AVIAN DISEASES 54:302–306, 2010
Research Note—
New Approach to Delist Highly Pathogenic Avian Influenza Viruses from BSL3+ Select
Agents to BSL2 Non-Select Status for Diagnostics and Vaccines
Samadhan J. Jadhao and David L. SuarezA
Southeast Poultry Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, 934 College Station Road,
Athens, GA 30605
Received 18 May 2009; Accepted and published ahead of print 6 October 2009
SUMMARY. Highly pathogenic avian influenza viruses (AIVs) are Select Agents in the United States and are required to be
handled in bio-containment level-3 enhanced (BSL3+) facilities. Using a reverse genetics system, we attenuated a highly pathogenic
virus, with the goal of making it low pathogenic and having it delisted as a Select Agent so that it could be handled in a biocontainment level-2 facility for diagnostic or vaccine production applications. We utilized two approaches to attenuate the target
AIV by mutating the highly pathogenic hemagglutinin (HA) cleavage site to be low pathogenic and by replacing the full-length NS
gene segment with a naturally truncated 124–amino acid NS1 coding gene from A/turkey/Oregon/73 (H7N3) virus (tkOR71
trNS1). To delist an AIV so that it can be handled in a BSL2 facility, the amino acid sequence of the HA cleavage site of the rescued
virus must be confirmed to be compatible with a low-pathogenic AIV; it should not plaque in cell culture without supplementation
of exogenous trypsin; and intravenous pathotyping in 4–6-wk-old specific-pathogen-free chickens must confirm that the virus is
low pathogenic. The candidate A/duck/Vietnam/Baclieu/09/07 (rH5N1/PR8/trNS1) virus with five PR8 internal genes, tkOR71
trNS1 gene, and A/chicken/Indonesia/7/03 N1 neuraminidase gene was constructed. The virus was shown to not plaque in cell
culture without addition of trypsin. The virus was low pathogenic in the standard intravenous pathotyping test (IVPI 5 0) and also
caused no disease in a separate intranasal inoculation test in 4-wk-old specific-pathogen-free chickens, thus demonstrating that the
virus is suitable for deselection.
RESUMEN. Nota de Investigación—Enfoque nuevo para la eliminación de un virus de influenza aviar de alta patogenicidad de
la lista de agentes selectos BSL3+ para ser incluido en el estatus no selecto BSL2 con fines de diagnóstico y elaboración de vacunas.
Los virus de alta patogenicidad de influenza aviar (AIVs, por su siglas en inglés) son Agentes Infecciosos Selectos en los Estados
Unidos y requieren para su manejo de instalaciones con un nivel de biocontención tipo 3 extra (BSL3+, por sus siglas en inglés).
Usando un sistema de reversión genética, se atenuó un virus de alta patogenicidad con el objetivo de hacerlo de baja patogenicidad y
lograr que se eliminara de la lista de agentes selectos para de esta manera, poder manejarlo con fines diagnósticos o para la
producción de vacunas en instalaciones de un nivel de biocontención 2 (BSL2, por sus siglas en inglés). Se utilizaron dos enfoques
para la atenuación del virus seleccionado, mediante mutación en el sitio de disociación de la hemaglutinina (HA, por sus siglas en
inglés) de alta patogenicidad, para convertirlo en un virus de baja patogenicidad, o por el reemplazo del segmento completo del
gene NS por un gene truncado de manera natural que codifica para una proteı́na NS1 de 124 aminoácidos y que pertenece al virus
A/pavo/Oregon/73 (H7N3), (gene tkOR71 trNS1). Para retirar de la lista de agentes selectos a un virus de influenza aviar y de esta
manera poder manejarlo en instalaciones tipo BSL2, la secuencia de aminoácidos del sitio de disociación de la hemaglutinina del
virus rescatado debe ser confirmada como compatible con un virus de influenza aviar de baja patogenicidad; éste no debe formar
placas en cultivo celular sin la suplementación de tripsina exógena y la tipificación de la patogenicidad mediante inoculación
intravenosa en aves libres de patógenos especı́ficos de 4 a 6 semanas debe confirmar que el virus es de baja patogenicidad. Se
construyó un virus candidato, el virus A/pato/Vietnam/Baclieu/09/07 (rH5N1/PR8/trNS1) con cinco genes internos de PR8, con
el gene tkOR71 trNS1, y el gene de la neuraminidasa N1 del virus A/pollo/Indonesia/7/03. El virus no produjo placas en los
cultivos celulares sin la adición de tripsina. El virus se comportó como un virus de baja patogenicidad en la prueba estándar de
tipificación de la patogenicidad por inoculación intravenosa mostrando un ı́ndice de patogenicidad intravenosa (IVPI, por sus siglas
en inglés) de cero y no causó enfermedad después de la inoculación intranasal en pollos libres de patógenos especı́ficos de cuatro
semanas de edad, lo que demostró que el virus era apto para ser deseleccionado.
