Phylogenetic analysis of the promoter element 2 of paramyxo- and filoviruses.

Paramyxo- and filovirus genomes are equipped with bipartite promoters at their 3' ends to initiate RNA synthesis. The two elements, the primary promoter element 1 (PE1) and the secondary promoter element 2 (PE2), are separated by a spacer region that must be precisely a multiple of 6 nucleotides (nts), indicating these viruses adhere to the "rule of six." However, our knowledge of PE2 has been limited to a narrow spectrum of virus species. In this study, a comparative analysis of 1,647 paramyxoviral genomes from a public database revealed that the paramyxovirus PE2 can be clearly categorized into two distinct subcategories: one marked by C repeats at every six bases (exclusive to the subfamily Orthoparamyxovirinae) and another characterized by CG repeats every 6 nts (observed in the subfamilies Avulavirinae and Rubulavirinae). This unique pattern collectively mirrors the evolutionary lineage of these subfamilies. Furthermore, we showed that PE2 of the Rubulavirinae, with the exception of mumps virus, serves as part of the gene-coding region. This may be due to the fact that the Rubulavirinae are the only paramyxoviruses that cannot propagate without RNA editing. Filoviruses have three to eight consecutive uracil repeats every six bases (UN5) in PE2, which is located in the 3' end region of the genome. We obtained PE2 sequences from 2,195 filoviruses in a public database and analyzed the sequence conservation among virus species. Our results indicate that the continuity of UN5 hexamers is consistently maintained with a high degree of conservation across virus species. IMPORTANCE: The genomic intricacies of paramyxo- and filoviruses are highlighted by the bipartite promoters-promoter element 1 (PE1) and promoter element 2 (PE2)-at their 3' termini. The spacer region between these elements follows the "rule of six," crucial for genome replication. By a comprehensive analysis of paramyxoviral genome sequences, we identified distinct subcategories of PE2 based on C and CG repeats that were specific to Orthoparamyxovirinae and Avulavirinae/Rubulavirinae, respectively, mirroring their evolutionary lineages. Notably, the PE2 of Rubulavirinae is integrated into the gene-coding region, a unique trait potentially linked to its strict dependence on RNA editing for virus growth. This study also focused on the PE2 sequences in filovirus genomes. The strict conservation of the continuity of UN5 among virus species emphasizes its crucial role in viral genome replication.

Reverse Genetics Systems for the De Novo Rescue of Diverse Members of Paramyxoviridae.

Paramyxoviruses place significant burdens on both human and wildlife health; while some paramyxoviruses are established within human populations, others circulate within diverse animal reservoirs. Concerningly, bat-borne paramyxoviruses have spilled over into humans with increasing frequency in recent years, resulting in severe disease. The risk of future zoonotic outbreaks, as well as the persistence of paramyxoviruses that currently circulate within humans, highlights the need for efficient tools through which to interrogate paramyxovirus biology. Reverse genetics systems provide scientists with the ability to rescue paramyxoviruses de novo, offering versatile tools for implementation in both research and public health settings. Reverse genetics systems have greatly improved over the past 30 years, with several key innovations optimizing the success of paramyxovirus rescue. Here, we describe the significance of such advances and provide a generally applicable guide for the development and use of reverse genetics systems for the rescue of diverse members of Paramyxoviridae.

Global genetic diversity of measles virus (Paramyxoviridae: Morbillivirus: Morbillivirus hominis): historical aspects and current state.

Monitoring the circulation of the measles virus and studying its genetic diversity is an important component of the measles elimination program. A methodological approach to molecular genetic studies and their interpretation in the measles surveillance was developed in the early 2000s. During its development, clear areas of circulation of each genotype of the virus were identified, therefore, the determination of viruses' genotypes was proposed to monitor circulation and identify transmission pathways. However, in the future, due to a significant decrease in the number of active genotypes, an approach based on sub-genotyping was proposed: determining not only the genotype of the virus, but also its genetic lineage/genetic variant. The Global Measles and Rubella Laboratory Network (GMRLN) systematically monitors the circulation of the measles virus at the sub-genotypic level, depositing the results in a specialized database MeaNS2. It is this database that is the most complete and reliable source of information about the genetic characteristic of measles viruses. This review presents both historical information and the latest data on the global genetic diversity of the measles virus.

