Protection from Severe Influenza Virus Infections in Mice Carrying the Mx1 Influenza Virus Resistance Gene Strongly Depends on Genetic Background

Abstract

Influenza virus infections represent a serious threat to human health. Both extrinsic and intrinsic factors determine the severity of influenza. The MX dynamin-like GTPase 1 (Mx1) gene has been shown to confer strong resistance to influenza A virus infections in mice. Most laboratory mouse strains, including C57BL/6J, carry nonsense or deletion mutations in Mx1 and thus a nonfunctional allele, whereas wild-derived mouse strains carry a wild-type Mx1 allele. Congenic C57BL/6J (B6-Mx1(r/r)) mice expressing a wild-type allele from the A2G mouse strain are highly resistant to influenza A virus infections, to both mono- and polybasic subtypes. Furthermore, in genetic mapping studies, Mx1 was identified as the major locus of resistance to influenza virus infections. Here, we investigated whether the Mx1 protective function is influenced by the genetic background. For this, we generated a congenic mouse strain carrying the A2G wild-type Mx1 resistance allele on a DBA/2J background (D2-Mx1(r/r)). Most remarkably, congenic D2-Mx1(r/r) mice expressing a functional Mx1 wild-type allele are still highly susceptible to H1N1 virus. However, pretreatment of D2-Mx1(r/r) mice with alpha interferon protected them from lethal infections. Our results showed, for the first time, that the presence of an Mx1 wild-type allele from A2G as such does not fully protect mice from lethal influenza A virus infections. These observations are also highly relevant for susceptibility to influenza virus infections in humans.

Importance: Influenza A virus represents a major health threat to humans. Seasonal influenza epidemics cause high economic loss, morbidity, and deaths each year. Genetic factors of the host strongly influence susceptibility and resistance to virus infections. The Mx1 (MX dynamin-like GTPase 1) gene has been described as a major resistance gene in mice and humans. Most inbred laboratory mouse strains are deficient in Mx1, but congenic B6-Mx1(r/r) mice that carry the wild-type Mx1 gene from the A2G mouse strain are highly resistant. Here, we show that, very unexpectedly, congenic D2-Mx1(r/r) mice carrying the wild-type Mx1 gene from the A2G strain are not fully protected against lethal influenza virus infections. These observations demonstrate that the genetic background is very important for the protective function of the Mx1 resistance gene. Our results are also highly relevant for understanding genetic susceptibility to influenza virus infections in humans.

FIG 1

D2-Mx1^r/r mice were highly susceptible to H1N1 virus (PR8) infections whereas B6-Mx1^r/r mice were resistant. Eight- to 12-week-old female mice (D2-Mx1^r/r, B6-Mx1^r/r, D2-Mx1^−/−, and B6-Mx1^−/−) were infected intranasally with 2 × 10³ FFU of PR8 (H1N1) influenza A virus. Body weight loss (A) and survival rates (B) were monitored over a period of 14 days. Mice that lost 30% or more of the starting body weight were sacrificed and recorded as dead. Data represent mean percentages of body weight change (± SEM) compared to starting body weight (100%). Differences in body weight loss were significant between D2-Mx1^r/r and B6-Mx1^r/r mice after day 3 p.i. (P < 0.0001, nonparametric Mann-Whitney test). Survival rates were significantly different between D2-Mx1^r/r and B6-Mx1^r/r mice (P < 0.0001, log rank Mantel-Cox test). n, number of mice per group.

FIG 2

Chemokine and cytokine levels in BAL fluid of D2-Mx1^r/r mice exhibit stronger inflammatory responses than do those of B6-Mx1^−/− mice. Female D2-Mx1^r/r (circles) and B6-Mx1^r/r (squares) mice were infected with 2 × 10³ FFU of PR8F intranasally. Bronchoalveolar lavage (BAL) fluid was collected from mock-infected control mice at day 3 posttreatment and from infected mice at day 3 and day 5 p.i. The concentration of chemokines and cytokines was determined by using the mouse cytokine/chemokine magnetic bead panel (Mcytomag-70K) from Millipore. At each time point, five biological replicates were analyzed.

FIG 3

Confirmation of functional wild-type Mx1 in D2-Mx1^r/r mice by outcrossing to B6-Mx1^−/− mice. Congenic D2-Mx1^r/r mice were outcrossed to B6-Mx1^−/− mice, and the phenotypes of the resulting F1 mice [(B6 × D2(B6).A2G-Mx1^r/−)F1 or reciprocal crosses] were compared to the phenotype of F1 mice derived from an outcross of B6-Mx1^r/r to D2-Mx1^−/− [(D2 × B6.A2G-Mx1^r/−)F1 or reciprocal cross]. F1 mice from both crosses did not show significant differences in body weight loss (A) or survival (B) (Mann-Whitney U test for body weight change analysis and log rank Mantel-Cox test for survival curves). n, number of mice per group.

FIG 4

Upregulation of Ifnb1 and Mx1 in congenic D2-Mx1^r/r mice after influenza A virus infection. Eight- to 12-week-old female D2-Mx1^r/r and B6-Mx1^r/r mice were inoculated with 2 × 10³ FFU of PR8 H1N1 virus or PBS intranasally. On day 3 postinoculation, lung homogenates were prepared and levels of Ifnb1 (A) and Mx1 (B) mRNA expression were measured by quantitative RT-PCR and compared to PBS-treated controls. Infection with PR8 H1N1 virus induced comparable fold changes of Ifnb1 and Mx1 expression levels in both D2-Mx1^r/r and B6-Mx1^r/r mice (P = 0.5303 for Ifnb1 and P = 0.4346 for Mx1, two-tailed Student's t test, n = 3).

