Antivirals in medical biodefense.

The viruses historically implicated or currently considered as candidates for misuse in bioterrorist events are poxviruses, filoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses and a number of arboviruses causing encephalitis, including alpha- and flaviviruses. All these viruses are of concern for public health services when they occur in natural outbreaks or emerge in unvaccinated populations. Recent events and intelligence reports point to a growing risk of dangerous biological agents being used for nefarious purposes. Public health responses effective in natural outbreaks of infectious disease may not be sufficient to deal with the severe consequences of a deliberate release of such agents. One important aspect of countermeasures against viral biothreat agents are the antiviral treatment options available for use in post-exposure prophylaxis. These issues were adressed by the organizers of the 16th Medical Biodefense Conference, held in Munich in 2018, in a special session on the development of drugs to treat infections with viruses currently perceived as a threat to societies or associated with a potential for misuse as biothreat agents. This review will outline the state-of-the-art methods in antivirals research discussed and provide an overview of antiviral compounds in the pipeline that are already approved for use or still under development.

PI4KB on Inclusion Bodies Formed by ER Membrane Remodeling Facilitates Replication of Human Parainfluenza Virus Type 3.

Many positive-strand RNA viruses remodel the endomembrane to form specialized replication organelles. However, knowledge regarding whether negative-strand RNA viruses take advantage of intracellular membranes for replication is limited. Here we show that a negative-strand RNA virus, human parainfluenza virus type 3 (HPIV3), remodels the endoplasmic reticulum (ER) membrane to form inclusion bodies (IBs), whereby the phosphoprotein (P) of HPIV3 recruits phosphatidylinositol 4-kinase beta (PI4KB) to IBs to generate PI4P, creating a PI4P-enriched microenvironment to promote HPIV3 replication. In addition, we find that human respiratory syncytial virus (HRSV) also takes advantage of the ER to form IBs and that these IBs are also enriched with PI4P. The nucleoprotein of HRSV recruits PI4KB to IBs. These results suggest that paramyxoviruses also exploit the host endomembrane to form IBs and that PI4KB is recruited by viral proteins to enrich IBs with PI4P to facilitate viral replication.

Analysis of Paramyxovirus Transcription and Replication by High-Throughput Sequencing.

We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo(dT)-selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyze the abundance of viral mRNAs because genomic RNA copurifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depleted of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analyzed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5, or mumps virus and determined the abundances of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of non-poly(A) mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses.IMPORTANCE High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs.

Characterization of Teviot virus, an Australian bat-borne paramyxovirus.

Bats are the reservoir hosts for multiple viruses with zoonotic potential, including coronaviruses, paramyxoviruses and filoviruses. Urine collected from Australian pteropid bats was assessed for the presence of paramyxoviruses. One of the viruses isolated was Teviot virus (TevPV), a novel rubulavirus previously isolated from pteropid bat urine throughout the east coast of Australia. Here, we further characterize TevPV through analysis of whole-genome sequencing, growth kinetics, antigenic relatedness and the experimental infection of ferrets and mice. TevPV is phylogenetically and antigenically most closely related to Tioman virus (TioPV). Unlike many other rubulaviruses, cell receptor attachment by TevPV does not appear to be sialic acid-dependent, with the receptor for host cell entry being unknown. The infection of ferrets and mice suggested that TevPV has a low pathogenic potential in mammals. Infected ferrets seroconverted by 10 days post-infection without clinical signs of disease. Furthermore, infected ferrets did not shed virus in any respiratory secretions, suggesting a low risk of onward transmission of TevPV. No productive infection was observed in the mouse infection study.

Mumps: An Emergency Medicine-Focused Update.

BACKGROUND: Mumps is a Paramyxoviridae virus. This disease was rampant prior to introduction of the measles, mumps, and rubella vaccine, resulting in decreased incidence. This disease has demonstrated several outbreaks. OBJECTIVE: This review provides a focused evaluation of mumps, an update on outbreaks, management recommendations, and ways to decrease transmission. DISCUSSION: Clusters of mumps outbreaks continue to occur. The virus is a paramyxovirus, a single-stranded RNA virus. The vaccine can provide lifelong immunity if administered properly, though prior to 1967 and introduction of the vaccine, the virus was common. In the past decade, there have been several notable outbreaks. Humans are the only known hosts, with disease spread through exposure to droplets and saliva. Factors affecting transmission include age, compromised immunity, time of year, travel, and vaccination status. Upper respiratory symptoms, fever, and headache are common, with unilateral or bilateral parotitis, and the virus may spread to other systems. Diagnosis is clinical, though polymerase chain reaction and immunoglobulin testing are available. This review provides several recommendations for vaccine in pregnancy, patients living in close quarters, health care personnel, and those immunocompromised. Treatment is generally supportive, with emphasis on proper isolation to prevent widespread outbreaks. Although reporting regulations and procedures vary by state, mumps is reportable in most states. CONCLUSIONS: Mumps is an easily spread virus. Although vaccination is the most effective way to prevent transmission, early recognition of the disease is crucial. As an emergency physician, it is important to recognize the clinical presentation, recommended testing, treatment, and isolation procedures.

