How does the polymerase of non-segmented negative strand RNA viruses commit to transcription or genome replication?

The Mononegavirales, or non-segmented negative-sense RNA viruses (nsNSVs), includes significant human pathogens, such as respiratory syncytial virus, parainfluenza virus, measles virus, Ebola virus, and rabies virus. Although these viruses differ widely in their pathogenic properties, they are united by each having a genome consisting of a single strand of negative-sense RNA. Consistent with their shared genome structure, the nsNSVs have evolved similar ways to transcribe their genome into mRNAs and replicate it to produce new genomes. Importantly, both mRNA transcription and genome replication are performed by a single virus-encoded polymerase. A fundamental and intriguing question is: how does the nsNSV polymerase commit to being either an mRNA transcriptase or a replicase? The polymerase must become committed to one process or the other either before it interacts with the genome template or in its initial interactions with the promoter sequence at the 3´ end of the genomic RNA. This review examines the biochemical, molecular biology, and structural biology data regarding the first steps of transcription and RNA replication that have been gathered over several decades for different families of nsNSVs. These findings are discussed in relation to possible models that could explain how an nsNSV polymerase initiates and commits to either transcription or genome replication.

Specific Residues in the C-Terminal Domain of the Human Metapneumovirus Phosphoprotein Are Indispensable for Formation of Viral Replication Centers and Regulation of the Function of the Viral Polymerase Complex.

Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A hallmark of HMPV infection is the formation of membraneless, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures, and both proteins are required for the viral polymerase to synthesize RNA during infection. HMPV P is a homotetramer with regions of intrinsic disorder and has several known and predicted phosphorylation sites of unknown function. In this study, we found that the P C-terminal intrinsically disordered domain (CTD) must be present to facilitate IB formation with HMPV N, while either the N-terminal intrinsically disordered domain or the central oligomerization domain was dispensable. Alanine substitution at a single tyrosine residue within the CTD abrogated IB formation and reduced coimmunoprecipitation with HMPV N. Mutations to C-terminal phosphorylation sites revealed a potential role for phosphorylation in regulating RNA synthesis and P binding partners within IBs. Phosphorylation mutations which reduced RNA synthesis in a reporter assay produced comparable results in a recombinant viral rescue system, measured as an inability to produce infectious viral particles with genomes containing these single P mutations. This work highlights the critical role HMPV P plays in facilitating a key step of the viral life cycle and reveals the potential role for phosphorylation in regulating the function of this significant viral protein. IMPORTANCE Human metapneumovirus (HMPV) infects global populations, with severe respiratory tract infections occurring in infants, the elderly, and the immunocompromised. There are currently no FDA-approved therapeutics available to prevent or treat HMPV infection. Therefore, understanding how HMPV replicates is vital for the identification of novel targets for therapeutic development. During HMPV infection, viral RNA synthesis proteins localize to membraneless structures called inclusion bodies (IBs), which are sites of genome replication and transcription. The HMPV phosphoprotein (P) is necessary for IBs to form and for the virus to synthesize RNA, but it is not known how this protein contributes to IB formation or if it is capable of regulating viral replication. We show that the C-terminal domain of P is the location of a molecular interaction driving IB formation and contains potential phosphorylation sites where amino acid charge regulates the function of the viral polymerase complex.

Genomic analysis of canine pneumoviruses and canine respiratory coronavirus from New Zealand.

