NP, PB1, and PB2 viral genes contribute to altered replication of H5N1 avian influenza viruses in chickens.
ABSTRACT The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.
- SourceAvailable from: Pablo R Murcia[Show abstract] [Hide abstract]
ABSTRACT: Avian influenza (AI) viruses of the H7 subtype have the potential to evolve into highly pathogenic (HP) viruses that represent a major economic problem for the poultry industry and a threat to global health. However, the emergence of HPAI viruses from low pathogenic (LPAI) progenitor viruses is currently poorly understood. To investigate the origin and evolution of one of the most important avian influenza epidemics described in Europe, we investigated the evolutionary and spatial dynamics of the entire genome of 109 H7N1 (46 LPAI and 63 HPAI) viruses collected during Italian H7N1 outbreaks between March 1999 and February 2001. Phylogenetic analysis revealed that the LPAI and HPAI epidemics shared a single ancestor, that the HPAI strains evolved from the LPAI viruses in the absence of reassortment, and that there was a parallel emergence of mutations among HPAI and later LPAI lineages. Notably, an ultra-deep sequencing analysis demonstrated that some of the amino acid changes characterizing the HPAI virus cluster were already present with low frequency within several individual viral populations from the beginning of the LPAI H7N1 epidemic. A Bayesian phylogeographic analysis revealed stronger spatial structure during the LPAI outbreak, reflecting the more rapid spread of the virus following the emergence of HPAI. The data generated in this study provide the most complete evolutionary and phylogeographic analysis of epidemiologically intertwined high and low pathogenic viruses undertaken to date, and highlight the importance of implementing prompt eradication measures against LPAI to prevent the appearance of viruses with fitness advantages and unpredictable pathogenic properties.Importance The Italian H7 avian influenza (AI) epidemic of 1999-2001 was one of the most important AI outbreaks described in Europe. H7 viruses have the ability to evolve into HP (highly pathogenic) forms from LP (low pathogenic) precursors, although the mechanisms underlying this evolutionary transition are only poorly understood. We combined epidemiological information, whole-genome sequence data and ultra-deep sequencing approaches to provide the most complete characterization of the evolution of HPAI from LPAI viruses undertaken to date. Our analysis revealed that the LPAI viruses were the direct ancestors of the HPAI strains and identified low frequency minority variants with HPAI mutations that were present in the LPAI samples. Spatial analysis provided key information for the design of effective control strategies for AI at both local and global scales. Overall, this work highlights the importance of implementing rapid eradication measures to prevent the emergence of novel influenza viruses with severe pathogenic properties.Journal of Virology 02/2014; · 4.65 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Recent reports of the detection of the zoonotic low pathogenic avian influenza (LPAI) H7N9 viruses in healthy pigeons have again put the spotlight on the potential role of pigeons and doves in the transmission of avian influenza between infected poultry and humans. A surge in studies followed the highly pathogenic avian influenza (HPAI) H5N1 epidemic, and this review collates the new data on AIV in pigeons and doves, both from a surveillance perspective, as well as the results of numerous clinical studies. Collectively, results of 32 field studies representing 24 countries across four continents indicate an antibody prevalence of 8.01% in pigeons and doves but only 0.37% of the total was associated with exposure to the same serotype as a highly pathogenic avian influenza (HPAI) outbreak occurring in poultry at the time. Only 1.1% of 6155 columbids sampled tested positive for the virus, and only 9/6155 (0.15%) viruses were detected in regions that were experiencing outbreaks of a notifiable serotype at the time.In 22 experimental infection studies with HPAI and LPAI viruses since 1944, only 26/715 (3.64%) mortalities were reported, and these could usually be associated with excessive doses of inoculum, which would induce fatal inflammatory responses. Since seroconversion and virus detection was demonstrated in many of these studies, albeit without clinical signs in most cases, it is clear that columbids are susceptible to infection, but ineffective propagators and disseminators of the virus, ie. “dead end” hosts for AIVs, even HPAI. Viruses are shed in minute quantities from both the choana and in the faeces for a short duration but titers are below the minimum threshold require to infect other speciesVeterinary Microbiology 06/2014; · 2.73 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Since 1997, several epizootic avian influenza viruses (AIVs) have been transmitted to humans, causing diseases and even deaths. The recent emergence of severe human infections with AIV (H7N9) in China has raised concerns about efficient interpersonal viral transmission, polygenic traits in viral pathogenicity and the management of newly emerging strains. The symptoms associated with viral infection are different in various AI strains: H5N1 and newly emerged H7N9 induce severe pneumonia and related complications in patients, while some H7 and H9 subtypes cause only conjunctivitis or mild respiratory symptoms. The virulence and tissue tropism of viruses as well as the host responses contribute to the pathogenesis of human AIV infection. Several preventive and therapeutic approaches have been proposed to combat AIV infection, including antiviral drugs such as M2 inhibitors, neuraminidase inhibitors, RNA polymerase inhibitors, attachment inhibitors and signal-transduction inhibitors etc. In this article, we summarize the recent progress in researches on the epidemiology, clinical features, pathogenicity determinants, and available or potential antivirals of AIV.Acta Pharmacologica Sinica 10/2013; 34(10):1257-1269. · 2.35 Impact Factor
JOURNAL OF VIROLOGY, May 2008, p. 4544–4553
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 9
NP, PB1, and PB2 Viral Genes Contribute to Altered Replication of
H5N1 Avian Influenza Viruses in Chickens?‡
Jamie L. Wasilenko,1Chang Won Lee,2,3† Luciana Sarmento,1Erica Spackman,1
Darrell R. Kapczynski,1David L. Suarez,1and Mary J. Pantin-Jackwood1*
Southeast Poultry Research Laboratory, USDA-Agricultural Research Service, Athens, Georgia1; Department of
Food Animal Health Research Program, OARDC, The Ohio State University, Wooster, Ohio2; and
Department of Veterinary Preventive Medicine, College of Veterinary Medicine,
The Ohio State University, Columbus, Ohio3
Received 12 December 2007/Accepted 15 February 2008
The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic,
although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of
influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different
virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to
generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were
used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP
genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes.
