Bovine parainfluenza virus type 3 (BPIV3) fusion and hemagglutinin-neuraminidase glycoproteins make an important contribution to the restricted replication of BPIV3 in primates.
ABSTRACT This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the HPIV3 F and HN genes (rBPIV3-F(H)HN(H)) were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determinants of its host range restriction of replication in rhesus monkeys. rBPIV3-F(H)HN(H) replicated in rhesus monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Furthermore, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F(H)HN(H) conferred a level of protection against challenge with HPIV3 that was indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-F(H)HN(H) as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.
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ABSTRACT: Human parainfluenza virus type 3 (HPIV3) is a common cause of upper and lower respiratory tract illness in infants and young children. Live-attenuated cold-adapted HPIV3 vaccines have been evaluated in infants but a suitable interval for administration of a second dose of vaccine has not been defined. HPIV3-seronegative children between the ages of 6 and 36 months were randomized 2:1 in a blinded study to receive two doses of 10(5) TCID50 (50% tissue culture infectious dose) of live-attenuated, recombinant cold-passaged human PIV3 vaccine (rHPIV3cp45) or placebo 6 months apart. Serum antibody levels were assessed prior to and approximately 4-6 weeks after each dose. Vaccine virus infectivity, defined as detection of vaccine-HPIV3 in nasal wash and/or a≥4-fold rise in serum antibody titer, and reactogenicity were assessed on days 3, 7, and 14 following immunization. Forty HPIV3-seronegative children (median age 13 months; range 6-35 months) were enrolled; 27 (68%) received vaccine and 13 (32%) received placebo. Infectivity was detected in 25 (96%) of 26 evaluable vaccinees following doses 1 and 9 of 26 subject (35%) following dose 2. Among those who shed virus, the median duration of viral shedding was 12 days (range 6-15 days) after dose 1 and 6 days (range 3-8 days) after dose 2, with a mean peak log10 viral titer of 3.4PFU/mL (SD: 1.0) after dose 1 compared to 1.5PFU/mL (SD: 0.92) after dose 2. Overall, reactogenicity was mild, with no difference in rates of fever and upper respiratory infection symptoms between vaccine and placebo groups. rHPIV3cp45 was immunogenic and well-tolerated in seronegative young children. A second dose administered 6 months after the initial dose was restricted in those previously infected with vaccine virus; however, the second dose boosted antibody responses and induced antibody responses in two previously uninfected children.Vaccine 10/2013; · 3.49 Impact Factor
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ABSTRACT: A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (Bernstein et al., Pediatr Infect Dis J 2012;31:109-114; Yang et al., Vaccine 2013;31:2822-2827). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the 1(st) (pre-N), 2(nd) (N-P), 3(rd) (P-M), and 6(th) (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the 1(st) to the 6(th) position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts (URT, LRT) of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the 2(nd), 3(rd), and 6(th) positions conferred increased temperature-sensitivity: this was greatest for the 3(rd) position and was the most attenuating in vivo. Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo, but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN ORF, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity.Importance The research findings presented in this manuscript will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young children. Moreover, this knowledge has general application to the development and clinical evaluation of other mononegavirus vectors and vaccines.Journal of Virology 01/2014; · 4.65 Impact Factor
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ABSTRACT: Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV-3) are the major causes of lower respiratory tract infection in infants and young children. RSV is also responsible for a significant amount of morbidity and mortality in the elderly, the immunosuppressed and those with chronic cardiac or pulmonary disease. Despite extensive research, no effective antiviral therapy or vaccine is currently available for either virus. Over the past 20 years a number of different approaches for developing vaccines for RSV and HPIV-3 have been tested, with varying degrees of success. However, as it is not yet possible to identify one strategy that yields a vaccine preparation suitable for all high-risk populations, a multi-faceted approach to RSV and HPIV-3 vaccine development is still necessary. Ribavirin and immunoglobulin preparations with high titres of RSV-specific neutralizing antibodies are licensed for the treatment and prevention of RSV infection, but neither is cost-effective or easy to administer. New clinically effective agents are needed to diminish the impact of RSV and HPIV-3. This article will review the currently available means for controlling the two viruses, the search for new antiviral agents, and future prospects for preventing and treating RSV and HPIV-3 infections.International Journal of Pharmaceutical Medicine 01/2001; 15(4).
JOURNAL OF VIROLOGY,
Oct. 2000, p. 8922–8929Vol. 74, No. 19
Bovine Parainfluenza Virus Type 3 (BPIV3) Fusion and
Hemagglutinin-Neuraminidase Glycoproteins Make an
Important Contribution to the Restricted
Replication of BPIV3 in Primates
ALEXANDER C. SCHMIDT,1,2* JOSEPHINE M. MCAULIFFE,1ANNE HUANG,1SONJA R. SURMAN,1
JANE E. BAILLY,1WILLIAM R. ELKINS,1PETER L. COLLINS,1BRIAN R. MURPHY,1
AND MARIO H. SKIADOPOULOS1
Laboratory of Infectious Disease, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20892,1Department of
Pediatrics, Freie Universita ¨t Berlin, 12200 Berlin, Germany2
Received 22 March 2000/Accepted 14 July 2000
This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein
genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of
nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F
and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the
HPIV3 F and HN genes (rBPIV3-FHHNH) were generated to assess the effect of glycoprotein substitution on
replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric
viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their
parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal
proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level
similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determi-
nants of its host range restriction of replication in rhesus monkeys. rBPIV3-FHHNHreplicated in rhesus
monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and
HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Further-
more, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype
in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of
BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract
of rhesus monkeys, rBPIV3-FHHNHconferred a level of protection against challenge with HPIV3 that was
indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-
FHHNHas a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.
