 DIAGNOSTIC NOTES
Diagnosis of swine influenza
Bruce H. Janke, DVM, PhD
Janke BH. Diagnosis of swine influenza. Swine Health Prod.
2000;8(2):79-84. This article is also available in PDF
format. This Diagnostic Note has been peer refereed.
Veterinary Diagnostic Laboratory, Iowa State University, Ames,
Iowa 50011
Summary
In recent years, swine influenza has become recognized as a
significant contributor to the porcine respiratory disease complex
(PRDC), which is causing severe problems in many swine operations.
Infection with swine influenza virus (SIV) is not always clinically
evident and diagnostic tests often must be conducted to detect
the presence of the virus, especially in cases of enzootic infection.
A variety of assays are available to detect SIV or the antibody
induced by the virus. The appearance of antigenically variant
H1N1 strains and, more recently, of new H3N2 subtype strains in
swine populations in the United States has raised questions about
cross-protection and about our ability to detect the new strains
with currently available diagnostic tests. In this review, we
present information to update the reader on the methods available
to detect swine influenza, to differentiate among SIV strains,
and on the optimum use of these tests.
 Keywords: swine,
swine influenza virus, diagnosis, H3N2 subtype
Received: October 20, 1999
Accepted: January 13, 2000
Historically, swine influenza
has been considered relatively easy to diagnose. In its classical
epizootic form, which induces a harsh barking cough and high fever,
the infection could be identified on the basis of clinical signs
alone. Because the disease passed through herds very quickly with
only short-lived effects, the infection was not considered a significant
problem and treatment was usually deemed unnecessary.1 Recently,
an enzootic form of the disease has appeared in large operations
as a part of the porcine respiratory disease complex (PRDC).2
The ubiquitous presence of the virus and the significant accompanying
losses incurred due to respiratory disease has increased the importance
of infections. Perhaps this is best highlighted by the demand
for a vaccine.
The enzootic form of infection is not clinically dramatic or
unique, and diagnostic testing is often necessary to detect infection.
To further complicate matters, new strains of swine influenza
virus (SIV) have appeared in swine populations in the United States
and Canada during the last decade, derived through the well-defined
processes of antigenic drift and shift which have been described
for influenza viruses that affect other species. Antigenically
variant or atypical swine influenza viruses were identified first
in Canada and later in the United States in the late 1980s and
early 1990s.3,4 Within the past year, an even more
dramatic and important change has occurred in the United States:
a new subtype (H3N2) was identified as a significant pathogen
in swine. This is the first occurrence of a new subtype of SIV
in the United States since 1918.5
Some of the most dramatic epizootics of H3N2 infections observed
recently occurred in vaccinated pregnant gilts and sows that subsequently
aborted in high numbers. However, abortion appears to result from
the high fevers induced by infection of naive dams rather than
through direct infection of the fetus. There is one report of
isolation of virus directly from a single porcine fetus from a
sow that aborted during an outbreak,6 but previous
research on H1N1 SIV has indicated that the virus does not leave
the respiratory tract to any great extent and usually will not
be found in aborted fetuses.7 Similar studies have
not yet been conducted with H3N2 virus, but at this time, there
is no reason to believe this subtype will act any differently.
Once an animal has recovered and become immune, the virus is cleared
from that animal (i.e., no carrier state has been identified);
thus, sows that abort should not have subsequent reproductive
problems. Ongoing reproductive problems reported in herds that
suffered through an abortion epizootic of H3N2 SIV infection may
have been due to incomplete herd immunity with subsequent abortions
occurring in gilts or sows that did not become infected during
the initial epizootic.
Reports of atypical H1N1 SIV led to concerns about our ability
to detect variant strains with the diagnostic tests already in
place and whether the amount of cross-protection afforded pigs
that were vaccinated or had been infected with classical strains
was adequate. Dramatic epizootics due to infection with the new
H3N2 strains have occurred, even in herds heavily immunized with
a vaccine considered efficacious against the H1N1 strains. Apparently,
these vaccines offer little cross-protection against infection
with H3N2 strains. This lack of cross-reaction has renewed concerns
about our ability to detect both H3N2 and H1N1 infections with
the current antibody-dependent techniques.
