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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 8, 2001 as doi:10.1096/fj.01-0115fje. |
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2
Departments of
* Nutrition and
Pediatrics2, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7220, USA;
Nestlé Research Center, Lausanne, Switzerland; and
USDA, ARS, Beltsville Human Nutrition Research Center, Beltsville, Maryland
2Correspondence: Department of Pediatrics, 535 Burnett-Womack, CB #7220, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7220, USA. E-mail: melinda_beck{at}unc.edu
SPECIFIC AIM
To determine whether host selenium (Se) deficiency can induce changes in the genome of a replicating influenza virus such that a normally mild virus converts into a more virulent strain and to characterize such genomic changes.
PRINCIPAL FINDINGS
1. Replication of a mild strain of influenza virus in Se-deficient
mice results in a novel virulent strain that causes severe lung
pathology even when passed into Se-adequate mice
Se-deficient mice developed much more severe lung pathology
postinfection with influenza virus than Se-adequate infected mice. To
determine whether host factors or viral factors were responsible for
the increased pathogenicity of influenza virus that had replicated in
Se-deficient mice, a passage experiment was performed. We infected
groups of Se-adequate and Se-deficient mice with influenza
A/Bangkok/1/79 (H3N2). At 5 days postinfection, the mice were killed
and virus was recovered from the lungs. Five separate isolates from
each group of mice were used to inoculate five individual Se-adequate
mice. Se-adequate mice infected with a virus recovered from
Se-deficient mice developed severe lung pathology (mean
score±SD: 3.8±0.4). This was in stark contrast to
Se-adequate mice infected with a virus recovered from Se-adequate mice:
only a mild lung pathology developed (mean score±SD:
1.5±0.4), indistinguishable from that seen in the lungs of Se-adequate
mice infected with the original stock virus (Fig. 1
). These results strongly suggested that the influenza virus had
undergone a genetic change when replicating in a Se-deficient host.
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2. Compared with the original stock virus, few mutations were found
in the hemagglutinin (HA) or neuraminidase (NA) genes of viruses
recovered from either Se-deficient or Se-adequate influenza-infected
mice
Influenza viruses contain eight different single-stranded RNA
segments that code for viral proteins. To determine whether any
mutations had occurred in the virus that had replicated in Se-deficient
mice, we selected three viruses isolated from Se-adequate mice and
three viruses isolated from Se-deficient mice for sequencing. Because
HA and NA are surface proteins associated with virulence and are
responsible for antigenic changes of the virus, it was expected that
any mutations observed would be found in these genes. However,
sequencing of the HA gene revealed only one or two randomly distributed
nucleotide (nt) changes in viruses isolated from Se-adequate mice and
only two or three random nt changes in viruses isolated from
Se-deficient mice as compared to our stock virus. Two of the mutations
were silent and the remaining nt changes resulted in amino acid
substitutions. At all other sequenced sites of the HA gene, the
isolates were identical to the stock virus and to one another. One
isolate from a Se-deficient animal had no nt changes.
Sequencing of the NA gene revealed one nt change in each of two isolates from Se-adequate mice. Both changes led to amino acid substitutions. The third isolate from a Se-adequate mouse had no changes when compared with the stock virus. One isolate from a Se-deficient mouse contained 2 nt changes from the stock virus, each of which resulted in an amino acid substitution. The other isolate from a Se-deficient mouse did not differ from the stock virus.
3. Compared with the original stock virus, multiple mutations were
found in the M1 matrix protein gene of viruses recovered from
Se-deficient influenza-infected mice
Segment 7 of the influenza virus genome codes for two matrix
proteins, M1 and M2, which are associated with viral virulence. M1 is
strictly an internal protein
;0;1;2;ruletorule;\-30;;;cell;cell>whereas M2 forms an ion
channel that is exposed on the virion surface. In contrast to the HA
and NA genes, multiple mutations were found in the M1 matrix protein
gene of viral isolates recovered from Se-deficient mice vs. those
recovered from Se-adequate mice. As shown in Table 1
, the sequence of the M1 protein gene determined from the virus that had
replicated in Se-deficient mice had 29 nt changes compared with the
stock virus. Seven of these nt changes resulted in amino acid changes.