Disruption of the parental virus’ functional polymerase complexes by exchanging PB1 or PB2 genes decreased
viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP
gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death
times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and
inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral
replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the
pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have
shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.
The eight-segment negative-sense single-strand RNA ge-
nome of influenza A viruses provides for the possibility of
genetic reassortment between different influenza viruses that
contributes to the natural evolution of these viruses. Wild
aquatic birds are known to be natural reservoirs for avian
influenza viruses (AIVs) (58), and coinfection of these avian
hosts occurs commonly (49). Live poultry markets, where many
birds of different species share close quarters, can also contrib-
ute to the reassortment between different influenza viruses
because of the potential for dual infection.
Reassortment of one or more viral gene segments can lead
to attenuation and plays a major role for influenza viruses
when crossing host barriers (13, 33, 51, 56). Just as reassort-
ment can lead to attenuation, the converse is also true, and the
emergence of viruses with increased pathogenicity or increased
host range was an important occurrence in past outbreaks and
is also likely in future outbreaks. Of particular interest is the
role of reassortment in past human influenza outbreaks in
which avian gene segments reassorted with human viruses,
resulting in the pandemic outbreaks of 1957 and the pandemic
outbreak of 1968, in which avian hemagglutinin (HA), NA, and
PB1 genes and HA and PB1 avian genes, respectively, under-
went reassortment with circulating human viruses (29, 46).
The host tropism and pathogenicity of influenza viruses are
considered to be multigenic, and the primary virulence factor is
the presence of multiple basic amino acids or the insertion of
amino acids at the HA cleavage site (8, 9, 25, 47). Reverse
genetic techniques and classical reassortment studies have elu-
cidated several genes and specific mutations as being impor-
tant contributors in host range determination and the patho-
genicity of influenza viruses in mammals. In addition to the HA
cleavage site, a lysine residue at amino acid 627 in PB2 has
been found in most influenza viruses pathogenic to humans
and also influences virulence in mice (22, 36, 50, 54, 55). The
alternate splice product of the PB1 gene, PB1-F2, has also
been linked to the increased virulence of the 1918 virus in mice
(14). PA and NS1 have also been shown to contribute to
pathogenicity for mice, and NS1 also influences the pathoge-
nicity of influenza viruses in pigs (16, 32, 45, 48, 52).
Despite what is known about the virulence of influenza vi-
ruses in mammals, the role of the genes important for host
tropism and the virulence of AIVs in avian species remains
largely undetermined. Currently, pathogenesis studies of AIVs
in chickens clearly indicate that the proteolytic cleavage site of
HA (22, 25, 27) and changes in the NS1 protein (11, 33) are
important determinants of virulence. Recently it was reported
that PA and PB1 gene mutations were responsible for differ-
ences between nonpathogenic and highly pathogenic viral
clones in ducks (26), and mixtures of polymerase components
* Corresponding author. Mailing address: Southeast Poultry Re-
search Laboratory, USDA-ARS, 934 College Station Road, Athens,
GA 30605. Phone: (706) 546-3419. Fax: (706) 546-3161. E-mail: Mary
† Present address: Department of Food Animal Health Research
Program, Ohio Agricultural Research and Development Center, The
Ohio State University, 1680 Madison Avenue, Wooster, OH 44691-
‡ Supplemental material for this article may be found at http://jvi
?Published ahead of print on 27 February 2008.
from different viruses have been found to lead to the attenu-
ation of AIVs in chickens (43). However, the K627 mutation of
PB2, important for virulence in mammals, does not seem to
influence AIV virulence in avian cell lines (31).
In this study, we used reverse genetics to generate two H5N1
AI recombinant viruses with different pathogenicity levels in
chickens. Single-gene reassortant viruses were generated in
order to better understand the role of gene reassortment
events as a way of generating variant AIVs and to study the
role of each gene segment in viral pathogenicity in chickens.
Our results indicate that the HA, NS, NP, and M2 proteins are
important contributing factors in the increased virulence of our
reassortant viruses in chickens. Three genes, PB1, PB2, and
NP, impacted replication of the reassortant viruses, emphasiz-
ing the importance of these proteins in the replication process.
In addition, infection with the reassortant viruses affected host
gene expression levels of alpha interferon (IFN-?), the ortho-
myxovirus resistance gene 1 (Mx1), IFN-?, and/or inducible
nitric oxide synthase (iNOS), suggesting that the regulation of
host genes may contribute to the differences in replication
observed in chickens.
MATERIALS AND METHODS
Generation of infectious reassortant viruses. Two high-pathogenicity recom-
binant H5N1 AIVs were derived from A/Egret/Hong Kong/757.2/02 and
A/Chicken/Indonesia/7/03 as follows and are referred to as rEgret and rIndo,
respectively. RNA was extracted from virus stocks of the wild-type viruses, after
propagation in embryonating chicken eggs (ECEs), using Trizol LS (Invitrogen,
Inc., Carlsbad, CA) according to the manufacturer’s instructions. Transcription
and expression plasmids were constructed as previously described (38). 293T
cells were transfected with 1 ?g of each of the eight transcription and four
protein expression plasmids and 11 ?l of Lipofectamine 2000 (Invitrogen) in a
2-ml volume of OptiMEM I (Invitrogen). Cells were washed after 4 h at 37°C,
and medium was replaced with Dulbecco’s modified Eagle medium I supple-
mented with 10% fetal bovine serum (Invitrogen) for 72 h. ECEs were inoculated
with 100 ?l of the cell supernatant. Virus was harvested from the allantoic fluid
of eggs 36 to 48 h after inoculation and titrated in ECEs. Titration end points
were calculated by the method of Reed and Muench (42). In addition to the
reconstitution of the parental strains by reverse genetics, six single-gene reassor-
tants containing one of the rEgret genes were produced in the rIndo background.
