Canine Distemper Virus Pathogenesis in the Ferret Model.

Canine distemper virus (CDV) is a highly contagious pathogen within the morbillivirus genus infecting a wide range of different carnivore species. The virus shares most biological features with other closely related morbilliviruses, including clinical signs, tissue tropism, and replication cycle in the respective host organisms.In the laboratory environment, experimental infections of ferrets with CDV were established as a potent surrogate model for the analysis of several aspects of the biology of the human morbillivirus, measles virus (MeV). The animals are naturally susceptible to CDV and display severe clinical signs resembling the disease seen in patients infected with MeV. As seen with MeV, CDV infects immune cells and is thus associated with a strong transient immunosuppression. Here we describe several methods to evaluate viral load and parameters of immunosuppression in blood-circulating immune cells isolated from CDV-infected animals.

PEtOxylated Interferon-α2a Bioconjugates Addressing H1N1 Influenza A Virus Infection.

Influenza A viruses (IAV), including the pandemic 2009 (pdm09) H1N1 or avian influenza H5N1 virus, may advance into more pathogenic, potentially antiviral drug-resistant strains (including loss of susceptibility against oseltamivir). Such IAV strains fuel the risk of future global outbreaks, to which this study responds by re-engineering Interferon-α2a (IFN-α2a) bioconjugates into influenza therapeutics. Type-I interferons such as IFN-α2a play an essential role in influenza infection and may prevent serious disease courses. We site-specifically conjugated a genetically engineered IFN-α2a mutant to poly(2-ethyl-2-oxazoline)s (PEtOx) of different molecular weights by strain-promoted azide-alkyne cyclo-addition. The promising pharmacokinetic profile of the 25 kDa PEtOx bioconjugate in mice echoed an efficacy in IAV-infected ferrets. One intraperitoneal administration of this bioconjugate, but not the marketed IFN-α2a bioconjugate, changed the disease course similar to oseltamivir, given orally twice every study day. PEtOxylated IFN-α2a bioconjugates may expand our therapeutic arsenal against future influenza pandemics, particularly in light of rising first-line antiviral drug resistance to IAV.

Overcoming the Barrier of the Respiratory Epithelium during Canine Distemper Virus Infection.

Canine distemper virus (CDV) is a highly contagious pathogen and is known to enter the host via the respiratory tract and disseminate to various organs. Current hypotheses speculate that CDV uses the homologous cellular receptors of measles virus (MeV), SLAM and nectin-4, to initiate the infection process. For validation, here, we established the well-differentiated air-liquid interface (ALI) culture model from primary canine tracheal airway epithelial cells. By applying the green fluorescent protein (GFP)-expressing CDV vaccine strain and recombinant wild-type viruses, we show that cell-free virus infects the airway epithelium mainly via the paracellular route and only after prior disruption of tight junctions by pretreatment with EGTA; this infection was related to nectin-4 but not to SLAM. Remarkably, when CDV-preinfected DH82 cells were cocultured on the basolateral side of canine ALI cultures grown on filter supports with a 1.0-µm pore size, cell-associated CDV could be transmitted via cell-to-cell contact from immunocytes to airway epithelial cultures. Finally, we observed that canine ALI cultures formed syncytia and started to release cell-free infectious viral particles from the apical surface following treatment with an inhibitor of the JAK/STAT signaling pathway (ruxolitinib). Our findings show that CDV can overcome the epithelial barrier through different strategies, including infection via immunocyte-mediated transmission and direct infection via the paracellular route when tight junctions are disrupted. Our established model can be adapted to other animals for studying the transmission routes and the pathogenicity of other morbilliviruses. IMPORTANCE Canine distemper virus (CDV) is not only an important pathogen of carnivores, but it also serves as a model virus for analyzing measles virus pathogenesis. To get a better picture of the different stages of infection, we used air-liquid interface cultures to analyze the infection of well-differentiated airway epithelial cells by CDV. Applying a coculture approach with DH82 cells, we demonstrated that cell-mediated infection from the basolateral side of well-differentiated epithelial cells is more efficient than infection via cell-free virus. In fact, free virus was unable to infect intact polarized cells. When tight junctions were interrupted by treatment with EGTA, cells became susceptible to infection, with nectin-4 serving as a receptor. Another interesting feature of CDV infection is that infection of well-differentiated airway epithelial cells does not result in virus egress. Cell-free virions are released from the cells only in the presence of an inhibitor of the JAK/STAT signaling pathway. Our results provide new insights into how CDV can overcome the barrier of the airway epithelium and reveal similarities and some dissimilarities compared to measles virus.

