Veronika von Messling

A ferret model of canine distemper virus virulence and immunosuppression.

ABSTRACT Canine distemper virus (CDV) infects many carnivores, including ferrets and dogs, and is the member of the Morbillivirus genus most easily amenable to experimentation in a homologous small-animal system. To gain insights into the determinants of CDV pathogenesis, we isolated a strain highly virulent for ferrets by repeated passaging in these animals. Sequence comparison of the genome of this strain with that of its highly attenuated precursor revealed 19 mutations distributed almost evenly in the six genes. We then recovered a virus from a cDNA copy of the virulent CDV strains consensus sequence by using a modified reverse genetics system based on B cells. We infected ferrets with this virus and showed that it fully retained virulence as measured by the timing of rash appearance, disease onset, and death. Body temperature, leukocyte number, lymphocyte proliferation activity, and cell-associated viremia also had similar kinetics. We then addressed the question of the relative importance of the envelope and other viral constituents for virulence. Viruses in which the envelope genes (matrix, fusion, and hemagglutinin) of the virulent strain were combined with the other genes of the attenuated strain caused severe rash and fever even if the disease onset was delayed. Viruses in which the nucleocapsid, polymerase, and phosphoprotein genes (coding also for the V and C proteins) of the virulent strain were combined with the envelope genes of the attenuated strain caused milder signs of disease. Thus, virulence-inducing mutations have accumulated throughout the genome.

The Hemagglutinin of Canine Distemper Virus Determines Tropism and Cytopathogenicity

ABSTRACT Canine distemper virus (CDV) and measles virus (MV) cause severe illnesses in their respective hosts. The viruses display a characteristic cytopathic effect by forming syncytia in susceptible cells. For CDV, the proficiency of syncytium formation varies among different strains and correlates with the degree of viral attenuation. In this study, we examined the determinants for the differential fusogenicity of the wild-type CDV isolate 5804Han89 (CDV5804), the small- and large-plaque-forming variants of the CDV vaccine strain Onderstepoort (CDVOS and CDVOL, respectively), and the MV vaccine strain Edmonston B (MVEdm). The cotransfection of different combinations of fusion (F) and hemagglutinin (H) genes in Vero cells indicated that the H protein is the main determinant of fusion efficiency. To verify the significance of this observation in the viral context, a reverse genetic system to generate recombinant CDVs was established. This system is based on a plasmid containing the full-length antigenomic sequence of CDVOS. The coding regions of the H proteins of all CDV strains and MVEdm were introduced into the CDV and MV genetic backgrounds, and recombinant viruses rCDV-H5804, rCDV-HOL, rCDV-HEdm, rMV-H5804, rMV-HOL, and rMV-HOS were recovered. Thus, the H proteins of the two morbilliviruses are interchangeable and fully functional in a heterologous complex. This is in contrast with the glycoproteins of other members of the familyParamyxoviridae, which do not function efficiently with heterologous partners. The fusogenicity, growth characteristics, and tropism of the recombinant viruses were examined and compared with those of the parental strains. All these characteristics were found to be predominantly mediated by the H protein regardless of the viral backbone used.

Receptor (SLAM [CD150]) recognition and the V protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus.

ABSTRACT Experimental infections of ferrets with canine distemper virus (CDV) recapitulate many hallmarks of measles: rash, high fever, viremia, depression of delayed-type hypersensitivity responses, lowered leukocyte counts, and reduced lymphocyte proliferation activity. To understand how a morbillivirus invades the host and causes immunosuppression, we generated CDV either unable to recognize one of the receptors or incapable of expressing either one or both of the candidate interferon antagonist proteins V and C. Variants of these viruses expressing green fluorescent protein were also generated. Striking similarities between CDV infection of ferrets and human immunodeficiency virus host invasion were documented: first, massive early replication in the gut-associated lymphatic tissue, including intestinal Peyers patches, followed by extensive infection of lymphatic organs, including thymus and circulating lymphocytes. Moreover, T cells were selectively depleted. Thus, CDV takes advantage of mucosal surfaces for host invasion and lymphocytes for swift dissemination. A CDV unable to recognize the signaling lymphocytic activation molecule (SLAM [CD150]) that is expressed in lymphocytes and other immune cells did not spread. A V-defective CDV multiplied with reduced efficiency in lymphocytes and did not inhibit the interferon and cytokine responses. Protein C affected the severity of rash and digestive symptoms elicited by V-defective CDV, but it was dispensable for the invasion of the lymphatic organs. These findings prove formally that SLAM recognition is necessary for morbillivirus virulence. They also reveal how two viral proteins affect pathogenesis: V sustains the swift lymphocyte-based invasion of mucosal tissue and lymphatic organs, whereas C sustains subsequent infection phases.

