JOURNAL OF VIROLOGY, June 2011, p. 5394–5405
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 11
Mutations in the Fusion Protein Cleavage Site of Avian Paramyxovirus
Serotype 2 Increase Cleavability and Syncytium Formation but
Do Not Increase Viral Virulence in Chickens?
Madhuri Subbiah,1† Sunil K. Khattar,1† Peter L. Collins,2and Siba K. Samal1*
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland,1and Laboratory of
Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland2
Received 30 December 2010/Accepted 17 March 2011
Avian paramyxovirus serotype 2 (APMV-2) is one of the nine serotypes of APMV, which infect a wide variety
of avian species around the world. In this study, we constructed a reverse genetics system for recovery of
infectious recombinant APMV-2 strain Yucaipa (APMV-2/Yuc) from cloned cDNA. The rescued recombinant
virus (rAPMV-2) resembled the biological virus in growth properties in vitro and in pathogenicity in vivo. The
reverse genetics system was used to analyze the role of the cleavage site of the fusion (F) protein in viral
replication and pathogenesis. The cleavage site of APMV-2/Yuc (KPASR2F) contains only a single basic
residue (position ?1) that matches the preferred furin cleavage site [RX(K/R)R2]. (Underlining indicates the
basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.) Contrary to what
would be expected for this cleavage sequence, APMV-2 does not require, and is not augmented by, exogenous
protease supplementation for growth in cell culture. However, it does not form syncytia, and the virus is
avirulent in chickens. A total of 12 APMV-2 mutants with F protein cleavage site sequences derived from APMV
serotypes 1 to 9 were generated. These sites contain from 1 to 5 basic residues. Whereas a number of these
cleavage sites are associated with protease dependence and lack of syncytium formation in their respective
native viruses, when transferred into the APMV-2 backbone, all of them conferred protease independence,
syncytium formation, and increased replication in cell culture. Examination of selected mutants during a
pulse-chase experiment demonstrated an increase in F protein cleavage compared to that for wild-type
APMV-2. Despite the gains in cleavability, replication, and syncytium formation, analysis of viral pathogenicity
in 9-day-old embryonated chicken eggs, 1-day-old chicks, and 2-week-old chickens showed that the F protein
cleavage site mutants did not exhibit increased pathogenicity and remained avirulent. These results imply that
structural features in addition to the cleavage site play a major role in the cleavability of the F protein and the
activity of the cleaved protein. Furthermore, cleavage of the F protein is not a determinant of APMV-2
pathogenicity in chickens.
Paramyxoviruses are pleomorphic, enveloped viruses con-
taining a nonsegmented, negative-sense, single-stranded
RNA genome. These viruses have been isolated from a wide
variety of mammalian and avian species around the world
(20). All paramyxoviruses isolated from avian species are
classified into the genus Avulavirus, representing the avian
paramyxoviruses (APMV), and the genus Metapneumovirus,
representing the avian metapneumoviruses in the family
Paramyxoviridae. APMV have been divided into nine differ-
ent serotypes (APMV-1 to -9) based on hemagglutination
inhibition (HI) and neuraminidase inhibition (NI) assays (1).
APMV-1, which includes all strains of Newcastle disease virus
(NDV), has been characterized extensively because virulent
NDV strains cause severe disease in chickens. The complete
genome sequence and reverse genetics systems are available
for the following representative NDV strains: lentogenic
strains LaSota (28, 30) and B1 (24), mesogenic strains Beau-
dette C (18) and Anhinga (13), and velogenic strains Hert/33
(11), ZJ1 (21), and Texas GB (unpublished data). As an initial
step toward characterizing the other APMV serotypes, com-
plete genome sequences of one or more representative strains
of APMV serotypes 2 to 9 were recently determined (8, 19, 25,
26, 33, 34, 36, 38).
APMV-2 was first isolated from a diseased chicken in 1956
in Yucaipa, CA (7). Since then, many APMV-2 strains have
been isolated from domestic poultry and from free-range, cap-
tive, and wild birds around the world (2). APMV-2 infections
have been reported in chickens in the United States, Canada,
Russia, Japan, Israel, India, Saudi Arabia, and Costa Rica and
in turkeys in the United States, Canada, Israel, France, and
Italy (4). The infection is more prevalent in turkeys than in
chickens (6). APMV-2 infection was shown to affect hatchabil-
ity and poult yield in turkeys (5). APMV-2 was found to cause
a drop in egg production in commercial layer and broiler
breeder farms in Scotland (37). APMV-2 strains have also
frequently been isolated from passerine and psittacine birds.
Surveillance of wild birds has indicated that APMV-2 infec-
tions are more frequent in passerines (3, 35).
The genome of APMV-2 strain Yucaipa (APMV-2/Yuc) is
14,904 nucleotides (nt) in length and contains a 55-nt leader
sequence at the 3? end and a 154-nt trailer sequence at the 5?
* Corresponding author. Mailing address: Virginia-Maryland Regional
College of Veterinary Medicine, University of Maryland, College Park,
MD 20742. Phone: (301) 314-6813. Fax: (301) 314-6855. E-mail: ssamal
† These authors contributed equally to the work presented in this
?Published ahead of print on 30 March 2011.
end. The genome consists of genes that encode nucleocapsid
protein (N), phosphoprotein (P), matrix protein (M), fusion
protein (F), hemagglutinin-neuraminidase protein (HN), and
large protein (L). The genes are flanked on either side by
highly conserved transcription start and stop sequences and
have intergenic sequences of various lengths. Similarly to that
for other paramyxoviruses, the P gene contains a putative ed-
iting site for the production of V and W proteins (36). Thus,
the APMV-2 genome is broadly similar to that of NDV.
