2008), age and weight of chickens (Tsukamoto
et al., 2007), air circulation and the rearing system of a
chicken house. Also ambient humidity and temperature
affect transmission of influenza viruses to a greater extent
than initially thought (Lowen et al., 2006, 2007, 2008). In a
guinea pig model, both cold and dry conditions favour
aerosol transmission, whereas high temperature (30 uC) or
high humidity (80 %) blocks the transmission.
Understanding the phenotypic determinants of the transmissibility of AI viruses in chickens is a prerequisite for
better control of AI outbreaks in poultry farms. Previous
studies have provided valuable insights into the transmission mechanisms of AI viruses in chickens (Alexander
et al., 1986; Okamatsu et al., 2007; Swayne & Slemons,
2008; Tsukamoto et al., 2007; van der Goot et al., 2003;
Westbury et al., 1981). These studies suggest that the
important phenotypic determinants of the virus are
pathogenicity, amount of virus shed into the environment,
duration of virus shedding and chicken infectious doses.
However, the phenotypic determinants are still obscure
because multiple factors affect transmissibility of AI viruses
in chickens such as virus strain (adaptation or pathogenicity) (Forman et al., 1986; Swayne & Slemons, 2008; van
der Goot et al., 2003), bird infectious dose (Swayne &
Currently, it is thought that LP viruses can transmit to
chickens more efficiently than HPAI viruses because
chickens infected with HPAI viruses have early deaths
and the virus has a short excretion time (Alexander,
2007a). Westbury et al. (1981) reported that HP viruses
spread slowly and failed to infect all chickens that had
contact, whereas LP viruses spread quickly and infected all
chickens that had contact. Our previous studies also
support this hypothesis that transmission of a H5N1
HPAI virus is inefficient when a small number of infected
chickens are used (Tsukamoto et al., 2007), while LPAI
virus transmits efficiently (Okamatsu et al., 2007). Before
HPAI outbreaks, the LPAI viruses usually spread broadly in
the field. On the other hand, results from one paper clearly
indicated that the HPAI virus transmits more efficiently
than the LPAI virus (van der Goot et al., 2003). Studies on
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Phenotypes influencing transmissibility of AIV
the association between virus excretion kinetics and
the transmissibility in chickens infected with AI viruses
are limited (Forman et al., 1986). Therefore, further
studies on the transmission mechanisms in chickens are
necessary.
A wireless thermo-sensor is a tool to monitor fever and the
exact time of death of each chicken infected with the HPAI
virus (Suzuki et al., 2009). In this study, we used the
wireless thermo-sensor to compare the transmissibility of
three H5N1 HPAI viruses, of varied pathogenicity, in
chickens by precisely monitoring the body temperature
kinetics or the time of death of inoculated and control
chickens.
Three H5N1 HPAI viruses were used in this study: A/
chicken/Yamaguchi/7/2004 (H5N1) (genotype V, clade 25) (CkYM7), A/chicken/Miyazaki/K11/2007 (genotype Z,
clade 2-2) (H5N1) (CkMZ11) and A/duck/Yokohama/
aq 10/2003 (H5N1) (clade 6) (DkYK10). The most virulent
virus, CkYM7, isolated from a chicken during the outbreak
in Japan (Mase et al., 2005b) causes only severe depression
and no fever with a short time to death (Suzuki et al.,
2009). The intermediate virulent virus, CkMZ11, isolated
from a chicken farm, induces slight gross lesions with an
intermediate time to death. The least virulent virus,
DkYK10, isolated in Japan from duck meat imported from
China (Mase et al., 2005a), causes severe clinical signs,
severe gross lesions, and high fever with a long time to
death (Suzuki et al., 2009). The amino acid sequences at
the haemagglutinin cleavage sites of CkYM7, CkMZ11
and DkYK10 are PQRERRKKR, PQGERRRKKR and
PQRERRRKKR, respectively. These viruses were propagated in the allantoic membrane of 10-day-old embryonated chicken eggs, and the 50 % egg infective dose (EID50)
was determined by the method of Reed and Muench
(1938). The HPAI viruses were handled in a biosafety level
(BSL) 3 facility, following the biosafety manual for HPAI
viruses. All animal experiments were approved by the
Animal Ethics Committee of our institute.