Bovine parainfluenza virus type 3 (BPIV3) is restricted in
replication in the respiratory tract of rhesus monkeys, chim-
panzees, and humans, and it is being evaluated as a vaccine
against human PIV3 (HPIV3) (8, 10, 12, 26, 27). HPIV3 and
BPIV3 are closely related enveloped, nonsegmented, negative-
strand RNA viruses within the Respirovirus genus of the Para-
myxoviridae family (2, 10). The two viruses are 25% related
antigenically by cross-neutralization studies (8), and they share
neutralization epitopes on their fusion (F) and hemagglutinin-
neuraminidase (HN) surface glycoproteins (9, 30). HPIV3 and
BPIV3 are essentially identical in genome organization (2).
Both viruses encode nine proteins: the nucleoprotein (N),
phosphoprotein (P), and large polymerase protein (L) are nu-
cleocapsid-associated proteins; the C, D, and V accessory pro-
teins are proteins of unknown function encoded by the P
mRNA or by an edited version thereof; the M protein is an
internal matrix protein; and the F and HN glycoproteins are
protective antigens of the virus that induce neutralizing anti-
bodies (10, 14). The amino acid sequence identities of the HN
and F proteins of HPIV3 and BPIV3 are 79 and 75%, respec-
A study to define the genetic basis of the host range restric-
tion of replication of BPIV3 in the respiratory tract of primates
was previously initiated by constructing and characterizing a
recombinant HPIV3 (rHPIV3) in which the N open reading
frame (ORF) was replaced by that of its BPIV3 counterpart
(1). The resulting chimeric virus, here referred to as rHPIV3-
NB, replicated efficiently in vitro but was restricted in replica-
tion in the upper respiratory tract of rhesus monkeys, identi-
fying the N protein as an independent determinant of the host
range restriction of BPIV3 in rhesus monkeys (1). In this study,
the contribution of the F and HN genes to the attenuation of
BPIV3 for rhesus monkeys was examined by generating and
characterizing two reciprocal BPIV3/HPIV3 chimeras. In one
chimera, the F and HN genes of HPIV3 were replaced with
their BPIV3 counterparts, resulting in a recombinant desig-
nated rHPIV3-FBHNB. The reciprocal chimeric recombinant
(rBPIV3-FHHNH) was constructed by replacing the F and HN
genes of a recombinant BPIV3 (rBPIV3) with their HPIV3
counterparts. The F and HN genes were exchanged as pairs
because of the known requirement for the presence of homol-
ogous F and HN proteins of PIVs for full functional activity
(13, 21, 41). The replication of the two chimeric PIV3 recom-
binants was evaluated in vitro and also in vivo in the respiratory
tract of rhesus monkeys. The findings of this study identify the
BPIV3 F and HN genes as major contributors to the restricted
* Corresponding author. Mailing address: LID, NIAID, NIH, Bldg.
7, Rm. 130, 7 Center Dr. MSC 0720, Bethesda, MD 20892. Phone:
(301) 496-3490. Fax: (301) 496-8312. E-mail: firstname.lastname@example.org.
replication of the BPIV3 in nonhuman primates, demonstrate
that one or more additional BPIV3 genes contribute to this
host range phenotype, and identify rBPIV3-FHHNH, which
possesses attenuating BPIV3 sequences as well as the antigenic
specificity of HPIV3, as a promising candidate for a vaccine
MATERIALS AND METHODS
Viruses and cells. HEp-2 and simian LLC-MK2 monolayer cell cultures were
maintained in minimal essential medium (Life Technologies, Gaithersburg, Md.)
supplemented with 5% fetal bovine serum (Summit Biotechnology, Fort Collins,
Colo.), 50 ?g of gentamicin sulfate per ml, and 4 mM glutamine (Life Technol-
The wild-type BPIV3 strain Kansas/15626/84 (clone 5-2-4, lot BPI3-1) (BPIV3
Ka) was previously described (4, 27). The HPIV3 JS wild type, its recombinant
version (rHPIV3), and rHPIV3 containing the BPIV3 Ka N ORF in place of the
HPIV3-N ORF (rHPIV3-NB) were also described previously (1, 15). PIVs were
propagated at 32°C in LLC-MK2 cells (ATCC CCL-7) as previously described
(20). The modified vaccinia virus strain Ankara (MVA) recombinant that ex-
presses bacteriophage T7 RNA polymerase (MVA-T7) was generously provided
by L. Wyatt and B. Moss (44).
Construction of antigenomic cDNAs encoding BPIV3/HPIV3 recombinants.