The envelope of paramyxoviruses contains two surface gly-
coproteins, the hemagglutinin-neuraminidase (HN) protein,
which is responsible for attachment to the host cell, and the
fusion (F) protein, which mediates fusion of the viral envelope
with the cell membrane. The F protein is synthesized as an
inactive precursor (F0) and is cleaved into two biologically
active, disulfide bonded F2-F1 subunits by host cell protease
(20). Cleavage of the F protein is necessary for virus entry and
cell-to-cell fusion. The F protein cleavage site is a well-char-
acterized determinant of NDV pathogenicity in chickens (17,
23, 27, 28). The F protein of mesogenic and velogenic strains of
NDV typically contains a polybasic cleavage site [(R/K)RQ(R/
K)R2F)] that contains the preferred recognition site for furin
[RX(K/R)R2], which is an intracellular protease present in a
wide range of cells and tissues. (Underlining indicates the basic
amino acids at the F protein cleavage site, and the arrow
indicates the site of cleavage.) Consequently, the F protein of
these strains can be cleaved in different tissues, making it
possible for virulent strains to spread systemically. In con-
trast, avirulent NDV strains typically have basic residues at
the ?1 and ?4 positions in the cleavage site [(G/E)(K/
R)Q(G/E)R2L)] and depend on secretory protease (or, in cell
culture, added trypsin or chicken egg allantoic fluid) for cleav-
age. This limits the replication of avirulent strains to the re-
spiratory and enteric tracts, where the secretory protease is
The putative F cleavage site of APMV-2/Yuc (93DKPASR2F99)
has two basic residues (underlined), of which only the basic
residue in the ?1 position conforms to the preferred furin
cleavage site. Conversely, the F1 subunit of strain Yucaipa
begins with a phenylalanine residue, as is characteristic of
virulent NDV strains, rather than a leucine residue, as is seen
in most avirulent NDV strains (9). APMV-2 strain Yucaipa
replicates in a wide range of cells without the addition of
exogenous protease, and the inclusion of protease does not
improve the efficiency of replication (36). This is incongruent
with the lack of the preferred furin motif, although it was
not known whether efficient intracellular cleavage indeed
occurred. Interestingly, APMV-2 produces single-cell infec-
tions and does not cause syncytium formation, a hallmark of
paramyxovirus cytopathic effect (CPE). Also, APMV-2/Yuc is
highly attenuated in chickens, which is incongruent with its
independence from exogenous protease. Thus, questions re-
mained about the cleavability of the F protein of APMV-2/Yuc
and its role in infectivity and pathogenicity.
To investigate the role of the F protein cleavage site in repli-
cation and pathogenesis of APMV-2, we have developed a re-
verse genetics system for APMV-2/Yuc. The full-length APMV-2
cDNA clone was used to generate 10 APMV-2 mutants whose
F protein cleavage sites were derived from APMV-1 to -9, as
well as an 11th mutant in which the only difference from the
recombinant virus (rAPMV-2) was a change in the terminal
residue of the F1 subunit from phenylalanine to leucine. All of
the F protein cleavage site mutants replicated efficiently and
maintained the mutations after propagation in embryonated
chicken eggs. The 10 mutants containing cleavage sites derived
from the other APMV serotypes exhibited protease indepen-
dence, syncytium formation, and increased replication in vitro.
However, the mutations did not change the avirulent nature of
APMV-2/Yuc as determined by mean death time (MDT) in
9-day-old embryonated chicken eggs, by intracerebral patho-
genicity index (ICPI) in 1-day-old chicks, and by natural route
of infection in 2-week-old chickens. These results suggest that
the cleavage site sequence is not a major determinant for
cleavability of the F protein of APMV-2 and virulence of
APMV-2 in chickens.
MATERIALS AND METHODS
Viruses and cells. APMV-2 strain Yucaipa (APMV-2/Yuc) was obtained from
the National Veterinary Services Laboratory, Ames, IA. The virus was grown in
the allantoic cavities of 9-day-old embryonated, specific-pathogen-free (SPF)
chicken eggs. The allantoic fluid was collected 3 days postinoculation (dpi), and
the hemagglutination (HA) titer was determined using 0.5% chicken erythro-
cytes (RBC) at room temperature. The modified vaccinia virus strain Ankara
expressing T7 RNA polymerase (MVA) was grown in primary chicken embryo
fibroblasts (DF1). DF1, human epidermoid carcinoma (HEp-2), and Madin-
Darby canine kidney (MDCK) cell lines were grown in Dulbecco’s modified
Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and main-
tained in DMEM with 5% FBS. The African green monkey kidney Vero cell line
was grown in Eagle’s minimal essential medium (EMEM) containing 10% FBS
and maintained in EMEM with 5% FBS. In experiments that required supple-
mentation of exogenous protease for cleavage of the F protein, either 1 ?g/ml of
acetyl trypsin (Invitrogen) or 5% chicken egg allantoic fluid was used. The ability
of the viruses to produce plaques was tested in DF1, Vero, and MDCK cells
under 0.8% methylcellulose overlay. Plaques were visualized by immunoper-
oxidase staining using a polyclonal antiserum raised against APMV-2/Yuc in
Construction of plasmids expressing support proteins and the full-length
antigenome. Support plasmids pN, pP, and pL were constructed to individually
express the N, P, and L proteins, respectively. cDNAs bearing the open reading
frames (ORFs) of the N, P, and L genes (positions 141 to 1514, 1681 to 2880, and
7938 to 14666 in the complete genomic sequence, respectively) were cloned
under the control of the T7 RNA polymerase promoter in vector pGEM7Z (N
and P genes) or pTM1 (L gene). A full-length cDNA of the APMV-2 genome
was constructed in plasmid pBR322/dr/Yuc. This plasmid is a modified form of
plasmid pBR322 in which the fragment between the EcoRI and PstI sites was
removed and replaced by a 72-nt oligonucleotide linker. An 84-nt hepatitis delta
virus (HDV) antigenome ribozyme sequence and a T7 RNA polymerase tran-
scription termination signal (10) were inserted into the downstream end of this
linker. The full-length cDNA clone (pAPMV-2) expressing the complete 14,904-
nt-long antigenome of APMV-2/Yuc was constructed as six fragments that were
generated by reverse transcription-PCR (RT-PCR) of RNA from APMV-2/Yuc-
infected cells. To facilitate construction, a total of five unique restriction enzyme
sites were created by mutating 10 nt without changing any amino acids (Fig. 1).
These fragments were sequentially cloned into the pBR322/dr/Yuc plasmid be-
tween the T7 promoter and the HDV antigenome ribozyme sequence (Fig. 1). In
the full-length cDNA, the F ORF is flanked by unique NotI and PacI enzyme
sites, which made it possible to readily substitute mutated F genes. A total of 11
F protein cleavage site mutants (Table 1) were generated by overlapping PCR.
The mutated fragments were digested with NotI and PacI enzymes and were
used to replace the corresponding fragments in the full-length cDNA of
pAPMV-2. The full-length cDNAs of all fusion protein cleavage site mutants
were sequenced in their entirety using an ABI 3130xl genetic analyzer (Applied
Recovery of recombinant viruses. Infectious virus was recovered from the
cDNAs as previously described (10, 14, 18). Briefly, HEp-2 cells were grown
overnight to 70% confluence in six-well culture plates and were cotransfected
with 5 ?g of the respective full-length cDNA plasmid, 3 ?g of pN, 2 ?g of pP, and
1 ?g of pL by using 15 ?l of Lipofectamine 2000 (Invitrogen). Along with the
transfection mixture, 1 focus-forming unit per cell of recombinant vaccinia virus
VOL. 85, 2011 EFFECTS OF APMV-2 F PROTEIN MUTATIONS IN CHICKENS5395
expressing T7 RNA polymerase (MVA) was added. The transfection mixture was
replaced after 6 h with DMEM containing 5% FBS. Two days after transfection,
the HEp-2 cells were scraped into the medium and frozen and thawed three
times, and the resulting supernatant was inoculated into the allantoic cavities of
9-day-old embryonated, SPF chicken eggs. The allantoic fluid was harvested 3 dpi
and tested for HA activity. Allantoic fluid with a positive HA titer was used for
the isolation of the viral RNA followed by sequence confirmation of the F
protein cleavage site. The recovered parental and mutant viruses were passaged
three times in 9-day-old embryonated chicken eggs, and their stability was ver-
ified by sequencing the complete F ORF of each mutant virus. Additionally, the
HN ORF of each mutant virus was sequenced after three passages to ensure that
there were no mutations in the HN protein.