The 50 % chicken infective dose (CID50) is the minimal
infectious dose required for chicken infection and determined by the method of Reed and Muench (1938). Since
those of CkYM7 and DkYK10 are described previously
(Suzuki et al., 2009), the CID50 of CkMZ11 was
determined. Serial 10-fold dilutions of CkMZ11, containing 101.0–106.0 EID50 0.1 ml21, were inoculated intranasally
into four 4-week-old specific-pathogen-free (SPF) chickens
wearing a wireless thermo-sensor. The sensor developed for
this study was composed of a sensor (1.2 g) and battery
(1.8 g), and was attached to the surface of the abdominal
skin of each chicken using sports tape. The sensor signal
was continuously recorded by a computer every 20 s from
1 day before virus inoculation to the end of the experiment
(7 days). Body temperature and time of death of each
chicken were estimated as described previously (Suzuki et
al., 2009). The presence of clinical signs and gross lesions
was observed three times a day until all of the chickens died
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or for 7 days. Infection of each chicken was defined based
on observations of clinical signs and gross lesions, kinetic
curves of body temperature, and the time of death to
prevent the artificial transmission by handling chickens for
viral sampling. The average fevers and mean time of death
(MTD) of each group are summarized in Table 1, and the
CID50 of CkMZ11 was determined to be 102.5 EID50, which
was the same as CkYM7 and DkYK10 (Suzuki et al., 2009).
Fever in chickens infected with CkYM7 and CkMZ11 was
slight, whereas it was high in those infected with DkYK10.
MTD became shorter in proportion with increasing virus
inoculum, while fever was consistent in each virus strain
and not influenced by the virus amount. All of the
surviving chickens were shown to be free from haemagglutination inhibition antibodies to CkMZ11 and no virus
was isolated from lungs by using embryonated chicken
eggs.
Transmissibility from inoculated chickens to control
contact or separated chickens was compared among three
HPAI viruses in a negative-pressure isolator. The isolator
was divided into compartments A and B by a stainless wire
mesh (1 cm square) to prevent direct contact of chickens
(Tsukamoto et al., 2007). A total of 18 4-week-old chickens
per strain wearing the sensor was divided into inoculated,
contact and separated groups. The five inoculated and five
contact chickens were placed in compartment A, while
eight separated chickens were put in compartment B. The
ambient temperature and humidity of the BSL3 room was
25 uC and 40 %, respectively, and feed and water were
supplied automatically. To maintain the chance of virus
transmission, corrugated paper was set on a mesh floor in
each compartment to prevent the dropping of faeces to the
bottom floor of the isolator. The five chickens in the
inoculated group were intranasally administered with 106.0
EID50 0.1 ml21 of each virus and returned to compartment
A. Then, the isolators were kept closed throughout the
experimental period (20 days) to prevent the artificial or
accidental transmission of the virus to chickens by
handling. Also, dead chickens were left in the isolators
throughout the experimental period to keep the transmission condition the same as field outbreaks. Time of death
of each chicken was determined as described previously
(Suzuki et al., 2009), but in case of a broken sensor the
time of death was estimated roughly by the clinical
observations (* in Fig. 1).
Transmissibility in chickens was clearly different among the
three HPAI viruses, and all of the chickens from the
CkYM7, CkMZ11 and DkYK10 groups were dead within
four, seven and 16 days after inoculation, respectively
(Fig. 1). Observations of clinical signs and gross lesions and
fever kinetic curves analysis confirmed that all chicken
deaths were caused by the infection with these HPAI
viruses and not associated with accidental death.
CkYM7 transmitted the quickest of the three viruses to
contact and separated chickens (Fig. 1a). The MTDs of the
inoculated, contact and separated groups were 33.0±5.2,
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2303
K. Suzuki and others
Table 1. MTD and fever of 4-week-old chickens after inoculation with different amounts of HPAI viruses
The four 4-week-old SPF chickens in each isolator were attached with a wireless thermo-sensor and inoculated intranasally with each dose of virus.
These chickens were observed three times a day for 7 days. Sera collected from the veins of the wing of surviving chickens did not contain
antibodies to AI viruses, as shown by an agar gel precipitation test.
Amount of virus inoculated in each chicken (EID50)
Virus*
Feverd
MTD§
CID50D
101
102
103
104
105
106
CkYM7
0±0.3
0±0.5
0.7±0.8
1.0±0.4
1.1±0.2
0.9±0.2
CkMZ11
DkYK10
0±0.2
0±0.2
0±0.2
0±0.2
1.2±0.4
2.7±0.1
1.0±0.6
2.1±0.3
1.1±0.7
2.4±0.6
1.0±0.2
2.4±0.3
CkYM7
.168
.168
56.5±4.0
48.5±1.7
40.2±1.0
36.0±2.4
102.5
CkMZ11
DkYK10
.168
.168
.168
.168
79.8±19.1
110.5±24.6
70.5±2.6
97.1±16.3
53±3.9
96.8±6.6
54.3±5.7
87.4±6.0
102.5
102.5
Reference
Suzuki et al.
