University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
Public Health Resources Public Health Resources
2001
Host nutritional selenium status as a driving force
for in"uenza virus mutations
Heather K. Nelson
University of NC at Chapel Hill
Qing Shi
University of NC at Chapel Hill
Peter Van Dael
Nestlé Research Center, Lausanne, Switzerland
Eduardo J. Schi!rin
Nestlé Research Center, Lausanne, Switzerland
Stephanie Blum
Nestlé Research Center, Lausanne, Switzerland
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Nelson, Heather K.; Shi, Qing; Van Dael, Peter; Schi<rin, Eduardo J.; Blum, Stephanie; Barclay, Denis; Levander, Orville A.; and Beck,
Melinda A., "Host nutritional selenium status as a driving force for in>uenza virus mutations" (2001). Public Health Resources. 454.
h?p://digitalcommons.unl.edu/publichealthresources/454
The FASEB Journal
express article 10.1096/fj.01-0115fje. Published online June 8, 2001.
Host nutritional selenium status as a driving force for
influenza virus mutations
Heather K. Nelson,* Qing Shi,
†
Peter Van Dael,
‡
Eduardo J. Schiffrin,
‡
Stephanie Blum,
‡
Denis
Barclay,
‡
Orville A. Levander,
§
and Melinda A. Beck*
,†
*Departments of Nutrition and
†
Pediatrics, University of NC at Chapel Hill;
‡
Nestlé Research
Center, Lausanne, Switzerland; and
§
USDA, ARS, Beltsville Human Nutrition Research Center,
Beltsville, MD
Corresponding author: Melinda A. Beck, Ph.D., Department of Pediatrics, 535 Burnett-Womack,
CB #7220, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7220. Email:
melinda_beck@unc.edu
ABSTRACT
Previous work from our laboratory has demonstrated that infection with influenza
A/Bangkok/1/79 (H3N2), a relatively mild strain of the virus, caused much more severe
pneumonitis in selenium (Se)-deficient mice than in Se-adequate mice. Here we report that the
increased virulence observed in the Se-deficient mice is due to mutations in the influenza virus
genome, resulting in a more virulent genotype. Most of the mutations occurred in the gene for
the M1 matrix protein, an internal protein that is thought to be relatively stable. A total of 29
nucleotide changes were observed in this gene, and all 29 changes were identical in three
separate isolates taken from three different Se-deficient mice. In contrast, only one to three
mutations were seen in the genes for the hemagglutinin or neuraminidase proteins, surface
antigens that are known to be highly variable. Once the mutations have occurred, even hosts with
normal nutritional status are susceptible to the newly virulent strain. This work, in conjunction
with our earlier work with coxsackievirus, shows that specific nutritional deficiencies can have a
profound impact on the genome of RNA viruses. Poor nutritional status in the host may
contribute to the emergence of new viral strains.
Key words: selenium
•
influenza virus
•
oxidative stress
•
mutations
•
quasispecies
n a global basis, infections with influenza virus cause hundreds of thousands of deaths
and millions of illnesses each year (1). In the United States alone, influenza kills more
than 20,000 persons and hospitalizes more than 110,000 patients annually (2). Influenza
virus readily undergoes mutation and genomic reassortment, thus making it difficult for the host
immune system to recognize new strains. Previous work with coxsackieviruses demonstrated a
rapid mutation to or selection of virulent genomes from avirulent genomes in selenium (Se)-
deficient mice (3). Se is a nutritionally essential trace element required for the activity of a
number of selenoenzymes, including the antioxidant enzyme, glutathione peroxidase (4). Mice
deficient in Se developed myocarditis when infected with a normally benign strain of the
coxsackievirus. This increase in virulence was due to six point mutations that occurred in virus
O
This document is a U.S. government work and
is not subject to copyright in the United States.
replicating in Se-deficient mice. Once the mutations had occurred, even mice of normal Se status
were now susceptible to the increased virulence of the mutated virus.
The purpose of this study was to determine whether poor host Se status could similarly alter the
genome of an influenza virus by increasing its rate of mutation. Our previous demonstrated that
Se-deficient mice infected with influenza A/Bangkok/1/79 (H3N2) developed more severe lung
pathology than infected Se-adequate mice did (5). Here we report that this increase in virulence
was due to a change in the viral genotype.
MATERIALS AND METHODS
Mice
Three-week-old C57Bl/6J male mice (Jackson Laboratories, Bar Harbor, ME) were housed
4/cage in the University of North Carolina animal facility and provided with food and water
daily. Mice were fed specified diets for 4 wk before virus inoculation. All mice were
maintained under protocols approved by the Institutional Animal Review Board of the University
of North Carolina.