(i) Construction of a cDNA to recover rBPIV3. A full-length cDNA was con-
structed to encode the complete 15,456-nucleotide (nt) antigenomic RNA of
BPIV3 Ka (GenBank accession no. AF178654), with the exception of nt 21 (T to
G) and 23 (C to T) (2). The nucleotides differing at each position were both
observed in wild-type BPIV3 Ka virus populations with similar frequencies. The
cDNA was assembled from four subclones derived from reverse transcription
(RT) of viral RNA (2), using the SuperScript II preamplification system (Life
Technologies) and PCR amplification with a High Fidelity PCR kit (Clontech
Laboratories, Palo Alto, Calif.). The RT-PCR products were cloned into mod-
ified pUC19 plasmids (New England Biolabs, Beverly, Mass.), using the follow-
ing internal restriction enzyme recognition sites: SmaI (BPIV3 Ka sequence
position nt 186), PstI (nt 2896), MluI (nt 6192), SacII (nt 10452), and BspLU11I
(nt 15412). Multiple subclones of the antigenomic cDNA were sequenced using
a Perkin-Elmer ABI 310 sequencer with dRhodamine terminator cycle sequenc-
ing (Perkin-Elmer Applied Biosystems, Warrington, United Kingdom), and only
those matching the consensus sequence of BPIV3 Ka (2) were used for assembly
of the full-length clone. The 3? and 5? ends of BPIV3 Ka had been cloned
previously (2). Assembly of the full-length cDNA took place in the previously
described p(Right) vector (15), which we modified to contain a new polylinker
with restriction enzyme recognition sites for XhoI, SmaI, MluI, SacII, EcoRI,
HindIII, and RsrII. The full-length cDNA clone pBPIV3(184) contained the
following elements in 3?-to-5? order: a T7 promoter followed by two nonviral
guanosine residues, the complete antigenomic sequence of BPIV3 Ka, a hepatitis
delta virus ribozyme, and a T7 polymerase transcription terminator, as previously
described (1, 15).
(ii) Construction of rHPIV3-FBHNBand rBPIV3-FHHNH. Unique restriction
enzyme recognition sites were introduced into the BPIV3 antigenomic cDNA
and into the previously described HPIV3 antigenomic cDNA p3/7(131)2G (15)
to facilitate the exchange of the F and HN genes between BPIV3 and HPIV3
cDNAs. Using the transformer site-directed mutagenesis protocol from Clon-
tech, SgrAI restriction sites were introduced in the downstream noncoding region
of the M gene at position 4811 of the rBPIV3 sequence and position 4835 of the
rHPIV3 JS sequence (GenBank accession no. Z11575). The sequence was
changed from TCCAACATTGCA to TCCACCGGTGCA in rBPIV3 and from
CGGACGTATCTA to CGCACCGGTGTA in rHPIV3 (recognition sites un-
derlined). BsiWI restriction sites were introduced in the downstream noncoding
region of the HN gene at nt 8595 of the rBPIV3 sequence and at nt 8601 of the
rHPIV3 JS sequence. The sequence was changed from GATATAAAGA to
GACGTACGGA in rBPIV3 to give pBPIVs(107) and from GACAAAAGGG to
GACGTACGGG in rHPIV3 to give pHPIVs(106). The F and HN genes were
exchanged between pBPIVs(107) and pHPIV3s(106) by digestion of each with
SgrAI and BsiWI, gel purification of the fragments, and assembly of the appro-
priate fragments into the two full-length cDNAs. The HPIV3 backbone bearing
the BPIV3 F and HN genes, designated pHPIV(215), encoded 15,480 nt of viral
sequence, of which nt 4835 to 8619 came from BPIV3, and it was used to derive
rHPIV3-FBHNB(Fig. 1). The BPIV3 backbone bearing the HPIV3 F and HN
genes, designated pBPIV(215), encoded 15,438 nt of viral sequence, of which nt
4811 to 8577 came from HPIV3, and it was used to derive rBPIV3-FHHNH(Fig.
BPIV3 support plasmids for recovery of virus from cDNA. Support plasmids
encoding the BPIV3 Ka N, P, and L genes were assembled in modified pUC19
vectors and then cloned into the previously described pTM vector (15). To place
the individual genes immediately downstream of the T7 promoter in the pTM
vector, an NcoI site was introduced at the start codon of the N, P, and L ORFs
by site-directed mutagenesis. The NcoI restriction site and a naturally occurring
restriction site downstream of each ORF (SpeI for N, HincII for P, and BspLU11
for L) was used for cloning into pTM. After cloning, the NcoI site in pTM(N) was
mutagenized back to the original sequence to restore the correct amino acid
assignment in the second codon. In pTM(P) and pTM(L), the amino acid se-
quence encoded by the ORF was not altered by the introduction of NcoI sites.
Transfection. HEp-2 cells (approximately 1.5 ? 106cells per well of a six-well
plate) were grown to 90% confluence and transfected with 0.2 ?g of the BPIV3
support plasmids pTM(N) and pTM(P), and 0.1 ?g of pTM(L), along with 5 ?g
of the full-length antigenomic cDNA and 12 ?l LipofectACE (Life Technolo-
gies). Each transfection mixture also contained 1.5 ? 107PFU of MVA-T7, as
previously described (15). The cultures were incubated at 32°C for 12 h before
the medium was replaced with minimal essential medium (Life Technologies)
containing 10% fetal bovine serum. The supernatants were harvested after in-
cubation at 32°C for an additional 3 days, passaged onto LLC-MK2 cell mono-
layers in 25-cm2flasks, and incubated for 5 days at 32°C. Virus present in the
supernatant was plaque purified sequentially three times prior to amplification
Molecular characterization of recovered chimeric recombinants. The pres-
ence of the heterologous F and HN genes in the bovine or human PIV3 back-
bone was confirmed in plaque-purified recombinant viruses by RT-PCR of viral
RNA isolated from infected cells as previously described (2), using a primer pair
that recognized conserved sequences in rBPIV3 and rHPIV3. The generation of
each PCR product was dependent on the inclusion of reverse transcriptase,
indicating that each was derived from viral RNA and not from contaminating
cDNA (data not shown). This yielded similarly sized fragments (nt 4206 to 9035
in rBPIV3, nt 4224 to 9041 in rHPIV3, nt 4206 to 9017 in rBPIV3-FHHNH, and
nt 4224 to 9059 in rHPIV3-FBHNB) which were then digested with EcoRI and
analyzed by electrophoresis on a 1% agarose gel as previously described (2). The
nucleotide sequence flanking the introduced SgrAI and BsiWI restriction sites in
each virus was confirmed by sequencing the corresponding RT-PCR product.