Regulation of host gene expression by J paramyxovirus.

Paramyxoviruses are negative-sense, single-stranded RNA viruses that are associated with numerous diseases in humans and animals. J paramyxovirus (JPV) was first isolated from moribund mice (Mus musculus) with hemorrhagic lung lesions in Australia in 1972. In 2016, JPV was classified into the newly established genus Jeilongvirus. Novel jeilongviruses are being discovered worldwide in wildlife populations. However, the effects of jeilongvirus infection on host gene expression remains uncharacterized. To address this, cellular RNA from JPV-infected mouse fibroblasts was collected at 2, 4, 8, 12, 16, 24, and 48 hours post-infection (hpi) and were sequenced using single-end 75 base pairs (SE75) sequencing chemistry on an Illumina NextSeq platform. Differentially expressed genes (DEGs) between the virus-infected replicates and mock replicates at each timepoint were identified using the Tophat2-Cufflinks-Cuffdiff protocol. At 2 hpi, 11 DEGs were identified in JPV-infected cells, while 1,837 DEGs were detected at 48 hpi. A GO analysis determined that the genes at the earlier timepoints were involved in interferon responses, while there was a shift towards genes that are involved in antigen processing and presentation processes at the later timepoints. At 48 hpi, a KEGG analysis revealed that many of the DEGs detected were involved in pathways that are important for immune responses. qRT-PCR verified that Rtp4, Ifit3, Mx2, and Stat2 were all upregulated during JPV infection, while G0s2 was downregulated. After JPV infection, the expression of inflammatory and antiviral factors in mouse fibroblasts changes significantly. This study provides crucial insight into the different arms of host immunity that mediate Jeilongvirus infection. Understanding the pathogenic mechanisms of Jeilongvirus will lead to better strategies for the prevention and control of potential diseases that may arise from this group of viruses.

RNA sequencing of avian paramyxovirus (Paramyxoviridae, Avulavirinae) isolates from wild mallards in Belgium, 2021: complete genomes and coinfections.

We used untargeted RNA sequencing to characterize three Avulavirinae isolates from pooled samples obtained from wild mallards in Belgium in 2021. The complete genome sequences of two avian Orthoavulavirus-1 (AOAV-1) strains and one avian Paraavulavirus-4 (APMV-4) strain were determined confirming hemagglutination inhibition testing of the virus isolates. In addition, the applied sequencing strategy identified an avian influenza virus (AIV) coinfection in all three virus isolates, confirming weak-positive AIV realtime RT-PCR results from the original sample material. In one AOAV-1 isolate, partial sequences covering all genome segments of an AIV of subtype H11N9 could be de novo assembled from the sequencing data. Besides an AIV coinfection, RNA metagenomic data from the APMV-4 isolate also showed evidence of Alpharetrovirus and Megrivirus coinfection. In total, two AOAV-1 of Class II, genotype I.2 and one APMV-4 complete genome sequences were assembled and compared to publicly available sequences, highlighting the importance of surveillance for poultry pathogens in wild birds. Beyond the insights from full genome characterization of virus isolates, untargeted RNA sequencing strategies provide additional insights in the RNA virome of clinical samples as well as their derived virus isolates that are particularly useful when targeting wild avifauna reservoirs of poultry pathogens.

[Intranasal vaccine against COVID-19 based on a recombinant variant of the Sendai virus (Paramyxoviridae: Respirovirus) strain Moscow].