Key words: avian influenza virus, reverse genetics, NS1 gene, avian influenza vaccine, select agents
Abbreviations: AIV 5 avian influenza virus; bp 5 base pair; BSL-3+ 5 bio-containment level-3; ECE 5 embryonated chicken
eggs; EID50 5 50% egg infectivity; HA 5 hemagglutinin; MDCK 5 Madin Darby Canine Kidney; PBS 5 phosphate-buffered
saline; RT-PCR 5 reverse transcriptase–PCR; SPF 5 specific pathogen free
Highly pathogenic H5N1 avian influenza viruses (AIVs) have
become endemic in poultry in several Asian and at least one African
country, resulting in unprecedented economic loss to poultry
farmers and zoonotic infections in people, leading to fatal illnesses
(1,2,3,5,7,9,10,11,12,16,20,22,23,27,28,29). Under these circumA
Corresponding author. E-mail: david.suarez@ars.usda.gov
stances, control and prevention of avian influenza in poultry is a high
priority. In addition to the traditional culling approach, preventive
vaccination for avian influenza in poultry has gained importance
because it has yielded some success in controlling the disease and
because of the increasing acceptance of vaccination as a control tool
in some affected Asian countries. The success of preventive
vaccination programs depends in part on the availability of vaccine
strains that are antigenically matched to the circulating highly
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H5N1 avian influenza virus with truncated NS1
pathogenic AIV field strains (14,25). Because naturally circulating
low pathogenic AIVs provide a poor antigenic match against the
circulating Asian H5N1 lineage of viruses, the use of plasmid-based
reverse genetics and site-directed mutagenesis has become indispensable as a way to produce antigenically matched vaccine strains. A
vaccine seed strain can be attenuated by mutating the hemagglutinin
(HA) cleavage site so that it has a low pathogenic cleavage site, and
this approach allows the virus to remain antigenically matched to the
parent strain, because the cleavage site is distantly located in relation
to neutralizing sites on the HA protein (17,24). Alternative methods
of attenuation have been described, including changes to the NS1
protein. One study characterized a variant of A/turkey/Oregon/71
(H7N3) that had a naturally truncated 124–amino acid NS1
protein, compared with the usual 230–amino acid NS1 protein. The
variant virus with the truncated NS1 protein did not efficiently
block the induction of type I interferon, which the parent virus with
the full-length NS1 gene did, and it exhibited an attenuated
pathotype in chickens and poorly transmitted to in-contact sentinel
chickens (6,26).
Highly pathogenic AIVs are considered Select Agents in the
United States, and the live virus is required to be handled in an
enhanced bio-containment level-3 (BSL-3+) facility. Although an
HPAI virus can be attenuated to be low pathogenic using reverse
genetics, these viruses are still considered Select Agents and must be
handled under BSL-3 conditions, which limits how they can be used.