Type I IFN Triggers RIG-I/TLR3/NLRP3-dependent Inflammasome Activation in Influenza A Virus Infected Cells

Influenza A virus (IAV) triggers a contagious and potentially lethal respiratory disease. A protective IL-1β response is mediated by innate receptors in macrophages and lung epithelial cells. NLRP3 is crucial in macrophages; however, which sensors elicit IL-1β secretion in lung epithelial cells remains undetermined. Here, we describe for the first time the relative roles of the host innate receptors RIG-I (DDX58), TLR3, and NLRP3 in the IL-1β response to IAV in primary lung epithelial cells. To activate IL-1β secretion, these cells employ partially redundant recognition mechanisms that differ from those described in macrophages. RIG-I had the strongest effect through a MAVS/TRIM25/Riplet–dependent type I IFN signaling pathway upstream of TLR3 and NLRP3. Notably, RIG-I also activated the inflammasome through interaction with caspase 1 and ASC in primary lung epithelial cells. Thus, NS1, an influenza virulence factor that inhibits the RIG-I/type I IFN pathway, strongly modulated the IL-1β response in lung epithelial cells and in ferrets. The NS1 protein derived from a highly pathogenic strain resulted in increased interaction with RIG-I and inhibited type I IFN and IL-1β responses compared to the least pathogenic virus strains. These findings demonstrate that in IAV-infected lung epithelial cells RIG-I activates the inflammasome both directly and through a type I IFN positive feedback loop.

Severe seasonal influenza in ferrets correlates with reduced interferon and increased IL-6 induction.

Even though ferrets are one of the principal animal models for influenza pathogenesis, the lack of suitable immunological reagents has so far limited their use in host response studies. Using recently established real-time PCR assays for a panel of ferret cytokines, we analyzed the local ferret immune response to human influenza isolates of the H1N1 and H3N2 subtypes that varied in their virulence. We observed that the severity of clinical signs correlated with gross- and histopathological changes in the lungs and was subtype-independent. Strains causing a mild disease were associated with a strong and rapid innate response and upregulation of IL-8, while severe infections were characterized by a lesser induction of type I and II interferons and strong IL-6 upregulation. These findings suggest that more virulent strains may interfere more efficiently with the host response at early disease stages.

Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion.

ABSTRACT Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.

Efficiency of Measles Virus Entry and Dissemination through Different Receptors

ABSTRACT The efficiency with which different measles virus (MV) strains enter cells through the immune cell-specific protein SLAM (CD150) or other receptors, including the ubiquitous protein CD46, may influence their pathogenicity. We compared the cell entry efficiency of recombinant MV differing only in their attachment protein hemagglutinin (H). We constructed these viruses with an additional gene expressing an autofluorescent reporter protein to allow direct detection of every infected cell. A virus with a wild-type H protein entered cells through SLAM two to three times more efficiently than a virus with the H protein of the attenuated strain Edmonston, whereas cell entry efficiency through CD46 was lower. However, these subtle differences were amplified at the cell fusion stage because the wild-type H protein failed to fuse CD46-expressing cells. We also proved formally that a mutation in H protein residue 481 (asparagine to tyrosine) results in improved CD46-specific entry. To define the selective pressure exerted on that codon, we monitored its evolution in different H protein backgrounds and found that several passages in CD46-expressing Vero cells were necessary to shift it in the majority of the MV RNA. To verify the importance of these observations for human infections, we examined MV entry into peripheral blood mononuclear cells and observed that viruses with asparagine 481 H proteins infect these cells more efficiently.