Susceptibility of Well-Differentiated Airway Epithelial Cell Cultures from Domestic and Wild Animals to Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, and the number of worldwide cases continues to rise. The zoonotic origins of SARS-CoV-2 and its intermediate and potential spillback host reservoirs, besides humans, remain largely unknown. Because of ethical and experimental constraints and more important, to reduce and refine animal experimentation, we used our repository of well-differentiated airway epithelial cell (AEC) cultures from various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. We observed that SARS-CoV-2 replicated efficiently only in monkey and cat AEC culture models. Whole-genome sequencing of progeny viruses revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat AEC cultures. Our findings, together with previous reports of human-to-animal spillover events, warrant close surveillance to determine the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19: Protection of mice after a single immunization.

Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.

C Protein is Essential for Canine Distemper Virus Virulence and Pathogenicity in Ferrets.

Paramyxoviruses, including members of the genus Morbillivirus, express accessory proteins with ancillary functions during viral replication. One of these, the C protein, is expressed from an alternate open reading frame (ORF) located in the P gene. The measles virus (MeV) C protein has been implicated in modulation of interferon signaling, but has more recently been shown to play a vital role in regulation of viral transcription and replication, preventing the excessive production of double-stranded RNA. Failure to do so, as seen with C-deficient MeV, leads to early activation of innate immune responses resulting in restriction of viral replication and attenuation in the host. One puzzling aspect of morbillivirus C protein biology has been the finding that a C-deficient canine distemper virus (CDV) generated with a similar mutagenesis strategy displayed no attenuation in ferrets, an animal model commonly used to evaluate CDV pathogenesis. To resolve how virus lacking this protein could maintain virulence, we re-visited the CDV C protein and found that truncated C proteins are expressed from the CDV gene using alternative downstream start codons even when the first start codon was disrupted. We introduced an additional point mutation abrogating expression of these truncated C proteins. A new CDV with this mutation was attenuated in vitro and led to increased activation of protein kinase R. It was also strongly attenuated in ferrets, inducing only mild disease in infected animals, thus replicating the phenotype of C-deficient MeV. Our results demonstrate the crucial role of morbillivirus C proteins in pathogenesis.IMPORTANCE The measles (MeV) and canine distemper viruses (CDV) express accessory proteins that regulate the host immune response and enhance replication. The MeV C protein is critical in preventing the generation of excess immunostimulatory double-stranded RNA. C protein-deficient MeV is strongly attenuated compared to wild-type virus, whereas CDV with a similarly disrupted C open reading frame is fully pathogenic. Here we show that CDV can compensate the disrupting mutations by expression of truncated, but apparently functional C proteins from several alternative start codons. We generated a new recombinant CDV that does not express these truncated C protein. This virus was attenuated both in cell culture and in ferrets, and finally resolves the paradox of the MeV and CDV C proteins, showing that both in fact have similar functions important for viral pathogenesis.

Avian Influenza A Virus Infects Swine Airway Epithelial Cells without Prior Adaptation.

Pigs play an important role in the interspecies transmission of influenza A viruses (IAV). The porcine airway epithelium contains binding sites for both swine/human IAV (α2,6-linked sialic acids) and avian IAV (α2,3-linked sialic acids) and therefore is suited for adaptation of viruses from other species as suggested by the "mixing vessel theory". Here, we applied well-differentiated swine airway epithelial cells to find out whether efficient infection by avian IAV requires prior adaption. Furthermore, we analyzed the influence of the sialic acid-binding activity and the virus-induced detrimental effects. Surprisingly, an avian IAV H1N1 strain circulating in European poultry and waterfowl shows increased and prolonged viral replication without inducing a strong innate immune response. This virus could infect the lower respiratory tract in our precision cut-lung slice model. Pretreating the cells with poly (I:C) and/or JAK/STAT pathway inhibitors revealed that the interferon-stimulated innate immune response influences the replication of avian IAV in swine airway epitheliums but not that of swine IAV. Further studies indicated that in the infection by IAVs, the binding affinity of sialic acid is not the sole factor affecting the virus infectivity for swine or human airway epithelial cells, whereas it may be crucial in well-differentiated ferret tracheal epithelial cells. Taken together, our results suggest that the role of pigs being the vessel of interspecies transmission should be reconsidered, and the potential of avian H1N1 viruses to infect mammals needs to be characterized in more detail.