A virus can be delisted as a Select Agent in the United States if it
meets certain requirements. In brief, the HA cleavage site must be
changed so that it is compatible with a low pathogenic virus; it must
be shown that the virus can no longer grow in cell culture without
supplementation of exogenous trypsin in cell culture medium; and,
finally, the virus must be tested in the standard intravenous
pathotyping test in chickens, per World Organization for Animal
Health guidelines. If a virus meets all of these conditions, it may be
considered for deselection. In addition to the change at the HA
cleavage, we considered it beneficial to include additional attenuation factors in the virus to increase the safety of working with live
virus in BSL-2 facilities. The use of a truncated NS1 gene of A/
turkey/Oregon/71 (H7N3) was selected as a way to attenuate the
virus in animals; this selection will still allow the virus to be grown to
high titer in 9-to-10-day-old embryonating chicken eggs. Based on
this hypothesis, we used a reverse genetics approach to incorporate
both changes: the H5 HA cleavage site mutation and a truncated
NS1 protein in the recombinant H5N1 virus. Further, we evaluated
the rescued virus for its ability to replicate in chickens in an attempt
to have the virus deselected.
MATERIALS AND METHODS
Reverse transcriptase–PCR (RT-PCR) and nucleotide sequencing.
The AIVs used in this study were obtained from the Southeast Poultry
Research Laboratory (USDA-ARS, Athens, GA). The H5 HA gene of A/
duck/Vietnam/Baclieu/09/07 (H5N1) virus was reverse transcribed into
complementary DNA using influenza A virus 12 mer primer
AGCAAAAGCAGG and AMV reverse transcription system (Promega,
Inc., Madison, WI). Full-length H5 HA was PCR amplified using
BsmBI HA forward 59-TATTCGTCTCAGGGAGCAAAAGCAGGGG-39 and BsmBI HA reverse 59-TATTCGTCTCGCTGTAGTAGAAACAAGGGTGTTTT-39 primers and PfuUltraH II Fusion HS
DNA Polymerase (Stratagene, Inc., Cedar Creek, TX). The H5 HA
gene amplicon was restriction digested with BsmBI enzyme and cloned
into a bi-directional 425–base pair (bp) promoter chicken RNA
polymerase I reverse genetics vector (Jadhao and Suarez, unpubl. data).
Automated nucleotide sequencing of the H5 HA gene was performed
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using gene-specific primers and a PRISM Ready Reaction DyeDeoxy
Terminator Cycle Sequencing kit (PerkinElmer, Foster City, CA) on a
3730 PerkinElmer machine. Using site-directed mutagenesis PCR, the
highly pathogenic H5 HA cleavage sequence PQREGRRKKR/GLF was
mutated to low pathogenic PQRETR/GLF cleavage site. The mutated H5
HA cleavage site sequence was also confirmed by nucleotide sequencing.
The AIV A/turkey/Oregon/71 (H7N3), with a truncated NS1 protein of
only 124 amino acids, was used as the source to clone the NS gene. As was
the case with the H5 HA gene, the NS gene segment of A/turkey/Oregon/
71 (H7N3) virus variant was amplified using primers BsmBI NS forward
59-TATTCGTCTCAGGGAGCAAAAGCAGGGTG-39 and BsmBI NS
reverse 59-TATTCGTCTCGCTGTAGTAGAAACAAGGGTGTTTT39 and cloned in the reverse genetic vector. The NS segment was
sequenced to confirm the truncated NS1 124–amino acid length. The NS
gene segment-specific primers were used to perform one-step RT-PCR to
demonstrate truncated NS segment in the recombinant H5N1 (rH5N1/
PR8/trNS1) virus.