(2009)
This study
Suzuki et al.
(2009)
Suzuki et al.
(2009)
This study
Suzuki et al.
(2009)
*CkYM7, A/chicken/Yamaguchi/7/2004 (H5N1); CkMZ11, A/chicken/Miyazaki/K11/2007 (H5N1); DkYK10, A/duck/Yokohama/aq10/2003
(H5N1).
DCID50, 50 % chicken infectious dose.
dFever, difference in degrees of body temperature before and after virus inoculation in each chicken was calculated and the average of four chickens
is shown above.
§MTD, mean time of death given in hours after virus inoculation.
64.9±7.7 and 82.8±14.2 h, respectively. Airborne transmission may have occurred in three of eight separated
chickens (D in Fig. 1a), since the MTD of the three chickens
(66.2 h) was similar to that of the five contact chickens
(64.9 h). The MTD of the five remaining separated
chickens was 92.8 h. Transmission to contact chickens or
separated chickens occurred all at once in CkYM7 and the
transmission interval was around 30 h.
CkMZ11 spread at a moderate speed, and the MTDs of the
inoculated, contact and separated groups were 51.4±3.3,
101.6±9.6 and 147.4±25.1 h, respectively (Fig. 1b). One
separated chicken died at 98.3 h post-inoculation, probably
through airborne transmission (D in Fig. 1b), whereas the
other five separated chickens died later (MTD 5155.6 h).
The CkMZ11 also transmitted simultaneously to contact or
separated chickens, and the transmission interval was
around 50 h.
On the other hand, a different pattern of transmission
emerged for DkYK10 (Fig. 1c). The death times varied
widely among chickens, especially in separated chickens.
The MTDs of the inoculated, contact and separated groups
were 84.4±17.2, 165.2±26.1 and 285.5±67.3 h, respectively. One separated chicken (D in Fig. 1c) died earlier than
others, probably through airborne transmission because
the time of death (166.3 h) was similar to that of contact
chickens (165.2 h). We found that some chickens in the
group pecked the feathers, skin or blood of dead chickens,
suggesting these behaviours may contribute to the spread
of DkYK10 to chickens.
2304
To determine the association between virus excretion and
transmissibility in chickens, we compared the virus
excretion kinetics from chickens infected with one of the
three H5N1 HPAI viruses. Five chickens were inoculated
intranasally with CkYM7, CkMZ11 or DkYK10 at 106.0
EID50 0.1 ml21, and oropharyngeal and cloacal swabs were
collected at each time point and titrated for virus infectivity
with embryonated chicken eggs. The titres were expressed
as EID50 ml21 by the method of Reed and Muench (1938).
The virus titres of both oropharyngeal and cloacal swabs
continuously increased after inoculation and peaked at the
time of death in all three strains (Fig. 2). The peak virus
yields in both swabs differed considerably among virus
strains, and those from oropharyngeal swabs were 105.8±1.0
EID50 ml21 for CkYM7, 104.7±0.2 EID50 ml21 for CkMZ11
and 102.5±1.7 EID50 ml21 for DkYK10. In addition, the
time for virus titres of both swabs to reach to CID50 (102.5
EID50 in all three viruses) was earliest in CkYM7 followed
by CkMZ11 and then DkYK10.
The swab sampling to determine the infection of control
birds made the chickens panic, which resulted in stirring
up the dust in the isolator, leading to artificial transmission. Also, the basic reproduction number (R0) serves as an
indicator to determine how many control animals are
infected from one infected animal. If R0 is .1 disease can
spread, while if R0 is ,1 transmission chains will fade out.
However, this method is not applicable when infection
does not occur from one infected chicken as shown in
CkYM7 (Tsukamoto et al., 2007) or all control animals are
infected because the R0 is ‘. In contrast, the sensor system
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Journal of General Virology 91
Phenotypes influencing transmissibility of AIV
Fig. 2. Kinetic curves of mean virus titres in (a) oropharyngeal and
(b) cloacal swabs collected sequentially from chickens inoculated
with one of the HPAI viruses. Each of the five 4-week-old SPF
chickens was inoculated intranasally with 106.0 EID50 0.1 ml”1 of
CkYM7 (#), CkMZ11 (h) or DkYK10 (g). Mean virus titres of
oropharyngeal and cloacal swabs are expressed as EID50
ml”1±SEM. The dotted line indicates CID50 (103.5 EID50 ml”1) of
each HPAI virus.