Replication of HPIV3/BPIV3 chimeras in cell culture. The multicycle growth
kinetics of BPIV3 Ka, rHPIV3-FBHNB, rBPIV3-FHHNH, rHPIV3-NB, and
rHPIV3 in LLC-MK2 cells were determined by infecting cells in triplicate at a
multiplicity of infection (MOI) of 0.01 and harvesting samples at 24-h intervals
over a 6-day period, as previously described (34). Samples were flash-frozen and
titered in a single assay on LLC-MK2 cell monolayers in 96-well plates at 32°C,
as described elsewhere (16).
Monkey studies. Rhesus monkeys, which were seronegative for PIV3 as de-
termined by hemagglutination inhibition (HAI) assay (8), were inoculated intra-
nasally and intratracheally in groups of two or four animals with 10550% tissue
culture infectious doses (TCID50) of BPIV3 Ka, rHPIV3-FBHNB, rBPIV3-
FHHNH, rHPIV3-NB, or rHPIV3 per ml. Nasopharyngeal swabs were collected
daily on days 1 to 11 and on day 13. Tracheal lavage samples were collected on
FIG. 1. Genomes of the rHPIV3-FBHNBand rBPIV3-FHHNHchimeras and of the parent viruses, rHPIV3 JS and BPIV3 Ka, shown schematically (not to scale).
The F and HN genes were exchanged as a single restriction fragment between rHPIV3 and rBPIV3, using SgrAI and BsiWI sites that had been introduced preceding
the M and HN gene end sequences, respectively.
VOL. 74, 2000 BOVINE PIV3 F AND HN GENES ATTENUATE HUMAN PIV38923
days 2, 4, 6, 8, and 10 postinfection. Individual samples were flash-frozen and
stored at ?70°C until all samples were available for titration. Virus in the
specimens was titered on LLC-MK2 cell monolayers in 24- and 96-well plates as
previously described (16). Sera collected from monkeys on days 0 and 28 were
tested by HAI assay using HPIV3 JS and BPIV3 Ka as antigens, as previously
described (8). On day 28 postinoculation, the monkeys were challenged intrana-
sally and intratracheally with 106TCID50of HPIV3 JS per site. Nasopharyngeal
swab samples were collected on days 3, 4, 5, 6, 7, and 8, and tracheal lavage
samples were collected on days 4, 6, and 8 postchallenge. Samples were titered
in a single assay as described above. Serum was collected on day 28 postchal-
Recovery of rBPIV3 and BPIV3/HPIV3 chimeras from cDNA.
A complete BPIV3 antigenomic cDNA, designated pBPIV
(184), was constructed to encode the consensus sequence of
BPIV3 Ka, with the exception of nt 21 (T to G) and 23 (C to
T) (2). This BPIV3 antigenomic cDNA was further modified
by the introduction of unique SgrAI and BsiWI sites into the
downstream noncoding regions of the M and HN genes, re-
spectively. The same restriction sites were introduced into the
downstream noncoding regions of the M and HN genes of a
previously described complete HPIV3 antigenomic cDNA, p3/
7(131)2G (15). The F and HN glycoprotein genes of HPIV3
and BPIV3 were swapped by exchanging this SgrAI-BsiWI
restriction fragment. A direct exchange of entire genes was
anticipated to be well tolerated because of the high level of
sequence conservation between the cis-acting signals of BPIV3
and HPIV3 (2). The HPIV3 antigenomic cDNA bearing the
BPIV3 F and HN genes was designated pHPIV(215), and the
BPIV3 antigenomic cDNA bearing the HPIV3 F and HN
genes was designated pBPIV(215).