Generation of recombinant AIV using reverse genetics. The
chicken RNA polymerase I 425 promoter-based bi-directional plasmidbased reverse genetics system was constructed for A/Puerto Rico/8/34
(H1N1) strain (Jadhao and Suarez, unpubl. data), similar to the 250–bp
chicken RNA polymerase I promoter-based bi-directional plasmid-based
reverse genetics system described previously by Massin et al. (18). To
generate a recombinant virus, the mutated low pathogenic RETR
cleavage site coding H5-subtype HA gene was derived from A/duck/
Vietnam/Baclieu/09/07 (H5N1) virus, the N1-subtype neuraminidase
gene was from A/chicken/Indonesia/7/03 (H5N1) virus, and the
internal genes (PB2, PB1, PA, NP, and M), with the exception of the
NS gene, originated from the A/Puerto Rico/8/34 (H1N1) strain. The
NS gene segment was derived from the A/turkey/Oregon/71 (H7N3)
virus variant coding a truncated 124–amino acid NS1 protein. The DF1 cell line, procured from American Type Culture Collection and
maintained at Southeast Poultry Research Laboratory (USDA-ARS,
Athens, GA), was used for transfection. The recombinant rH5N1/PR8/
trNS1 AIV was rescued in chicken fibroblast (DF-1) cells using an 8 bidirectional chicken RNA polymerase I promoter-driven plasmid-based
reverse genetics system. The transfection procedure was similar to
previously described protocols (18,19), but with a few modifications.
Briefly, 1 mg each of the bi-directional plasmid and 16 ml of
Lipofectamine 2000 (Invitrogen, Inc., Carlsbad, CA) was used to
transfect a six-well plate dish of chicken fibroblast (DF-1) cells. After
incubation for 72 hr in 37 C/5% CO2 incubator, the transfection
supernatants were inoculated in 9-day-old embryonated specificpathogen-free (SPF) chicken eggs. The allantoic fluids were harvested
after 3–4 days from the eggs and tested for virus rescue by
hemagglutination and egg infectivity assay (13). The sterility of the
allantoic fluid with HA-positive test was ascertained by inoculation of
thioglycolate broth tubes at 37 C for 1 wk. The stock virus was
produced by a second limiting dilution passage in the SPF embryonated
chicken eggs (ECE).
Confirmation of low pathogenic HA cleavage site and truncated
NS1 gene. The H5 HA and NS gene segment of the reverse genetics
rescued H5N1 virus was amplified using gene-specific primers (primer
sequence available upon request) in a one-step RT-PCR (Qiagen, Inc.,
Valencia, CA). The H5 HA cleavage site low pathogenic RETR
sequence was confirmed by direct nucleotide sequencing, as described
earlier in this paper. The truncated NS segment of 700-bp size was
confirmed by 1% agarose gel electrophoresis.
Growth properties in Madin Darby Canine Kidney (MDCK)
cell culture. The plaque-forming ability of the recombinant A/duck/
Vietnam/Baclieu/09/07 (rH5N1/PR8/trNS1) virus strain with mutated
low pathogenic RETR cleavage site sequence and truncated NS1 gene
from A/turkey/Oregon/71 (H7N3) virus variant was tested in MDCK
cell line in the presence and absence of exogenous L-(tosylamido-2phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin. The
confluent MDCK cell cultures in the six-well culture plates were
infected with 10-fold dilutions of the virus in Dulbecco’s Minimum
Essential Medium at 37 C/5% CO2 atmosphere. After 1 hr, medium
was removed and overlaid with 0.9% agar prepared in phenol red free
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S. J. Jadhao and D. L. Suarez
MEM medium with or without 1 mg/ml TPCK-treated trypsin. The
plates were incubated at 37 C/5% CO2 atmosphere for 4 days and fixed
with 1% neutral formalin for at least 24 hr. Following removal of agar,
the fixed cell cultures were stained with 0.05% neutral red to better
visualize the plaques.