AIMS: To isolate canine respiratory coronavirus (CRCoV) and canine pneumovirus (CnPnV) in cell culture and to compare partial genomic sequences of CRCoV and CnPnV from New Zealand with those from other countries. METHODS: Oropharyngeal swab samples from dogs affected by canine infectious respiratory disease syndrome that were positive for CnPnV (n = 15) or CRCoV (n = 1) by virus-specific reverse transcriptase quantitative PCR (RT-qPCR) in a previous study comprised the starting material. Virus isolation was performed in HRT-18 cells for CRCoV and RAW 264.7 and Vero cells for CnPnV. The entire sequence of CnPnV G protein (1,266 nucleotides) and most (8,063/9,707 nucleotides) of the 3' region of CRCoV that codes for 10 structural and accessory proteins were amplified and sequenced. The sequences were analysed and compared with other sequences available in GenBank using standard molecular tools including phylogenetic analysis. RESULTS: Virus isolation was unsuccessful for both CRCoV and CnPnV. Pneumovirus G protein was amplified from 3/15 (20%) samples that were positive for CnPnV RNA by RT-qPCR. Two of these (NZ-048 and NZ-049) were 100% identical to each other, and 90.9% identical to the third one (NZ-007). Based on phylogenetic analysis of the G protein gene, CnPnV NZ-048 and NZ-049 clustered with sequences from the USA, Thailand and Italy in group A, and CnPnV NZ-007 clustered with sequences from the USA in group B. The characteristics of the predicted genes (length, position) and their putative protein products (size, predicted structure, presence of N- and O-glycosylation sites) of the New Zealand CRCoV sequence were consistent with those reported previously, except for the region located between open reading frame (ORF)3 (coding for S protein) and ORF6 (coding for E protein). The New Zealand virus was predicted to encode 5.9 kDa, 27 kDa and 12.7 kDa proteins, which differed from the putative coding capacity of this region reported for CRCoV from other countries. CONCLUSIONS: This report represents the first characterisation of partial genomic sequences of CRCoV and CnPnV from New Zealand. Our results suggest that the population of CnPnV circulating in New Zealand is not homogeneous, and that the viruses from two clades described overseas are also present here. Limited conclusions can be made based on only one CRCoV sequence, but the putative differences in the coding capacity of New Zealand CRCoV support the previously reported variability of this region. The reasons for such variability and its biological implications need to be further elucidated.

Respiratory Syncytial Virus, Human Metapneumovirus, and Parainfluenza Virus Infections in Lung Transplant Recipients: A Systematic Review of Outcomes and Treatment Strategies.

BACKGROUND: Respiratory syncytial virus (RSV), parainfluenza virus (PIV), and human metapneumovirus (hMPV) are increasingly associated with chronic lung allograft dysfunction (CLAD) in lung transplant recipients (LTR). This systematic review primarily aimed to assess outcomes of RSV/PIV/hMPV infections in LTR and secondarily to assess evidence regarding the efficacy of ribavirin. METHODS: Relevant databases were queried and study outcomes extracted using a standardized method and summarized. RESULTS: Nineteen retrospective and 12 prospective studies were included (total 1060 cases). Pooled 30-day mortality was low (0-3%), but CLAD progression 180-360 days postinfection was substantial (pooled incidences 19-24%) and probably associated with severe infection. Ribavirin trended toward effectiveness for CLAD prevention in exploratory meta-analysis (odds ratio [OR] 0.61, [0.27-1.18]), although results were highly variable between studies. CONCLUSIONS: RSV/PIV/hMPV infection was followed by a high CLAD incidence. Treatment options, including ribavirin, are limited. There is an urgent need for high-quality studies to provide better treatment options for these infections.

A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein.

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 µM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection. We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

Chloroquine and pyrimethamine inhibit the replication of human respiratory syncytial virus A.

Human respiratory syncytial virus (hRSV) is a major cause of respiratory illness in young children and can cause severe infections in the elderly or in immunocompromised adults. To date, there is no vaccine to prevent hRSV infections, and disease management is limited to preventive care by palivizumab in infants and supportive care for adults. Intervention with small-molecule antivirals specific for hRSV represents a good alternative, but no such compounds are currently approved. The investigation of existing drugs for new therapeutic purposes (drug repositioning) can be a faster approach to address this issue. In this study, we show that chloroquine and pyrimethamine inhibit the replication of human respiratory syncytial virus A (long strain) and synergistically increase the anti-replicative effect of ribavirin in cellulo. Moreover, chloroquine, but not pyrimethamine, inhibits hRSV replication in the mouse model. Our results show that chloroquine can potentially be an interesting compound for treatment of hRSV infection in monotherapy or in combination with other antivirals.