Diets
Diets were purchased from Harlan Teklad (Indianapolis, IN). Se was added to the adequate diets
as sodium selenite. The Se level of the mouse diets was determined by continuous flow hydride
generation atomic absorption spectrometry after acid digestion. The analysis was validated
against NIST 1549 nonfat milk powder (National Institute of Standards and Technology,
Gaithersburg, MD). The Se content of the experimental diets was determined to be 154 ± 8
µ
g
Se/kg for the Se-adequate diet and below the instrumental detection limit of 2.7
µ
g Se/kg for the
Se-deficient diet.
Virus
Influenza A/Bangkok/1/79 (H3N2) was propagated in 10-day-old embryonated hen’s egg. The
virus was collected in the allantoic fluid and titered by both hemagglutination (6) and 50% tissue
culture infectious dose on Madin-Darby canine kidney cells (7). Stock virus was aliquoted in
0.5-mL volumes and stored at –80
°
C until needed.
Infection of mice
Mice were lightly anesthetized with an intraperitoneal injection of ketamine (0.022 mg) and
xylazine (0.0156 mg). Following anesthesia, 10 hemagglutination units (HAUs) of influenza
A/Bangkok/1/79 in 0.05 mL of phosphate-buffered saline were instilled intranasally, and the
mice were allowed to recover from the anesthesia.
Histopathology of lungs
The right lung was removed, inflated with Optimal Cutting Temperature (OCT, Sigma, St. Louis,
MO), diluted in phosphate-buffered saline, embedded in OCT, and immediately frozen on dry
ice. Sections (6
µ
m) were cut on a cryostat and fixed and stained with hematoxylin-eosin. The
extent of inflammation was graded without knowledge of the experimental variables by two
independent investigators. Grading was performed semiquantitatively according to the relative
degree (from lung to lung) of inflammatory infiltration. The scoring was as follows: 0, no
inflammation; 1+, mild influx of inflammatory cells with cuffing around vessels; 2+, increased
inflammation with approximately 25–50% of the total lung involved; 3+, severe inflammation
involving 50–75% of the lung; and 4+, almost all lung tissue contains inflammatory infiltrates.
Determination of lung virus titers
One-quarter of the left lung (cut on the long-axis) was removed immediately after the mice were
killed and frozen in liquid nitrogen. The lung tissue was weighed and ground, using a Tenbroeck
tissue grinder, in a small volume of RPMI 1640 (Fisher Scientific, Pittsburgh, PA). Ground
tissues were then centrifuged at 2000
×
g for 15 min and the supernate recovered and titered by
hemagglutination (6).
Viral passage
Mice were divided into two groups at weaning and fed either a Se-adequate or a Se-deficient diet
(Harlan Teklad). Both groups of mice were infected with 10 HAUs of influenza A/Bangkok/1/79
(stock virus) intranasally following 4 weeks of consuming their respective diets. At day 5
postinfection, the mice were killed and one lobe of the lung was inflated and embedded in OCT.
The remaining lobes were snap frozen in liquid nitrogen. Five randomly chosen lung samples
from each diet group were weighed, ground in 150
µ
L of minimal essential media, and titered by
hemagglutination. Ten HAUs of each recovered virus were used to inoculate five individual Se-
adequate mice at 7 wk of age. Mice were killed at 6 days postinfection, and their lungs were
examined for histopathology.
Viral sequencing
RNA was isolated from infected Madin-Darby canine kidney cells by freeze/thaw, centrifugation
of the suspension, and addition of NaCl (final concentration 0.38 M) and 7% polyethylene
glycol. Following an overnight incubation at 4
°
C, the cell lysate was centrifuged and the pellet
resuspended in lysis buffer (0.5 M EDTA; 1.0 M Tris-HCl , pH 8.0; 5.0 M NaCl; 14.2 M
β
-
mercaptoethanol; 10% SDS; 10mg/ml proteinase K) and incubated at 50
°
C for 1 h. Viral RNA
was extracted from the samples with one phenol/chloroform extraction followed by one
chloroform extraction. Glycogen, 10 M ammonium acetate, and ethanol were added, and the
samples were kept at –80
°
C for 45 min. Samples were then centrifuged at 16,000
×
g for 25 min,
followed by a 75% ethanol wash of the pellet. The RNA pellet was air-dried for 15 min and
stored at –80
°
C. cDNA was made by using random hexameric primers, following the Promega
(Madison, WI) Reverse Transcription System manufacturer’s instructions (Catalog # A3500).
Primers were designed for amplification of the matrix (M), HA, and neuraminidase (NA) genes.
PCR was performed with the following primer sets: M (accession number: K01140): 5'-
GCACAGAGACTTGAAGATGT-3' and 3'-ATAGACTTTGGCACTCCTTC-5'; HA1