All 29 nt changes were identical in all three isolates from
Se-deficient mice. One isolate from a Se-deficient mouse had five
additional nt changes in addition to the 29 nt differences shared among
all three isolates (Table 1)
. The sequence of the M1 protein gene
of virus isolated from Se-adequate mice had no nt changes whatsoever
compared with the stock virus.
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For the M2 protein gene, no changes were found in viruses recovered from Se-deficient mice. One isolate from a Se-adequate mouse had one nt change (nt 785), and this nucleotide change led to an amino acid change. The other two isolates were identical to the stock virus.
CONCLUSIONS AND SIGNIFICANCE
Influenza A viruses are highly contagious and are responsible for considerable morbidity and mortality worldwide. New strains of influenza arise each year not only because of the propensity for its RNA segments to reassort, but also because of the high mutation rate of the genes that encode its surface antigens. Selenium is a component of the peroxide-destroying enzyme glutathione peroxidase, and a dietary deficiency in Se leads to increased oxidative stress in the host due to a loss of this antioxidant protection. Because a host Se deficiency had been shown earlier to increase the mutation rate of a Picornavirus, coxsackievirus B3, we theorized that a decrease in host Se status might do the same for the influenza virus. If the oxidative stress status of the host altered the genome of a virus outside the Picornavirus family, this would suggest that RNA viruses in general may be susceptible to oxidative damage. This could provide a novel mechanism for the emergence of viral diseases.
The fact that the nucleotide changes were found predominantly in the M1 gene rather than the HA or the NA genes was unexpected. The HA and the NA proteins are exposed on the surface of the virion and are associated with antigenic changes of the virus. It was expected that any mutations found would be in the HA and/or the NA genes. The gene for the M1 protein generally is stable among influenza A viruses. This stability is thought to be due to M1 being an internal protein and thus not subject to immune pressure from antibodies. The fact that the HA and NA genes were relatively stable in our study whereas the M1 gene had a large number of changes makes our findings even more intriguing.
Our experiments did not involve evolution of the influenza virus in
multiple host mice over time, which is influenced by the immune
pressure exerted by the host. Instead, our results reflect changes in
the virus that occurred during its replication cycles in a single
mouse. Thus, as diagrammed in Fig. 2
, it seems likely that the heightened oxidative stress in the
Se-deficient host during the viral replication cycle contributed to the
increased mutation rate of the matrix gene. Once these changes in the
genome occurred, then even mice with normal Se status were susceptible
to the increased virulence of the mutated virus.
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There are several possible explanations for the change in the viral genome isolated from Se-deficient mice. One is that direct oxidative damage to the viral genome may have occurred due to the increased oxidative stress present in the Se-deficient mice. A second mechanism for the viral genetic change is one of selection. The RNA-dependent polymerase of RNA viruses is highly error prone and lacking in proofreading capabilities. Therefore, within an individual host, an RNA virus exists as a heterogeneous mixture of closely related viruses called quasispecies. Within this mixture is the dominant, or consensus, sequence. The mutations found in the Se-deficient animals may have been generated by a selection process in which a new consensus sequence became dominant. The selection of the new consensus sequence may have been facilitated by the increased oxidative stress status of the host due to a deficiency in Se.
Our work points to the considerable influence that host nutritional status may exert on a viral pathogen. Together with the coxsackievirus model, our data on the influenza virus suggest that many RNA viruses may be susceptible to nutritionally induced oxidative damage. We propose that the nutritional status of the host should be considered when exploring mutations and mutation rates of viruses. Finally, our results demonstrate a unique mechanism by which viruses can mutate and point to the importance of antioxidant protection against viral disease.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0115fje ; to cite
this article, use FASEB J. (June 8, 2001) 10.1096/fj.01-0115fje 
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