Incomplete genetic reconstitution of B cell pools contributes to prolonged immunosuppression after measles.

Measles is a disease caused by the highly infectious measles virus (MeV) that results in both viremia and lymphopenia. Lymphocyte counts recover shortly after the disappearance of measles-associated rash, but immunosuppression can persist for months to years after infection, resulting in increased incidence of secondary infections. Animal models and in vitro studies have proposed various immunological factors underlying this prolonged immune impairment, but the precise mechanisms operating in humans are unknown. Using B cell receptor (BCR) sequencing of human peripheral blood lymphocytes before and after MeV infection, we identified two immunological consequences from measles underlying immunosuppression: (i) incomplete reconstitution of the naïve B cell pool leading to immunological immaturity and (ii) compromised immune memory to previously encountered pathogens due to depletion of previously expanded B memory clones. Using a surrogate model of measles in ferrets, we investigated the clinical consequences of morbillivirus infection and demonstrated a depletion of vaccine-acquired immunity to influenza virus, leading to a compromised immune recall response and increased disease severity after secondary influenza virus challenge. Our results show that MeV infection causes changes in naïve and memory B lymphocyte diversity that persist after the resolution of clinical disease and thus contribute to compromised immunity to previous infections or vaccinations. This work highlights the importance of MeV vaccination not only for the control of measles but also for the maintenance of herd immunity to other pathogens, which can be compromised after MeV infection.

Comparative Loss-of-Function Screens Reveal ABCE1 as an Essential Cellular Host Factor for Efficient Translation of Paramyxoviridae and Pneumoviridae.

Paramyxoviruses and pneumoviruses have similar life cycles and share the respiratory tract as a point of entry. In comparative genome-scale siRNA screens with wild-type-derived measles, mumps, and respiratory syncytial viruses in A549 cells, a human lung adenocarcinoma cell line, we identified vesicular transport, RNA processing pathways, and translation as the top pathways required by all three viruses. As the top hit in the translation pathway, ABCE1, a member of the ATP-binding cassette transporters, was chosen for further study. We found that ABCE1 supports replication of all three viruses, confirming its importance for viruses of both families. More detailed characterization revealed that ABCE1 is specifically required for efficient viral but not general cellular protein synthesis, indicating that paramyxoviral and pneumoviral mRNAs exploit specific translation mechanisms. In addition to providing a novel overview of cellular proteins and pathways that impact these important pathogens, this study highlights the role of ABCE1 as a host factor required for efficient paramyxovirus and pneumovirus translation.IMPORTANCE The Paramyxoviridae and Pneumoviridae families include important human and animal pathogens. To identify common host factors, we performed genome-scale siRNA screens with wild-type-derived measles, mumps, and respiratory syncytial viruses in the same cell line. A comparative bioinformatics analysis yielded different members of the coatomer complex I, translation factors ABCE1 and eIF3A, and several RNA binding proteins as cellular proteins with proviral activity for all three viruses. A more detailed characterization of ABCE1 revealed its essential role for viral protein synthesis. Taken together, these data sets provide new insight into the interactions between paramyxoviruses and pneumoviruses and host cell proteins and constitute a starting point for the development of broadly effective antivirals.

Nipah virus induces two inclusion body populations: Identification of novel inclusions at the plasma membrane.

