Evaluation of pneumonia virus of mice as a possible human pathogen

Abstract

Pneumonia virus of mice (PVM), a relative of human respiratory syncytial virus (RSV), causes respiratory disease in mice. There is serologic evidence suggesting widespread exposure of humans to PVM. To investigate replication in primates, African green monkeys (AGM) and rhesus macaques (n = 4) were inoculated with PVM by the respiratory route. Virus was shed intermittently at low levels by a subset of animals, suggesting poor permissiveness. PVM efficiently replicated in cultured human cells and inhibited the type I interferon (IFN) response in these cells. This suggests that poor replication in nonhuman primates was not due to a general nonpermissiveness of primate cells or poor control of the IFN response. Seroprevalence in humans was examined by screening sera from 30 adults and 17 young children for PVM-neutralizing activity. Sera from a single child (6%) and 40% of adults had low neutralizing activity against PVM, which could be consistent with increasing incidence of exposure following early childhood. There was no cross-reaction of human or AGM sera between RSV and PVM and no cross-protection in the mouse model. In native Western blots, human sera reacted with RSV but not PVM proteins under conditions in which AGM immune sera reacted strongly. Serum reactivity was further evaluated by flow cytometry using unfixed Vero cells infected with PVM or RSV expressing green fluorescent protein (GFP) as a measure of viral gene expression. The reactivity of human sera against RSV-infected cells correlated with GFP expression, whereas reactivity against PVM-infected cells was low and uncorrelated with GFP expression. Thus, PVM specificity was not evident. Our results indicate that the PVM-neutralizing activity of human sera is not due to RSV- or PVM-specific antibodies but may be due to low-affinity, polyreactive natural antibodies of the IgG subclass. The absence of PVM-specific antibodies and restriction in nonhuman primates makes PVM unlikely to be a human pathogen.

Fig 1

Multistep growth kinetics of rPVM, rPVMΔNS1, rPVMΔNS2, and rPVMΔNS12 in A549 cells. Duplicate monolayer cultures of human A549 airway epithelial cells were infected at an MOI of 0.1 PFU/cell with the indicated virus. Supernatant aliquots were taken at 24-h intervals postinfection and replaced with an equivalent volume of fresh medium. Viral titers were determined by plaque assay in duplicate for each time point. Data are means with standard errors, although in many cases the error bars are obscured by the symbols because the errors were very small. The limit of detection is 0.7 log₁₀ PFU per ml (dashed line). Data are representative of two independent experiments.

Fig 2

Analysis of the type I IFN response in A549 cells infected with rPVM, rPVMΔNS1, rPVMΔNS2, or rPVMΔNS12 by qRT-PCR and ELISA. Triplicate cultures of A549 cells were mock infected, infected with sucrose gradient-purified rPVM, rPVMΔNS1, rPVMΔNS2, or rPVMΔNS12 at an MOI of 3 PFU/cell, or infected with sucrose gradient-purified UV-inactivated rPVMΔNS2. Cell cultures were harvested at the indicated time points, the medium supernatants were clarified and used for quantification of viral titers and secreted cytokines, and the cell pellets were processed to purify total cell-associated RNA. (A) PVM titers (log₁₀ PFU per ml) at 64 h postinfection measured by plaque assay of medium supernatants (9). (B) Levels of cell-associated PVM F and N RNA measured by qRT-PCR. (C) Levels of cell-associated IFN-β and IFN-α RNA measured by qRT-PCR (top) and levels of IFN-β and IFN-α protein in medium supernatants measured by ELISA (bottom). (D) Levels of cell-associated ISG56, IRF7, and CXCL10 RNA measured by qRT-PCR (top) and level of CXCL10 protein in medium supernatants measured by ELISA (bottom). All qRT-PCR results are expressed as difference (fold) relative to values in cells 24 h after inoculation with UV-inactivated rPVMΔNS2.

Fig 3

RSV- and PVM-neutralizing antibodies in human serum samples from 30 adults and 17 infants and young children. PRNT₆₀ were measured by plaque reduction neutralization assays against PVM and RSV. Sera from RSV- and PVM-infected AGM served as monospecific controls and also provided evaluation of possible cross-neutralization. Sera with a PRNT₆₀ of ≥5.3 log₂ (≥1:40) were considered seropositive (dashed line). Data points for PVM-seropositive adult donors are circled. Some donors are identified by numbered dots to allow comparison of titers of RSV- and PVM-neutralizing antibodies. Association between titers of RSV- and PVM-neutralizing antibodies in human sera among adults was calculated using a Wilcoxon signed rank test (***, P < 0.001). Data are representative of two (human sera) and three (AGM sera) independent experiments.

Fig 4

Evaluation of cross-protection between RSV and PVM in the mouse model. Six-week-old BALB/c mice in groups of 15 were immunized by intranasal inoculation (80-μl volume) under light methoxyflurane (Metofane) anesthesia on day 0 with L15 medium (as the mock-infected control) or with RSV (500,000 PFU), rPVM ΔNS2 (2,000 PFU), or rPVM-GFP (10 PFU). On day 4 (RSV and rPVMΔNS2 groups) or day 11 (rPVM-GFP group), 5 mice from each group were euthanized, and virus titers in lung homogenates (log₁₀ PFU per g of lung) were determined by plaque assay (top). On day 28, 5 animals from each group were challenged by intranasal administration of PVM (2,000 PFU) (middle), and the remaining 5 animals in each group were challenged by intranasal administration of RSV (500,000 PFU) (bottom). Lungs were harvested 4 days postchallenge, and virus titers were determined by plaque titration. Group means are indicated by solid lines. The limit of detection was 1.7 log₁₀ PFU per g of tissue (dashed line).