rBPIV3, rHPIV3-FBHNB, and rBPIV3-FHHNHchimeras
were recovered from the cDNAs pBPIV(184), pHPIV(215),
and pBPIV(215) after transfection of HEp-2 cells, and their
identities were confirmed by EcoRI digestion (Fig. 2). In each
case, the predicted unique fragment pattern was observed,
confirming the identity of the backbone and the inserted F and
In LLC-MK2 cells, the cytopathic effect (CPE) caused by
rBPIV3-FHHNHwas indistinguishable from that of HPIV3 JS
(condensed, rounded-up cells and small syncytia) but different
from that of BPIV3 (large multicellular syncytia), whereas the
CPE caused by rHPIV3-FBHNBwas identical to that caused by
BPIV3. Although this was not a systematic observation, the
differences in the cytopathology of the chimeric PIVs could
FIG. 2. Confirmation of the identity of recombinant viruses by RT-PCR of viral RNA and EcoRI digestion. RT-PCR products of viral RNA were prepared with
a primer pair that recognized conserved regions on either side of the F and HN genes in both BPIV3 and HPIV3. Digestion with EcoRI resulted in a unique pattern
of restriction fragments for each of the four viruses. In the schematic diagrams on the left, horizontal lines symbolize the amplified viral sequences and vertical bars
show the positions of EcoRI sites. The expected size (in nucleotides) of each restriction fragment is indicated above the line. Numbers below each line correspond to
sequence positions in the antigenomic RNA of BPIV3 Ka, HPIV3 JS (GenBank accession no. AF178654 and Z11575), or the indicated chimeric derivative. On the
right, a 1% agarose gel of the EcoRI digestion of PCR products confirms the identities of parental and chimeric viruses. The asterisks indicate gel bands that contain
comigrating restriction fragments. Positions of molecular weight markers (MW) are indicated in nucleotides.
8924 SCHMIDT ET AL.J. VIROL.
point to a cosegregation of CPE with the parental origin of the
F and HN genes.
BPIV3/HPIV3 chimeras replicate efficiently in cell culture.
The growth kinetics of rHPIV3-FBHNBand rBPIV3-FHHNH
were compared with those of their parental viruses by infecting
LLC-MK2 monolayers at an MOI of 0.01 and monitoring the
production of infectious virus. The kinetics and magnitude of
replication of the two chimeric viruses were comparable to
those of their HPIV3 or BPIV3 parental viruses (Fig. 3). This
suggested that BPIV3 and HPIV3 glycoproteins were compat-
ible with the heterologous PIV3 internal proteins.
The F and HN genes of the BPIV3/HPIV3 chimeras are
determinants of the host range restriction of replication of
BPIV3 Ka in the upper respiratory tract of rhesus monkeys.
rHPIV3-FBHNBand rBPIV3-FHHNHwere evaluated for the
ability to replicate in the upper and lower respiratory tract of
rhesus monkeys. Two questions were specifically addressed.
First, did the introduction of the BPIV3 F and HN genes into
HPIV3 restrict its replication in rhesus monkeys, as previously
shown for the BPIV3 N protein (1)? Second, did the introduc-
tion of the HPIV3 F and HN genes into BPIV3 increase its
replication in rhesus monkeys? If the predominant attenuating
mutations of BPIV3 were in genes other than the F and HN
genes, then one would expect little overall effect of the HPIV3-
BPIV3 glycoprotein exchange on replication of BPIV3 in rhe-
Each chimeric virus was administered intranasally and intra-
tracheally to rhesus monkeys at a dose of 105TCID50per site.
The level of replication of the chimeric viruses was compared
to that of the rHPIV3 and BPIV3 parental viruses and to that
of rHPIV3-NB(Table 1). Since the rHPIV3 parental virus
replicated to a low to moderate level in the lower respiratory
tract, meaningful comparisons between groups could be made
only for replication in the upper respiratory tract. The level of
replication of rHPIV3-FBHNBin the upper respiratory tract
was similar to that of its BPIV3 parent and substantially lower
than that of its HPIV3 parent (Table 1; Fig. 4A). This showed
that the BPIV3 glycoprotein genes contained one or more
major determinants of the host range attenuation phenotype of
BPIV3 for rhesus monkeys. The magnitudes and patterns of
replication of rHPIV3-FBHNBand rHPIV3-NBwere very sim-
ilar, indicating that the two bovine genetic elements attenuate
HPIV3 to a similar extent.
The rBPIV3-FHHNHchimera replicated significantly less
well than rHPIV3 in the upper respiratory tract (Table 1), and
it grouped with BPIV3 in a Duncan multiple range test. How-
ever, inspection of its pattern of replication in Fig. 4B sug-
gested that rBPIV3-FHHNHreplicated to a level intermediate
between that of its HPIV3 and BPIV3 parents. This interpre-
tation is supported by Friedman’s test of consistency of ranks
(40), which indicates that the median titers of HPIV3, rBPIV3-
FHHNH, and BPIV3 between days 3 and 8 postinfection are
significantly different (df 2 and 8; P ? 0.05). The observation
that the introduction of the HPIV3 F and HN proteins resulted
in an increase in the replication of BPIV3 in rhesus monkeys
indicates (i) that F and HN contain one or more determinants
of host range restriction in the upper respiratory tract and (ii)
that one or more genetic elements of BPIV3 that lie outside of
the F and HN genes, e.g., the N protein, also attenuate the
virus for rhesus monkeys.
The chimeric BPIV3 bearing HPIV3 glycoprotein genes in-
duces serum HAI antibody to HPIV3 and a high level of re-
sistance to HPIV3 challenge. rBPIV3-FHHNHhas important
features that make it a candidate live attenuated virus vaccine
against HPIV3, including attenuating genes from BPIV3 and
the antigenic specificity of HPIV3, i.e., the F and HN glyco-
proteins, which are the major protective antigens. Therefore,
its immunogenicity and protective efficacy against challenge
FIG. 3. Multicycle replication of chimeric and parental viruses in simian LLC-MK2 cells. Multicycle replication (MOI of 0.01) of the three chimeras rHPIV3-
FBHNB, rBPIV3-FHHNH, and rHPIV3-NBis compared with the replication of the BPIV3 Ka and rHPIV3 parents. Virus titers are shown as mean log10TCID50per
milliliter ? standard error of triplicate samples. The lower limit of detection of this assay is 10 TCID50, as indicated by the dotted horizontal line.