Zoonotic Potential of Emerging Paramyxoviruses: Knowns and Unknowns.

The risk of spillover of enzootic paramyxoviruses and the susceptibility of recipient human and domestic animal populations are defined by a broad collection of ecological and molecular factors that interact in ways that are not yet fully understood. Nipah and Hendra viruses were the first highly lethal zoonotic paramyxoviruses discovered in modern times, but other paramyxoviruses from multiple genera are present in bats and other reservoirs that have unknown potential to spillover into humans. We outline our current understanding of paramyxovirus reservoir hosts and the ecological factors that may drive spillover, and we explore the molecular barriers to spillover that emergent paramyxoviruses may encounter. By outlining what is known about enzootic paramyxovirus receptor usage, mechanisms of innate immune evasion, and other host-specific interactions, we highlight the breadth of unexplored avenues that may be important in understanding paramyxovirus emergence.

Human and Mouse Eosinophils Have Antiviral Activity against Parainfluenza Virus.

Respiratory viruses cause asthma exacerbations. Because eosinophils are the prominent leukocytes in the airways of 60-70% of patients with asthma, we evaluated the effects of eosinophils on a common respiratory virus, parainfluenza 1, in the lung. Eosinophils recruited to the airways of wild-type mice after ovalbumin sensitization and challenge significantly decreased parainfluenza virus RNA in the lungs 4 days after infection compared with nonsensitized animals. This antiviral effect was also seen in IL-5 transgenic mice with an abundance of airway eosinophils (NJ.1726) but was lost in transgenic eosinophil-deficient mice (PHIL) and in IL-5 transgenic mice crossed with eosinophil-deficient mice (NJ.1726-PHIL). Loss of the eosinophil granule protein eosinophil peroxidase, using eosinophil peroxidase-deficient transgenic mice, did not reduce eosinophils' antiviral effect. Eosinophil antiviral mechanisms were also explored in vitro. Isolated human eosinophils significantly reduced parainfluenza virus titers. This effect did not involve degradation of viral RNA by eosinophil granule RNases. However, eosinophils treated with a nitric oxide synthase inhibitor lost their antiviral activity, suggesting eosinophils attenuate viral infectivity through production of nitric oxide. Consequently, eosinophil nitric oxide production was measured with an intracellular fluorescent probe. Eosinophils produced nitric oxide in response to virus and to a synthetic agonist of the virus-sensing innate immune receptor, Toll-like receptor (TLR) 7. IFNγ increased expression of eosinophil TLR7 and potentiated TLR7-induced nitric oxide production. These results suggest that eosinophils promote viral clearance in the lung and contribute to innate immune responses against respiratory virus infections in humans.

Virus distribution and detection in corn snakes (Pantherophis guttatus) after experimental infection with three different ferlavirus strains.

Ferlaviruses are important pathogens of snakes. However, factors influencing the pathogenicity of individual isolates as well as optimal protocols for virus detection in tissues of infected snakes have been insufficiently studied. The objectives of this study were to compare virus detection using previously described PCR and cell culture protocols following infection with three genetically distinct ferlaviruses in corn snakes (Pantherophis guttatus) as a model species. Groups of 12 corn snakes were each inoculated intratracheally with a genogroup A, B, or C ferlavirus. Tracheal washes and cloacal swabs were tested for virus shedding on days 16 and 28. Three animals were each euthanized on days 4, 16, 28, and 49. Beside immunohistochemistry of lung tissue, several organs (lung, intestine, pancreas, kidney, brain) were tested for the presence of ferlavirus. Distinct differences were noted in the pathogenicity of the three viruses, with a genotype B isolate causing the greatest pathology. PCR was more sensitive in comparison to cell culture, but results varied depending on the tissues. Ferlaviruses spread rapidly into the tissues, including the brain. Overall average detection rate was 72%, and was highest on day 16. There were differences between the groups, with the most virulent strain causing 100% positive samples at the end of the study. Some snakes were able to clear the infection. Shedding via cloaca was seen only on day 28. For ante-mortem sampling, a tracheal wash sample is recommended, for post mortem diagnosis, a pooled organ sample should be tested.