In this manuscript, we present information to update the reader
on the methods available to detect swine influenza and to differentiate
among SIV strains, and provide guidance for the optimum use of
these tests.
Swine influenza virus
Influenza viruses are double-stranded RNA viruses that belong
to the family Orthomyxoviridae. The viruses are classified into
types A, B, and C according to the composition of nucleoproteins
and matrix proteins. The nucleoproteins support the nucleic acids
of the viral genome, and the matrix proteins line the inside of
the viral envelope.8 These proteins are relatively
less important for protective immunity when compared to the external
proteins. They are involved in cell-mediated immunity and recovery
from infection. These proteins are relatively conserved; i.e.,
they show only minimal variation among strains, and thus detection
tests aimed at finding these proteins will identify all of the
strains with which we are concerned. Nearly all swine influenza
viruses worldwide are type A viruses; only a few isolated reports
from Asia have indicated infection of swine with influenza viruses
of other types.
The external proteins that project from the surface of the
virus--the hemagglutinin (HA) and the neuraminidase (NA)--are
more significant for infection and immunity. The HA is responsible
for attachment of the virus to the host receptor and infection
of the host cell. This protein is also the major antigen against
which the host raises an antibody response. The NA protein functions
in release of progeny virions from the infected host cell, and
helps reduce self-agglutination of the virus. This protein has
a less prominent role as an immunogen. Fifteen HA proteins and
nine NA proteins have been identified in influenza viruses in
animals, birds, and humans. The degree of homology in the amino
acid sequence between HA proteins of different subtypes varies
from 25%-80%, while homology between HA proteins within the same
subtype is generally greater than 90%.9 Only H1 and
H3 hemagglutinins have been recovered from swine worldwide.
The H3N2 virus that appeared in North Carolina swine last year
appeared to be a reassortant virus that contained the genes for
all but one of the internal proteins derived from the pre-existing
H1N1 swine virus. The genes for the HA and NA proteins and one
internal protein, PB1, are thought to have been derived from a
recent human H3N2 strain.10 This event in itself is
somewhat unusual in that most infections of swine with H3N2 strains
in other parts of the world have been incidences of human strains
entering swine populations intact or by reassortants of human
and avian strains.8,11 Of interest, and perhaps concern,
is the fact that the H3N2 viruses from different parts of the
United States that have been studied to date differ in composition.
Like the original North Carolina isolate, three H3N2 viruses isolated
from swine in the Midwestern United States (Iowa, Minnesota, and
Texas) contain similar HA, NA, and PB1 genes from a human strain
and three of the internal genes from the classical swine H1N1
virus. However, the Midwest isolates also contain two internal
genes, PB2 and PA, that appear to have been derived from an avian
strain.10 The HA of the original North Carolina isolate
is more divergent from the more closely related Midwest isolates.
The HA1 portion of the North Carolina isolate differs from the
three Midwest isolates in 18-20 amino acids. The Midwest isolates
differ from each other in only two to four amino acids.
The significance of these differences is not clear, but the
fact that the few strains studied so far do not appear to be the
same is intriguing. A shift in subtype had not occurred in the
75 years that have passed since the disease was first identified
in the United States. When a new subtype did appear, it seemed
to spread very rapidly throughout the country. Were there multiple
incidences of co-infection and reassortment in separate swine
populations, or did additional changes occur as the new subtype
spread through naive populations to produce this variation? Retrospective
analysis of the composition of multiple isolates from different
regions of the country and from different times is expected to
provide more information.
In general, the H1 and H3 hemagglutinins are the hemagglutinins
that are most divergent from each other, sharing only approximately
25% homology.9 Thus, very little cross-reactivity between
antibodies for these proteins would be expected, which could affect
cross-protection in the field and antibody-based diagnostic assays.