Formation of cytoplasmic inclusion bodies (IBs) is a hallmark of infections with non-segmented negative-strand RNA viruses (order Mononegavirales). We show here that Nipah virus (NiV), a bat-derived highly pathogenic member of the Paramyxoviridae family, differs from mononegaviruses of the Rhabdo-, Filo- and Pneumoviridae families by forming two types of IBs with distinct localizations, formation kinetics, and protein compositions. IBs in the perinuclear region form rapidly upon expression of the nucleocapsid proteins. These IBperi are highly mobile and associate with the aggresome marker y-tubulin. IBperi can recruit unrelated overexpressed cytosolic proteins but do not contain the viral matrix (M) protein. Additionally, NiV forms an as yet undescribed IB population at the plasma membrane (IBPM) that is y-tubulin-negative but contains the M protein. Infection studies with recombinant NiV revealed that IBPM require the M protein for their formation, and most likely represent sites of NiV assembly and budding. The identification of this novel type of plasma membrane-associated IBs not only provides new insights into NiV biology and may open new avenues to develop novel antiviral approaches to treat these highly pathogenic viruses, it also provides a basis for a more detailed characterization of IBs and their role in virus assembly and replication in infections with other Mononegavirales.

Generation of therapeutic antisera for emerging viral infections.

The recent Ebola virus outbreak has highlighted the therapeutic potential of antisera and renewed interest in this treatment approach. While human convalescent sera may not be readily available in the early stages of an outbreak, antisera of animal origin can be produced in a short time frame. Here, we compared adjuvanted virus-like particles (VLP) with recombinant modified vaccinia virus Ankara and vesicular stomatitis virus (VSV), both expressing the Ebola virus antigens. The neutralizing antibody titers of rabbits immunized with adjuvanted VLPs were similar to those immunized with the replication-competent VSV, indicating that presentation of the antigen in its native conformation rather than de novo antigen expression is essential for production of functional antibodies. This approach also yielded high-titer antisera against Nipah virus glycoproteins, illustrating that it is transferable to other virus families. Multiple-step immunoglobulin G purification using a two-step 20-40% ammonium sulfate precipitation followed by protein A affinity chromatography resulted in 90% recovery of functionality and sustained in vivo stability. Adjuvanted VLP-based immunization strategies are thus a promising approach for the rapid generation of therapeutic antisera against emerging infections.

Canine Distemper Virus Spread and Transmission to Naive Ferrets: Selective Pressure on Signaling Lymphocyte Activation Molecule-Dependent Entry.

Upon infection, morbilliviruses such as measles virus, rinderpest virus, and canine distemper virus (CDV) initially target immune cells via the signaling lymphocyte activation molecule (SLAM) before spreading to respiratory epithelia through the adherens junction protein nectin-4. However, the roles of these receptors in transmission from infected to naive hosts have not yet been formally tested. To experimentally addressing this question, we established a model of CDV contact transmission between ferrets. We show here that transmission of wild-type CDV sometimes precedes the onset of clinical disease. In contrast, transmission was not observed in most animals infected with SLAM- or nectin-4-blind CDVs, even though all animals infected with the nectin-4-blind virus developed sustained viremia. There was an unexpected case of transmission of a nectin-4-blind virus, possibly due to biting. Another unprecedented event was transient viremia in an infection with a SLAM-blind virus. We identified three compensatory mutations within or near the SLAM-binding surface of the attachment protein. A recombinant CDV expressing the mutated attachment protein regained the ability to infect ferret lymphocytes in vitro, but its replication was not as efficient as that of wild-type CDV. Ferrets infected with this virus developed transient viremia and fever, but there was no transmission to naive contacts. Our study supports the importance of epithelial cell infection and of sequential CDV H protein interactions first with SLAM and then nectin-4 receptors for transmission to naive hosts. It also highlights the in vivo selection pressure on the H protein interactions with SLAM.IMPORTANCE Morbilliviruses such as measles virus, rinderpest virus, and canine distemper virus (CDV) are highly contagious. Despite extensive knowledge of how morbilliviruses interact with their receptors, little is known about how those interactions influence viral transmission to naive hosts. In a ferret model of CDV contact transmission, we showed that sequential use of the signaling lymphocytic activation molecule (SLAM) and nectin-4 receptors is essential for transmission. In one animal infected with a SLAM-blind CDV, we documented mild viremia due to the acquisition of three compensatory mutations within or near the SLAM-binding surface. The interaction, however, was not sufficient to cause disease or sustain transmission to naive contacts. This work confirms the sequential roles of SLAM and nectin-4 in morbillivirus transmission and highlights the selective pressure directed toward productive interactions with SLAM.