Ferret models of viral pathogenesis.

Emerging and well-known viral diseases remain one the most important global public health threats. A better understanding of their pathogenesis and mechanisms of transmission requires animal models that accurately reproduce these aspects of the disease. Here we review the role of ferrets as an animal model for the pathogenesis of different respiratory viruses with an emphasis on influenza and paramyxoviruses. We will describe the anatomic and physiologic characteristics that contribute to the natural susceptibility of ferrets to these viruses, and provide an overview of the approaches available to analyze their immune responses. Recent insights gained using this model will be highlighted, including the development of new prophylactic and therapeutic approaches. To provide decision criteria for the use of this animal model, its strengths and limitations will be discussed.

The L gene of J paramyxovirus plays a critical role in viral pathogenesis.

J paramyxovirus (JPV) was first isolated from moribund mice with hemorrhagic lung lesions in Australia in the 1970s. Recent sequencing of JPV (JPV-LW) confirms that JPV is a paramyxovirus with several unique features. However, neither JPV-LW nor a recombinant JPV based on its sequence (rJPV-LW) caused obvious illness in mice. In this work, we analyzed a different JPV isolate (JPV-BH), which behaved differently from JPV-LW; JPV-BH grew more slowly in Vero cells and had less of a cytopathic effect on tissue culture cells but caused severe disease in mice. We have determined the whole genome sequence of JPV-BH. There were several nucleotide sequence differences between JPV-BH and JPV-LW, one in the leader sequence, one in the GX gene, and three in the L gene. The high sequence identity between JPV-BH and JPV-LW suggests that JPV-BH and JPV-LW are the same virus strain but were obtained at different passages from different laboratories. To understand the roles of these nucleotide sequence differences in pathogenicity in mice, we generated a recombinant JPV-BH strain (rJPV-BH) and hybrid rJPV-BH strains with sequences from the leader sequence (rJPV-BH-Le-LW), the GX gene (rJPV-BH-GX-LW), and the L gene (rJPV-BH-L-LW) of JPV-LW and compared their pathogenicities in mice. We have found that rJPV-BH-L-LW was attenuated in mice, indicating that nucleotide sequence differences in the L gene play a critical role in pathogenesis.

Menangle virus, a pteropid bat paramyxovirus infectious for pigs and humans, exhibits tropism for secondary lymphoid organs and intestinal epithelium in weaned pigs.

This study is the first report of experimental infection and transmission of Menangle virus (MenPV) in pigs. Isolated in 1997 from piglets that were stillborn at a large commercial piggery in New South Wales, Australia, MenPV is a recently identified paramyxovirus of bat origin that causes severe reproductive disease in pigs and an influenza-like illness, with a rash, in humans. Although successfully eradicated from the infected piggery, the virus was only isolated from affected fetuses and stillborn piglets during the period of reproductive disease, and thus the mode of transmission between pigs was not established. To investigate the pathogenesis of MenPV, we undertook time-course studies in 6-week-old pigs following intranasal administration of a low-passage, non-plaque-purified isolate from the lung of an infected stillborn piglet. Viraemia was of short duration and low titre, as determined by real-time RT-PCR and virus isolation. Following an incubation period of 2-3 days, virus was shed in nasal and oral secretions, faeces and urine, typically for less than 1 week. Cessation of shedding correlated with the development of neutralizing antibodies in sera. Secondary lymphoid organs and intestine were identified, using quantitative real-time RT-PCR, as major sites of viral replication and dissemination, and this was confirmed by positive immunolabelling of viral antigen within various lymphoid tissues and intestinal epithelium. These data provide new insights into the pathogenesis of MenPV in weaned pigs, and will facilitate future control and eradication programmes should it ever re-emerge in the pig population.

Function of the small hydrophobic protein of J paramyxovirus.

At 18,954 nucleotides, the J paramyxovirus (JPV) genome is one of the largest in the family Paramyxoviridae, consisting of eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. To study the function of novel paramyxovirus genes in JPV, a plasmid containing a full-length cDNA clone of the genome of JPV was constructed. In this study, the function of the small hydrophobic (SH) protein of JPV was examined by generating a recombinant JPV lacking the coding sequence of the SH protein (rJPVΔSH). rJPVΔSH was viable and had no growth defect in tissue culture cells. However, more tumor necrosis factor alpha (TNF-α) was produced during rJPVΔSH infection, suggesting that SH plays a role in inhibiting TNF-α production. rJPVΔSH induced more apoptosis in tissue culture cells than rJPV. Virus-induced apoptosis was inhibited by neutralizing antibody against TNF-α, suggesting that TNF-α contributes to JPV-induced apoptosis in vitro. The expression of JPV SH protein inhibited TNF-α-induced NF-κB activation in a reporter gene assay, suggesting that JPV SH protein can inhibit TNF-α signaling in vitro. Furthermore, infection of mice with rJPVΔSH induced more TNF-α expression, indicating that SH plays a role in blocking TNF-α expression in vivo.