VOL. 74, 2000 BOVINE PIV3 F AND HN GENES ATTENUATE HUMAN PIV38925
with HPIV3 were examined. Rhesus monkeys were immunized
by infection with BPIV3 Ka, rHPIV3-FBHNB, rBPIV3-FH-
HNH, rHPIV3-NB, or rHPIV3. They were challenged 28 days
later with HPIV3 JS wild-type virus. Serum samples were taken
prior to the initial infection on day 0 and prior to the challenge
(Table 1). BPIV3 and rHPIV3-FBHNBinduced serum HAI
antibodies that reacted more efficiently with BPIV3 than
HPIV3, whereas the converse was the case for HPIV3 and
rBPIV3-FHHNH. Thus, the origin of the glycoprotein genes in
each virus determined whether the HAI antibody response was
directed predominantly against HPIV3 or against BPIV3. The
replication of challenge HPIV3 virus was significantly reduced
in the upper and lower respiratory tracts of previously immu-
nized monkeys (Table 2). Although the level of protective
efficacy against HPIV3 was not significantly different among
the different viruses, viruses bearing HPIV3 F and HN ap-
peared to be slightly more protective in the upper respiratory
tract than viruses bearing BPIV3 F and HN. This is in accor-
dance with the higher level of HPIV3-specific serum HAI
antibodies induced by viruses bearing HPIV3 F and HN.
The Jennerian approach to the development of live attenu-
ated viruses involves the use of a mammalian or avian virus to
immunize humans against an antigenically related human vi-
rus. The approach is named after Edward Jenner’s successful
use of vaccinia virus, a virus putatively of bovine origin, to
protect against smallpox in humans. Mammalian and avian
viruses that are well adapted to their natural host typically do
TABLE 1. The F and HN glycoprotein genes of BPIV3 contribute to its restricted replication in the respiratory tracts of rhesus monkeys
Mean peak virus titerc(log10TCID50/ml ? SE)
Serum HAI antibody titer (mean reciprocal
log2? SE) on day 28e[Duncan grouping] for:
4.7 ? 0.54 [A]
3.1 ? 0.58 [B]
3.0 ? 0.60 [B]
2.9 ? 0.28 [B]
2.6 ? 0.26 [B]
2.4 ? 0.37 [A]
1.6 ? 0.05 [A]
1.4 ? 0.19 [A]
2.0 ? 0.24 [A]
1.6 ? 0.10 [A]
9.5 ? 0.72 [A]
6.8 ? 0.63 [BC]
8.2 ? 0.48 [AB]
4.5 ? 0.29 [D]
5.5 ? 0.62 [CD]
6.8 ? 1.03 [B]
3.8 ? 0.63 [C]
6.5 ? 0.62 [B]
9.5 ? 0.65 [A]
9.2 ? 0.60 [A]
aMonkeys were inoculated intranasally and intratracheally with 105TCID50of virus in a 1-ml inoculum at each site. Two animals in the rHPIV3 group received
rHPIV3 with the SgrAI and BsiWI sites (rHPIV3s). There was no significant difference in the level of replication between rHPIV3 and rHPIV3s.
bThe groups with six animals contain four animals each from a previous rhesus study (1).
cMean of the peak virus titer for each animal in its group irrespective of sampling day.
dVirus titrations were performed on LLC-MK2 cells at 32°C. The limit of detectability of virus titer was 10 TCID50/ml. Mean viral titers were compared using a
Duncan multiple range test (? ? 0.05). Within each column, mean titers with different letters are statistically different. Titers indicated with two letters are not
significantly different from those indicated with either letter.
eThe titers on day 0 were ?2.0. Day 28 was the day of challenge with wild-type HPIV3.
fNasopharyngeal (NP) swab samples were collected on days 1 to 11 and on day 13 postinfection.
gTracheal lavage samples were collected on days 2, 4, 6, 8, and 10 postinfection.
FIG. 4. Mean titers of chimeric and parental viruses in nasopharyngeal swabs of infected rhesus monkeys over the course of infection. Virus titers are shown as mean
TCID50per milliliter in LLC-MK2 cells ? standard error for groups of four or six monkeys infected with the same virus. Data are from the same experiment as shown
in Table 1. (A) Mean titers of rHPIV3-FBHNBcompared to rHPIV3 and BPIV3 Ka titers; (B) mean rBPIV3-FHHNHtiters compared to those of BPIV3 Ka and
rHPIV3, which are the same values in panel A but are presented separately to facilitate comparison. Day 5 titers are not shown because they were much lower than
day 4 and day 6 titers, most likely due to technical problems during the sample collection.