Growth characteristics of F protein cleavage site mutant viruses. The growth
characteristics of the parental and mutant viruses were evaluated in DF1, Vero,
and MDCK cells with and without 5% allantoic fluid supplementation in the
medium. The ability of the mutant viruses to produce plaques was tested in DF1,
Vero, and MDCK cells under 0.8% methylcellulose overlay. The plaques were
immunostained using anti-APMV-2/Yuc polyclonal serum in DF1 and Vero cells
3 dpi or in MDCK cells 7 dpi depending on the onset of plaques. The ability of
mutant viruses to produce syncytia was tested in DF1 cells. The cells were fixed
with cold methanol 3 dpi.
The growth kinetics of the wild-type, parental, and mutant viruses was deter-
mined in DF1 cells. Briefly, DF1 cells grown in six-well plates were infected with
each virus at a multiplicity of infection (MOI) of 1 with or without 5% allantoic
fluid supplementation. After a 1-h adsorption, the infected cells were washed
with phosphate-buffered saline (PBS), the medium was replaced, and the cul-
tures were incubated at 37°C. At 24, 48, 72, 96, and 120 h postinfection, 200 ?l
of culture supernatants was collected and stored at ?70°C for virus titration, and
an equal volume of fresh medium was added back. Virus titers of the samples
were determined by serial endpoint assay on DF1 cells in 96-well plates, with
duplicate wells per virus per dilution. The infected cells were stained by an
immunoperoxidase method using the polyclonal antiserum raised against
APMV-2/Yuc in chickens. The virus titers (50% endpoint tissue culture infec-
tious dose [TCID50]/ml) were calculated using the method of Reed and Muench
Preparation of hyperimmune serum against the F protein of APMV-2 strain
Yucaipa. A keyhole limpet hemocyanin (KLH)-conjugated synthetic peptide (27
residues), representing amino acids 510 to 536 of the cytoplasmic tail of the F
protein of APMV-2 strain Yucaipa, was custom synthesized (Invitrogen). A
rabbit was injected subcutaneously with 0.5 mg of peptide in Freund’s complete
adjuvant. After 2 weeks, a booster immunization was given with 0.5 mg peptide
in Freund’s incomplete adjuvant, and 2 weeks later, the hyperimmune serum was
collected. Western blotting was performed using infected-cell lysates to confirm
the specificity of antiserum to the Yucaipa F protein (data not shown).
Pulse-chase labeling and radioimmunoprecipitation. Radioimmunoprecipita-
tions were performed as described previously (16). Briefly, DF1 cells were in-
fected with parental or mutant APMV-2 at an MOI of 10 for 12 h at 37°C, after
which time the cells were switched to methionine- and cysteine-free medium
(starvation medium). After 2 h, the medium was replaced by starvation medium
containing 100 ?Ci per ml of [35S]methionine-cysteine (EasyTag EXPRESS35S
protein labeling mix; PerkinElmer), and the cells were incubated at 37°C for 30
min (pulse). Subsequently, the medium was replaced by medium containing an
excess of unlabeled methionine and cysteine, and the cells were further incu-
bated for 0, 20, 40, and 60 min (chase). Cell lysates were prepared by lysing the
cells in radioimmunoprecipitation assay (RIPA) buffer (0.005 M Tris-HCl, 0.15
M NaCl, 1% sodium deoxycholate, 1% NP-40, 1 mM phenylmethylsulfonyl
fluoride [PMSF]), and the APMV-2 F protein was precipitated from the lysates
by incubation with antiserum specific for the APMV-2 F cytoplasmic tail and
analyzed by 10% SDS-polyacrylamide gel electrophoresis. Labeled proteins were
visualized by autoradiography. Autoradiographs were scanned, and densitomet-
ric analysis of the F0 and F1 protein bands was performed by using the Photo-
Pathogenicity tests. The pathogenicity of the mutant viruses was determined
by two internationally accepted pathogenicity tests (4). These were (i) the mean
death time (MDT) test in 9-day-old embryonated chicken eggs and (ii) the
intracerebral pathogenicity index (ICPI) test in 1-day-old chicks.
Briefly, for the MDT test, a series of 10-fold (10?6to 10?9) dilutions of fresh
allantoic fluid from infected eggs was made in sterile phosphate-buffered saline
(PBS), and 0.1 ml of each dilution was inoculated into the allantoic cavities of
five 9-day-old embryonated chicken eggs per dilution and incubated at 37°C.
Each egg was examined three times daily for 7 days, and the time of embryo
death, if any, was recorded. The minimum lethal dose (MLD) is the highest virus
dilution that causes all embryos inoculated with that dilution to die. The MDT is
the mean time in hours for the MLD to kill all of the inoculated embryos. The
MDT has been used to classify APMV-1 strains into the following categories:
velogenic strains (less than 60 h), mesogenic strains (60 to 90 h), and lentogenic
strains (more than 90 h).
For the ICPI test, 0.05 ml of a 1:10 dilution of fresh infective allantoic fluid for
FIG. 1. Generation of a full-length cDNA clone of APMV-2/Yuc. The full-length cDNA clone was constructed by assembling six subgenomic
fragments in pBR322/dr/Yuc, flanked on the upstream side by a T7 RNA polymerase promoter sequence and on the downstream side by the
hepatitis delta virus ribozyme sequence followed by a T7 terminator sequence. The restriction enzymes used in the assembly and their positions
are shown on either side of the subgenomic fragments. A total of 10 nucleotide changes were made to generate five unique restriction enzyme sites.
Four of these sites were created in untranslated regions (UTRs): substitutions C2923A, G2924A, T2925A, and G2926C created the PmeI site in
the downstream UTR of the P gene; G4154C created the NotI site in the upstream UTR of the F gene; G5971A and A5973T created the PacI
site in the downstream UTR of the F gene; and T7870C created the DraIII site in the downstream UTR of the HN gene. The fifth restriction site,
SacII, was created by two silent changes (A11321G and A11322C) within the L ORF.