Dissociation of paramyxovirus interferon evasion activities: universal and virus-specific requirements for conserved V protein amino acids in MDA5 interference.

The V protein of the paramyxovirus subfamily Paramyxovirinae is an important virulence factor that can interfere with host innate immunity by inactivating the cytosolic pathogen recognition receptor MDA5. This interference is a result of a protein-protein interaction between the highly conserved carboxyl-terminal domain of the V protein and the helicase domain of MDA5. The V protein C-terminal domain (CTD) is an evolutionarily conserved 49- to 68-amino-acid region that coordinates two zinc atoms per protein chain. Site-directed mutagenesis of conserved residues in the V protein CTD has revealed both universal and virus-specific requirements for zinc coordination in MDA5 engagement and has also identified other conserved residues as critical for MDA5 interaction and interference. Mutation of these residues produces V proteins that are specifically defective for MDA5 interference and not impaired in targeting STAT1 for proteasomal degradation via the VDC ubiquitin ligase complex. Results demonstrate that mutation of conserved charged residues in the V proteins of Nipah virus, measles virus, and mumps virus also abolishes MDA5 interaction. These findings clearly define molecular determinants for MDA5 inhibition by the paramyxovirus V proteins.

Viral entry mechanisms: the increasing diversity of paramyxovirus entry.

The paramyxovirus family contains established human pathogens such as the measles virus and human respiratory syncytial virus, as well as emerging pathogens including the Hendra and Nipah viruses and the recently identified human metapneumovirus. Two major envelope glycoproteins, the attachment protein and the fusion protein, promote the processes of viral attachment and virus-cell membrane fusion required for entry. Although common mechanisms of fusion protein proteolytic activation and the mechanism of membrane fusion promotion have been shown in recent years, considerable diversity exists in the family relating to receptor binding and the potential mechanisms of fusion triggering.

Role for the paramyxovirus genomic promoter in limiting host cell antiviral responses and cell killing.

The parainfluenza virus simian virus 5 (SV5) is a poor inducer of innate immune responses. In contrast, the naturally occurring SV5 variant Wake Forest parainfluenza virus (WF-PIV) activates the synthesis of proinflammatory cytokines and beta interferon (IFN-beta). Comparison of SV5 and WF-PIV genome sequences revealed nine nucleotide differences within the viral genomic promoter, including two substitutions (U5C and A14G) in the most highly conserved 3'-end promoter element. To test the consequences of these promoter variations, a recombinant SV5 mutant [Le-(U5C, A14G)] was engineered to harbor the two WF-PIV genomic promoter substitutions in an otherwise wild-type (WT) SV5 background. Human lung epithelial cells infected with the Le-(U5C, A14G) mutant had higher rates of viral protein synthesis and levels of mRNA than cells infected with WT SV5, but levels of genomic RNA were not changed. Unlike WT SV5, the Le-(U5C, A14G) mutant was a potent inducer of interleukin-6 and IFN-beta synthesis, despite expressing a functional V protein antagonist. Cytokine responses to Le-(U5C, A14G) infection were reduced either by small interfering RNA-mediated knockdown of retinoic acid-inducible gene I (RIG-I) or after infection of cells that were engineered to express the reovirus sigma3 double-stranded RNA-binding protein. Le-(U5C, A14G) induced cytopathic effects not seen with WT SV5, and the extent of cell killing correlated with elevated levels of viral F protein and cell-cell fusion. Our results support a model whereby the SV5 promoter has evolved to function at an attenuated level in order to limit (i) synthesis of aberrant RNAs which induce RIG-I-mediated responses and (ii) overproduction of mRNA for potentially toxic gene products, such as the F protein. Control of genomic promoter activity may be particularly important for viruses such as SV5, that express a V protein targeting mda-5 but do not encode antagonists such as the paramyxovirus C proteins, that specifically target RIG-I.

Contribution of the leader sequence to homologous viral interference among Sendai virus strains.