The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity.

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design.IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant viruses in cell culture but result in gradual viral attenuation in a lethal canine distemper virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxovirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxovirus family.

Cross-neutralisation of viruses of the tick-borne encephalitis complex following tick-borne encephalitis vaccination and/or infection.

The tick-borne encephalitis complex contains a number of flaviviruses that share close genetic homology, and are responsible for significant human morbidity and mortality with widespread geographical range. Although many members of this complex have been recognised for decades, licenced human vaccines with broad availability are only available for tick-borne encephalitis virus. While tick-borne encephalitis virus vaccines have been demonstrated to induce significant protective immunity, as determined by virus-neutralisation titres, vaccine breakthrough (clinical infection following complete vaccination), has been described. The aim of this study was to confirm the cross-neutralisation of tick-borne flaviviruses using mouse immune ascitic fluids, and to determine the magnitude of cross-neutralising antibody titres in sera from donors following tick-borne encephalitis vaccination, infection, and vaccine breakthrough. The results demonstrate that there is significant cross-neutralisation of representative members of the tick-borne encephalitis complex following vaccination and/or infection, and that the magnitude of immune responses varies based upon the exposure type. Donor sera successfully neutralised most of the viruses tested, with 85% of vaccinees neutralising Kyasanur forest disease virus and 73% of vaccinees neutralising Alkhumra virus. By contrast, only 63% of vaccinees neutralised Powassan virus, with none of these neutralisation titres exceeding 1:60. Taken together, the data suggest that tick-borne encephalitis virus vaccination may protect against most of the members of the tick-borne encephalitis complex including Kyasanur forest disease virus and Alkhumra virus, but that the neutralisation of Powassan virus following tick-borne encephalitis vaccination is minimal.

Nectin-4 Interactions Govern Measles Virus Virulence in a New Model of Pathogenesis, the Squirrel Monkey (Saimiri sciureus).

In addition to humans, only certain nonhuman primates are naturally susceptible to measles virus (MeV) infection. Disease severity is species dependent, ranging from mild to moderate for macaques to severe and even lethal for certain New World monkey species. To investigate if squirrel monkeys (Saimiri sciureus), which are reported to develop a course of disease similar to humans, may be better suited than macaques for the identification of virulence determinants or the evaluation of therapeutics, we infected them with a green fluorescent protein-expressing MeV. Compared to cynomolgus macaques (Macaca fascicularis) infected with the same virus, the squirrel monkeys developed more-severe immunosuppression, higher viral load, and a broader range of clinical signs typical for measles. In contrast, infection with an MeV unable to interact with the epithelial receptor nectin-4, while causing immunosuppression, resulted in only a mild and transient rash and a short-lived elevation of the body temperature. Similar titers of the wild-type and nectin-4-blind MeV were detected in peripheral blood mononuclear cells and lymph node homogenates, but only the wild-type virus was found in tracheal lavage fluids and urine. Thus, our study demonstrates the importance of MeV interactions with nectin-4 for clinical disease in the new and better-performing S. sciureus model of measles pathogenesis.IMPORTANCE The characterization of mechanisms underlying measles virus clinical disease has been hampered by the lack of an animal model that reproduces the course of disease seen in human patients. Here, we report that infection of squirrel monkeys (Saimiri sciureus) fulfills these requirements. Comparative infection with wild-type and epithelial cell receptor-blind viruses demonstrated the importance of epithelial cell infection for clinical disease, highlighting the spread to epithelia as an attractive target for therapeutic strategies.

Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens.

The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains.IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that received recombinant rabies viruses carrying only the CDV attachment protein according to the same immunization scheme died. Irrespective of the CDV antigens used, all animals developed protective titers against rabies virus, illustrating that a bivalent rabies virus-based vaccine against CDV induces protective immune responses against both pathogens.

Squalene-containing licensed adjuvants enhance strain-specific antibody responses against the influenza hemagglutinin and induce subtype-specific antibodies against the neuraminidase.