Respiratory Syncytial Virus Infection Promotes Necroptosis and HMGB1 Release by Airway Epithelial Cells.

Rationale: Respiratory syncytial virus (RSV) bronchiolitis causes significant infant mortality. Bronchiolitis is characterized by airway epithelial cell (AEC) death; however, the mode of death remains unknown.Objectives: To determine whether necroptosis contributes to RSV bronchiolitis pathogenesis via HMGB1 (high mobility group box 1) release.Methods: Nasopharyngeal samples were collected from children presenting to the hospital with acute respiratory infection. Primary human AECs and neonatal mice were inoculated with RSV and murine Pneumovirus, respectively. Necroptosis was determined via viability assays and immunohistochemistry for RIPK1 (receptor-interacting protein kinase-1), MLKL (mixed lineage kinase domain-like pseudokinase) protein, and caspase-3. Necroptosis was blocked using pharmacological inhibitors and RIPK1 kinase-dead knockin mice.Measurements and Main Results: HMGB1 levels were elevated in nasopharyngeal samples of children with acute RSV infection. RSV-induced epithelial cell death was associated with increased phosphorylated RIPK1 and phosphorylated MLKL but not active caspase-3 expression. Inhibition of RIPK1 or MLKL attenuated RSV-induced HMGB1 translocation and release, and lowered viral load. MLKL inhibition increased active caspase-3 expression in a caspase-8/9-dependent manner. In susceptible mice, Pneumovirus infection upregulated RIPK1 and MLKL expression in the airway epithelium at 8 to 10 days after infection, coinciding with AEC sloughing, HMGB1 release, and neutrophilic inflammation. Genetic or pharmacological inhibition of RIPK1 or MLKL attenuated these pathologies, lowered viral load, and prevented type 2 inflammation and airway remodeling. Necroptosis inhibition in early life ameliorated asthma progression induced by viral or allergen challenge in later life.Conclusions: Pneumovirus infection induces AEC necroptosis. Inhibition of necroptosis may be a viable strategy to limit the severity of viral bronchiolitis and break its nexus with asthma.

A molecular survey of canine respiratory viruses in New Zealand.

AIMS: The aim of this study was to identify viruses associated with canine infectious respiratory disease syndrome (CIRDS) among a population of New Zealand dogs. METHODS: Convenience samples of oropharyngeal swabs were collected from 116 dogs, including 56 CIRDS-affected and 60 healthy dogs from various locations in New Zealand between March 2014 and February 2016. Pooled samples from CIRDS-affected (n = 50) and from healthy (n = 50) dogs were tested for the presence of canine respiratory viruses using next generation sequencing (NGS). Individual samples (n = 116) were then tested by quantitative PCR (qPCR) and reverse transcriptase qPCR (RT-qPCR) for specific viruses. Groups were compared using Fisher's exact or χ2 tests. The effect of explanatory variables (age, sex, type of household, presence of viral infection) on the response variable (CIRDS-affected or not) was tested using RR. RESULTS: Canine pneumovirus (CnPnV), canine respiratory coronavirus (CRCoV), canine herpesvirus-1 (CHV-1), canine picornavirus and influenza C virus sequences were identified by NGS in the pooled sample from CIRDS-affected but not healthy dogs. At least one virus was detected by qPCR/RT-qPCR in 20/56 (36%) samples from CIRDS dogs and in 23/60 (38%) samples from healthy dogs (p = 0.84). CIRDS-affected dogs were most commonly positive for CnPnV (14/56, 25%) followed by canine adenovirus-2 (CAdV-2, 5/56, 9%), canine parainfluenza virus (CpiV) and CHV-1 (2/56, 4% each), and CRCoV (1/56, 2%). Only CnPnV (17/60, 28%) and CAdV-2 (14/60, 23%) were identified in samples from healthy dogs, and CAdV-2 was more likely to be detected healthy than diseased dogs (RR 0.38; 95% CI = 0.15-0.99; p = 0.045). CONCLUSIONS: The frequency of detection of viruses traditionally linked to CIRDS (CAdV-2 and CPiV) among diseased dogs was low. This suggests that other pathogens are likely to have contributed to development of CIRDS among sampled dogs. Our data represent the first detection of CnPnV in New Zealand, but the role of this virus in CIRDS remains unclear. On-going monitoring of canine respiratory pathogens by NGS would be beneficial, as it allows rapid detection of novel viruses that may be introduced to the New Zealand canine population in the future. Such monitoring could be done using pooled samples to minimise costs. CLINICAL RELEVANCE: Testing for novel respiratory viruses such as CnPnV and CRCoV should be considered in all routine laboratory investigations of CIRDS cases, particularly in dogs vaccinated with currently available kennel cough vaccines.