Sendai viruses (SeV) derived from persistent infection have a capacity to interfere with co-infected wild-type virus. Here we showed that interference was also caused by the laboratory strains Z and Nagoya. The leader mutations A(20)U and A(24)U related to viral adaptation from mice to chicken eggs significantly affected the capacity for viral interference, especially through genome amplification. Furthermore, recombinant SeV that possessed the mutations A(34)G and G(47)A, which are commonly found in the leader sequence of persistent infection-derived SeV strains, had an increased capacity for interference. Viral replication of human parainfluenza viruses 1, 2, and 3, but not the mumps virus or Newcastle disease virus, was suppressed by co-infection of a persistent infection-derived SeV strain, suggesting suppression of closely related human paramyxoviruses. These results indicate that homologous interference is partly dependent on the promoter sequence and further suggest involvement of promoter activity for genome amplification related to host factors in viral interference.

Pathophysiology and genetics of metabolic bone disorders characterized by increased bone turnover.

Bone is the most important supportive tissue in the human body, and in order to maintain its integrity, it is continuously renewed by a process called "remodeling". Paget's disease of bone (PDB), familial expansile osteolysis (FEO), expansile skeletal hyperphosphatasia (ESH), early-onset Paget's disease of bone (EOPDB), and juvenile Paget's disease (JPD) are all metabolic bone disorders characterized by accelerated bone remodeling. Histological studies have shown that bone-resorbing osteoclasts are the primary disease-causing cells in these disorders. In this review, we provide an overview of the clinical differences between diseases with increased bone turnover. Our main focus is on Paget's disease because this is, by far, the most common form of this type of disease. Molecular genetic studies of these disorders have revealed key players in bone remodeling and have provided further insights in signal transduction in osteoclasts. Moreover, a syndromal form of PDB has been characterized in which PDB is associated with inclusion body myopathy and frontotemporal dementia, pointing toward similar biological pathways in osteoclasts, muscle, and brain cells. However, several additional genes underlying conditions with increased bone turnover remain to be identified.

Full-length genome sequence and genetic relationship of two paramyxoviruses isolated from bat and pigs in the Americas.

Mapuera virus (MPRV) was isolated from a fruit bat in Brazil in 1979, but its host range and disease-causing potential are unknown. Porcine rubulavirus (PoRV) was identified as the aetiological agent of disease outbreaks in pigs in Mexico during early 1980s, but the origin of PoRV remains elusive. In this study, the completed genome sequence of MPRV was determined, and the complete genome sequence of PoRV was assembled from previously published protein-coding genes and the non-coding genome regions determined from this study. Comparison of sequence and genome organization indicated that PoRV is more closely related to MPRV than to any other members of the genus Rubulavirus. In the P gene coding region of both viruses, there is an ORF located at the 5' end of the P gene overlapping with the P protein coding region, similar to the C protein ORF present in most viruses of the subfamily Paramyxovirinae, but absent in other known rubulaviruses. Based on these findings, we hypothesise that PoRV may also originate from bats, and spillover events from bats to pigs, either directly or via an intermediate host, were responsible for the sporadic disease outbreaks observed in Mexico.

Role of sialic acid-containing molecules in paramyxovirus entry into the host cell: a minireview.

Sialic acid-containing compounds play a key role in the initial steps of the paramyxovirus life cycle. As enveloped viruses, their entry into the host cell consists of two main events: binding to the host cell and membrane fusion. Virus adsorption occurs at the surface of the host cell with the recognition of specific receptor molecules located at the cell membrane by specific viral attachment proteins. The viral attachment protein present in some paramyxoviruses (Respirovirus, Rubulavirus and Avulavirus) is the HN glycoprotein, which binds to cellular sialic acid-containing molecules and exhibits sialidase and fusion promotion activities. Gangliosides of the gangliotetraose series bearing the sialic acid N-acetylneuraminic (Neu5Ac) on the terminal galactose attached in alpha2-3 linkage, such as GD1a, GT1b, and GQ1b, and neolacto-series gangliosides are the major receptors for Sendai virus. Much less is known about the receptors for other paramyxoviruses than for Sendai virus. Human parainfluenza viruses 1 and 3 preferentially recognize oligosaccharides containing N-acetyllactosaminoglycan branches with terminal Neu5Acalpha2-3Gal. In the case of Newcastle disease virus, has been reported the absence of a specific pattern of the gangliosides that interact with the virus. Additionally, several works have described the use of sialylated glycoproteins as paramyxovirus receptors. Accordingly, the design of specific sialic acid analogs to inhibit the sialidase and/or receptor binding activity of viral attachment proteins is an important antiviral strategy. In spite of all these data, the exact nature of paramyxovirus receptors, apart from their sialylated nature, and the mechanism(s) of viral attachment to the cell surface are poorly understood.