8926 SCHMIDT ET AL. J. VIROL.
not replicate efficiently in humans and hence exhibit an atten-
uation phenotype based on host range restriction. At present,
we lack a thorough understanding of the genetic basis of this
form of host range restriction. However, animal viruses such as
vaccinia virus or bovine rotavirus that manifest host range
restriction in humans exhibit significant divergence of nucleo-
tide sequence from that of the corresponding human virus (32,
38), and it is reasoned that extensive sequence divergence of
this nature should lead to the genetic stability of the host range
attenuation phenotype following replication of the vaccine in
the foreign human host. The Jennerian approach to the devel-
opment of live attenuated viruses has been successfully em-
ployed to develop rotavirus vaccines. The rhesus rotavirus was
found to be attenuated in humans and protective against hu-
man serotype 3, to which it is antigenically related (25). A
second Jennerian rotavirus vaccine, based on the UK strain of
bovine rotavirus, is also being developed (7). Jennerian vac-
cines for PIV1 and for hepatitis A virus are attenuated and
immunogenic in nonhuman primates (18, 22). Another exam-
ple involves reassortant viruses that contain two gene segments
encoding the hemagglutinin and neuraminidase surface glyco-
proteins from a human influenza A virus and the six remaining
gene segments from an avian influenza A virus that were at-
tenuated in humans (5, 33, 39). This indicated that one or more
of the six gene segments of the avian virus attenuated the
avian-human influenza A viruses for humans. The genetic de-
terminants of this attenuation were mapped using reassortant
viruses possessing a single gene segment from an attenuating
avian influenza A virus and the remaining genes from a human
influenza A virus strain. It was shown that the nonstructural,
polymerase (PB1 and PB2), and M genes contributed to the
attenuation phenotype of avian influenza A viruses in humans
(6). In another study, the severe host range restriction of bo-
vine respiratory syncytial virus (BRSV) for replication in chim-
panzees was only slightly alleviated by replacement of the
BRSV F and G glycoproteins with their HRSV counterparts.
This indicated that F and G are involved in this host range
restriction, but that one or more additional BRSV genes are
also involved (3). This illustrates that more than one gene can
contribute to the host range restriction phenotype of a mam-
malian or avian virus in primates. We expect that multiple
determinants will typically specify the host range phenotype of
Jennerian vaccines, although this has not been well studied.
The present study sought to further explore the genetic basis
of attenuation manifested by the Jennerian BPIV3 vaccine
candidate for nonhuman primates. Previously, it was found
that introduction of the BPIV3 N ORF into the HPIV3 back-
ground resulted in a level of host range restriction nearly
equivalent to that of BPIV3. Here, we found that this was also
true for the F and HN genes of BPIV3 that were introduced
into the HPIV3 backbone as a set of two genes. Unfortunately,
we were unable to observe significant differences in replication
of chimeric and parental viruses for the lower respiratory tracts
of rhesus monkeys due to a low level of replication of HPIV3
wild-type virus at this site. Clearly, the rhesus monkey is lim-
ited in its ability to detect differences in replication of BPIV3
and HPIV3 for the lower respiratory tract, but previous studies
in humans of BPIV3 and HPIV3 candidate vaccines indicated
that the attenuation of these viruses for the upper respiratory
tract of rhesus monkeys correlated well with their attenuation
in both the upper and lower respiratory tract in seronegative
infants and children (8, 20, 27, 28).
The mechanisms responsible for the restricted replication of
the BPIV3/HPIV3 chimeras in rhesus monkeys are unknown,
but it is not surprising that the N and HN/F proteins have been
identified as attenuating elements since in other viral systems
these proteins are important determinants of host range (11,
29, 36, 37). There are several possible mechanisms by which
HN and F glycoproteins could be determinants of host range.
First, the balance of receptor binding and neuraminidase ac-
tivities of the HPIV3 and BPIV3 glycoproteins could be opti-
mized for the sialoglycoproteins and sialoglycolipids present in
the respiratory tracts of the hosts. Such receptors are known to
differ among hosts (11, 24). Second, the HN and F glycopro-
teins are known to interact with host cell proteins such as
chaperones and cytoskeletal proteins during transport and
folding, and their role in virus assembly could be optimized for
their host of origin (34, 35). Third, optimal cleavage activation
of F could be host cell specific (19, 23, 43). Fourth, the activity
of the neuraminidase can be modified by intracellular halide
ion concentration and other factors which could differ between
The importation of BPIV3 genes into a virulent HPIV3
backbone is useful to identify genes that are independent at-
tenuating genetic elements, but this analysis does not provide
information on the relative contribution that these genes make
to the overall attenuation of BPIV3 for primates. To accom-
plish this, one needs to start with BPIV3 and replace a single
attenuating genetic element, identified as indicated above, with
its HPIV3 counterpart. If the resulting BPIV3/HPIV3 chimeric
TABLE 2. Immunization of rhesus monkeys with BPIV3/HPIV3 chimeric recombinants induces resistance to challenge
with wild-type HPIV3 28 days later
Mean peak virus titercfollowing challenge
(log10TCID50/ml ? SE) [Duncan grouping]d
Serum HAI antibody titer (mean reciprocal log2? SE)
for HPIV3 [Duncan grouping] for:
On the day of challenge28 days after challenge
4.5 ? 0.33 [A]
2.3 ? 0.14 [B]
2.5 ? 0.25 [B]
2.3 ? 0.41 [B]
3.0 ? 0.14 [B]
2.9 ? 0.26 [B]
4.5 ? 0.19 [A]
1.2 ? 0.20 [B]
1.0 ? 0.48 [B]
1.4 ? 0.08 [B]
1.0 ? 0.0 [B]
1.3 ? 0.20 [B]
?212.0 ? 0.58 [A]
11.7 ? 0.21 [A]
10.5 ? 0.29 [AB]
11.5 ? 0.22 [A]
9.5 ? 0.87 [B]
9.3 ? 0.76 [B]
9.5 ? 0.72 [A]
6.8 ? 0.63 [BC]
8.2 ? 0.48 [AB]
4.5 ? 0.29 [D]
5.5 ? 0.62 [CD]
aEach previously immunized monkey and nonimmunized controls were challenged with 106TCID50of HPIV3 JS in a 1-ml inoculum at each site.