Fig 5

Western blot analysis of the 12 adult human sera with PVM-neutralizing activity. The presence of RSV- and PVM-specific antibodies in the 12 adult serum samples with significant PVM-neutralizing activity was investigated by Western blot analysis against sucrose-gradient-purified rRSV or rPVM. Other lanes contain control protein preparations and control antibodies to identify viral proteins. Protein samples were subjected to 4-to-12% SDS-PAGE gel electrophoresis under nonreducing and nondenaturing conditions and then transferred to a PVDF membrane. In the case of human test sera and AGM control sera, the serum dilutions used and the RSV or PVM log₂ PRNT₆₀ of the undiluted sera are indicated above each membrane strip. Note that the RSV- and PVM-seropositive AGM control sera were used at dilutions designed to have PRNT₆₀ similar to that of the human test sera. Data are from one of three independent experiments with similar results. (A) Reactivity with RSV proteins. The left and center panels (lanes a to g) show controls to identify viral protein species. Lanes a to c, rabbit polyclonal antibody raised against gradient-purified RSV (RSV-specific PAb) reacted with mock-infected cell lysate (a) and gradient-purified rRSV (b) and rPVM (c). Lane d, RSV F MAb 1129 (5) reacted with gradient-purified rRSV. Lanes e to g, RSV-specific AGM serum reacted with mock-infected cell lysate (e) and gradient-purified rPVM (f) and rRSV (g). The right panel shows results for the 12 human test sera reacted with blots prepared from gradient-purified rRSV. (B) Reactivity with PVM proteins. The left and center panels (lanes a to i) show controls to identify viral protein species. Lanes a to c, rabbit polyclonal antibody raised gradient purified PVM (PVM-specific PAb) reacted with lysates of cells that had been mock-transfected (a) or transfected with plasmid expressing the PVM N (b) or P protein (c). Lanes d and e, same PVM-specific PAb reacted with gradient-purified rRSV (d) or rPVM (e). Lane f, rabbit polyclonal antiserum raised against a recombinant vaccinia virus expressing the PVM F protein (PVM F-specific PAb) reacted with gradient-purified rPVM. Lanes g to i, PVM-specific AGM serum reacted with mock-infected cell lysate (g) and gradient-purified rPVM (h) and rRSV (i). The right panel shows results for the 12 human test sera reacted with gradient-purified rPVM.

Fig 6

Evaluation of the binding activity of selected human sera to RSV- and PVM-infected cells by flow cytometry. Vero cells were infected at an MOI of 1 with either RSV or PVM, each expressing GFP. Twenty-four hours postinfection, cells were detached with 1.0 mM EDTA. Infected cells were stained with selected human test sera or control antibodies, followed by secondary antibodies conjugated to Alexa 647. The cells were then fixed and analyzed by flow cytometry to compare the level of antibody binding (y axis) to GFP expression as a measure of viral gene expression (x axis). For each human or AGM serum, the reciprocal log₂ PRNT₆₀ for RSV and PVM are noted. Results are representative of three independent experiments. Antibody isotype controls are not shown. (A) Antibody controls. reactivity of RSV F-specific MAb 1169 (left) and PVM G-specific polyclonal antibody (right) with RSV-GFP- and PVM-GFP-infected cells (top and bottom, respectively). (B and C) Analysis of AGM (left) and human (right) sera for IgG (B) or IgM (C) antibodies that react with RSV-GFP- and PVM-GFP-infected cells. One example is shown for each of 6 categories of sera (columns from left to right): (i) preimmune AGM, (ii) RSV⁺ PVM⁻ AGM, (iii) RSV⁻ PVM⁺ AGM, (iv) nonimmune human (from an infant), (v) RSV⁺ PVM^lo adult human, and (vi) RSV⁻ PVM⁺ adult human. A total of 4 donors per category were tested, with similar results; the data shown are for one representative individual.

Fig 7

Evaluation of RSV- and PVM-neutralizing activity in human serum samples after partial depletion of IgG antibodies. Human sera previously identified as having PVM-neutralizing activity were passed through protein G Sepharose columns to partially remove IgG antibodies. Sera from RSV- and PVM-infected AGM served as monospecific controls. Some donors are identified by numbers, located below the lanes (A) and above the dots (B), to allow comparison between titers of neutralizing antibodies and level of IgG depletion. (B) PRNT₆₀ for PVM and RSV were measured before and after IgG depletion. Sera with a PRNT₆₀ of ≥5.3 log₂ (>1:40) were considered seropositive (dashed line). Data are from a single titration experiment; a second experiment provided similar results. (A) Analysis of IgG content by 4-to-12% SDS-PAGE gel electrophoresis under nonreducing and nondenaturing conditions, with commercially obtained purified human IgG as a marker. Gels were stained with Coomassie blue. The reductions in IgG band intensity for the numbered samples were as follows: 1, 80%; 2, 88%; 3, 76%; 4, 45%; 5, 41%; and 6, 79%. ****, P < 0.0001.