TABLE 1. Fusion (F) protein cleavage sites of different APMV
serotypes that were incorporated into the F protein cleavage
site of APMV-2
Recombinant virus Fusion protein cleavage sitea
rAPMV-2 (type 1v)....................................................RRQKR2F
rAPMV-2 (type 1av)..................................................GRQGR2L
rAPMV-2 (type 1 Africa)..........................................RRRRR2F
rAPMV-2 (type 3)......................................................RPRGR2L
rAPMV-2 (type 4)......................................................DIQPR2F
rAPMV-2 (type 5)......................................................KRKKR2F
rAPMV-2 (type 6)......................................................APEPR2L
rAPMV-2 (type 7)......................................................LPSSR2F
rAPMV-2 (type 8)......................................................YPQTR2L
rAPMV-2 (type 9)......................................................IREGR2I
aSegment III (as shown in Fig. 1) in pAPMV-2 was mutated by overlap PCR
to change the F protein cleavage site, digested using NotI and PacI sites, and
subcloned into the full-length cDNA to generate rAPMV-2 F protein cleavage
site mutants. The basic amino acids (K and R) at the F protein cleavage site are
shown in bold. The arrow indicates the site of cleavage.
5396 SUBBIAH ET AL.J. VIROL.
each virus was inoculated into groups of 10 1-day-old SPF chicks via the intra-
cerebral route. The birds were observed for clinical signs and mortality once
every 8 h for a period of 10 days. At each observation, the birds were scored as
follows: 0 if normal, 1 if sick, and 2 if dead. The ICPI is the mean score per bird
for all observations over the 10-day period. Highly virulent velogenic viruses give
values approaching 2, and avirulent or lentogenic strains give values close to 0.
Effect of the number of basic amino acids at the F protein cleavage site of
mutant viruses on pathogenicity in 2-week-old chickens. The effect of the num-
ber of basic amino acid residues at the F protein cleavage site on viral patho-
genesis and tissue tropism was determined by experimentally infecting 2-week-
old SPF chickens with mutant viruses that differed from one another by the
number of basic amino acids at their F protein cleavage site. The viruses com-
pared were rAPMV-2 (type 4), rAPMV-2, rAPMV-2 (type 3), rAPMV-2 (type
1v), and rAPMV-2 (type 5), which contain 1, 2, 3, 4, and 5 basic amino acids,
respectively, at their F protein cleavage sites. Briefly, groups of six 2-week-old
SPF chickens were inoculated with 0.2 ml of 28HA units of each virus by the
oculonasal route. The birds were observed daily and scored for any clinical signs
for 7 dpi. Three birds from each group were euthanized 3 and 7 dpi, and oral and
cloacal swabs were taken. The following tissues were collected 3 dpi for virus
isolation and immunohistochemistry: brain, lung, trachea, spleen, kidney, and
cecal tonsils. At 7 dpi, only brain, trachea, and lungs were collected for virus
isolation. The tissue samples were processed in three ways: (i) homogenates were
prepared for virus titration, (ii) samples were frozen for subsequent immuno-
histochemistry, and (iii) samples were fixed in formalin for subsequent histopa-
Virus isolation from swabs and virus titration of tissue sample. The oral and
cloacal swabs were collected in 1 ml of PBS containing antibiotics (2,000 units/ml
penicillin G, 200 ?g/ml of gentamicin sulfate, and 4 ?g/ml of amphotericin B;
Sigma Chemical Co., St. Louis, MO). The swab-containing tubes were centri-
fuged at 1,000 ? g for 20 min, and the supernatants were removed for virus
isolation. Virus isolation was performed by inoculating the supernatant into the
allantoic cavities of 9-day-old embryonated chicken eggs, and 3 dpi, the allantoic
fluid was tested for HA activity. The HA-positive samples were further con-
firmed by HI tests with APMV-2-specific antiserum. The virus titers in the tissue
samples were determined by the following method. Briefly, the tissue samples
were homogenized, and the supernatant was serially diluted and used to infect
DF1 cells, with duplicate wells per dilution. Infected wells were identified by
immunostaining, and the TCID50/ml was calculated using the method of Reed
and Muench (29).
Immunohistochemistry and histopathology. The frozen tissue samples col-
lected 3 dpi were sectioned at Histoserve, Inc. (MD). Briefly, the frozen sections
were rehydrated in three 10-min changes of PBS. The sections were fixed in
ice-cold acetone for 15 min at ?80°C and then washed three times in PBS and
blocked with 2% bovine serum albumin for 1 h at room temperature inside a
humidified chamber. The sections were incubated with a 1:500 dilution of the
primary polyclonal antiserum for 2 h at room temperature. After three washes
with PBS, the sections were incubated further with horseradish peroxidase
(HRP)-conjugated goat anti-chicken antibody for 30 min. After a final wash
cycle, the sections were incubated with 3-3?-diaminobenzidine tetrahydrochlo-
ride (DAB; Vector Laboratories) for 2 min, washed with distilled water, and
counterstained with hematoxylin (Vector Laboratories). Sections were mounted
with mounting medium and examined under a light microscope (Zeiss Axiovert
200M), and microphotographs were taken. For histopathology, tissue samples
were collected from the brain, trachea, lung, spleen, cecal tonsils, and kidney 3
dpi and fixed in 10% neutral buffered formalin. The fixed tissues were processed,
embedded with paraffin, sectioned, and then stained by hematoxylin and eosin
Development of an APMV-2 reverse genetics system and
construction of F protein cleavage site mutants. A cDNA
clone expressing the antigenome of APMV-2/Yuc was con-
structed from six cDNA segments that were synthesized by
RT-PCR from virion-derived genomic RNA (Fig. 1). The
cDNA segments were cloned in a sequential manner into the
low-copy-number plasmid pBR322/dr/Yuc between a T7 pro-
moter and the hepatitis delta virus ribozyme sequence. The
resulting APMV-2 cDNA in the plasmid pBR322/dr/Yuc is a
faithful copy of the published APMV-2/Yuc genome consensus
sequence (36) except for 10 silent nucleotide changes that were
introduced to create five new unique restriction enzyme sites
used in the construction (Fig. 1). This construct contains a T7
promoter that initiates a transcript with three extra G residues
at its 5? end, which increases the efficiency of T7 polymerase
transcription and does not interfere with recovery (18).
Eleven mutant derivatives with mutations in the F protein
cleavage site were constructed (Table 1). Specifically, the wild-
type APMV-2 F protein cleavage site (KPASR2F), which
contains two basic amino acids, was replaced with the naturally
occurring F protein cleavage sites of APMV serotypes 1 to 9
(Table 1), which contain from one to five basic amino acids.
The serotype 1 substitution was represented by three different
sequences: the “virulent” sequence RRQKR2F (type 1v), the
“avirulent” sequence GRQGR2L (type 1av), and the highly
basic virulent African strain sequence RRRRR2F (type 1
Africa) (Table 1). In addition, one construct, rAPMV-2 (F-L)
(Table 1), contained the APMV-2 cleavage site in which the
phenylalanine residue at the N-terminal end of the F1 subunit,
which is associated with improved cleavage and virulence in
APMV-1, was replaced by leucine, which is found in avirulent
strains of APMV-1. Of the panel of 11 cleavage site mutants,
leucine was present at the N terminus of the F1 subunit in five
cases: the rAPMV-2 (F-L) mutant noted above, the “avirulent”
APMV-1 mutant noted above, and the type 3, 6, and 8 mutants
(Table 1). These mutants were readily constructed using the
unique NotI and PacI sites flanking the F gene (Fig. 1).