bThe groups with six animals contain four animals each from a previous rhesus study (1).
cMean of peak virus titer for each animal in its group irrespective of sampling day.
dVirus titrations were performed on LLC-MK2 cells. The limit of detectability of virus titer was 10 TCID50/ml. Mean viral titers were compared using a Duncan
multiple range test (? ? 0.05). Within each column, mean titers with different letters are statistically different. Titers indicated with two letters are not significantly
different from those indicated with either letter.
eNasopharyngeal swab samples were collected on days 3 to 8 postchallenge.
fTracheal lavage samples were collected on days 4, 6, and 8 postchallenge.
VOL. 74, 2000 BOVINE PIV3 F AND HN GENES ATTENUATE HUMAN PIV38927
virus exhibits increased replicative capacity in primates, then
one can conclude that the gene makes a contribution to the
overall attenuation of BPIV3 for primates. To perform this
analysis, rBPIV3 was derived from cDNA and used to con-
struct a BPIV3/HPIV3 chimeric virus in which the F and HN
genes of BPIV3 were replaced with their HPIV3 counterparts.
The resulting chimeric recombinant rBPIV3-FHHNH, like its
rHPIV3-FBHNBcounterpart, replicated in vitro as well as its
parental viruses. This observation confirmed our assumption
that the highly conserved PIV3 gene-end, intergenic, and gene-
start cis-acting sequences (2) that were exchanged along
with the F and HN ORFs to generate rBPIV3-FHHNHand
rHPIV3-FBHNBwould be recognized by the heterologous
PIV3 polymerase complexes of the chimeric viruses. We had
also thought it likely that the F and HN exchange between
BPIV3 and HPIV3 would be compatible since the considerably
more divergent HPIV1 F and HN proteins were highly func-
tional in a HPIV3 background (42), and this was confirmed by
the undiminished capacity of the chimeric viruses for replica-
tion in vitro. rBPIV3-FHHNHreplicated in the upper respira-
tory tracts of rhesus monkeys to a level intermediate between
that of its HPIV3 and BPIV3 parents, indicating that the
BPIV3 F and HN genes make an independent contribution to
the overall attenuation of BPIV3 for primates, at least in the
upper respiratory tract. The overall attenuation of BPIV3 thus
is the sum of two or more genetic elements, one of which is the
set of F and HN genes and one of the others is possibly N (1).
Although BPIV3 itself is being evaluated as a vaccine virus
for HPIV3 (26, 27), it is only 25% related antigenically to
HPIV3 (8). Thus, the immunogenicity of BPIV3 against
HPIV3 would be improved if it could be modified to express
the protective F and HN antigens of HPIV3. rBPIV3-FHHNH
represents such a virus; in this study, immunization of rhesus
monkeys with rBPIV3-FHHNHinduced a higher level of anti-
body to HPIV3 than did immunization with BPIV3. Further-
more, rBPIV3-FHHNHconferred a level of protection against
replication of HPIV3 challenge in the upper and lower re-
spiratory tract that was statistically indistinguishable from
that conferred by a previous infection with rHPIV3. Simi-
larly, rHPIV3-NB, which is attenuated by the BPIV3 N protein
but possesses HPIV3 protective antigens, also induced a high
level of resistance to HPIV3 challenge, confirming our previ-
ous observations (1). Despite replicating to a similar level in
rhesus monkeys, rHPIV3-NBinduced higher levels of antibod-
ies to HPIV3 than rBPIV3-FHHNH, but the reasons for this
are not understood. Additional animals are being immunized
to determine whether this difference in immunogenicity is re-
rBPIV3-FHHNHreplicates to a higher level in rhesus mon-
keys than BPIV3, although it is significantly attenuated com-
pared to HPIV3. Since the level of replication of BPIV3 in
humans is low (27), this increase might be well tolerated by
vaccinees. Alternatively, it is possible that rBPIV3-FHHNH
might replicate in human infants to a level sufficiently high to
cause respiratory tract illness. However, the slight increase in
replication of rBPIV3-FHHNHin primates offers an opportu-
nity to use rBPIV3-FHHNHas a vector for other viral antigens.
Recently, it was shown that the importation of a measles virus
HA glycoprotein as an additional gene into an attenuated
HPIV3 vaccine candidate further attenuated the vaccine in
vivo (17). Thus, the slight increase in replication of rBPIV3-
FHHNHin monkeys over that of BPIV3 might be offset by the
addition of one or more foreign glycoprotein genes. The data
presented here further define the basis for the host range
restriction of BPIV3 for primates and identify rBPIV3-FHHNH
as a potential vaccine candidate and as a vector that deserves
We thank Robert Chanock for review of the manuscript and Kath-
ryn Hanley for help with statistical analysis. We also thank Anna
Durbin for providing the p(Right) vector and Ernest Williams as well
as Chris Cho for excellent technical support.
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