Synergism and Antagonism of Bacterial-Viral Coinfection in the Upper Respiratory Tract.

Streptococcus pneumoniae (the pneumococcus) is a leading cause of pneumonia in children under 5 years of age. Coinfection by pneumococci and respiratory viruses enhances disease severity. Little is known about pneumococcal coinfections with respiratory syncytial virus (RSV). Here, we developed a novel infant mouse model of coinfection using pneumonia virus of mice (PVM), a murine analogue of RSV, to examine the dynamics of coinfection in the upper respiratory tract, an anatomical niche that is essential for host-to-host transmission and progression to disease. Coinfection increased damage to the nasal tissue and increased production of the chemokine CCL3. Nasopharyngeal pneumococcal density and shedding in nasal secretions were increased by coinfection. In contrast, coinfection reduced PVM loads in the nasopharynx, an effect that was independent of pneumococcal strain and the order of infection. We showed that this "antagonistic" effect was absent using either ethanol-killed pneumococci or a pneumococcal mutant deficient in capsule production and incapable of nasopharyngeal carriage. Colonization with a pneumococcal strain naturally unable to produce capsule also reduced viral loads. The pneumococcus-mediated reduction in PVM loads was caused by accelerated viral clearance from the nasopharynx. Although these synergistic and antagonistic effects occurred with both wild-type pneumococcal strains used in this study, the magnitude of the effects was strain dependent. Lastly, we showed that pneumococci can also antagonize influenza virus. Taken together, our study has uncovered multiple novel facets of bacterial-viral coinfection. Our findings have important public health implications, including for bacterial and viral vaccination strategies in young children. IMPORTANCE Respiratory bacterial-viral coinfections (such as pneumococci and influenza virus) are often synergistic, resulting in enhanced disease severity. Although colonization of the nasopharynx is the precursor to disease and transmission, little is known about bacterial-viral interactions that occur within this niche. In this study, we developed a novel mouse model to examine pneumococcal-viral interactions in the nasopharynx with pneumonia virus of mice (PVM) and influenza. We found that PVM infection benefits pneumococci by increasing their numbers in the nasopharynx and shedding of these bacteria in respiratory secretions. In contrast, we discovered that pneumococci decrease PVM numbers by accelerating viral clearance. We also report a similar effect of pneumococci on influenza. By showing that coinfections lead to both synergistic and antagonistic outcomes, our findings challenge the existing dogma in the field. Our work has important applications and implications for bacterial and viral vaccines that target these microbes.

Murine Pneumonia Virus Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus from an Added Gene Is Highly Attenuated and Immunogenic in Rhesus Macaques.

Human respiratory syncytial virus (RSV) continues to be the leading viral cause of severe acute lower respiratory tract disease in infants and children worldwide. A licensed vaccine or antiviral drug suitable for routine use remains unavailable. Like RSV, Murine pneumonia virus (MPV) is a member of the genus Orthopneumovirus, family Pneumoviridae Humans are not normally exposed to MPV, and MPV is not cross-protective with RSV. We evaluated MPV as an RSV vaccine vector expressing the RSV fusion (F) glycoprotein. The RSV F open reading frame (ORF) was codon optimized, and the encoded RSV F protein was made identical to an early passage of RSV strain A2. The RSV F ORF was placed under the control of MPV transcription signals and inserted at the first (rMPV-F1), third (rMPV-F3), or fourth (rMPV-F4) gene position of a version of the MPV genome that contained a codon-pair-optimized polymerase (L) gene. The recovered viruses replicated in vitro as efficiently as the empty vector, with stable expression of RSV F protein. Replication and immunogenicity of rMPV-F1 and rMPV-F3 were evaluated in rhesus macaques following intranasal and intratracheal administration. Both viruses replicated at low levels in the upper and lower respiratory tracts, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high levels similar to those induced by wild-type RSV replicating to a 5- to 25-fold-higher titer. In conclusion, this study demonstrated that rMPV provides a highly attenuated yet immunogenic vector for the expression of RSV F protein, with potential application in RSV-naive and RSV-experienced populations.IMPORTANCE Human respiratory syncytial virus (RSV) is an important human pathogen that lacks a licensed vaccine or antiviral drug suitable for routine use. We describe here the evaluation of recombinant murine pneumonia virus (rMPV) as a live-attenuated vector that expresses the RSV F protein, the major RSV neutralization antigen, as an experimental RSV vaccine. The rMPV-RSV-F vectors expressing RSV F from the first, third, or fourth gene position were genetically stable and were not restricted for replication in vitro In contrast, the vectors exhibited highly attenuated replication in the respiratory tract of rhesus macaques, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high titers similar to those conferred by wild-type RSV. Given the lack of preexisting immunity to MPV in humans and the lack of cross-neutralization and cross-protection between MPV and RSV, an rMPV-vectored RSV vaccine should be immunogenic in both RSV-naive children and RSV-experienced adults.

Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

Intranasal immunisation with recombinant adenovirus vaccines protects against a lethal challenge with pneumonia virus of mice.

Pneumonia virus of mice (PVM) infection of BALB/c mice induces bronchiolitis leading to a fatal pneumonia in a dose-dependent manner, closely paralleling the development of severe disease during human respiratory syncytial virus infection in man, and is thus a recognised model in which to study the pathogenesis of pneumoviruses. This model system was used to investigate delivery of the internal structural proteins of PVM as a potential vaccination strategy to protect against pneumovirus disease. Replication-deficient recombinant human adenovirus serotype 5 (rAd5) vectors were constructed that expressed the M or N gene of PVM pathogenic strain J3666. Intranasal delivery of these rAd5 vectors gave protection against a lethal challenge dose of PVM in three different mouse strains, and protection lasted for at least 20 weeks post-immunisation. Whilst the PVM-specific antibody response in such animals was weak and inconsistent, rAd5N primed a strong PVM-specific CD8(+) T cell response and, to a lesser extent, a CD4(+) T cell response. These findings suggest that T-cell responses may be more important than serum IgG in the observed protection induced by rAd5N.

Sustained inflammation and differential expression of interferons type I and III in PVM-infected interferon-gamma (IFNγ) gene-deleted mice.

Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is detected in the airways in response to infection with the pneumovirus pathogen, pneumonia virus of mice (PVM; Family Paramyxoviridae), its role in promoting disease has not been fully explored. Here, we evaluate PVM infection in IFNγ(-/-) mice. Although the IFNγ gene-deletion has no impact on weight loss, survival or virus kinetics, expression of IFNß, IFNλ2/3 and IFN-stimulated 2-5' oligoadenylate synthetases was significantly diminished compared to wild-type counterparts. Furthermore, PVM infection in IFNγ(-/-) mice promoted prominent inflammation, including eosinophil and neutrophil infiltration into the airways and lung parenchyma, observed several days after peak virus titer. Potential mechanisms include over-production of chemoattractant and eosinophil-active cytokines (CXCL1, CCL11, CCL3 and IL5) in PVM-infected IFNγ(-/-) mice; likewise, IFNγ actively antagonized IL5-dependent eosinophil survival ex vivo. Our results may have clinical implications for pneumovirus infection in individuals with IFNγ signaling defects.

Detection of a pneumonia virus of mice (PVM) in an African hedgehog (Atelerix arbiventris) with suspected wobbly hedgehog syndrome (WHS).

A pneumonia virus of mice (PVM) from an African hedgehog (Atelerix arbiventris) with suspected wobbly hedgehog syndrome (WHS) was detected and genetically characterized. The affected hedgehog had a nonsuppurative encephalitis with vacuolization of the white matter, and the brain samples yielded RNA reads highly homogeneous to PVM strain 15 (96.5% of full genomic sequence homology by analysis of next generation sequencing). PVM antigen was also detected in the brain and the lungs immunohistochemically. A PVM was strongly suggested as a causative agent of encephalitis of a hedgehog with suspected WHS. This is a first report of PVM infection in hedgehogs.

CD8+ T-cell epitope mapping for pneumonia virus of mice in H-2b mice.

The paramyxovirus pneumonia virus of mice (PVM) is a rodent model of human respiratory syncytial virus (hRSV) pathogenesis. Here we characterized the PVM-specific CD8(+) T-cell repertoire in susceptible C57BL/6 mice. In total, 15 PVM-specific CD8(+) T-cell epitopes restricted by H-2D(b) and/or H-2K(b) were identified. These data open the door for using widely profiled, genetically manipulated C57BL/6 mice to study the contribution of epitope-specific CD8(+) T cells to PVM pathogenesis.

Novel pneumoviruses (PnVs): Evolution and inflammatory pathology.