Growth properties in ECE. The recombinant A/duck/Vietnam/
Baclieu/09/07 (rH5N1/PR8/trNS1) virus was diluted 10-fold in
phosphate-buffered saline (PBS) with 13 antibiotic and antimycotic
solution and inoculated at 0.1 ml via chorio-allantoic cavity route in 9day-old SPF-ECE (n 5 5/virus dilution) and incubated for 3–4 days.
The allantoic fluids were harvested from the chilled eggs and tested by
HA assay using 0.5% (v/v) chicken erythrocytes. Fifty percent egg
infectivity (EID50/ml) virus titers were determined by the Reed and
Muench method (21).
Pathogenicity of the recombinant virus in chickens. The animal
experiments were conducted with the approval and oversight of the
Southeast Poultry Research Laboratory Institutional Animal Care and
Use Committee. Eight 4-wk-old white leghorn SPF chickens were
inoculated by intravenous route or intranasal route with 0.2 ml of 1:10
dilution of the A/duck/Vietnam/Baclieu/09/07 (rH5N1/PR8/trNS1)
virus strain prepared in PBS (pH 7.2). The birds were housed in
negative-pressure Horsfal units fitted with HEPA filters in an enhanced
BSL-3 animal building. Water and feed were provided to the birds ad
libitum. The chickens were monitored for 10 days for the presence of
clinical signs or mortality, as required for AIV standard pathotyping.
Oro-pharyngeal swabs were collected in 1-ml PBS/antibiotic-antimycotic solution (pH 7.2) on day 2 and day 4 postchallenge. RNA was
extracted from the oro-pharyngeal swabs using the Trizol and magnetic
bead–based extraction protocol (8). Influenza A virus matrix genespecific real-time RT-PCR was performed to assess the extent of the oropharyngeal virus shedding (15). A/duck/Vietnam/Baclieu/09/07
(rH5N1/PR8/trNS1) virus aliquot with predetermined EID50 titer
was used for RNA extraction and was included to generate a standard
curve to determine the virus shedding in the oro-pharyngeal swabs of
chickens in the pathotyping experiment (15). To assess the seroconversion, serum samples were harvested on day 14 postchallenge, and an agar
gel precipitation test was performed using reagents procured from the
National Veterinary Services Laboratories in Ames, IA (4).
RESULTS AND DISCUSSION
The goal of this work was to produce an attenuated virus from a
highly pathogenic H5N1 virus using a protocol that would
potentially allow the virus to be deselected so that it could be safely
handled under BSL-2 conditions. Using a chicken RNA polymerase
I 425-bp promoter plasmid-based reverse genetics system, a virus was
rescued that contained an H5 HA gene from A/duck/Vietnam/
Baclieu/09/07 that had a mutated cleavage site characteristic of a low
pathogenic RETR sequence, a truncated NS1 gene from A/turkey/
r
Fig. 1. (a) Mutagenesis of highly pathogenic H5 HA cleavage site of
A/duck/Vietnam/Baclieu/09/07 (H5N1) virus to characteristic low
pathogenic RETR/GLF HA cleavage site amino acid sequence. Using
site-directed mutagenesis, amino acid sequence GRRK coding codons
were deleted from the HP H5 HA cleavage site; at 22 position AAG
coding for basic amino acid lysine (K) was mutated to ACC to code for
threonine (T); and at 27 GAG coding for glutamic acid (E) was
mutated to alternate codon GAA coding for glutamic acid (E) to obtain
characteristic low pathogenic RETR cleavage site. (b) Demonstration of
truncated 700-bp NS gene segment (lane 1) in recombinant A/duck/
Vietnam/Baclieu/09/07 (rH5N1/PR8/trNS1) virus by one-step RTPCR, as compared to the wild-type A/Puerto Rico/8/34 (H1N1) NS
segment (lanes 2 and 3). M1 5 100-bp ladder; M2 5 1-Kb ladder. (c)
Demonstration of inability to form plaques in MDCK cell culture in
absence of exogenous trypsin (top) and ability to form plaques in
presence of exogenous trypsin 4 days postinfection (bottom) with A/
duck/Vietnam/Baclieu/09/07 (rH5N1/PR8/trNS1) H5N1 virus containing characteristic low pathogenic H5 HA RETR/GLF cleavage site
amino acid sequence and truncated NS1 gene from A/turkey/Oregon/71
(H7N3) virus.