Interferon-Induced Transmembrane Protein 1 Restricts Replication of Viruses That Enter Cells via the Plasma Membrane.

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1-/- mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization.IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, and Ifitm1-/- mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections.

Phylogenetic evidence of a novel lineage of canine pneumovirus and a naturally recombinant strain isolated from dogs with respiratory illness in Thailand.

BACKGROUND: Canine pneumovirus (CPV) is a pathogen that causes respiratory disease in dogs, and recent outbreaks in shelters in America and Europe have been reported. However, based on published data and documents, the identification of CPV and its variant in clinically symptomatic individual dogs in Thailand through Asia is limited. Therefore, the aims of this study were to determine the emergence of CPV and to consequently establish the genetic characterization and phylogenetic analysis of the CPV strains from 209 dogs showing respiratory distress in Thailand. RESULTS: This study identified and described the full-length CPV genome from three strains, designated herein as CPV_CP13 TH/2015, CPV_CP82 TH/2016 and CPV_SR1 TH/2016, that were isolated from six dogs out of 209 dogs (2.9%) with respiratory illness in Thailand. Phylogenetic analysis suggested that these three Thai CPV strains (CPV TH strains) belong to the CPV subgroup A and form a novel lineage; proposed as the Asian prototype. Specific mutations in the deduced amino acids of these CPV TH strains were found in the G/glycoprotein sequence, suggesting potential substitution sites for subtype classification. Results of intragenic recombination analysis revealed that CPV_CP82 TH/2016 is a recombinant strain, where the recombination event occurred in the L gene with the Italian prototype CPV Bari/100-12 as the putative major parent. Selective pressure analysis demonstrated that the majority of the nucleotides in the G/glycoprotein were under purifying selection with evidence of positive selection sites. CONCLUSIONS: This collective information on the CPV TH strains is the first evidence of CPV emergence with genetic characterization in Thailand and as first report in Asia, where homologous recombination acts as a potential force driving the genetic diversity and shaping the evolution of canine pneumovirus.

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications.IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets.

The nonstructural proteins of Pneumoviruses are remarkably distinct in substrate diversity and specificity.

BACKGROUND: Interferon (IFN) inhibits viruses by inducing several hundred cellular genes, aptly named 'interferon (IFN)-stimulated genes' (ISGs). The only two RNA viruses of the Pneumovirus genus of the Paramyxoviridae family, namely Respiratory Syncytial Virus (RSV) and Pneumonia Virus of Mice (PVM), each encode two nonstructural (NS) proteins that share no sequence similarity but yet suppress IFN. Since suppression of IFN underlies the ability of these viruses to replicate in the host cells, the mechanism of such suppression has become an important area of research. This Short Report is an important extension of our previous efforts in defining this mechanism. RESULTS: We show that, like their PVM counterparts, the RSV NS proteins also target multiple members of the ISG family. While significantly extending the substrate repertoire of the RSV NS proteins, these results, unexpectedly, also reveal that the target preferences of the NS proteins of the two viruses are entirely different. This is surprising since the two Pneumoviruses are phylogenetically close with similar genome organization and gene function, and the NS proteins of both also serve as suppressors of host IFN response. CONCLUSION: The finding that the NS proteins of the two highly similar viruses suppress entirely different members of the ISG family raises intriguing questions of pneumoviral NS evolution and mechanism of action.