FIG 5

Resistance to lethal H1N1 virus infections is controlled by Mx1 copy number and genetic background. F1 mice of different Mx1 allele combinations and different C57BL/6J and DBA/2J background combinations were tested for susceptibility to PR8 H1N1 virus. Groups of 8- to 12-week-old female mice [F1(B6 × D2)-Mx1^r/r, F1(B6 × D2)-Mx1^r/−, B6-Mx1^r/−, and D2-Mx1^r/−] were infected intranasally with 2 × 10³ FFU of PR8 virus, and survival was monitored until day 14 p.i. Mice that lost 30% or more of the starting body weight were sacrificed and recorded as dead. For the F1(B6 × D2)-Mx1^r/− group, data from reciprocal crosses [(B6 × D2(B6).A2G-Mx1^r/−)F1 (n = 6) and (D2 × B6.A2G-Mx1^r/−)F1 (n = 10)] were combined. Two copies of the wild-type Mx1 locus increased resistance compared to that with one copy. Introduction of the DBA/2J background in hybrid F1(B6 × D2)-Mx1^r/r mice increased susceptibility, and the pure DBA/2J background in Mx1^r/r mice increased susceptibility further.

FIG 6

Restriction of virus replication is determined by the presence of a functional Mx1 allele and genetic background. Eight- to 12-week-old female B6-Mx1^r/r (A), D2-Mx1^r/r (B), B6-Mx1^−/− (C), D2-Mx1^−/− (D), and F1(B6 × D2)-Mx1^r/− (E) mice were infected intranasally with 2 × 10³ FFU of PR8 virus. Infectious virus particles in lung homogenates were determined by focus-forming assay at days 1, 3, and 5 p.i. Viral loads on day 1 p.i. were significantly different between infected mice that carried a DBA/2J genetic background and those that carried a C57BL/6J genetic background (B6-Mx1^r/r compared to D2-Mx1^r/r using Mann-Whitney test, P = 0.0022), and only mice carrying both a functional Mx1 allele and a C57BL/6J genetic background reduced viral loads from day 1 to day 3 p.i. D2-Mx1^r/r, B6-Mx1^r/r, and B6-Mx1^−/− mice, n = 6 per time point; D2-Mx1^−/− mice, n = 5 per time point; F1(B6 × D2)-Mx1^r/− mice, n = 3 per time point.

FIG 7

D2-Mx1^r/r mice were protected against infections with low-dose H1N1 (PR8) virus and partially protected against infections with H3N2 virus. Eight- to 12-week-old female mice [B6-Mx1^r/r, D2-Mx1^r/r, F1(B6 × D2)-Mx1^r/−, B6-Mx1^−/−, and D2-Mx1^−/−] were infected intranasally with 10 FFU of PR8 H1N1 virus (A) or with 2 × 10³ FFU of H3N2 virus (B). Survival rates were monitored over a period of 14 days p.i. Mice that lost 30% or more of the starting body weight were sacrificed and recorded as dead. Almost all infected D2-Mx1^r/r mice survived infection with low-dose PR8 virus. D2-Mx1^r/r mice were partially protected against H3N2 infections compared to B6-Mx1^−/− (log rank Mantel-Cox test, P = 0.0065). Also, all F1(B6 × D2)-Mx1^r/− mice with a hybrid C57BL/6J genetic background survived the H3N2 infections.

FIG 8

Upregulation of Mx1 in congenic D2-Mx1^r/r mouse strains after IFN-α treatment. D2-Mx1^r/r and B6-Mx1^r/r mice were treated with 50,000 IU of recombinant IFN-α intranasally. Levels of Mx1 mRNA expression were measured by quantitative RT-PCR and compared to those in PBS-treated controls. Treatment with IFN-α induces comparable fold increases of Mx1 expression in both D2-Mx1^r/r and B6-Mx1^r/r mice. No difference was detected between B6-Mx1^r/r and D2-Mx1^r/r mice (P = 0.4918, two-tailed Student's t test, n = 3).

FIG 9

Alpha interferon pretreatment rescues D2-Mx1^r/r mice. Eight- to 11-week-old female D2-Mx1^r/r mice were pretreated with 1 μg recombinant human alpha interferon B/D (IFN-α) intranasally 1 day prior to infection, and PBS was given as a mock control. All mice were subsequently infected intranasally with 2 × 10³ FFU of PR8 virus. (A) Survival rate was monitored for 14 days p.i. Mice that lost 30% or more of the starting body weight were sacrificed and recorded as dead. IFN-α-pretreated mice showed higher survival rates than did PBS-treated controls (log rank survival, P = 0.0019, n = 5 mice per group). (B) Virus particles in lung homogenates from IFN-α-pretreated and PBS-treated mice were determined in a focus-forming assay on day 1 p.i. Virus titers were significantly different between IFN-α- and PBS-pretreated groups (**, P = 0.01, Mann-Whitney U test). n = 5 mice per group.