Infection or vaccination against H1N1 strains would likely provide
very little protection against infection with H3N2. This appeared
to be the case in the vaccinated sows that were so severely affected
during the H3N2 epizootics in late 1998 and early 1999. Most of
the SIV detection tests used in veterinary diagnostic laboratories
use antibody directed against the conserved type-specific antigens
and so have been successful in detecting both H1N1 and H3N2 subtypes.
However, additional differential serologic tests have had to be
developed to detect seroconversion to the H3N2 strains.
The HA amino acid sequences in the H1 and H3 subtypes is sufficiently
different that cross-reacting antibodies would not be expected,
but the cross-reactivity between strains within the same subtype
is less predictable from assessment of amino acid sequence homology
alone. Variations in HA1 amino acid sequences between typical
and atypical H1N1 strains were never more than 15-18 amino acids,
resulting in homologies of 94%-96%.4,12 These minor
variations did not appear to have any affect on cross-protection
or to affect diagnostic test sensitivity.13 However,
some researchers who are well-acquainted with influenza, extrapolating
from experiences with H3 influenza viruses in other species, have
predicted that the H3 hemagglutinin in H3N2 strains will be less
stable than the H1 and that greater antigenic drift may occur
as the virus continues to move through swine populations.
How much drift is necessary before it has clinical and diagnostic
significance? The answer is unclear and may be affected more by
the location of changes on the protein than by the total sequence
change. Comparative serologic studies on the cross-reactivity
of strains may provide more clinically relevant clues than nucleic
acid and amino acid sequence studies. A network of collaborating
centers of influenza research established by the World Health
Organization to address international surveillance of influenza
activity annually examines information on currently circulating
human strains to advise on the appropriate strains for inclusion
in human influenza vaccines to be produced in the following year.14
Serologic data are used in these discussions.
Studies comparing the amino acid sequence of the HA from H3N2
isolates from Iowa, North Carolina, Minnesota, Texas, and Illinois
have indicated that these strains are 95%-98% homologous.10,15
From past studies with H1N1 strains,13 this degree
of homology might not be expected to greatly affect cross-protection,
unless the change at any particular point(s) in the amino acid
sequence markedly affected protein conformation. Such a critical
point mutation may have occurred because antiserum raised in caesarian-derived,
colostrum-deprived (CDCD) pigs against the original North Carolina
strain does not react with the Midwest strains in hemagglutination
inhibition tests. Antisera against the Midwest strains also does
not react with the North Carolina strain.16 Other isolates
resembling the Midwest isolates have subsequently been recovered
from swine in North Carolina, but no additional isolates similar
to the initial North Carolina strain have been found in that state.17
Whether strains resembling the original North Carolina isolate
are present elsewhere in the country has not yet been determined.
Diagnostic tests that detect virus, viral proteins (antigens),
or viral nucleic acid
Fluorescent antibody (FA) test
This test uses either antiserum prepared against whole virus
(polyclonal antibody) or an antibody against a specific protein
(monoclonal antibody), which is usually applied to frozen sections
of lung from pigs submitted for laboratory examination. The reagent
used in the FA test in most diagnostic laboratories is a polyclonal
antiserum offered by National Veterinary Services Laboratories,
which was prepared by injection of pigs with an H1N1 strain of
SIV initially isolated from swine in 1973 (A/Sw/Ia/73).
The animals from which the antibody was harvested were exposed
to whole virus, and antibody against both internal and external
proteins are in the antiserum. The reagent will detect both H1N1
and H3N2 strains, although some labs have reported that fluorescence
in lungs infected with H3N2 virus is less intense. This test can
be completed within several hours and results can be available
the same day if samples are submitted early enough in the day.
Fresh tissue should be chilled but not frozen.
Immunohistochemical (IHC) test
This test is similar in principle to the FA test but usually
refers to the use of antibody applied to sections of formalin-fixed
tissue. The sensitivities of the FA test and IHC test are similar,18
but the advantage of the IHC test is that it can be applied to
tissues optimally fixed at the time of necropsy without loss of
tissue integrity during transport to the laboratory. To reduce
background staining, monoclonal antibodies are usually used. The
antibodies used at the Veterinary Diagnostic Laboratory at Iowa
State University (ISU VDL) are directed against type A nucleoproteins
and thus will detect both H1N1 and H3N2 strains. We are currently
working to develop a differential IHC test that will allow us
to determine whether infection was with H1N1 or H3N2 strains,
eliminating the need to isolate virus. This test requires processing
of tissues into histopathologic sections, but results can be available
the next day if formalin-fixed tissues are submitted.