A previous report of a novel pneumovirus (PnV) isolated from the respiratory tract of a dog described its significant homology to the rodent pathogen, pneumonia virus of mice (PVM). The original PnV-Ane4 pathogen replicated in and could be re-isolated in infectious state from mouse lung but elicited minimal mortality compared to PVM strain J3666. Here we assess phylogeny and physiologic responses to 10 new PnV isolates. The G/glycoprotein sequences of all PnVs include elongated amino-termini when compared to the characterized PVMs, and suggest division into groups A and B. While we observed significant differences in cytokine production and neutrophil recruitment to the lungs of BALB/c mice in response to survival doses (50 TCID50 units) of representative group A (114378-10-29-KY-F) and group B (7968-11-OK) PnVs, we observed no evidence for positive selection (dN > dS) among the PnV/PnV, PVM/PnV or PVM/PVM G/glycoprotein or F/fusion protein sequence pairs.

Lactobacillus priming of the respiratory tract: Heterologous immunity and protection against lethal pneumovirus infection.

We showed previously that wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus species were fully (100%) protected against the lethal sequelae of infection with the virulent pathogen, pneumonia virus of mice (PVM), a response that is associated with diminished expression of proinflammatory cytokines and diminished virus recovery. We show here that 40% of the mice primed with live Lactobacillus survived when PVM challenge was delayed for 5months. This robust and sustained resistance to PVM infection resulting from prior interaction with an otherwise unrelated microbe is a profound example of heterologous immunity. We undertook the present study in order to understand the nature and unique features of this response. We found that intranasal inoculation with L. reuteri elicited rapid, transient neutrophil recruitment in association with proinflammatory mediators (CXCL1, CCL3, CCL2, CXCL10, TNF-alpha and IL-17A) but not Th1 cytokines. IFNγ does not contribute to survival promoted by Lactobacillus-priming. Live L. reuteri detected in lung tissue underwent rapid clearance, and was undetectable at 24h after inoculation. In contrast, L. reuteri peptidoglycan (PGN) and L. reuteri genomic DNA (gDNA) were detected at 24 and 48h after inoculation, respectively. In contrast to live bacteria, intranasal inoculation with isolated L. reuteri gDNA elicited no neutrophil recruitment, had minimal impact on virus recovery and virus-associated production of CCL3, and provided no protection against the negative sequelae of virus infection. Isolated PGN elicited neutrophil recruitment and proinflammatory cytokines but did not promote sustained survival in response to subsequent PVM infection. Overall, further evaluation of the responses leading to Lactobacillus-mediated heterologous immunity may provide insight into novel antiviral preventive modalities.

Eosinophils and their interactions with respiratory virus pathogens.

Eosinophils are implicated in the pathophysiology of respiratory virus infection, most typically in negative roles, such as promoting wheezing and bronchoconstriction in conjunction with virus-induced exacerbations of reactive airways disease and in association with aberrant hypersensitivity responses to viral vaccines. However, experiments carried out in vitro and in vivo suggest positive roles for eosinophils, as they have been shown to reduce virus infectivity in tissue culture and promote clearance of the human pathogen, respiratory syncytial virus in a mouse challenge model. The related natural rodent pathogen, pneumonia virus of mice (PVM), is highly virulent in mice, and is not readily cleared by eosinophils in vivo. Interestingly, PVM replicates in eosinophils and promotes cytokine release. The molecular basis of virus infection in eosinophils and its relationship to disease outcome is currently under study.

Deletion of nonstructural proteins NS1 and NS2 from pneumonia virus of mice attenuates viral replication and reduces pulmonary cytokine expression and disease.