Recovery of infectious parental and F protein cleavage site
mutant viruses. The rAPMV-2 wild-type parent and the 11 F
cleavage site mutants were recovered by transfection of the
respective antigenome plasmids into HEp-2 cells together with
plasmids encoding the N, P, and L proteins, necessary for viral
RNA replication and transcription. Plasmid transcription was
driven by T7 RNA polymerase supplied by T7 recombinant
vaccinia virus strain MVA. The supernatants from the trans-
fected HEp-2 monolayers were inoculated into the allantoic
cavities of 9-day-old embryonated chicken eggs. Allantoic fluid
was harvested 3 days after infection and tested for HA activity.
Allantoic fluid with a positive HA titer was used as a prelim-
inary viral stock. Part of this material was used to isolate viral
genomic RNA, which was subjected to RT-PCR and partial
sequence analysis of the F gene to confirm the sequence of the
F protein cleavage site. The parental virus and the F protein
cleavage site mutants were readily recovered from full-length
cDNA clones. None of these recombinant viruses required the
addition of exogenous protease during transfection and recov-
ery. Each of these viruses was passaged three times in 9-day-
old embryonated chicken eggs, and the sequences of the F and
HN ORFs were confirmed, showing that the introduced mu-
tations were maintained without any adventitious mutations.
Growth and cleavage characteristics of parental and F pro-
tein cleavage site mutant viruses in cell culture. All 12 recom-
binant viruses replicated in DF1, Vero, and MDCK cells, and
the supplementation by exogenous proteases or egg allantoic
fluid as a source of protease did not enhance growth of any of
the viruses. In general, all of the viruses replicated well in DF1
and Vero cells, while their growth pattern was comparatively
slow in MDCK cells. The parental recombinant virus resem-
bled its biological parent in growth characteristics, causing
single-cell infection in all three cell lines rather than forming
VOL. 85, 2011EFFECTS OF APMV-2 F PROTEIN MUTATIONS IN CHICKENS 5397
syncytia. Also, neither biological nor recombinant APMV-2
produced plaques under methylcellulose overlay in the pres-
ence or absence of exogenous proteases. In contrast, nearly all
of the F protein cleavage site mutant viruses, with the sole
exception of rAPMV-2 (F-L), gained the ability to produce
syncytia and plaques under methylcellulose overlay in the DF1
and Vero cells by 3 dpi and in MDCK cells by 7 dpi (Fig. 2A).
Among the 10 F cleavage site mutants that had gained the
ability to form syncytia and plaques, there was no difference in
morphology and size of plaques in DF1 and Vero cells. How-
ever, rAPMV-2 (type 1v), rAPMV-2 (type 3), rAPMV-2 (type
4), rAPMV-2 (type 5), and rAPMV-2 (type 9) produced
slightly larger plaques than did other mutants in MDCK cells.
There was no apparent difference in morphology of these
plaques. Further, the syncytia produced by rAPMV-2 (type 1
Africa), rAPMV-2 (type 3), and rAPMV-2 (type 9) were larger
in size than those produced by other mutants (Fig. 2B).
The replication kinetics of parental and F protein cleavage
site mutant viruses were compared in a multistep growth cycle.
Monolayers of DF1 cells were infected with the viruses, the
cells were washed 1 h later, and samples from the medium
overlay were collected at 24-h intervals and quantified in DF1
cells by the TCID50method. This analysis showed that all of
the syncytium-forming mutant viruses grew to a 10-fold-higher
titer than biological or recombinant wild-type APMV-2 by 3
dpi, whereas the single nonsyncytial mutant, rAPMV-2 (F-L),
replicated similarly to the wild-type parent (Fig. 3).
To examine whether the alterations in the amino acid se-
quence of the F protein cleavage site in the various mutants
indeed affected cleavage of the F0 protein, DF1 cells were
infected with parental rAPMV-2, which produces single-cell
infection, and some of the syncytium-forming cleavage site
mutants, specifically, rAPMV-2 (type 1v), rAPMV-2 (type 5),
and rAPMV-2 (type 1 Africa). The viral proteins in DF1-
infected cells were pulse-labeled for 30 min with Trans35S-
label and chased for 0, 20, 40, and 60 min in nonradioactive
medium. Cell lysates were prepared and subjected to immu-
noprecipitation with an antiserum specific for the cytoplasmic
tail of the APMV-2/Yuc F protein. Immunoprecipitates were
analyzed by SDS-PAGE under reducing conditions (Fig. 4A).
Autoradiographs of polyacrylamide gels were scanned by den-
sitometry, and the percentage of F protein cleavage at each
time point was calculated by dividing the amount of F1
protein by the total of F1 plus F0 proteins (Fig. 4B). The
most efficient cleavage of F protein occurred in rAPMV-2
(type 1 Africa), which contained five arginine (R) residues
at the cleavage site (RRRRR2F). Even at 0 min of chase,
the efficiency of F protein cleavage was 100% (Fig. 4A and
FIG. 2. (A) Plaque formation in DF1, Vero, and MDCK cell lines 3, 3, and 7 days postinfection, respectively, under methylcellulose overlay
in the absence of exogenous protease supplementation. The plaques were visualized by immunoperoxidase staining using polyclonal sera raised
against APMV-2/Yuc in chicken. (B) Syncytium formation in DF1 cells 3 days postinfection. The syncytia were visualized after the cells were fixed
with cold methanol. The morphology of DF1 cells infected with wild-type (wt) APMV-2 and rAPMV-2 (F-L) looked similar to that of DF1 cells
infected with rAPMV-2.
FIG. 3. Comparison of the kinetics of replication of biological wild-type APMV-2/Yuc, recombinant wild-type rAPMV-2, and the 11 fusion
cleavage site mutants of APMV-2 in DF1 cells. DF1 cells in six-well plates were infected in duplicate with wt and recombinant viruses at an MOI
of 1, and samples were taken from the culture supernatant at 24-h intervals until 120 h postinfection. Virus titers of the samples were determined
by serial endpoint dilution in 96-well cultures of DF1 cells, with infected wells detected by immunoperoxidase staining using a polyclonal antibody
against APMV-2/Yuc raised in chickens. Virus titers (TCID50/ml) were calculated by using the method of Reed and Muench (29). Values are
averages from three independent experiments, and error bars show standard deviations.
VOL. 85, 2011EFFECTS OF APMV-2 F PROTEIN MUTATIONS IN CHICKENS5399
B). The relative efficiencies of F protein cleavage in
rAPMV-2 (type 1v) (RRQKR2F) and rAPMV-2 (type 5)
(KRKKR2F) were 56 and 27% after 20 min of chase, com-
pared to 2% in parental rAPMV-2 (KPASR2F). The cleavage
efficiencies in rAPMV-2 (type 1v) and rAPMV-2 (type 5) in-
creased to 94 and 75% after 60 min of chase, compared to 43%
in parental rAPMV-2. These results showed that cleavage of
the F0 protein was more efficient in these syncytium-forming
APMV-2 mutants than in the nonsyncytial rAPMV-2 parent.