Antigen-capture ELISA
A commercial ELISA test (Directogen(TM) ; Becton
Dickinson Microbiology Systems; Sparks, Maryland) developed for
detection of influenza virus in throat or nasal swabs in humans
is also used in some veterinary laboratories. This test will detect
both H1N1 and H3N2 strains but will not differentiate between
subtypes. Studies on experimentally inoculated pigs suggest that
the ELISA is only slightly less sensitive than egg inoculation
in detecting virus shedding.19 In practice, however,
the test has not proven to be as sensitive as virus isolation
when applied to nasal swabs, and excess mucus or blood in the
sample can interfere with the test. The ELISA test has been successfully
used on swabs of small airways taken directly from lungs of pigs
at necropsy. Care should be taken to swab airways that lead from
affected lobules. Like FA tests, the ELISA tests can be conducted
relatively quickly, but they are considerably more expensive.
Freezing of samples may reduce the sensitivity of this test.
Polymerase chain reaction (PCR) tests
Nucleic acid primers have been developed in several research
laboratories, including the ISU VDL. These primers will hybridize
with and detect both H1N1 and H3N2 viruses and differentiate between
them. These probes have been used primarily to characterize virus
isolates. These probes could also be used directly on clinical
specimens without the necessity of isolation. The sensitivity
of such use has not yet been established.
In a preliminary study, primers against the conserved internal
nucleoprotein detected 100% of the infected lung tissues when
compared to virus isolation.20 Subtype-specific primers
used similarly in a multiplex PCR assay detected about 75% of
the infections. Information is not yet available on efficacy for
testing nasal swabs.
The reagents are more expensive and the procedures more labor-intensive
and time-consuming than with other tests. Test procedures require
2-3 days for completion.
Virus isolation
Historically, virus isolation of influenza viruses from most
host species has been conducted in embryonated chicken eggs. Because
of the expense of maintaining a continuous egg supply and the
labor-intensive procedures, not all laboratories have made this
diagnostic test routinely available. Isolation can be conducted
on nasal secretions and lung homogenate but the virus is not particularly
hardy and samples must be kept cool and moist. Secondary bacterial
infection also can interfere with virus isolation procedures by
killing the chick embryo before viral multiplication can occur.
Virus growth in eggs is determined by detecting hemagglutinating
activity in egg fluids 5 days after inoculation. Determining that
the hemagglutination activity is due to influenza virus infection
and identifying subtype is then conducted on egg allantoic fluids
by reaction with antisera or through PCR. These procedures take
more time than other tests and are better suited for characterizing
virus than for diagnosis. Cell cultures (MDCK cells or primary
porcine kidney cells) also are being used for isolation of influenza
viruses in some laboratories. When optimized, this method has
been reported to be as sensitive as egg inoculation.17
Virus isolation procedures take longer than other detection tests,
and success is dependent on the amount of virus present and quality
of the sample. With either method, allow 1-2 weeks for isolation
and characterization.
Diagnostic tests that detect circulating antibody to SIV (serology)
Hemagglutination inhibition test
The classic serologic test for detecting antibody against SIV
is the hemagglutination inhibition (HAI or HI) test. This test
detects circulating antibody that binds to the HA protein on the
surface of the virus, thus preventing the virus particles from
attaching to the surface of erythrocytes to form a virus-erythrocyte
meshwork (hemagglutination). The test is relatively simple and
can be completed within a few hours. Serum to be tested is mixed
with virus of known concentration and time is allowed for any
antibody present to react with this virus. Then the indicator
reagent (rooster or turkey erythrocytes) is added to determine
whether the virus in the test is still unbound and can agglutinate
the erythrocytes. The amount of antibody present in the serum
is determined by running serial two-fold dilutions of the serum
against the same concentration of virus. The titer is the dilution
at which there is no longer sufficient antibody present to prevent
or inhibit hemagglutination.