All experiments using HPAI H5N1 viruses were conducted using biosafety level
3 Ag (BSL-3 Ag) containment at SEPRL, USDA, in Athens, GA (5).
Sequencing of influenza virus genes. Genes from the rescued viruses were
sequenced to confirm the reassortment. Viral RNA was extracted from infectious
allantoic fluid from ECEs using Trizol LS reagent (Invitrogen). Gene sequences
were obtained by reverse transcription (RT)-PCR by use of a Qiagen One-Step
RT-PCR kit (Qiagen, Valencia, CA) using specific primers for each influenza
virus gene. The primer sequences and RT-PCR conditions used are available
upon request. PCR products were extracted from agarose gels, using a GenScript
QuickClean gel extraction kit (GenScript, Piscataway, NJ). An ABI BigDye
Terminator version 1.1 sequencing kit (Applied Biosystems, Foster City, CA) run
on a 3730xl DNA analyzer (Applied Biosystems) was used for sequencing PCR
products. The MegAlign program (Lasergene 7.1; DNAStar, Madison, WI) was
used to compare nucleotide sequences, using the Clustal W alignment algorithm.
In vivo characterization of reassortant viruses. In order to determine the
pathogenic phenotypes of the reassortant viruses in chickens, 2-week-old specific-
pathogen-free White Leghorn chickens (Gallus gallus domesticus) (from SEPRL
in-house flocks) were inoculated intranasally (IN) with the reassortant viruses
and evaluated for clinical signs for 10 days. The birds were housed in self-
contained isolation cabinets that were ventilated under negative pressure with
HEPA-filtered air and maintained under continuous lighting. The birds had ad
libitum access to feed and water. Each group, containing 11 birds, was inoculated
IN with 0.1 ml of an inoculum containing 10650% egg infective doses (EID50)/ml
of one of the viruses. Three birds from each group were euthanized and
necropsied at 2 days postinoculation (dpi). Gross lesions were recorded, and
tissue samples (lung, spleen, and brain) were collected separately from two birds
for virus isolation. Lung, bursa, kidney, adrenal gland, gonad, thymus, thyroid,
brain, liver, heart, ventriculus, pancreas, intestine, spleen, trachea, and thigh
muscle specimens were collected in 10% neutral buffered formalin solution from
the same birds. Sample birds, moribund birds, and all birds remaining at the end
of a 10-day period were humanely euthanized. Mean death time (MDT) was
calculated by determining the sum of the day of death for the chickens and
dividing it by the total number of dead chickens.
Virus isolation and titrations. Portions of the spleens, lungs, and brains from
two birds per group were collected at 2 dpi in brain heart infusion medium (BHI)
(BD Bioscience, Sparks, MD) and stored frozen at ?70°C. Titers of infectious
virus were determined as follows. Tissues were homogenized (10% [wt/vol]) and
diluted in BHI. One hundred microliters of the homogenate dilutions was inoc-
ulated into the allantoic cavity of ECEs. Titration end points were calculated by
the method of Reed and Muench (42) (n ? 2). The threshold of detection was
102.4EID50/g from tissues.
Histopathology and immunohistochemistry. Collected tissues fixed by submer-
sion in 10% neutral buffered formalin were routinely processed and embedded in
paraffin. Sections were made at 5 ?m and were stained with hematoxylin and
eosin. A duplicate 4-?m section was immunohistochemically stained by first
microwaving the sections in antigen retrieval citra solution (BioGenex, San
Ramon, CA) for antigen exposure. A 1:2,000 dilution of a mouse-derived mono-
clonal antibody (P13C11) specific for a type A influenza virus nucleoprotein
(developed at Southeast Poultry Research Laboratory, USDA) was applied and
allowed to incubate for 2 h at 37°C. The primary antibody was then detected by
the application of biotinylated goat anti-mouse immunoglobulin G secondary
antibody using a biotin-streptavidin detection system (supersensitive multilink
immunodetection system; BioGenex). Fast Red TR (BioGenex) served as the
substrate chromogen, and hematoxylin was used as a counterstain. All tissues
were systematically screened for microscopic lesions.
Total cellular RNA isolation. Total cellular RNA for the evaluation of gene
expression was prepared from spleen or lung tissues of chickens collected at 2
dpi. Tissue samples were homogenized in 3 ml minimal essential medium, Alpha
1? (Invitrogen), by passing the tissues through 100-?m cell strainers (BD Bio-
science, Sparks, MD), added to 6 ml Trizol, inverted, and stored at ?80°C.
Chloroform (1.2 ml) was added and spun, and the aqueous phase was added to
an equal volume of 70% ethanol. A Qiagen RNA midiprep kit (Qiagen) was used
to isolate the RNA from the aqueous phase-ethanol solution.
Semiquantitative analysis of mRNA gene expression. RT-PCR was carried out
using a Qiagen OneStep PCR kit (Qiagen) according to the manufacturer’s
instructions, using 100 ng of pooled cellular RNA per group (33.3 ng of total
RNA from three chickens per group) for each infected group 2 dpi in a 25-?l
reaction volume (n ? 1). The primers for IFN-? and ?-actin were previously
described (60). The IFN-?, Mx1, and iNOS primers were designed using nucle-
otide sequences from NCBI GenBank and are available upon request. ?-actin
served as an amplification and loading control. DNA was visualized by ethidium
bromide gel electrophoresis using an EDAS 290 imaging station (Kodak, New
Haven, CT). Band intensities were analyzed using Kodak 1D version 3.6.1 im-
aging software (Kodak). Band intensity values for each tissue were normalized to
the ?-actin values, and the values for control birds were arbitrarily set at 1.