INTRODUCTION: Intranasal vaccination using live vector vaccines based on non-pathogenic or slightly pathogenic viruses is the one of the most convenient, safe and effective ways to prevent respiratory infections, including COVID-19. Sendai virus is the best suited for this purpose, since it is respiratory virus and is capable of limited replication in human bronchial epithelial cells without causing disease. The aim of the work is to design and study the vaccine properties of recombinant Sendai virus, Moscow strain, expressing secreted receptor-binding domain of SARS-CoV-2 Delta strain S protein (RBDdelta) during a single intranasal immunization. MATERIALS AND METHODS: Recombinant Sendai virus carrying insertion of RBDdelta transgene between P and M genes was constructed using reverse genetics and synthetic biology methods. Expression of RBDdelta was analyzed by Western blot. Vaccine properties were studied in two models: Syrian hamsters and BALB/c mice. Immunogenicity was evaluated by ELISA and virus-neutralization assays. Protectiveness was assessed by quantitation of SARS-CoV-2 RNA in RT-PCR and histological analysis of the lungs. RESULTS: Based on Sendai virus Moscow strain, a recombinant Sen-RBDdelta(M) was constructed that expressed a secreted RBDdelta immunologically identical to natural SARS-CoV-2 protein. A single intranasal administration of Sen-RBDdelta(M) to hamsters and mice significantly, by 15 and 107 times, respectively, reduced replicative activity of SARS-CoV-2 in lungs of animals, preventing the development of pneumonia. An effective induction of virus-neutralizing antibodies has also been demonstrated in mice. CONCLUSION: Sen-RBDdelta(M) is a promising vaccine construct against SARS-CoV-2 infection and has a protective properties even after a single intranasal introduction.

Genomic Characterization of a Relative of Mumps Virus in Lesser Dawn Bats of Southeast Asia.

The importance of genomic surveillance on emerging diseases continues to be highlighted with the ongoing SARS-CoV-2 pandemic. Here, we present an analysis of a new bat-borne mumps virus (MuV) in a captive colony of lesser dawn bats (Eonycteris spelaea). This report describes an investigation of MuV-specific data originally collected as part of a longitudinal virome study of apparently healthy, captive lesser dawn bats in Southeast Asia (BioProject ID PRJNA561193) which was the first report of a MuV-like virus, named dawn bat paramyxovirus (DbPV), in bats outside of Africa. More in-depth analysis of these original RNA sequences in the current report reveals that the new DbPV genome shares only 86% amino acid identity with the RNA-dependent RNA polymerase of its closest relative, the African bat-borne mumps virus (AbMuV). While there is no obvious immediate cause for concern, it is important to continue investigating and monitoring bat-borne MuVs to determine the risk of human infection.

Discovery and characterization of novel paramyxoviruses from bat samples in China.

Many paramyxoviruses are responsible for a variety of mild to severe human and animal diseases. Based on the novel discoveries over the past several decades, the family Paramyxoviridae infecting various hosts across the world includes 4 subfamilies, 17 classified genera and 78 species now. However, no systematic surveys of bat paramyxoviruses are available from the Chinese mainland. In this study, 13,064 samples from 54 bat species were collected and a comprehensive paramyxovirus survey was conducted. We obtained 94 new genome sequences distributed across paramyxoviruses from 22 bat species in seven provinces. Bayesian phylodynamic and phylogenetic analyses showed that there were four different lineages in the Jeilongvirus genus. Based on available data, results of host and region switches showed that the bat colony was partial to interior, whereas the rodent colony was exported, and the felines and hedgehogs were most likely the intermediate hosts from Scotophilus spp. rather than rodents. Based on the evolutionary trend, genus Jeilongvirus may have originated from Mus spp. in Australia, then transmitted to bats and rodents in Africa, Asia and Europe, and finally to bats and rodents in America.

Classification of new morbillivirus and jeilongvirus sequences from bats sampled in Brazil and Malaysia.