Fig. 1. Time of death of each chicken in the (a) CkYM7, (b)
CkMZ11 and (c) DkYK10 groups are shown. Black, black dotted
and grey dotted lines indicate inoculated, contact and separated
groups of chickens, respectively. *Time of death was roughly
estimated by clinical observation of chickens (three times a day)
because the sensors were broken; 3chickens were infected
probably by airborne transmission.
is useful to compare the transmissibility of HPAI viruses by
monitoring the exact time of death of each chicken with
minimum artificial or accidental transmission. Our study
demonstrates that transmissibility of HPAI viruses in
chickens was directly associated with the time taken for
the virus titre in excreted body fluids to reach the minimal
infectious dose and the amount of virus excreted, but not
with virus shedding time or MTD. Despite the early death
of infected chickens, the most virulent CkYM7 was quickly
and largely excreted from chickens and resulted in quick
transmission to chickens compared with the other two
HPAI viruses. Also, the CID50 serves as a phenotypic
determinant for transmissibility in chickens (Swayne &
Slemons, 2008). Since three HPAI virus strains tested
had the same CID50, the present study shows that two
http://vir.sgmjournals.org
phenotypes identified in this study may play a key role in
transmissibility of HPAI viruses in chickens. Our study also
shows that a swab virus kinetic or swab virus titration at
the time of death, in addition to CID50 of the strain, are
appropriate methods to determine the transmissibility of
AI viruses in chickens.
The present study suggests that HPAI viruses may transmit
more efficiently than LPAI viruses in chickens; however,
this conclusion seems inconsistent with the current
understanding of transmission (Alexander, 2007a, b;
Alexander et al., 1986; Swayne & Halvorso, 2003). The
HPAI virus, A/duck/Victoria/75 (H7N6), fails to infect all
contact chickens, whereas the LPAI virus, A/duck/Victoria/
76 (H7N6), spreads to all contact birds (Westbury et al.,
1981). This discrepancy may be explained by the difference
in tissue tropism between the two AI viruses. Some HPAI
viruses can replicate more efficiently in the brain and/or
heart rather than in respiratory and/or digestive tracts
(Swayne, 1997, 2007), whereas LPAI viruses replicate only
in these tracts. Such HPAI viruses cause early death of
chickens in spite of low virus shedding from tracts. The
swab virus titre of DkYK10, which was equivalent to that of
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2305
K. Suzuki and others
LPAI viruses, was maintained at less than CID50 just before
the time of death, but DkYK10 caused early death of
chickens probably by the efficient replication in the brain
(unpublished data). Since the MTD of A/duck/Victoria/75
(7 days) (Westbury et al., 1981) is similar to DkYK10
(5.6 days) (data not shown), it is likely that A/duck/
Victoria/75 (HPAI virus) may not replicate well in the
respiratory and/or digestive tracts. Thus, tissue tropism of
HPAI viruses may influence transmissibility. Also, the
transmission mechanisms of AI viruses in chickens may be
more complex than the mammalian model, in which there
is positive association between virus excretion and transmissibility in the animals (Lowen et al., 2006, 2007, 2008;
Maines et al., 2006).
Maines, T. R., Chen, L. M., Matsuoka, Y., Chen, H., Rowe, T., Ortin, J.,
Falcon, A., Nguyen, T. H., Mai, L. Q. & other authors (2006). Lack of
Acknowledgements
Reed, L. & Muench, H. (1938). A simple method of estimating fifty
This work was funded by the Core Research for Evolutional Science
and Technology (CREST) programme of the Japan Science and
Technology Agency (JST). Authors’ contributions: K. S. carried out
most of the experiments and T. T. supported the animal experiments.
H. O. and T. I. participated in the sensor data analyses. All members
were involved in experimental design. K. T. supervised, coordinated
and finalized the manuscript. All authors read and approved the
manuscript.
transmission of H5N1 avian-human reassortant influenza viruses in a
ferret model. Proc Natl Acad Sci U S A 103, 12121–12126.