Statistical analyses. Data were analyzed using Prism v.5.01 software (Graph-
Pad Software, Inc., San Diego, CA), and values are expressed as the mean ?
standard error of the mean. The Kaplan-Meier survival rate data were analyzed
using the log rank test, and one-way analysis of variance with the Tukey-Kramer
posttest was used to analyze MDTs. Statistical significance was set at a P value of
Pathogenicity of the H5N1 recombinant viruses. The two-
parent recombinant H5N1 viruses used in this study were de-
rived from A/Chicken/Indonesia/7/03 and A/Egret/Hong Kong/
757.2/02 using reverse genetics. Table 1 shows the comparison of
the amino acid sequences of the resulting recombinant viruses,
rIndo and rEgret. The recombinant viruses that we generated
have a PA gene with the same amino acid sequence, but the
remaining viral genes have amino acid sequence differences
(see Table S1 in the supplemental material). When adminis-
tered to chickens IN, the rIndo virus resulted in a mortality of
6 of 8 and an MDT of 6.1 days, while the rEgret virus resulted
in a higher mortality (8 of 8) and a shorter MDT (3.25 days)
(Table 2). MDTs (Table 2) and survival rates were significantly
VOL. 82, 2008 REASSORTANT H5N1 AVIAN INFLUENZA VIRUSES IN CHICKENS4545
different (P ? 0.05) upon infection with rIndo and rEgret
compared to the control group and also to each other (Fig.
1A). Chickens inoculated with rEgret presented histological
lesions in tissues, which included diffuse interstitial pneumonia
in the lung, moderate tracheitis, multifocal splenic necrosis,
moderate cardiac degeneration, and multifocal nonsupurative
encephalitis among others (Fig. 2; Table 3). AIV antigen was
present in blood vessel endothelial cells in most tissues, tissue
macrophages, cardiac and skeletal muscle myocytes, neurons
and glial cells in the brain, pancreatic acinar cells, respiratory
epithelia of the trachea, and adrenal corticotrophic cells. In
contrast, chickens inoculated with rIndo presented lesions only
in the trachea and lungs, and viral antigen was primarily de-
tected in the cells of these tissues and infrequently in the liver,
spleen, and brain (Table 3). Virus titers in the lung tissue show
that the rEgret virus had slightly higher titers than the rIndo
virus, although there was variation in the rIndo virus titers (Fig.
The expression levels of four different immune-related genes
in spleen and lung tissues were compared 2 days after chal-
lenge with the different recombinant viruses (Fig. 4 and 5).
Infection with the rEgret virus resulted in increased expression
of IFN-?, IFN-?, iNOS, and Mx1 in the infected chicken spleen
tissues, but in the lung tissues, Mx1 was only slightly upregu-
lated compared to the controls. Only IFN-? gene expression,
in both tissues, was upregulated upon infection with rIndo
compared to the control. These data suggest that the recom-
FIG. 1. Survival after IN inoculation with the reassortant viruses.
Two-week-old chickens were inoculated with 0.1 ml of an inoculum
containing 106EID50/ml of the reassortant viruses, and mortality was
monitored for 10 days. Parent virus and control survival analysis
(A) and reassortant virus survival analysis (B). All groups are statisti-
cally different from rEgret and rIndo/rEgret NP (P ? 0.05) as deter-
mined by the log rank test. Explanation of footnotes in figure key: a,
survival is significantly different from the control group and also from
the rEgret group; b, survival is significantly different from all groups
and the control group; c, survival is significantly different from the
control group but not the rIndo group; d, survival is not significantly
different from the control group or the rIndo group; e, survival is
significantly different from the rIndo group but not the control group.
TABLE 1. Amino acid sequence comparisons of recombinant A/Egret/Hong Kong/757.2/02 and recombinant A/Chicken/Indonesia/7/03 viruses
Amino acid sequence
No. of different
Differing amino acid positions
22 ? 20-amino-acid
105, 132, 221, 251, 288, 292, 309, 339, 389, 394
110, 158, 215, 372, 400
8, 110, 111, 136, 140, 145, 194, 231, 498, 504, 549
22, 184, 400, 406, 423
8, 19, 29, 42, 48, 49–68, 74, 95, 100, 105, 224, 257,
258, 267, 270, 313, 338, 341, 346, 355, 378, 386,
10, 31, 64
7, 21, 48, 73, 112, 148, 200, 202, 209
arIndo virus NA contains a 20-amino-acid stalk deletion as well as 22 other amino acid differences.
TABLE 2. Morbidity, mortality, and MDT resulting from IN
inoculation of 2-week-old chickens with 0.1 ml of inoculum
containing 106EID50/ml of the reassortant viruses
No. of sick/
aThe MDTs of all groups were significantly different from that of the control
bMDTs were significantly different from the rIndo group.
cMDTs were significantly different from the rEgret group.
dTwo birds survived. One presented no clinical signs; the other one presented
only mild hemorrhages in the shanks and combs.
eThe surviving birds presented only mild hemorrhages in the shanks and
4546WASILENKO ET AL.J. VIROL.
binants induce different host responses and/or are able to dif-
ferentially suppress host gene response.
Six single-gene reassortants containing one of the rEgret
HA, PB2, PB1, NS, NP, or M genes were generated in the
rIndo background. We were unable to generate the rIndo/
rEgret NA reassortant due to the limited replication of this
reassortant virus in 293T cells or ECEs. The rest of the reas-
sortant viruses grew to high titers in ECEs (?106.6EID50/ml).
Inoculation with the single-gene reassortant viruses resulted in
variable morbidity, mortality, and MDTs (Table 2; Fig. 1). The
effect of the single-gene reassortments on pathogenicity of the
viruses in chickens is discussed below.
Exchange of the HA gene resulted in increased mortality,
expanded tissue range, and differential host gene expression
levels. The HA genes of both our H5N1 recombinant parent
viruses contain identical multiple basic amino acid motifs ad-
jacent to the HA0 cleavage site. However, there were 11 other
amino acid differences outside of the cleavage site of the HA
gene (Table 1). In order to evaluate the contribution of the HA
gene in the pathogenesis of these viruses, we exchanged the
HA gene of the rIndo virus with the rEgret virus HA gene. The
resulting virus, referred to as rIndo/rEgret HA, resulted in
increased mortality (8 of 8) over the rIndo parent virus (6 of 8).