As part of a broad One Health surveillance effort to detect novel viruses in wildlife and people, we report several paramyxovirus sequences sampled primarily from bats during 2013 and 2014 in Brazil and Malaysia, including seven from which we recovered full-length genomes. Of these, six represent the first full-length paramyxovirid genomes sequenced from the Americas, including two that are the first full-length bat morbillivirus genome sequences published to date. Our findings add to the vast number of viral sequences in public repositories, which have been increasing considerably in recent years due to the rising accessibility of metagenomics. Taxonomic classification of these sequences in the absence of phenotypic data has been a significant challenge, particularly in the subfamily Orthoparamyxovirinae, where the rate of discovery of novel sequences has been substantial. Using pairwise amino acid sequence classification (PAASC), we propose that five of these sequences belong to members of the genus Jeilongvirus and two belong to members of the genus Morbillivirus. We also highlight inconsistencies in the classification of Tupaia virus and Mòjiang virus using the same demarcation criteria and suggest reclassification of these viruses into new genera. Importantly, this study underscores the critical importance of sequence length in PAASC analysis as well as the importance of biological characteristics such as genome organization in the taxonomic classification of viral sequences.

Type I and Type II Interferon Antagonism Strategies Used by Paramyxoviridae: Previous and New Discoveries, in Comparison.

Paramyxoviridae is a viral family within the order of Mononegavirales; they are negative single-strand RNA viruses that can cause significant diseases in both humans and animals. In order to replicate, paramyxoviruses-as any other viruses-have to bypass an important protective mechanism developed by the host's cells: the defensive line driven by interferon. Once the viruses are recognized, the cells start the production of type I and type III interferons, which leads to the activation of hundreds of genes, many of which encode proteins with the specific function to reduce viral replication. Type II interferon is produced by active immune cells through a different signaling pathway, and activates a diverse range of genes with the same objective to block viral replication. As a result of this selective pressure, viruses have evolved different strategies to avoid the defensive function of interferons. The strategies employed by the different viral species to fight the interferon system include a number of sophisticated mechanisms. Here we analyzed the current status of the various strategies used by paramyxoviruses to subvert type I, II, and III interferon responses.

Discovery and Evolutionary Analysis of a Novel Bat-Borne Paramyxovirus.

Paramyxoviruses are a group of RNA viruses, such as mumps virus, measles virus, Nipah virus, Hendra virus, Newcastle disease virus, and parainfluenza virus, usually transmitted by airborne droplets that are predominantly responsible for acute respiratory diseases. In this paper, we identified a novel paramyxovirus belonging to genus Jeilongvirus infecting 4/112 (3.6%) bats from two trapping sites of Hainan Province of China. In these animals, the viral RNA was detected exclusively in kidney tissues. This is the first full-length Jeilongvirus genome (18,095 nucleotides) from bats of genus Hipposideros, which exhibits a canonical genome organization and encodes SH and TM proteins. Results, based on phylogenic analysis and genetic distances, indicate that the novel paramyxovirus formed an independent lineage belonging to genus Jeilongvirus, representing, thus, a novel species. In addition, the virus-host macro-evolutionary analysis revealed that host-switching was not only a common co-phylogenetic event, but also a potential mechanism by which rats are infected by bat-origin Jeilongvirus through cross-species virus transmission, indicating a bat origin of the genus Jeilongvirus. Overall, our study broadens the viral diversity, geographical distribution, host range, and evolution of genus Jeilongvirus.

Detection of novel paramyxoviruses in Chaerephon bat species in Nigeria and phylogenetics of paramyxoviruses co-evolution with bats in Africa.