Furthermore, the results showed that not only the number but
also the type of basic residue influences the cleavage of F0
protein. For example, rAPMV-2 (type 1 Africa) and rAPMV-2
(type 5) both contain five basic residues, but they vary greatly
in cleavage efficiency. rAPMV-2 (type 1 Africa) contains five R
residues, whereas rAPMV-2 (type 5) contains lysine (K) in the
?2, ?3, and ?5 positions. These results suggest that R is
preferred over K for cleavage by host cell proteases.
Pathogenicity of parental and F protein cleavage site mu-
tant viruses in chicken embryos and 1-day-old chicks. The
pathogenicity of the biological wild-type APMV-2, recombi-
nant wild-type rAPMV-2, and 11 F protein cleavage site mu-
tant viruses was determined by the mean death time (MDT)
test in 9-day-old embryonated chicken eggs and by the intra-
cerebral pathogenicity index (ICPI) test in 1-day-old chicks
(data not shown). Similarly to biological wild-type APMV-2,
rAPMV-2 and the mutant viruses did not cause death of
chicken embryos within the standard 7-day (168-h) time limit
for the assay, and thus the MDT for all of the viruses is scored
as ?168 h. However, it was observed that, when the incubation
was extended to 10 days, the syncytial mutant viruses caused
embryo deaths at lower dilutions (10?2to 10?3) than did the
biological wild-type APMV-2, rAPMV-2, and rAPMV-2 (F-L)
viruses (data not shown). This suggested that, while the syncy-
tial mutant viruses remained avirulent by the standard MDT
assay, they were marginally more virulent than the wild-type
viruses or the rAPMV-2 (F-L) mutant in embryonated
chicken eggs during extended incubation and at lower dilu-
tion. All of the recombinant viruses had ICPI values of zero,
resembling wild-type APMV-2. Both tests suggested that the
cleavage site of the F protein and the ability to form syncytia
did not significantly affect the pathogenicity of APMV-2 in
Pathogenicity of parental and F protein cleavage site mu-
tant viruses in 2-week-old chickens. The effect of the F protein
cleavage site on viral pathogenesis was further studied by ex-
perimentally infecting 2-week-old SPF chickens with selected
mutant viruses that varied in number of basic amino acids at
the F protein cleavage site and, in one case, by the presence of
L as the first residue of the F1 subunit. The following viruses
were chosen for comparison: rAPMV-2 (type 4) (DIQPR2F),
rAPMV-2 (KPASR2F), rAPMV-2 (type 3) (RPRGR2L),
rAPMV-2 (type 1v) (RRQKR2F), and rAPMV-2 (type 5)
(KRKKR2F). These contained 1, 2, 3, 4, and 5 basic amino
acid residues, respectively, at the F protein cleavage site, and
the rAPMV-2 (type 3) mutant contained L at the terminus of
F1. The birds were infected with 0.2 ml of 28HA units of
FIG. 4. Proteolytic cleavage of parental and mutant APMV-2 F0 proteins. (A) Cleavage of the F0 proteins of selected viruses was
examined by pulse-chase radiolabeling and immunoprecipitation. DF1 cells were infected at an MOI of 10 for 12 h at 37°C. Cells were
washed and incubated in medium lacking methionine and cysteine for 2 h. Infected cells were pulse-labeled with EXPRESS35S label
(PerkinElmer) for 30 min and then chased in nonradioactive medium containing excess methionine and cysteine for 0, 20, 40, and 60 min
as described in Materials and Methods. Cell lysates were prepared, and the F protein was immunoprecipitated with polyclonal antiserum
against the cytoplasmic tail of the F protein, followed by incubation with Staphylococcus aureus cells. The precipitated proteins were analyzed
by SDS-PAGE in the presence of reducing agent, and labeled proteins were visualized by autoradiography. The positions of precursor F0
and the cleavage product F1 are indicated by arrows. (B) The results from panel A were scanned, and the amounts of F0 and F1 proteins
were quantified by densitometry (using the Photoshop program). The amount of F1 protein as a percentage of total F protein (F1 plus F0)
was calculated to yield the percent cleavage.
5400SUBBIAH ET AL. J. VIROL.
infective fresh allantoic fluid by the oculonasal route. The birds
were observed daily for 7 days postinfection. Three birds from
each group were sacrificed on day 3 and the remaining on day
7. Oral and cloacal swabs were taken upon sacrifice and ana-
lyzed for viral shedding. In addition, tissue samples were taken
on day 3 from the brain, lung, trachea, spleen, kidney, and
cecal tonsil and on day 7 from the brain, lung, and trachea.
These were analyzed for virus titer, immunohistochemistry of
viral antigens, and histopathology.
There were no apparent clinical signs of illnesses in any of
the infected groups throughout the study period. The oral or
cloacal viral shedding was inconsistent due to low titer, and
there was no significant difference in viral shedding between
the parental and F protein cleavage site mutant viruses either
3 or 7 dpi (data not shown). Histopathological examinations of
tissue samples collected 3 dpi revealed similar microscopic
findings in all of the tested recombinant viruses. This is illus-
trated with representative viruses in Fig. 5. Specifically, the
trachea showed mild lymphocytic tracheitis, with epithelial at-
tenuation and regeneration (Fig. 5b). In the lungs, mild to
moderate multifocal lymphohistiocytic, perivascular, and inter-
stitial pneumonia was observed (Fig. 5d), and in the spleen,
there was minimal lymphoid depletion (Fig. 5f). Microscopic
lesions were not found in any of the other tissues.
The replication of parental and mutant viruses was quanti-
fied in the tissue samples taken 3 (Fig. 6A) and 7 (Fig. 6B) dpi.
None of the viruses was detected in the brain either 3 or 7 dpi.
The titers of virus in the other tissues showed no consistent
pattern with regard to the F protein cleavage site. At 3 dpi, for
example, rAPMV-2 (type 4), with a single basic residue, was
reduced in titer compared to the parental rAPMV-2 virus,
which has two basic residues, in the lungs, trachea, spleen, and
FIG. 5. Histopathology in sections of trachea, lung, and spleen harvested from 2-week-old chickens 3 days after inoculation with parental
rAPMV-2 or the rAPMV-2 (type 4), rAPMV-2 (type 3), rAPMV-2 (type 1v), or rAPMV-2 (type 5) mutant by the oculonasal route. Chickens were
mock infected (a, c, and e) or infected with parental rAPMV-2 virus (b, d, and f). Histopathology in sections of chicken tissues infected with mutant
viruses looked similar to that for tissues infected with parental virus. Sections were stained with hematoxylin and eosin (magnification, ?400).