Exchange of the HA gene resulted in MDTs and survival rates
that were not significantly different from those of the rIndo
group (Fig. 1B). However, viral antigen staining of tissues from
infected birds showed the presence of the virus in many organs,
with an expanded tissue range with immunohistochemical
staining for AIV in the heart, thymus, kidney, and pancreas
(Table 3). Virus titers in the spleen, lung, and brain tissues,
which had immunohistochemistry results comparable to those
of rIndo, showed that rIndo/rEgret HA had titers similar to
those of the rIndo parent virus in those tissues (Fig. 3). mRNA
expression levels of the immune-related genes showed that
rIndo/rEgret HA virus infection induced gene expression of
IFN-? and Mx1 in the spleen at levels similar to those of the
rEgret parent virus (Fig. 4). Expression levels of IFN-? and
iNOS in spleen tissue were more similar to the expression
levels of the controls. Gene expression in lung tissue showed
only increased expression levels of Mx1 over those of the rIndo
and rEgret parent viruses upon infection with rIndo/rEgret HA
Exchange of the NS gene increases mortality and the tissue
range of the reassortant virus. Our recombinant viruses, rIndo
and rEgret, have 9 amino acid sequence differences in NS1 and
1 difference in the NS2 protein (Table 1). Exchange of the NS
gene resulted in increased mortality (8 of 8) compared to the
rIndo parent (6 of 8) (Table 2). Infection with rIndo/rEgret NS
did not result in a significant difference in MDT (Table 2) or
survival rate (Fig. 1) compared to the rIndo parent. Viral
antigen distribution in tissues was expanded and differed from
the distribution of the parent rIndo virus among tissues and
was more similar to the rIndo/rEgret HA virus distribution
(Table 3). Exchange of the NS gene did not result in a repli-
FIG. 2. Experimental studies of chickens that were IN inoculated
with AIV H5N1 recombinant viruses; representative microscopic
lesions and immunohistologic findings. Photomicrographs (?200) of
tissue sections stained with hematoxylin and eosin (A, C, E, and G) or
by immunohistochemistry to demonstrate AIV (B, D, F, H, I, and J).
(A) Histiocytic interstitial pneumonia in a 2-week-old chicken inocu-
lated with rEgret, 2 dpi, with congestion and fibrous exudates in the
airways. (B) AI viral antigen (red, arrows) in macrophages and air
capillary and blood vessel endothelium in lung tissue of the same
chicken. (C) Spleen tissue of a 2-week-old chicken inoculated with
rEgret, 2 dpi; mild focal splenitis. (D) AI viral antigen (red, arrow) in
macrophages and vascular endothelial cells in spleen tissue of the same
chicken. (E) Severe interstitial pneumonia in a 2-week-old chicken
inoculated with rIndo/Egret NP, 2 dpi. There is congestion, mononu-
clear infiltrates, and serofibrinous exudates filling air capillaries.
(F) Diffuse staining (red) for AI viral antigen in the endothelium and
infiltrating macrophages in lung tissue of the same chicken.
(G) Splenic necrosis in a 2-week-old chicken inoculated with rIndo/
Egret NP, 2 dpi. (H) AI viral antigen (red) in macrophages in spleen
tissue of the same chicken. (I) AI viral antigen (red) in brain tissue of
a 2-week-old chicken inoculated with rIndo/Egret NP, 2 dpi. Staining
present in neurons and glial cells of brain tissue. (J) AI viral antigen
(red) in the myocardial cells of the heart of a 2-week-old chicken
inoculated with rIndo/Egret NP, 2 dpi.
VOL. 82, 2008 REASSORTANT H5N1 AVIAN INFLUENZA VIRUSES IN CHICKENS 4547
cation advantage in spleen and lung tissues, as the virus titers
remained at the rIndo parent levels (Fig. 3). Titers of rIndo/
rEgret NS in brain tissue were slightly elevated over those of
rIndo and rEgret. Expression of IFN-? was less than that of the
rIndo virus in both spleen and lung tissues, while iNOS expres-
sion levels were similar to the control levels in both tissues
(Fig. 4 and 5). IFN-? could not be detected in lung tissue, and
levels in spleen tissue were similar to the rIndo parent levels.
Only the Mx1 gene expression levels were increased over those
of rIndo in both spleen and lung tissues.
The M2 protein contributes to the expanded tissue range of
the reassortant virus. The rIndo and rEgret viruses share iden-
tical M1 protein sequences but differ in three amino acids in
the M2 spliced gene product (Table 1). Therefore, the changes
in pathogenicity that we saw when the M gene was exchanged
are due to the differences in the M2 protein alone. Exchanging
the M gene in rIndo led to increased mortality (8 of 8) com-
pared to the rIndo parent virus (6 of 8) (Table 2) and expanded
distribution of viral antigen in tissues (Table 3). Virus titers of
the rIndo/rEgret M virus in spleen tissue remained just above
the detection limit. Titers in lung tissue were lower than the
rIndo titers, and titers in brain tissue were at the level of rEgret
and rIndo despite the lack of viral antigen staining in the brain
tissue of rIndo/rEgret M-infected chickens (Fig. 3). The MDT
of rIndo/rEgret M-infected chickens was not significantly dif-
ferent from either rIndo or rEgret infection (Table 2), and the
survival rates were also not significantly different from infec-
tion with the rIndo parent (Fig. 1B). Despite the altered patho-
genesis observed with rIndo/rEgret M infection, mRNA gene
expression in the spleen tissue of infected chickens 2 dpi did
not differ greatly from the expression in chickens infected with
the rIndo parent virus. Gene expression of Mx1 and IFN-? in
lung tissue showed slight upregulation over levels in chickens
with rIndo infection (Fig. 5).
The nucleoprotein is important for viral replication and
pathogenicity of the reassortant virus. There are five amino
acid differences in the NP protein between the rIndo and
rEgret viruses (Table 1). The exchange of the NP gene resulted
FIG. 3. Virus titers in spleen, lung, and brain tissues. Tissues were taken 2 dpi and homogenized to a 10% (wt/vol) final concentration in BHI
medium. A portion (100 ?l) of 10-fold dilutions of the homogenates was inoculated into 10-day-old ECEs, and log10EID50/g was calculated. Values
are the mean ? standard error (n ? 2) with the exception of the rIndo/rEgret PB1 lung titer (n ? 1). The threshold of detection is 2.4 log10
EID50/gram of tissue.