Bat paramyxoviruses (PmV) are a diverse group of viruses and include zoonotic viruses such as henipaviruses. Members of this group in other continents have been associated with severe respiratory and neurological infections in animals and humans. Furthermore, despite the richness of diverse bat species that can transmit this virus in African countries like Nigeria, there is very scanty information as to the presence and co-evolution of paramyxoviruses in bats. There is a need for continuous surveillance of zoonotic viruses and their biological reservoirs as this will help in the prevention and management of pathogens' spillovers. This study detected novel paramyxoviruses in Chaerephon nigeriae bat species found in Badagry, Lagos. Phylogenetic analyses of paramyxovirus sequences' co-evolution with frugivorous and insectivorous bats circulating in African countries were also performed using sequences of African origin available in the Database of Bat-Associated Viruses (DBatVir: http://www.mgc.ac.cn/DBatVir/). Oral swabs (n = 18) and blood samples (n = 32) were collected from C. nigeriae bats in Badagry, Lagos. The L gene of bat paramyxovirus was detected in all oral swabs using PCR techniques. Six of the amplicons were successfully sequenced. Estimated phylogenies placed the sequences in close relationship with those isolated from insectivorous bats. Phylogenetic analyses of previously sequenced isolates in the African region showed the likelihood of different co-evolution mechanisms of paramyxoviruses with frugivorous bats compared with insectivorous bats. This may be due to codon usage bias of the L gene. Spatial distribution of paramyxoviruses in African countries showed limited ongoing surveillance of this virus in the continent, especially in southern and northern countries. Extensive surveillance of paramyxoviruses with possible zoonotic potentials among bat species in the continent is recommended. This will provide further insights into co-evolution as well as prevent possible spillover into the human population.

Evolution and Diversity of Bat and Rodent Paramyxoviruses from North America.

Paramyxoviruses are a diverse group of negative-sense, single-stranded RNA viruses of which several species cause significant mortality and morbidity. In recent years the collection of paramyxovirus sequences detected in wild mammals has substantially grown; however, little is known about paramyxovirus diversity in North American mammals. To better understand natural paramyxovirus diversity, host range, and host specificity, we sought to comprehensively characterize paramyxoviruses across a range of diverse cooccurring wild small mammals in southern Arizona. We used highly degenerate primers to screen fecal and urine samples and obtained a total of 55 paramyxovirus sequences from 12 rodent species and 6 bat species. We also performed Illumina transcriptome sequencing (RNA-seq) and de novo assembly on 14 of the positive samples to recover a total of 5 near-full-length viral genomes. We show there are at least two clades of rodent-borne paramyxoviruses in Arizona, while bat-associated paramyxoviruses formed a putative single clade. Using structural homology modeling of the viral attachment protein, we infer that three of the five novel viruses likely bind sialic acid in a manner similar to other respiroviruses, while the other two viruses from heteromyid rodents likely bind a novel host receptor. We find no evidence for cross-species transmission, even among closely related sympatric host species. Taken together, these data suggest paramyxoviruses are a common viral infection in some bat and rodent species present in North America and illuminate the evolution of these viruses. IMPORTANCE There are a number of viral lineages that are potential zoonotic threats to humans. One of these, paramyxoviruses have jumped into humans multiple times from wild and domestic animals. We conducted one of the largest viral surveys of wild mammals in the United States to better understand paramyxovirus diversity and evolution.

SARS-CoV-2 spike protein displays sequence similarities with paramyxovirus surface proteins; a bioinformatics study.

Recent emergence of SARS-CoV-2 and associated COVID-19 pandemic have posed a great challenge for the scientific community. In this study, we performed bioinformatic analyses on SARS-CoV-2 protein sequences, trying to unravel potential molecular similarities between this newly emerged pathogen with non-coronavirus ssRNA viruses. Comparing the proteins of SARS-CoV-2 with non-coronavirus positive and negative strand ssRNA viruses revealed multiple sequence similarities between SARS-CoV-2 and non-coronaviruses, including similarities between RNA-dependent RNA-polymerases and helicases (two highly-conserved proteins). We also observed similarities between SARS-CoV-2 surface (i.e. spike) protein with paramyxovirus fusion proteins. This similarity was restricted to a segment of spike protein S2 subunit which is involved in cell fusion. We next analyzed spike proteins from SARS-CoV-2 "variants of concern" (VOCs) and "variants of interests" (VOIs) and found that some of these variants show considerably higher spike-fusion similarity with paramyxoviruses. The 'spike-fusion' similarity was also observed for some pathogenic coronaviruses other than SARS-CoV-2. Epitope analysis using experimentally verified data deposited in Immune Epitope Database (IEDB) revealed that several B cell epitopes as well as T cell and MHC binding epitopes map within the spike-fusion similarity region. These data indicate that there might be a degree of convergent evolution between SARS-CoV-2 and paramyxovirus surface proteins which could be of pathogenic and immunological importance.