Histopathological examinations of tissue samples revealed similar microscopic findings in all tested recombinant viruses. (b) In the infected
trachea, minimal to mild attenuation and flattening of the tracheal epithelium, with reduction and loss of cilia, were observed. There was loss of
normal columnar epithelial architecture in these regions, with mild epithelial hyperplasia and multifocal replacement by low cuboidal epithelial
cells (arrows). Low numbers of individually apoptotic cells were seen within the epithelium in these regions. There was mild, multifocal,
subepithelial infiltrate of lymphocytes and fewer macrophages in the lamina propria. In summary, minimal to mild, multifocal lymphocytic
tracheitis with epithelial attenuation and regeneration was noted. (d) In the infected lung, small to moderate numbers of lymphocytes and few
macrophages were seen infiltrating around blood vessels and within the interstitium. Inflammatory cells formed dense perivascular aggregates that
extend into the interstitium, with small numbers of inflammatory cells multifocally infiltrating into the lamina propria subjacent to the airway
epithelium. Small numbers of individually apoptotic cells were present in inflammatory aggregates. Mild to moderate, multifocal, lymphohistio-
cytic, perivascular and interstitial pneumonia was observed. (f) In the infected spleen, the periarteriolar sheaths and white pulp regions exhibited
minimally reduced numbers of lymphocytes. There was also minimal lymphoid depletion.
VOL. 85, 2011 EFFECTS OF APMV-2 F PROTEIN MUTATIONS IN CHICKENS5401
kidney. However, the same was true of rAPMV-2 (type 1v),
which has four basic amino acids at the F protein cleavage site.
These three viruses had similar titers in the cecal tonsils. At 3
dpi, the replication of rAPMV-2 (type 3), with three unpaired
basic amino acids at the F protein cleavage site, showed a
slightly increased virus titer in lungs, trachea, spleen, and kid-
ney compared to that for the rAPMV-2 parent but no signifi-
cant difference in titer in the cecal tonsil. At 3 dpi, the repli-
cation of the rAPMV-2 (type 5) mutant, with five basic amino
acids at the F protein cleavage site, showed an increased virus
titer in trachea compared to that for the rAPMV-2 parent, but
there was no significant difference in titer in the spleen and
kidney, and the viral titers in the lungs and cecal tonsil were
reduced (Fig. 6A). At 7 dpi, rAPMV-2 (type 4), rAPMV-2
(type 1v), and rAPMV-2 (type 5) had reduced virus titers in the
upper respiratory tract (trachea) and lower respiratory tract
(lungs), whereas rAPMV-2 (type 3) had a reduced virus titer in
trachea, without any significant difference of titer in lung (Fig.
6B). Thus, there was no clear relationship between virus titer
and sequence of the F protein cleavage site. Viral antigens
were detected by immunohistochemistry in tissue samples that
were also positive by virus titration, which is shown for
rAPMV-2 in Fig. 7. This confirmed that the detection of virus
in harvested tissue indeed was associated with infection of the
organ. All infected birds were seropositive by 7 dpi as observed
by the HI test, with no differences associated with specific
viruses (data not shown).
In paramyxoviruses, proteolytic processing of the F protein
is a prerequisite for the generation of mature infectious virus.
There are nine serotypes of APMV within the genus Avulavirus
in the family Paramyxoviridae. Virulent strains of APMV-1
(NDV) have multibasic cleavage sites that are recognized and
cleaved by furin, a ubiquitous intracellular protease whose
preferred cleavage sequence is RX(R/K)R2. These viruses
are able to replicate in most cell types and cause systemic
infection. The avirulent strains of APMV-1 have one or occa-
sionally two unpaired basic amino acids that lack the furin
motif and are cleaved by trypsin-like extracellular proteases.
Hence, these viruses are restricted mostly to the respiratory
and gastrointestinal tracts, and they require supplementation
with exogenous proteases for in vitro growth. It has been shown
that the F protein cleavage site is a major determinant of
APMV-1 virulence in chickens (27, 28, 31). In contrast, the
contribution of the F protein cleavage site to the pathogenicity
of APMV-2 to -9 is unknown. Each of these APMV serotypes
has been isolated from a different avian species. The natural
host(s) of these viruses is not clearly defined. APMV-2 is
endemic among passerines and causes severe respiratory
illness in parrots but only mild respiratory illness in chick-
ens. APMV-5 causes high mortality in budgerigars but is ap-
athogenic in chickens. APMV serotypes 3, 4, 6, 7, 8, and 9 also
cause mild or inapparent disease in chickens. It is not known
whether the avirulence of APMV-2 to -9 in chickens is due to
the F protein cleavage site sequence of these serotypes.
The F protein cleavage site sequences vary widely among the
APMV serotypes. The F protein cleavage sites of APMV-4
(DIQPR2F) and APMV-7 (LPSSR2F) contain a single basic
amino acid residue, and that of APMV-2 (KPASR2F) has two
unpaired basic amino acids. However, in each case, only the R
residue in the ?1 position matches the furin cleavage se-
quence. These three serotypes contain phenylalanine at the F1
terminal end and do not require exogenous protease supple-
mentation for growth in cell culture (25, 36, 38). APMV-5
(KRKKR2F) contains five basic residues in the F cleavage
site and does not require exogenous protease supplementation
for growth in cell culture (34). The requirement of exoge-
nous protease supplementation for APMV-6 (APEPR2L)
varies with strains. The other APMV serotypes, APMV-3
(IREGR2I), require exogenous protease supplementation for in
vitro growth. It is noteworthy that the cleavage sites of APMV-3,
-6, and -8 have a leucine instead of a phenylalanine as the first
residue of the F1 subunit, which is also found in avirulent strains
of APMV-1. The presence of leucine at this position has been
associated with reduced cleavability of the APMV-1 F protein
The goal of this study was to evaluate the role of amino acid
sequence at the F protein cleavage site of APMV-2 in repli-
cation, formation of syncytia and plaques, and pathogenicity.
In order to mutate the F protein cleavage site, a reverse ge-
netics system for APMV-2 was developed for the first time.
This system will be useful to study the function of the APMV-2
FIG. 6. Virus titers from the indicated tissues harvested 3 (A) or 7
(B) dpi from 2-week-old chickens infected with F protein cleavage site
mutant viruses. Chickens were inoculated with 0.2 ml of 28HA units of
rAPMV-2, rAPMV-2 (type 4), rAPMV-2 (type 3), rAPMV-2 (type 1v),
or rAPMV-2 (type 5) by the oculonasal route. Each group is repre-
sented by 3 birds on each day. Titers are shown as mean log10TCID50/g
of tissue, and error bars show standard deviations.
5402 SUBBIAH ET AL.J. VIROL.
structural features and macromolecules in replication and
pathogenesis. We generated 12 recombinant APMV-2 F pro-
tein cleavage site mutants: 11 mutants containing the F protein
cleavage site of naturally occurring APMV serotypes, and 1
mutant where the phenylalanine at the N-terminal end of the
F1 subunit was replaced by a leucine residue.