The ability of the test to detect antibody against a particular
strain of virus in the field depends on the antigenic relationship
between the field virus strain that induced antibody and the virus
strain used in the test. Veterinary diagnostic laboratories in
the United States have used classic type A subtype H1N1 virus
in developing and running their routine SIV HI serology tests
because that was the only virus of concern. The virus used in
this test by most veterinary laboratories in the United States
is the A/Sw/Ia/73 (H1N1), supplied by National Veterinary Services
Laboratories. With the identification of antigenically variant
type A subtype H1N1 strains, there was concern that these standard
tests would not be able to detect antibody against these variants.
Studies were conducted at the ISU VDL comparing HI test results
with antibody induced in pigs with classical and antigenically
variant H1N1 strains against homologous and heterologous strains
used in the test. Results indicated that the degree of antigenic
variation in the HA proteins between classic or typical H1N1 strains
and atypical or antigenically variant H1N1 strains did not affect
the ability of the test to detect antibody against the other H1N1
strains. Sensitivity of the test was not reduced.13
Unexpectedly, similar studies with the original North Carolina
H3N2 isolate and the Midwest H3N2 isolates have indicated that
antibody induced by the North Carolina virus does not cross react
with the Midwest strains and vice versa. 16
The HI test also is considered a relatively sensitive test
as the HA protein is quite antigenic and stimulates high circulating
antibody concentrations. Titers of 1:40 or less may include nonspecific
reactions; titers of 1:80 and above are considered positive and
specific. Antibody can be detected within 5-7 days of infection
with many pigs exhibiting titers reaching 1:80 by 1 week postinfection
(PI) and peaking at 1:320-1:640 by 2-3 weeks PI. Antibody concentrations
will remain high for several weeks before beginning to decline
(Table 1 and Table 2). Passive antibody in pigs in infected
herds will disappear in most pigs by 6 weeks of age. Vaccination
of sows prefarrowing will prolong this passive antibody until
about 16 weeks of age (Table
3).21
There is very little cross-reaction between the HA proteins
of H1N1 and H3N2 subtype strains of SIV, and thus the standard
HI test using the H1N1 strains will not detect antibody against
H3N2 strains. Some difficulties have been encountered in developing
a similar HI test simply by using the H3N2 virus in the test.
More manipulation of the reagents has been necessary, but a differential
test is now available. Laboratories in the Midwest that have developed
the test have been using a Midwest (Texas or Iowa) strain. At
this point, studies suggest that the test does not have cross-reactivity
problems with H1N1 strains and will detect antibody against any
of the Midwest strains. However, the test will not detect antibody
induced by the original North Carolina strain. Because heat treatment
of the serum is conducted before use in the H1N1 test and because
such treatment will interfere with its use in the H3N2 test, you
should decide whether to test for one or both viruses at the time
of submission. Serum tested for antibody against H3N2 can be subsequently
tested for antibody against H1N1 but not vice versa.
Enzyme-linked immunosorbent assay for antibody
An ELISA test for SIV antibody has been previously developed
in a few research laboratories, but studies comparing the results
of this test with the H1 test indicated poor correlation, and
the test has not been used routinely in most veterinary diagnostic
laboratories. More recently, development of an SIV ELISA has been
undertaken by the company that successfully produced such tests
for PRV and PRRSV, and this test may be commercially available
soon.22, 23 Such tests offering differential screening
of H1N1 and H3N2 antibody would be very beneficial.
Samples for diagnosis of SIV infection
Nasal swabs
Nasal swabs from acutely affected pigs can be used for virus
isolation. You should select pigs with high fevers and clear nasal
discharge for such samples. Most pigs will shed virus for 5-7
days after infection. Swabs with synthetic fiber (rayon or dacron)
tips should be used as cotton will inactivate the virus. Swabs
also should be kept moist and cool to prevent desiccation and
inactivation of the virus. Culturettes(TM) (Baxter Healthcare
Corporation, Deerfield, Illinois) with crush bulbs to keep the
tips moist work well. Other swabs can be broken off into small
vials or snap cap tubes with physiologic saline or cell culture
media. Freezing may have a slight negative effect on use of these
swabs for virus isolation and should only be done if swabs cannot
be delivered to the laboratory in a timely manner.
