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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The three faces of paramyxovirus attachment proteins.

Enveloped viruses encode membrane-associated glycoproteins that direct the initial stages of virus infection. These usually oligomeric structures bind virions to cell surface receptors and, subsequently, direct fusion of viral membranes with cellular membranes. These structures are also the primary targets of neutralizing antibody as well as potential targets for antiviral agents. In several systems, solving the structure of a virus surface glycoprotein has been enormously valuable to our understanding of virus entry and the mechanisms of entry inhibition. The recent report of the structure of a paramyxovirus attachment protein should clarify the mechanism of cell entry by these viruses.

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.

Comparative pathology of the diseases caused by Hendra and Nipah viruses.

Information on the pathogenesis and transmissibility of Hendra and Nipah viruses was obtained by comparing their histopathology. Both viruses induced syncytial cells in vascular tissues and they were primarily vasotropic and/or neurotropic, generating interstitial pneumonia or encephalitis. Nipah virus in pigs was also epitheliotropic in respiratory epithelium and thus contagious.

The natural history of Hendra and Nipah viruses.

Pteropid bats (flying foxes), species of which are the probable natural host of both Hendra and Nipah viruses, occur in overlapping populations from India to Australia. Ecological changes associated with land use and with animal husbandry practices appear most likely to be associated with the emergence of these two agents.

Nipah virus--a potential agent of bioterrorism?

Nipah virus, a newly emerging deadly paramyxovirus isolated during a large outbreak of viral encephalitis in Malaysia, has many of the physical attributes to serve as a potential agent of bioterrorism. The outbreak caused widespread panic and fear because of its high mortality and the inability to control the disease initially. There were considerable social disruptions and tremendous economic loss to an important pig-rearing industry. This highly virulent virus, believed to be introduced into pig farms by fruit bats, spread easily among pigs and was transmitted to humans who came into close contact with infected animals. From pigs, the virus was also transmitted to other animals such as dogs, cats, and horses. The Nipah virus has the potential to be considered an agent of bioterrorism.

Newer viral encephalitides.

BACKGROUND: Viral encephalitis occurs in epidemic settings or is sporadic. New encephalitis patterns reflect the roles that biologic reservoirs and vectors play in determining virus-human interactions. "New" viral encephalitis can also result from human host modifications that increase susceptibility to neuroinvasive viral infection. REVIEW SUMMARY: Three human viruses, Nipah virus, Human Herpesvirus-6, and West Nile virus, present examples of how "new" viral encephalitides emerge in a specific geographic region or clinical setting. Nipah virus encephalitis emerged after the molecular evolution of a new zoonotic viral genus within the Paramyxovirinae family. Human herpesvirus-6 encephalitis has emerged in the immune suppressed human host harboring this ubiquitous but typically benign herpesvirus. West Nile virus encephalitis has emerged in the Western hemisphere after apparent abrupt translocation of this mosquito-borne virus to a distant geographic region with immunologically naive avian and human hosts. CONCLUSION: While the clinical features of these viral encephalitides are somewhat distinct, they each emerged as the result of human-derived factors that altered the biologic dynamic between humans and their viral pathogens.