Neutrophil extracellular traps cause airway obstruction during respiratory syncytial virus disease.

Human respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract disease (LRTD) in young children worldwide. Extensive neutrophil accumulation in the lungs and occlusion of small airways by DNA-rich mucus plugs are characteristic features of severe RSV-LRTD. Activated neutrophils can release neutrophil extracellular traps (NETs), extracellular networks of DNA covered with antimicrobial proteins, as part of the first-line defence against pathogens. NETs can trap and eliminate microbes; however, abundant NET formation may also contribute to airway occlusion. In this study, we investigated whether NETs are induced by RSV and explored their potential anti-viral effect in vitro. Second, we studied NET formation in vivo during severe RSV-LRTD in infants and bovine RSV-LRTD in calves, by examining bronchoalveolar lavage fluid and lung tissue sections, respectively. NETs were visualized in lung cytology and tissue samples by DNA and immunostaining, using antibodies against citrullinated histone H3, elastase and myeloperoxidase. RSV was able to induce NET formation by human neutrophils in vitro. Furthermore, NETs were able to capture RSV, thereby precluding binding of viral particles to target cells and preventing infection. Evidence for the formation of NETs in the airways and lungs was confirmed in children with severe RSV-LRTD. Detailed histopathological examination of calves with RSV-LRTD showed extensive NET formation in dense plugs occluding the airways, either with or without captured viral antigen. Together, these results suggest that, although NETs trap viral particles, their exaggerated formation during severe RSV-LRTD contributes to airway obstruction.

Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization.

Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32 °C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negative-strand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.

Coding-complete genome sequences of two strains of canine pneumovirus derived from dogs with upper respiratory disease in the United States.

Canine pneumovirus was detected by RT-qPCR in 2022 from nasal swabs collected from two dogs with upper respiratory disease in a shelter in Louisiana, United States. The genomes from the designated strains CPnV USA/LA/2022/124423 and USA/LA/2022/123696 were sequenced and show the closest similarity to the pneumonia virus of mice J3666.

Imaging analysis reveals budding of filamentous human metapneumovirus virions and direct transfer of inclusion bodies through intercellular extensions.

IMPORTANCE: Human metapneumovirus is an important respiratory pathogen that causes significant morbidity and mortality, particularly in the very young, the elderly, and the immunosuppressed. However, the molecular details of how this virus spreads to new target cells are unclear. This work provides important new information on the formation of filamentous structures that are consistent with virus particles and adds critical new insight into the structure of extensions between cells that form during infection. In addition, it demonstrates for the first time the movement of viral replication centers through these intercellular extensions, representing a new mode of direct cell-to-cell spread that may be applicable to other viral systems.

Structures and Mechanisms of Nonsegmented, Negative-Strand RNA Virus Polymerases.

The nonsegmented, negative-strand RNA viruses (nsNSVs), also known as the order Mononegavirales, have a genome consisting of a single strand of negative-sense RNA. Integral to the nsNSV replication cycle is the viral polymerase, which is responsible for transcribing the viral genome, to produce an array of capped and polyadenylated messenger RNAs, and replicating it to produce new genomes. To perform the different steps that are necessary for these processes, the nsNSV polymerases undergo a series of coordinated conformational transitions. While much is still to be learned regarding the intersection of nsNSV polymerase dynamics, structure, and function, recently published polymerase structures, combined with a history of biochemical and molecular biology studies, have provided new insights into how nsNSV polymerases function as dynamic machines. In this review, we consider each of the steps involved in nsNSV transcription and replication and suggest how these relate to solved polymerase structures.