Mase, M., Eto, M., Tanimura, N., Imai, K., Tsukamoto, K., Horimoto, T.,
Kawaoka, Y. & Yamaguchi, S. (2005a). Isolation of a genotypically
unique H5N1 influenza virus from duck meat imported into Japan
from China. Virology 339, 101–109.
Mase, M., Tsukamoto, K., Imada, T., Imai, K., Tanimura, N.,
Nakamura, K., Yamamoto, Y., Hitomi, T., Kira, T. & other authors
(2005b). Characterization of H5N1 influenza A viruses isolated
during the 2003–2004 influenza outbreaks in Japan. Virology 332,
167–176.
Okamatsu, M., Saito, T., Yamamoto, Y., Mase, M., Tsuduku, S.,
Nakamura, K., Tsukamoto, K. & Yamaguchi, S. (2007). Low
pathogenicity H5N2 avian influenza outbreak in Japan during the
2005–2006. Vet Microbiol 124, 35–46.
percent endpoints. Am J Hyg 27, 493–497.
Senne, D. A. (2007). Avian influenza in North and South America,
2002–2005. Avian Dis 51, 167–173.
Suzuki, K., Okada, H., Itoh, T., Tada, T., Mase, M., Nakamura, K.,
Kubo, M. & Tsukamoto, K. (2009). Association of increased
pathogenicity of Asian H5N1 highly pathogenic avian influenza
viruses in chickens with highly efficient viral replication accompanied
by early destruction of innate immune responses. J Virol 83, 7475–
7486.
Swayne, D. E. (1997). Pathobiology of H5N2 Mexican avian influenza
References
virus infections of chickens. Vet Pathol 34, 557–567.
Alexander, D. J. (2007a). An overview of the epidemiology of avian
influenza. Vaccine 25, 5637–5644.
Alexander, D. J. (2007b). Summary of avian influenza activity in
Europe, Asia, Africa, and Australasia, 2002–2006. Avian Dis 51, 161–
166.
Alexander, D. J., Parsons, G. & Manvell, R. J. (1986). Experimental
assessment of the pathogenicity of eight avian influenza A viruses of
H5 subtype for chickens, turkeys, ducks and quail. Avian Pathol 15,
647–662.
Swayne, D. E. (2007). Understanding the complex pathobiology of
high pathogenicity avian influenza viruses in birds. Avian Dis 51, 242–
249.
Swayne, D. E. & Halvorso, D. A. (2003). Influenza. In Diseases of
Poultry, 11th edn, pp. 135–160. Edited by Y. M. Saif, J. H. Barnes, J. R.
Glisson, A. M. Fadly, L. R. McGougald & D. E. Swayne. Ames, Iowa:
Iowa State Press.
Swayne, D. E. & Slemons, R. D. (2008). Using mean infectious dose
Forman, A. J., Parsonson, I. M. & Doughty, W. J. (1986). The
of high- and low-pathogenicity avian influenza viruses originating
from wild duck and poultry as one measure of infectivity and
adaptation to poultry. Avian Dis 52, 455–460.
pathogenicity of an avian influenza virus isolated in Victoria. Aust
Vet J 63, 294–296.
Tsukamoto, K., Imada, T., Tanimura, N., Okamatsu, M., Mase, M.,
Mizuhara, T., Swayne, D. & Yamaguchi, S. (2007). Impact of different
Lowen, A. C., Mubareka, S., Tumpey, T. M., Garcia-Sastre, A. &
Palese, P. (2006). The guinea pig as a transmission model for human
influenza viruses. Proc Natl Acad Sci U S A 103, 9988–9992.
husbandry conditions on contact and airborne transmission of H5N1
highly pathogenic avian influenza virus to chickens. Avian Dis 51,
129–132.
Lowen, A. C., Mubareka, S., Steel, J. & Palese, P. (2007). Influenza
van der Goot, J. A., Koch, G., de Jong, M. C. & van Boven, M. (2003).
virus transmission is dependent on relative humidity and temperature. PLoS Pathog 3, 1470–1476.
Transmission dynamics of low- and high-pathogenicity A/Chicken/
Pennsylvania/83 avian influenza viruses. Avian Dis 47, 939–941.
Lowen, A. C., Steel, J., Mubareka, S. & Palese, P. (2008). High
Westbury, H. A., Turner, A. J. & Amon, L. (1981). Transmissibility of
temperature (30 uC) blocks aerosol but not contact transmission of
influenza virus. J Virol 82, 5650–5652.
two avian influenza A viruses (H7N6) between chickens. Avian Pathol
10, 481–487.
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