TABLE 3. Severity of histological lesions and distribution of viral antigen in tissues from chickens IN inoculated with recombinant AIVsa
Histological lesions/viral antigen stainingb
rEgretrIndorIndo/Egret HArIndo/Egret NSrIndo/Egret MrIndo/Egret NPrIndo/Egret PB2rIndo/Egret-PB1
aTissues were taken 2 dpi and were immunohistochemically stained with antibodies to AIV nucleoprotein to visualize the viral antigen.
bLesions were scored as follows: ?, no lesions; ?, mild; ??, moderate; ???, severe lesions. The intensity of viral antigen staining in each section was scored as
follows: ?, no antigen staining; ?, infrequent; ??, common; ???, widespread staining.
4548WASILENKO ET AL.J. VIROL.
in an increase in mortality (8 of 8) compared to the rIndo
parent (Table 2). A significant decrease in MDT (2.3 days)
over the rIndo parent infection (6.1 days) was seen; however,
there was no significant difference in MDT compared to the
rEgret parent (Table 2). Survival rates of birds infected with
rIndo/rEgret NP were significantly different than the survival
rates of all other groups, including rEgret (Fig. 1). Infection
with the rIndo/rEgret NP virus also greatly increased viral
antigen staining in nearly all tissues tested at 2 dpi (Table 3;
Fig. 2). Titers from rIndo/rEgret NP-infected tissues demon-
strate that the replication advantage conferred by the exchange
of the NP gene resulted in increased viral titers in spleen, lung,
and brain tissues compared to all of the other viruses tested
(Fig. 3). IFN-? mRNA expression levels were similar to those
of the rIndo parent virus in both spleen and lung tissues, but
there was a sharp increase in IFN-? and iNOS as well as a
slight upregulation of Mx1 expression in both tissues compared
to gene expression resulting from infection with the rIndo
parent (Fig. 4 and 5).
PB1 or PB2 exchange results in decreased replication and
pathogenicity of the reassortant viruses. When either the
rEgret PB2 gene or PB1 gene was exchanged in the rIndo virus,
a decrease in mortality (3 of 8 and 2 of 8, respectively) was seen
compared to infection with rIndo (6 of 8) (Table 2). The two
viruses differed by 5 amino acids in the PB1 and 1 amino acid
in the PB1-F2 protein, and the PB2 proteins differed by 10
amino acids (Table 1). The MDTs of both viruses were not
significantly different from that of the rIndo parent virus (Fig.
1). The survival rate of birds infected with rIndo/rEgret PB2
and PB1 viruses was not different from the control group
survival rate, but survival of birds infected with rIndo/rEgret
PB1 was also different from the rIndo parent survival rate (Fig.
Viral replication was minimal and was consistent with viral
antigen staining. Viral titers were very low with rIndo/rEgret
PB2 infection, slightly above the threshold of detection in
spleen and lung tissues and at the threshold for brain tissue.
The rIndo/rEgret PB1 virus titers were at, or below, the thresh-
old of detection in all three tissues (Fig. 3). Infection with
rIndo/rEgret PB1 also resulted in decreased mRNA expression
of several immune-related genes compared to rIndo infection
(Fig. 4 and 5), most notably Mx1 in lung and brain tissues and
iNOS in spleen tissue. Infection with rIndo/rEgret PB1 re-
sulted in increased IFN-? gene expression levels in lung tissue
(Fig. 5) but not in spleen tissue (Fig. 4). Both rIndo/rEgret PB2
and rIndo/rEgret PB1 infection resulted in a marked down-
regulation of Mx1 in both spleen and lung tissues of infected
chickens compared to the control expression levels. As with
rIndo/rEgret PB1, rIndo/rEgret PB2 infection resulted in in-
creased expression levels of IFN-? in lung tissue but remained
FIG. 4. Semiquantitative analysis of differential mRNA gene expression in the spleen tissue of chickens infected with AIV recombinants. Total
cellular RNA was extracted from spleen tissue collected 2 dpi from three chickens. Equal amounts of RNA from the three chickens per group were
pooled prior to RT-PCR analysis. Analysis of the pooled RNA was carried out using different primer sets with ?-actin as an amplification and
loading control (n ? 1). Bands were quantified, and intensities shown were normalized to the ?-actin control. Gene expression of control birds
was arbitrarily set to 1.
VOL. 82, 2008REASSORTANT H5N1 AVIAN INFLUENZA VIRUSES IN CHICKENS 4549
at the same level as the rIndo parent virus in spleen tissue, as
did the iNOS expression levels in both tissues (Fig. 4 and 5).
The PA gene was not reassorted because there were no amino
acid differences between the two viruses.
The rIndo and rEgret viruses, although having a high se-
quence similarity (?91%) for all eight gene segments, had a
marked difference in virulence levels in chickens. Using reverse
genetics, a series of variant viruses differing by a single gene
segment was created in order to explore the contribution of
individual viral genes to viral pathogenesis in chickens. Unex-
pectedly, the reassortment of the NP gene resulted in the
biggest differences in virulence and replication. Some increase
in pathogenicity was seen with the exchange of the HA, NS,
and M gene segments, while the PB1 and PB2 reassortants had
impaired replication resulting in low virulence. Alterations in
host gene expression in response to infection with the reassor-
tant viruses suggest that the viruses use different mechanisms
to evade host responses.
HA is known to be an important determinant in the viru-
lence of AIV (9, 22, 47, 59). The HA genes of both H5N1
recombinant parent viruses contain the same multiple basic
amino acid motif adjacent to the cleavage site. However, there
were 11 other amino acid differences between viruses outside
of the cleavage site of the HA gene, 5 of them localized in
the receptor binding domain. The increased pathogenicity of
the rIndo/rEgret HA virus over the rIndo parent may therefore
be attributed to mutations outside of the multiple basic cleav-
age site, possibly in the receptor binding site.