Pneumonia virus of mice (PVM) strain 15 causes fatal pneumonia in mice and provides a convenient model for human respiratory syncytial virus pathogenesis and immunobiology. We prepared PVM mutants lacking the genes for nonstructural proteins NS1 and/or NS2. In Vero cells, which lack type I interferon (IFN), deletion of these proteins had no effect on the efficiency of virus growth. In IFN-competent mouse embryo fibroblasts, wild-type (wt) PVM and the DeltaNS1 virus grew efficiently and strongly inhibited the IFN response, whereas virus lacking NS2 was highly attenuated and induced high levels of IFN and IFN-inducible genes. In BALB/c mice, intranasal infection with wt PVM caused overt disease that began on day 6 and was lethal by day 9 postinoculation. In comparison, DeltaNS1 induced transient, reduced disease, and DeltaNS2 and DeltaNS12 caused no disease. Thus, NS1 and NS2 are virulence factors, with NS2 being a major antagonist of the type I IFN system. The pulmonary titers of wt PVM and DeltaNS1 were high on day 3 and increased further by day 6; in addition, expression of IFN and representative proinflammatory cytokines/chemokines and T lymphocyte-related cytokines was undetectable on day 3 but increased dramatically by day 6 coincident with the onset of disease. The titers of DeltaNS2 and DeltaNS12 were somewhat lower on day 3 and decreased further by day 6; in addition, these viruses induced a more circumscribed set of cytokines/chemokines (IFN, interleukin-6 [IL-6], and CXCL10) that were detected on day 3 and had largely subsided by day 6. Lung immunohistology revealed abundant PVM-positive pneumocytes and bronchial and bronchiolar epithelial cells in wt PVM- and DeltaNS1-infected mice on day 6 compared to few PVM-positive foci with DeltaNS2 and DeltaNS12. These results indicate that severe PVM disease is associated with high, poorly controlled virus replication driving the expression of high levels of pulmonary IFN and a broad array of cytokines/chemokines. In contrast, in the absence of NS2, there was an early, transient innate response involving moderate levels of IFN, IL-6, and CXCL10 that restricted virus replication and prevented disease.

Mutational analysis of the gene start sequences of pneumonia virus of mice.

The transcriptional start sequence of pneumonia virus of mice is more variable than that of the other pneumoviruses, with five different nine-base gene start (GS) sequences found in the PVM genome. The sequence requirements of the PVM gene start signal, and the efficiency of transcriptional initiation of the different virus genes, was investigated using a reverse genetics approach with a minigenome construct containing two reporter genes. A series of GS mutants were created, where each of the nine bases of the gene start consensus sequence of a reporter gene was changed to every other possible base, and the resulting effect on initiation of transcription was assayed. Nucleotide positions 1, 2 and 7 were found to be most sensitive to mutation whilst positions 4, 5 and 9 were relatively insensitive. The L gene GS sequence was found to have only 20% of the activity of the consensus sequence whilst the published M2 gene start sequence was found to be non-functional. A minigenome construct in which the two reporter genes were separated by the F-M2 gene junction of PVM was used to confirm the presence of two alternative, functional, GS sequences that could both drive the transcription of the PVM M2 gene.

Identification of a novel virulence factor in recombinant pneumonia virus of mice.

Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (Delta G) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the Delta G mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

Coupled translation of the second open reading frame of M2 mRNA is sequence dependent and differs significantly within the subfamily Pneumovirinae.

Coupled translation, first described in the M2 gene of pneumovirus respiratory syncytial virus (RSV), is an alternative mechanism of translational initiation in which the ribosomes which translate the first (M2-1) open reading frame (ORF) move a short distance upstream after termination and reinitiate translation from a second (M2-2) overlapping ORF. Here, we show that the same mechanism occurs in two closely related viruses, avian pneumovirus (APV) and pneumonia virus of mice (PVM), although with markedly different efficiencies. To identify the reasons for the variation in efficiency of coupled expression between RSV and APV, we used chimeric M2-1 genes containing different lengths of the M2-1 ORF from each virus. An essential component allowing coupled expression in the chimeras was a segment from the RSV M2-1 coding region containing a high degree of secondary structure. Additional sequences at the 5' end of the RSV M2-1 ORF also promoted coupled translation when the region with high levels of secondary structure was present. These data indicate that at least two distant parts of the mRNA transcript, together with a suitable overlapping region, are involved in the coupling process. Replacement of the last 102 nucleotides of the RSV M2-1 ORF with the equivalent APV sequence showed identical levels of coupled translation. Thus, the overlapping region can direct the ribosome back onto the start codon of the second ORF while the upstream coding sequence of the M2-1 ORF determines the levels of coupled expression.

Complete sequence of the RNA genome of pneumonia virus of mice (PVM).