H5N1 avian influenza virus with truncated NS1
Oregon/71 (H7N3) virus variant, the N1 gene from chicken/
Indonesia/07/03, and the remaining genes (PB2, PB1, PA, NP, and
M) from A/Puerto Rico/34 (H1N1) strain in the gene constellation.
To clearly identify the recombinant virus, a list of the origins of all
gene segments was included in the virus name, viz. A/duck/Vietnam/
Baclieu/09/07 H5 HA 3 A/chicken/Indonesia/7/03 N1 NA 3 A/
Puerto Rico/8/34 MA, NP, PA, PB1, PB2 3 A/turkey/Oregon/71
NS truncated.
The recombinant A/duck/Vietnam/Baclieu/09/07 (rH5N1/PR8/
trNS1) virus H5 HA gene was confirmed to have a low pathogenic
cleavage site of RETR/GLF (Fig. 1a) by sequencing. The RT-PCR
test using NS segment-specific primers indicated the presence of a
truncated NS segment of approximately 700 bp (Fig. 1b). The
recombinant virus did not form plaques in MDCK cell culture in
the absence of exogenous supplementation of TPCK-treated trypsin
(Fig. 1c). However, the same virus formed clear plaques in MDCK
cells in 4 days when the agar overlay was supplemented with
exogenous TPCK-treated trypsin (Fig. 1c). The growth property of
A/duck/Vietnam/Baclieu/09/07 (rH5N1/PR8/trNS1) virus in 9day-old ECE indicated that it grows to infectivity titers of 106
EID50/ml and HA titers as high as 512. Negative testing of the
recombinant A/duck/Vietnam/Baclieu/09/07 (rH5N1/PR8/trNS1)
virus RNA by real-time RT-PCR for Newcastle disease virus ensured
that the viral aliquots were free of this viral pathogen.
The intravenous pathotyping test, required for deselection of the
virus, and the intranasal infectivity test indicated that the virus did
not cause clinical signs over a period of 14 days in inoculated
chickens. No sero-conversion was found by the influenza A virusspecific agar gel immunodiffusion assay both in intravenous and
intranasally inoculated chickens. The influenza A virus matrix gene
real-time RT-PCR test on the oro-pharyngeal swabs did not detect
viral shedding on days 2 or 4 postchallenge. The agar gel
immunodiffusion assay and negative viral shedding data indicate
that the recombinant A/duck/Vietnam/Baclieu/09/07 (rH5N1/PR8/
trNS1) virus either did not replicate or replicated only poorly in
chickens and would be a safer alternative from which to produce
diagnostic antigens for handling in the laboratories using BSL-2
containment levels.
The data from this rescued virus demonstrate that the virus is
attenuated and meets the standards for being deselected. Regulatory
approval is still required before a virus can be deselected. Upon U.S.
Department of Agriculture approval, the mutated nonpathogenic
recombinant H5N1 viruses would be transferred to the BSL-2
laboratory for further work. This study describes a protocol to
generate more attenuated exotic rH5N1/PR8/trNS1 AIVs to handle
in the laboratories with lower or BSL-2 containment levels.
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ACKNOWLEDGMENTS
We thank Dr. Ruben Donis (Centers for Disease Control and
Prevention, Atlanta, GA) for providing the MDCK cell line. The
excellent technical assistance of Aniko Zsak, Suzanne DeBlois, and Joan
Beck is gratefully acknowledged for this ongoing research project. This
research project is being supported by U.S. Department of Agriculture
grant 901-6612-480, and there is an Interagency agreement with the
Centers for Disease Control and Prevention (908-6612-820).