Nasal swabs also can be used to collect samples for use in
the antigen-detection ELISA kits. Excess mucus or blood on the
swabs can interfere with successful use of the test. One lab has
reported that this test has not worked as well on nasal swabs
as on swabs directly applied to small airways in lung tissue.
PCR diagnostic tests also have been applied to nasal swab samples
but the relative sensitivity of this analysis versus VI and ELISA
has not been evaluated. Freezing also may adversely affect the
use of swabs in the ELISA test.
Lung tissue
Fresh and fixed lung collected at necropsy from pigs with respiratory
disease are the most commonly used samples for diagnosis. Swine
influenza virus initially infects the epithelium lining the airways,
and the resulting lesion is predominantly a bronchopneumonia characterized
by multiple coalescing foci of lobular consolidation in cranioventral
portions of lung. These areas should be submitted for diagnostic
evaluation. Fresh tissue (chilled, not frozen) can be used for
FA test and VI studies, and formalin-fixed tissue for IHC test
and histopathologic examination. Experimental studies have indicated
that peak virus load in the airways is present at 24 hours after
infection even before gross lesions develop.24 Virus
usually can still be detected in bronchioles and alveoli at 48-72
hours after infection. In many pigs, very little virus may be
found by FA or IHC by 72 hours PI, and distribution is often quite
focal.
Histopathologic examination can demonstrate lesions suggestive
of SIV infection for about 2 weeks after infection. By 3 weeks
PI, recovery is almost complete. The later that lungs are examined
after infection, the more difficult the lesion becomes to evaluate.
Porcine circovirus can induce bronchiolar damage similar to that
induced by SIV and both Mycoplasma hyopneumoniae and SIV
infections induce significant peribronchiolar and perivascular
lymphocytic cuffing. Samples from more than one pig are recommended
to address the diagnostic difficulties imposed by the focal nature
of the lesions and the pig-to-pig variation in timing of infection,
especially in cases of enzootic respiratory disease.
Serum samples
Serum samples for serology studies should be collected at least
a week after infection is suspected to have occurred. Most pigs
will have titers of at least 1:80 at a week after infection and
high titers (1:320-1:640) should be expected in at least some
of the pigs sampled 14-21 days after the group was infected. Paired
samples may be necessary in vaccinated herds.
Abortion
Direct isolation of virus from nasal swabs of sows that are
acutely ill or performing serologic studies on affected groups
are the preferred methods to determine whether SIV is involved
in reproductive problems. Attempts to isolate SIV from fetuses
is likely to be unrewarding.
Acknowledgements
I would like to thank the following people for providing information
for this report: K-J Yoon (Iowa State University); S Swenson (National
Veterinary Service Laboratory); J Collins, S Goyal, K Rossow (University
of Minnesota); D Johnson, P Sweet, L Fawcett (South Dakota State
University), C Olsen (University of Wisconsin), G Erickson (University
of North Carolina), G Stevenson (Purdue University), R Sibbel
(Schering-Plough).
References
1. Easterday BC, Hinshaw VS. Swine influenza. In: Diseases
of Swine. Leman AD, et al.(eds), 7th ed., Iowa
State University Press, Ames, Iowa. 1992;349-357.
2. Janke BH. Classic swine influenza. Large Anim Prac.
1998;19:24-29.
3. Dea S, Bilodeau R, Sauvageau C, et al. Antigenic variant
of swine influenza virus causing proliferative and necrotizing
pneumonia in pigs. J Vet Diagn Invest. 1992;4:380-392.
4. Olsen CW, McGregor MW, Cooley J, et al. Antigenic and genetic
analysis of a recently isolated H1N1 swine influenza virus. Am
J Vet Res. 1993;54:1630-1636.