Pneumonia virus of mice (PVM) is an enveloped RNA-containing virus of Family Paramyxoviridae. Sequences had been determined previously for a number of PVM genes, although these represented cloned cDNAs rather than consensus sequences. Sequences were not available for the 3' -leader and 5' -trailer regions that constitute the genome termini or for the large polymerase L gene that accounts for 43% of the genome. Also, the available sequences were from an attenuated variant of strain 15, whereas the present study analyzed the version of strain 15 that is available from the American Type Culture Collection (ATCC) and is highly virulent in mice. Analysis of unclosed RT-PCR products yielded a complete consensus sequence of 14,886 nt (GenBank accession number AY729016). Of the regions for which sequences had been previously reported for the non-pathogenic strain, there were 13 nucleotide differences and 10 amino acid differences compared to the present consensus sequence for the virulent isolate. The various genes of PVM shared 29-62% nucleotide sequence identity and 10-60% amino acid sequence identity with human or bovine respiratory syncytial virus (HRSV and BRSV), its closest relatives.

Genome sequence of the non-pathogenic strain 15 of pneumonia virus of mice and comparison with the genome of the pathogenic strain J3666.

Pneumonia virus of mice (PVM) is a member of the subfamily Pneumovirinae and is the closest known relative of respiratory syncytial virus. Both viruses cause pneumonia in their respective hosts. Here, the genome sequences of two strains of PVM, non-pathogenic strain 15 and pathogenic strain J3666, are reported. Comparison of the genome sequences revealed 59 nucleotide differences between the two strains, 37 of which were coding. The nucleotide differences were spread throughout the genome, affecting cis-acting regulatory regions and seven of the ten genes. Development of a reverse-genetics system for PVM should allow further elucidation of the functional importance of the genetic differences between the two strains identified here.

Detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis.

Sendai virus may induce acute respiratory tract disease in laboratory mice and is a common contaminant of biological materials. Pneumonia virus of mice (PVM) also infects the respiratory tract and, like Sendai virus, may induce a persistent wasting disease syndrome in immunodeficient mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proven useful for detection of Sendai virus and PVM immunodeficient animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, fnRT-PCR assays specific for Sendai virus and PVM were developed by targeting primer andprobe sequences to unique regions of the Sendai virus nucleocapsid (NP) gene and the PVM attachment (G) gene, respectively. The Sendai virus and PVM fnRT-PCR assays detected only Sendai virusand PVM , respectively. Neither assay detected other viruses of the family Paramyxoviridae or other RNA viruses that naturally infect rodents. The fnRT-PCR assays detected as little as 10 fg of Sendai virus RNA and one picogram of PVM RNA, respectively, andthe Sendai virus fnRT-PCR assay had comparable sensitivity when directly compared with the mouse antibody production test. The fnRT-PCR assays were also able to detect viral RNA in respiratory tract tissues and cage swipe specimens collected from experimentally inoculated C.B-17 severe combined immunodeficient mice, but did not detect viral RNA in age- and strain-matched mock-infected mice. In conclusion, these fnRT-PCR assays offer potentially high-throughput diagnostic assays to detect Sendai virus and PVM in immunodeficient mice, and to detect Sendai virus in contaminated biological materials.

Identification of amino acids that are critical to the processivity function of respiratory syncytial virus M2-1 protein.

The M2-1 protein of respiratory syncytial virus (RSV) is a transcription processivity factor that is essential for virus replication. The function of RSV M2-1 protein can be examined by using an RSVlacZ minigenome assay in vitro since the expression of the lacZ gene is dependent on M2-1. The M2-1 protein of pneumonia virus of mice (PVM), also a member of the Pneumovirus genus, functions poorly in the RSVlacZ minigenome assay despite conservation of the Cys(3)-His(1) motif at its N terminus and an overall 40% amino acid identity with RSV M2-1. To identify the amino acids responsible for the differences between these two proteins, two chimeric proteins were constructed. The RSV/PVM (RP) M2-1 chimera that contains the N-terminal 30 amino acids from RSV and the remaining C-terminal 148 amino acids from PVM maintained a level of activity at an ca. 36% of RSV M2-1. However, the PVM/RSV (PR) M2-1 chimera with the N-terminal 29 amino acids from PVM and 164 amino acids from RSV had an activity of <5% of RSV M2-1, indicating that the functional determinants are mainly located in the N terminus of M2-1. Mutagenesis of the N terminus of PR M2-1 and RSV M2-1 identified that Leu-16 and Asn-17 of RSV M2-1 are critical to the M2-1 function. In addition, several charged residues in the N terminus of RSV M2-1 also contributed to the functional integrity of M2-1.