Since we were not able to rescue the rIndo/rEgret NA reas-
sortant, the role of NA could not be explored in this study. One
possible reason may be that HA-NA incompatibility is respon-
sible for inefficient replication of the rIndo/rEgret NA reassor-
tant virus in 293T cells or ECEs. The HA and NA proteins of
influenza work together to efficiently bind and release virus
from the cells during replication, and it is known that HA-NA
incompatibility can result in inefficient viral replication or ag-
gregation on the cell (10, 44). The importance of a balanced
HA-NA relationship often results in the coevolution of NA
along with changes in HA of influenza viruses to maintain a
functional unit (34, 35, 57). There is some evidence the com-
mon chicken-adapted NA stalk deletion may result in steric
hindrance causing inefficient virus release (34). The rIndo
chicken AIV has a 20-amino-acid NA stalk deletion, while
rEgret virus NA does not carry this deletion. The importance
of possible HA-NA incompatibility warrants further study.
Previous studies have shown that the M gene from A/Mal-
lard/78 attenuates the H3N2 human viruses in squirrel mon-
keys and humans (13, 56). M has not previously been identified
as an important virulence factor in pathogenesis in chickens.
The matrix protein gene consists of spliced mRNA products
encoding M1, an internal structural protein, and M2, an inte-
FIG. 5. Semiquantitative analysis of differential mRNA gene expression in lung tissue from chickens infected with AIV recombinants. Total
cellular RNA was extracted from spleen and lung tissues from three chickens 2 dpi. Equal amounts of RNA from the three chickens per group
were pooled prior to RT-PCR analysis. Analysis was carried out using different primer sets with ?-actin as an amplification and loading control.
Bands were quantified, and intensities shown were normalized to the ?-actin control. Gene expression of control birds was arbitrarily set to 1.
4550WASILENKO ET AL.J. VIROL.
gral membrane ion-channel protein (58). Our recombinant
viruses have identical M1 protein sequences allowing us to
further attribute that the increased pathogenicity was due to
the three amino acid changes in the M2 sequence. The M2
ion-channel protein is thought to stabilize the HA proteins,
especially H5 and H7 HA proteins with multiple basic cleavage
sites, by maintaining the pH so that the conformation of HA
into the low-pH form does not occur prematurely (12). The
mechanism by which rEgret M2 increases the pathogenicity of
the rIndo virus may be the result of the increased stabilization
of rIndo HA so that attachment and replication of the rIndo/
rEgret M virus are more efficient, therefore allowing the
spread to a larger range of tissues than was seen with the viral
antigen staining (Table 3). However, viral replication data
from lung and spleen tissues do not appear to support this
explanation, as we did not see higher titers of rIndo/rEgret M
than of rIndo in those tissues in infected chickens (Fig. 3).
However, titers were measured at only one time point and may
not reflect titers throughout infection.
The effect of the M2 protein on virulence has been widely
studied, as it relates to resistance to the antiviral drug aman-
tadine, which blocks M2-ion channel activity (23). One of these
studies reported that the S31N mutation increased the viru-
lence of the A/WSN/33 virus in mice as well as conferred
amantadine resistance (1), and another study reported that the
S31N mutation reduced the activity of A/Chicken/Germany/34
M2 (20). Based on these previous studies, we expected that the
rIndo virus containing an N at position 31 of M2 would result
in a more virulent phenotype for chickens; however, that is not
the case. When rEgret M2 with the reportedly less virulent S at
position 31 replaced rIndo M2, the rIndo/rEgret M virus
showed increased virulence and tissue distribution. It is possi-
ble that one of the other two amino acid changes in M2 has a
greater contribution to virulence or that the other mutations
suppress the S31N mutation in rIndo M2, resulting in a less
virulent strain. The mechanism by which M2 increases the
virulence of rIndo/rEgret M needs further exploration.
Influenza NP is important in the packaging of the viral RNA
and has been shown to be involved in many aspects of viral
replication (41). Small interfering RNA against NP resulted in
decreased viral replication in ECEs and in mice (18, 19). It has
previously been demonstrated that NP directly interacts with
viral PB2, PB1, other viral NPs, and also other cellular factors
(7, 41). Previous studies have shown viral replication to be
more efficient when the NP and polymerase genes are derived
from the same virus, indicating viral replication requires the
proper combination of genes to function properly (37, 43). The
NP gene has also been shown to be important for host range
restriction for A/Mallard/78 or A/Pintail/Alberta/119/79 and
has also been shown to attenuate the H3N2 human viruses in
squirrel monkeys (13, 51, 56). NP has not previously been
identified as a sole virulence or replication factor in the patho-
genesis of AI in chickens. We show here that the exchange of
NP was sufficient to greatly increase replication, tissue tropism,
and virulence and alter the expression of selected host genes in
chickens. The mechanism by which NP increases virulence
remains unclear. Our reassortant viruses have five differences
in the NP amino acid sequence. One mutation, at position 22,
spans both the RNA binding and PB2-1 binding domains of NP
(41). Three of the other mutations (positions 400, 406, and
423) are located in the overlapping regions of the NP-2 and
PB2-3 binding domains, suggesting that it is either the NP or
PB2 binding function that is resulting in the increased replica-
tion (41). One possibility is that the altered NP-PB2 interaction
of the rIndo/rEgret NP virus results in more efficient binding in
the polymerase complex that aids increased replication, allow-
ing the virus to spread more efficiently to all tissues, resulting
in a more severe systemic disease.