We were able to recover all of the above-mentioned F pro-
tein cleavage site mutants without exogenous protease supple-
mentation, suggesting that the mutations did not adversely
affect the functions of F protein. Irrespective of the F protein
cleavage site or the type of cell line used, none of the APMV-2
F protein cleavage site mutants required exogenous protease
supplementation for in vitro growth, resembling the wild-type
APMV-2 virus. This was particularly surprising in the case of
mutants containing cleavage sites from the APMV-1 avirulent
strain, APMV-3, APMV-8, and APMV-9, because these cleav-
age sites require exogenous protease when present in their
respective natural F proteins. This suggests that APMV-2 con-
tains one or more structural features outside the cleavage site
that promotes cleavage by intracellular protease. This might
also account for the ability of wild-type APMV-2 to replicate
even though its F protein cleavage site contains only two basic
residues, one of which is consistent with the preferred furin
A second phenotype was the ability to form syncytia. Wild-
type APMV-1, -3, and -5 produce syncytia in infected cells,
whereas APMV-2, -4, and -6 to -9 cause single-cell infection.
Similarly to wild-type APMV-2, the rAPMV-2 and rAPMV-2
(F-L) mutants caused single-cell infections and did not form
syncytia or plaques. Surprisingly, however, all of the other F
protein cleavage site mutants produced syncytia and plaques in
DF1, Vero, and MDCK cells, even if the cleavage site involved
(e.g., APMV-4, -6, and -9) does not confer syncytium or plaque
formation in its native virus. In particular, the cleavage sites of
APMV-4, -6, -7, and -8, which contain only a single basic amino
acid, and the cleavage sites of avirulent APMV-1 and -9, which
contain two basic unpaired amino acids, also caused syncytium
formation in the absence of exogenous protease when present
within the APMV-2 F protein. One possibility for these results
could be an alteration of conformation of APMV-2 F protein
around the cleavage site due to change in amino acid, leading
to more efficient recognition by intracellular proteases and
hence cleavage and exposure of the amino-terminal F1 subunit
for cell-to-cell fusion. Alternatively, the F protein does not
need much help from HN to mediate fusion, and it may not
require as much cleaved F to get the virus to fuse with cells and
subsequently cause infection. This could also explain the dif-
ference in syncytium formation due to the good cleavage mu-
tants having sufficient F protein present to push past a thresh-
old needed to induce cell-cell fusion. It is known that cell-cell
FIG. 7. Immunohistochemistry of indicated organs 3 days after inoculation with rAPMV-2. Viral antigen was visualized by DAB using
polyclonal chicken serum raised against APMV-2/Yuc as the primary antibody followed by counterstaining with hematoxylin.
VOL. 85, 2011 EFFECTS OF APMV-2 F PROTEIN MUTATIONS IN CHICKENS5403
fusion differs from virus-cell fusion in the amount of fusion
activity needed to get the two pairs of membranes to merge. To
verify that the amino acid modifications at the F protein cleav-
age site indeed led to cleavage of F0 protein, pulse-chase
experiments with the parental and a few selected syncytium-
forming mutant viruses were performed. The results showed
that the F protein of parental rAPMV-2 is cleaved relatively
slowly but that the efficiency of cleavage is greatly enhanced in
the mutants that were examined. Further, our growth kinetics
results showed that syncytium formation increased the repli-
cation of the mutant viruses 10-fold, suggesting that the en-
hanced growth was probably due to increased cell-to-cell
spread of the virus.
Thus, incorporation of the cleavage sites of the other APMV
serotypes into APMV-2 resulted in growth that was indepen-
dent of added protease, was increased 10-fold, and conferred
the ability to form syncytia and plaques. The effects were sim-
ilar for the various cleavage sites irrespective of the number of
basic residues or the presence of phenylalanine versus leucine
at the N terminus of the F1 subunit. These results led to the
expectation that virulence in vivo would be enhanced, based on
the well-known example of APMV-1, for which the sequence
at the F protein cleavage site is a major determinant of viru-
lence (27, 28, 31). Surprisingly, however, all of the viruses
retained the avirulent phenotype of the APMV-2 parent. In all
cases, the MDT values of APMV-2 F protein cleavage site
mutants were more than 168 h and the ICPI values were zero,
similar to that of the wild-type APMV-2. This suggests that the
F protein cleavage site does not play an important role in the
virulence of APMV-2.
In addition, the effect of number of basic amino acids at the
F protein cleavage site on pathogenesis was studied in 2-week-
old chickens. We chose five mutants that varied from one
another in number of basic amino acid residues at the F protein
cleavage site, i.e., rAPMV-2 (type 4), rAPMV-2, rAPMV-2 (type
3), rAPMV-2 (type 1v), and rAPMV-2 (type 5), with 1, 2, 3, 4,
and 5 basic amino acids, respectively, at the F protein cleavage
site. Our results did not show any significant difference in viral
replication and tissue tropism among the F protein cleavage
site mutants, suggesting no direct correlation between the
number of basic amino acids at the F protein cleavage site and
the pathogenicity of APMV-2. Again, these results are consis-
tent with the interpretation that the F protein cleavage site is
not a major determinant of virulence in APMV-2.
We have shown previously that altering the F protein cleav-
age site of an avirulent strain to that of a neurovirulent strain
of NDV did not convert the avirulent strain into a neuroviru-
lent strain after a natural route of infection (27). Although the
biological activities of the fusion protein and growth charac-
teristics of the virus in vivo improved over those for the avir-
ulent parental strain, the complete spectrum of virulence phe-
notype could not be achieved by modifying the F cleavage site.
Further, it has been shown that other proteins of NDV, such as
the HN, V, NP, P, and L proteins, are responsible for virulence
along with the F protein (12, 15, 27, 32). Therefore, it is
possible that all of these proteins along with the F protein
might contribute to the virulence of APMV-2, which requires
In conclusion, we found that replacement of the F protein
cleavage site of APMV-2 with that of any of the other APMV
serotypes was associated with replication independent of
added protease, the ability to form syncytia and plaques, and
increased viral replication. It may be that these substitutions
caused a conformational change leading to increased efficiency
of F protein cleavage and function. However, none of these
changes increased the virulence or changed the tropism of the
virus. Thus, the cleavage and function of the F protein do not
appear to be important factors in the virulence of APMV-2.
We thank Daniel Rockemann for his technical assistance and help.
We thank Arthur Samuel for his help with handling of chickens during
the animal experiments and Heather Shive (NIH) for her help with the
interpretation of H&E-stained slides. We also thank Bernie Moss
(NIAID, NIH) for providing the T7 recombinant vaccinia virus.
This research was supported by NIAID contract no. N01A060009
(85% support) and the NIAID NIH Intramural Research Program
The views expressed herein do not necessarily reflect the official
policies of the Department of Health and Human Services, nor does
mention of trade names, commercial practices, or organizations imply
endorsement by the U.S. Government.
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