While seasonal influenza vaccines are usually non-adjuvanted, H1N1pdm09 vaccines were formulated with different squalene-containing adjuvants, to enable the reduction of antigen content thus increasing the number of doses available. To comparatively assess the effects of these adjuvants on antibody responses against matched and mismatched strains, and to correlate antibody levels with protection from disease, ferrets were immunized with 2µg of commercial H1N1pdm09 vaccine antigen alone or formulated with different licensed adjuvants. The use of squalene-containing adjuvants increased neutralizing antibody responses around 100-fold, and resulted in a significantly reduced viral load after challenge with a matched strain. While all animals mounted strong total antibody responses against the homologous H1N1 hemagglutinin (HA) protein, which correlated with the respective neutralizing antibody titers, no reactivity with the divergent H3, H5, H7, and H9 proteins were detected. Only the adjuvanted vaccines also induced antibodies against the neuraminidase (NA) protein, which were able to also recognize NA proteins from other N1 carrying strains. These findings not only support the use of squalene-containing adjuvants in dose-sparing strategies but also support speculations that the induction of NA-specific responses associated with the use of these adjuvants may confer partial protection to heterologous strains carrying the same NA subtype.

Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

Nipah Virus Matrix Protein Influences Fusogenicity and Is Essential for Particle Infectivity and Stability.

UNLABELLED: Nipah virus (NiV) causes fatal encephalitic infections in humans. To characterize the role of the matrix (M) protein in the viral life cycle, we generated a reverse genetics system based on NiV strain Malaysia. Using an enhanced green fluorescent protein (eGFP)-expressing M protein-deleted NiV, we observed a slightly increased cell-cell fusion, slow replication kinetics, and significantly reduced peak titers compared to the parental virus. While increased amounts of viral proteins were found in the supernatant of cells infected with M-deleted NiV, the infectivity-to-particle ratio was more than 100-fold reduced, and the particles were less thermostable and of more irregular morphology. Taken together, our data demonstrate that the M protein is not absolutely required for the production of cell-free NiV but is necessary for proper assembly and release of stable infectious NiV particles. IMPORTANCE: Henipaviruses cause a severe disease with high mortality in human patients. Therefore, these viruses can be studied only in biosafety level 4 (BSL-4) laboratories, making it more challenging to characterize their life cycle. Here we investigated the role of the Nipah virus matrix protein in virus-mediated cell-cell fusion and in the formation and release of newly produced particles. We found that even though low levels of infectious viruses are produced in the absence of the matrix protein, it is required for the release of highly infectious and stable particles. Fusogenicity of matrixless viruses was slightly enhanced, further demonstrating the critical role of this protein in different steps of Nipah virus spread.

Comparative pathogenesis of Alkhumra hemorrhagic fever and Kyasanur forest disease viruses in a mouse model.

Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV) are genetically closely-related, tick-borne flaviviruses that cause severe, often fatal disease in humans. Flaviviruses in the tick-borne encephalitis (TBE) complex typically cause neurological disease in humans whereas patients infected with KFDV and AHFV predominately present with hemorrhagic fever. A small animal model for KFDV and AHFV to study the pathogenesis and evaluate countermeasures has been lacking mostly due to the need of a high biocontainment laboratory to work with the viruses. To evaluate the utility of an existing mouse model for tick-borne flavivirus pathogenesis, we performed serial sacrifice studies in BALB/c mice infected with either KFDV strain P9605 or AHFV strain Zaki-1. Strikingly, infection with KFDV was completely lethal in mice, while AHFV caused no clinical signs of disease and no animals succumbed to infection. KFDV and high levels of pro-inflammatory cytokines were detected in the brain at later time points, but no virus was found in visceral organs; conversely, AHFV Zaki-1 and elevated levels of cytokines were found in the visceral organs at earlier time points, but were not detected in the brain. While infection with either virus caused a generalized leukopenia, only AHFV Zaki-1 induced hematologic abnormalities in infected animals. Our data suggest that KFDV P9605 may have lost its ability to cause hemorrhagic disease as the result of multiple passages in suckling mouse brains. However, likely by virtue of fewer mouse passages, AHFV Zaki-1 has retained the ability to replicate in visceral organs, cause hematologic abnormalities, and induce pro-inflammatory cytokines without causing overt disease. Given these striking differences, the use of inbred mice and the virus passage history need to be carefully considered in the interpretation of animal studies using these viruses.