PB1 has been shown to be associated with the high patho-
genicity of some H5N1 viruses in ducks (26), and the alternate
splice product, PB1-F2, has been shown to be important in the
virulence of human fatal case viruses in mice (14, 61). PB2 has
been shown to contribute to the virulence of AIV, with a lysine
residue at amino acid 627 being linked to the increased viru-
lence of H5N1 viruses (22). Neither of our recombinant viruses
has the lysine at amino acid 627 in PB2, suggesting that this
residue is not important for pathogenesis in chickens. The
polymerase complex (PA, PB2, and PB1) has been shown to
work inefficiently when the components are derived from dif-
ferent host viruses (13, 31, 37, 45), indicating that this relation-
ship is vital to replication and that these proteins may adapt
together to maintain functionality (39). While both of the re-
combinant viruses that we used were avian in origin, some
incompatibility may occur when the polymerase complex com-
ponents are derived from two different parent viruses. Further
studies will help determine the role of compatibility among the
polymerase genes in viral replication.
In accordance with previous studies, we found that PB1 and
PB2 are important for efficient virus replication (Table 3; Fig.
3). PB1 interacts with PB2, PA, and NP, and the multiple
binding interactions may explain the lower titers of the rIndo/
rEgret PB1 virus. The PB1 gene of more than one human
pandemic virus is known to be derived from avian viruses (29,
46). None of the five amino acid differences are in known
binding domains of PB1. For PB2, analysis of the binding
domains of PB2 indicates that the NP, PB1, and cap-binding
(24, 40) functions could be affected by the 105, 132, 221, 251,
389, or 394 amino acid changes that fall in those binding
domains. Disrupting the functional polymerase complex unit is
not favored for optimal replication efficiencies, as seen by the
decreased distribution, decreased tissue staining, and de-
creased viral titers in tissues (Table 3; Fig. 3).
Regulation of host gene expression may in part explain the
mechanism used by some of the reassortant viruses to evade
the host immune response. IFN-?, a cytokine previously shown
to have an antiviral effect on influenza viruses (33), was down-
regulated in both lung and spleen tissues infected by rIndo/
rEgret HA and rIndo/rEgret NS compared to the level in lung
and spleen tissues infected by the rIndo parent (Fig. 3). Down-
regulation of antiviral IFN-? may be one of the mechanisms
that these viruses use to evade the host responses and may
contribute to the increased replication of the viruses in tissues,
leading to the increased viral antigen staining that we saw
(Table 3). Infection with rIndo/rEgret PB1 and rIndo/rEgret
PB2 viruses resulted in upregulation of IFN-? in lung tissue
(Fig. 5). The increased IFN-? levels may explain why these
reassortants do not show increased virulence and have de-
creased replication in tissues (Table 3 and Fig. 3). The upregu-
lation of IFN-? in the lungs may have been enough to fight
infection at the site and prevent the systemic spread of the
VOL. 82, 2008 REASSORTANT H5N1 AVIAN INFLUENZA VIRUSES IN CHICKENS4551
virus. The D92E NS1 mutation has been shown to increase
resistance to IFN in pigs (48); however, none of our recombi-
nant viruses harbors this mutation. The fact that the replication
of the rEgret and rIndo viruses in the spleen was not affected
by IFN-? upregulation suggests it is one of many factors that
the host can regulate to inhibit influenza infection.
Upregulation of the Mx gene has been shown to increase
with IFN-? expression, and there is some evidence that this
may help combat influenza in mammals (17, 21). In a chicken
cell line, chicken Mx1 did not result in antiviral effects against
influenza (6); however, it was later found that Mx1 is a poly-
morphic gene and that the Mx protein from different breeds of
chickens can in fact have antiviral properties against influenza
(30). Our results indicate that Mx1 is strongly downregulated in
the lungs and spleens of rIndo/rEgret PB1- and rIndo/rEgret
PB2-infected chickens (Fig. 4 and 5), yet there is no replicative
advantage in downregulating Mx1 for these viruses (Fig. 3;
Table 3). Therefore, Mx1 gene expression levels in the lung and
spleen did not appear to correlate with the virulence of our
recombinants in chickens and support previous findings that
chicken Mx may not have antiviral activity in all chicken breeds
IFN-? has been shown to increase the secretion of reactive
oxygen species such as nitric oxide (NO) (2, 4, 15, 28, 53), and
there is some evidence that NO has antiviral properties (3, 15,
28). In the spleen, rEgret and rIndo/rEgret NP infection re-
sulted in an increase in iNOS and IFN-? gene expression.
However, only the rIndo/rEgret NP virus showed substantial
viral replication in the spleen. The increased NO production
may reflect the rapid replication of the rIndo/rEgret NP virus
and the host’s attempt to prevent the replication. The short
MDTs and increased tissue lesions resulting from infection
with these viruses may be due to the NO (Table 3; Fig. 2).
In particular, rIndo/rEgret NP caused extremely short MDTs
and massive replication of the virus in tissues (Table 2 and
Fig. 3). The rIndo/rEgret NP virus not only increased IFN-?
and iNOS expression in the spleen but was the only virus
studied that upregulated these genes in the lungs as well, which
also may explain in part the increased virulence of this strain in
In summary, the pathogenicity of AIVs is clearly due to a
polygenic effect, and in this study, single-gene changes led to
restriction or increased replication of AIVs in chickens. Reas-
sortment events can result in a number of different gene com-
binations; however, we, and others, have shown that all possi-
ble combinations of genes in reassortant viruses are not
necessarily viable. From the remaining viable gene reassor-
tants, we were able to determine the effects of single-gene
changes on the replication and pathogenesis of AIVs in chick-
ens. While the precise role of chicken immune response factors
is not clear, changes in the host gene expression of several
genes involved in immunity are influenced by infection with the
reassortant viruses and may be contributing factors in the
pathogenicity of AIVs in chickens. Based on the results ob-
tained in this study, the next step is to investigate the role of
individual amino acids in each of the genes shown to affect
AIV pathogenicity, as well as to continue exploring the effect
of certain gene combinations.
This work was supported by USDA, ARS CRIS, project 6612-
The authors thank Diane Smith, Carlos Estevez, Patti Miller, Kristin
Zaffuto, Melissa Scott, and the SAA sequencing facility for technical
assistance and Roger Brock for animal care assistance.
Mention of trade names or commercial products in the manuscript
is solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department of
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