First Detection and Genetic Characterization of Swine Orthopneumovirus from Domestic Pig Farms in the Republic of Korea.

Novel swine orthopneumovirus (SOV) infections have been identified in pigs in the USA and some European countries but not in Asian countries, including South Korea, to date. The current study reports the first SOV infections in four domestic pig farms located in four provinces across South Korea. The detection rate of SOV in oral fluid samples using qRT-PCR was 4.4% (14/389), indicating the presence of the virus in pigs at commercial farms in Korea. Two complete genome sequences and one glycoprotein (G) gene sequence were obtained from SOV-positive samples. The complete genome analysis of KSOV-2201 and KSOV-2202 strains showed 98.2 and 95.4% homologies with a previously reported SOV, and the phylogenetic tree exhibited a high correlation with a previously reported SOV strain from the US and a canine pneumovirus (CPnV) strain from China. Based on the genetic analysis of the viral G gene, the murine pneumonia virus (MPV)-like orthopneumoviruses (MLOVs) were divided into two genogroups (G1 and G2). Seventeen CPnVs and two feline pneumoviruses were grouped into G1, while the Korean SOV strains identified in this study were grouped into G2 along with one SOV and two CPnVs. These results will contribute to expanding our understanding of the geographical distribution and genetic characteristics of the novel SOV in the global pig population.

The HRA2pl fusion peptide exerts in vitro antiviral activity against human respiratory paramyxoviruses and pneumoviruses.

Acute respiratory infections are a group of diseases caused by viruses, bacteria, and parasites that mainly affect children until the age of 5 and immunocompromised senior adults. In Mexico, these infections are the main cause of morbidity in children, with more than 26 million cases of respiratory infections reported by the Secretariat of Health, in 2019. The human respiratory syncytial virus (hRSV), the human metapneumovirus (hMPV), and the human parainfluenza-2 (hPIV-2) are responsible for many respiratory infections. Currently, palivizumab, a monoclonal antibody against the fusion protein F, is the treatment of choice against hRSV infections. This protein is being studied for the design of antiviral peptides that act by inhibiting the fusion of the virus and the host cell. Therefore, we examined the antiviral activity of the HRA2pl peptide, which competes the heptad repeat A domain of the F protein of hMPV. The recombinant peptide was obtained using a viral transient expression system. The effect of the fusion peptide was evaluated with an in vitro entry assay. Moreover, the effectiveness of HRA2pl was examined in viral isolates from clinical samples obtained from patients with infections caused by hRSV, hMPV, or hPIV-2, by evaluating the viral titer and the syncytium size. The HRA2pl peptide affected the viruses' capacity of entry, resulting in a 4-log decrease in the viral titer compared to the untreated viral strains. Additionally, a 50% reduction in the size of the syncytium was found. These results demonstrate the antiviral potential of HRA2pl in clinical samples, paving the way toward clinical trials.

Enhanced broad spectrum in vitro antiviral efficacy of 3-F-4-MeO-Bn, 3-CN, and 4-CN derivatives of lipid remdesivir nucleoside monophosphate prodrugs.

Broad spectrum oral antivirals are urgently needed for the early treatment of many RNA viruses of clinical concern. We previously described the synthesis of 1-O-octadecyl-2-O-benzyl-glycero-3-phospho-RVn (V2043), an orally bioavailable lipid prodrug of remdesivir nucleoside (RVn, GS-441524) with broad spectrum antiviral activity against viruses with pandemic potential. Here we compared the relative activity of V2043 with new RVn lipid prodrugs containing sn-1 alkyl ether or sn-2 glycerol modifications. We found that 3-F-4-MeO-Bn, 3-CN-Bn, and 4-CN-Bn sn-2 glycerol modifications improved antiviral activity compared to V2043 when tested in vitro against clinically important RNA viruses from 5 virus families. These results support the continued development of V2043 and sn-2 glycerol modified RVn lipid prodrugs for the treatment of a broad range of RNA viruses for which there are limited therapies.