6. Madec F, Kaiser C, Gourreau JM, et al. Consequences pathologiques
d'un episode grippal severe (virus swine A/H1N1) dans les conditions
naturelles chez la truie non immune en debut de gestation. Comp
Immun Microbiol Infect Dis. 1989;12:17-27.
7. Brown TT, Mengeling WL, Pirtle EE. Failure of swine influenza
virus to cause transplacental infection of porcine fetuses.
Am J Vet Res. 1982;43:817-819.
8. Webster RG, Bean WJ, Gorman OT, et al. Evolution and ecology
of influenza A viruses. Microbiol Rev. 1992;56:152-179.
9. Murphy BR, Webster RG. Orthomyxoviruses. In: Virology.
Fields BN, Knipe DM, et al. (eds), Raven Press, Ltd, New York.
1990;1091-1152.
10. Zhou NN, Senne DA, Landgraf JS, Swenson SL, Erickson G,
Rossow K, Liu L, Yoon K-J, Krauss S, Webster RG. Genetic reassortment
of avian, swine and human influenza A viruses in American pigs.
J Virol. 1999;73:8851-8856.
11. Castrucci MR, Donatelli I, Sidoli L, et al. Genetic reassortment
between avian and human influenza A viruses in Italian pigs. Virol.
1993;193:503-506.
12. Rekik MR, Arora DJS, Dea S. Genetic variation in swine
influenza virus A isolate associated with proliferative and necrotizing
pneumonia in pigs. J Clin Microbiol. 1994;32:515-518.
14. Watson JM. Surveillance of influenza. In: Textbook of
Influenza. Nicholson KG, Webster RG, Hay AJ (eds), Blackwell
Science Ltd., Oxford. 1998;207-216.
18. Vincent LL, Janke BH, Paul PS, Halbur PG. A monoclonal-antibody-based
immunohistochemical method for the detection of swine influenza
virus in formalin-fixed, paraffin-embedded tissues. J Vet Diagn
Invest.1997;9:191-195.
References-nonrefereed
5. Janke BH, Yoon KJ, Halbur PG, Sorden SD, Senne DA, Swenson
SL, Schmitt B. Isolation of a new subtype (H3N2) of swine influenza
virus from pigs with respiratory disease in the north central
U.S. Proc IX Internatl Symp World Assoc Vet Lab Diagn.
1999:51.
13. Janke BH. Significance of antigenic variation in swine
influenza virus for serodiagnosis. Proc Sw Dis Conf Pract.
1997:7-14.
15. Junker DE, Weston CQ, Valencia DM, et al. Molecular characterization
of the hemagglutinin and neuraminidase genes from an H3N2 swine
influenza virus isolate recovered from the U.S. Proc Leman
Sw Conf. 1999;26(Suppl):5.
16. Swenson SL, et al. Experimental infection of pigs with
H3N2 swine influenza virus. Proc AAVLD. 1999:42.
17. Erickson G, et al. New approaches to swine influenza virus
isolation. Proc AAVLD. 1999:45
19. Swenson S, et al. A comparison of two enzyme immunoassays
with egg inoculation for the detection of swine influenza virus
from nasal swabs and lungs. Proc AASP. 1996;97
20. Harmon K, Krauss S, Yoon KJ. Development of a PCR based
differential test for H1N1 and H3N2 swine influenza viruses. Proc
AAVLD. 1999:44.
21. Thacker BJ, Cain CM, Janke BH, Kinker DR. Swine influenza
virus passive antibody levels in pigs from vaccinated or nonvaccinated
sows. Sw Res Reports. ISU, 1998.
22. Velek K, Hogan S, Liauw H. Development of an ELISA for
the detection of antibodies to swine influenza virus in swine
sera. Proc AAVLD. 1999;105.
23. Cornaglia E, Laganiere G, Lallier R, et al. A new competitive
ELISA for serological monitoring of swine influenza. Proc AAVLD.
1999:41
24. Janke BH, et al. Proc IX Internatl Symp World Assoc
Vet Lab Diagn. 1999:52
|