Intranasal Administration of Alpha Interferon Reduces Seasonal Influenza A Virus Morbidity in Ferrets

ABSTRACT The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, we assessed the protective potential of exogenous IFN-α against seasonal and highly pathogenic influenza viruses in ferrets. Intranasal treatment with IFN-α several hours before infection with the H1N1 influenza A virus strain A/USSR/90/77 reduced viral titers in nasal washes at least 100-fold compared to mock-treated controls. IFN-treated animals developed only mild and transient respiratory symptoms, and the characteristic fever peak seen in mock-treated ferrets 2 days after infection was not observed. Repeated application of IFN-α substantially increased the protective effect of the cytokine treatment. IFN-α did not increase survival after infection with the highly pathogenic H5N1 avian influenza A virus strain A/Vietnam/1203/2004. However, viral titers in nasal washes were significantly reduced at days 1 and 3 postinfection. Our study shows that intranasal application of IFN-α can protect ferrets from seasonal influenza viruses, which replicate mainly in the upper respiratory tract, but not from highly pathogenic influenza viruses, which also disseminate to the lung. Based on these results, a more intensive evaluation of IFN-α as an emergency drug against pandemic influenza A is warranted.

An oncolytic measles virus engineered to enter cells through the CD20 antigen

We have earlier shown that attenuated measles virus (MV) has therapeutic potential as a replicating oncolytic virus in models of non-Hodgkins lymphoma (NHL). In the current study, we investigated whether we could obtain replicating MVs capable of entering CD20(+) target cells through an interaction between a single-chain (scFv) anti-CD20 antibody and the CD20 antigen, a target of considerable clinical relevance in NHL. We replaced the H envelope glycoprotein of MV by an H-scFv anti-CD20 fusion protein with and without a protease-cleavable linker. Biochemical analysis of purified virions confirmed that the modified H proteins were incorporated into the viral particles with efficiency similar to unmodified H. Experiments employing CHO cells and CHO cells expressing human CD20 indicated that the MVH alpha CD20 viruses were able to replicate well in CHOCD20 but not CHO cells. MVH alpha CD20 or a nonmodified control MV were administered systemically to immunodeficient mice bearing bilateral human tumor xenografts, one side with and the other side without CD20 expression. Growth of CD20(+) tumors was retarded by MVH alpha CD20 as compared with the control virus. The viruses had equivalent effects on the CD20(-) tumors. Thus we have demonstrated that the entry of a replicating oncolytic virus can be mediated through an interaction between a highly clinically relevant single-chain antibody and its target antigen, and we have shown that this interaction enhances in vivo oncolytic activity.

Oncolytic Efficacy and Enhanced Safety of Measles Virus Activated by Tumor-Secreted Matrix Metalloproteinases

Cancer cells secrete matrix metalloproteinases (MMP) that degrade the extracellular matrix and are responsible for some hallmarks of malignant cancer. Many viruses, including a few currently used in oncolytic virotherapy clinical trials, depend on intracellular proteases to process their proteins and activate their particles. We show here for measles virus (MV) that particle activation can be made dependent of proteases secreted by cancer cells. The MV depends on the intracellular protease furin to process and activate its envelope fusion (F) protein. To make F protein activation cancer cell specific, we introduced hexameric sequences recognized by an MMP and identified the mutant proteins most effective in fusing MMP-expressing human fibrosarcoma cells (HT1080). We showed that an MMP inhibitor interferes with syncytia formation elicited by mutant F proteins and confirmed MMP-dependent cleavage by Edman degradation sequence analysis. We generated recombinant MVs expressing the modified F proteins in place of furin-activated F. These viruses spread only in cells secreting MMP. In nude mice, an MMP-activated MV retarded HT1080 xenograft growth as efficiently as the furin-activated MV vaccine strain. In MV-susceptible mice, the furin-activated virus caused lethal encephalitis upon intracerebral inoculation, whereas the MMP-activated did not. Thus, MV particle activation can be made dependent of proteases secreted by cancer cells, enhancing safety. This study opens the perspective of combining targeting at the particle activation, receptor recognition, and selective replication levels to improve the therapeutic index of MV and other viruses in ongoing clinical trials of oncolysis.