Highly Potent Host-Specific Small-Molecule Inhibitor of Paramyxovirus and Pneumovirus Replication with High Resistance Barrier.

Multiple enveloped RNA viruses of the family Paramyxoviridae and Pneumoviridae, like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine. Therefore, there is an urgent need for treatment options to counteract these viral infections. The development of antiviral drugs in general stands as a huge challenge due to the rapid emergence of viral escape mutants. Here, we disclose the discovery of a small-molecule antiviral, compound 1 (ZHAWOC9045), active against several pneumo-/paramyxoviruses, including MeV, NiV, CDV, RSV, and parainfluenza virus type 5 (PIV-5). A series of mechanistic characterizations revealed that compound 1 targets a host factor which is indispensable for viral genome replication. Drug resistance profiling against a paramyxovirus model (CDV) demonstrated no detectable adaptation despite prolonged time of investigation, thereby mitigating the rapid emergence of escape variants. Furthermore, a thorough structure-activity relationship analysis of compound 1 led to the invention of 100-times-more potent-derivatives, e.g., compound 2 (ZHAWOC21026). Collectively, we present in this study an attractive host-directed pneumoviral/paramyxoviral replication inhibitor with potential therapeutic application. IMPORTANCE Measles virus, respiratory syncytial virus, canine distemper virus, and Nipah virus are some of the clinically significant RNA viruses that threaten substantial number of lives each year. Limited to no availability of treatment options for these viral infections makes it arduous to handle the outbreaks. This highlights the major importance of developing antivirals to fight not only ongoing infections but also potential future epidemics. Most of the discovered antivirals, in clinical trials currently, are virus targeted, which consequently poses the challenge of rapid emergence of escape variants. Here, we present compound 1 (ZHAWOC9045), discovered to target viral replication in a host-dependent manner, thereby exhibiting broad-spectrum activity against several members of the family Pneumo-/Paramyxoviridae. The inability of viruses to mutate against the inhibitor mitigated the critical issue of generation of escape variants. Importantly, compound 1 was successfully optimized to a highly potent variant, compound 2 (ZHAWOC21026), with a promising profile for pharmacological intervention.

Eptesicus fuscus Orthorubulavirus, a Close Relative of Human Parainfluenza Virus 4, Discovered in a Bat in South Dakota.

Bats are a reservoir for many zoonotic viruses and host large numbers of genetically diverse species in the families Rhabdoviridae, Coronaviridae, and Paramyxoviridae. Viruses from these families have repeatedly spilled over to humans in recent decades, causing significant clinical disease and deaths. Here, metagenomic sequencing of a big brown bat (Eptesicus fuscus) submitted for rabies testing due to human exposure identified a novel paramyxovirus, Eptesicus fuscus orthorubulavirus (EfORV), in South Dakota, United States. The nearly complete 15,814-nucleotide genome shared 72% identity with that of human parainfluenza virus 4 (HPIV4), a virus that causes significant clinical disease, typically bronchiolitis and pneumonia, in children less than 2 years of age. Phylogenetic analysis confirmed a close evolutionary history between EfORV and HPIV4, reminiscent of other orthorubulaviruses with highly similar bat and mammalian species, including conspecific human and bat mumps virus, mammalian parainfluenza virus 5 and bat Alston virus, and porcine La Piedad Michoacán virus and bat Mapuera virus. These results support the idea that bats are a reservoir for diverse paramyxoviruses with closely shared evolutionary histories, compared with a number of significant human pathogens, and expand the range of bat paramyxoviruses to North America. Given the propensity of paramyxoviruses to overcome species barriers, additional surveillance and characterization of EfORV are warranted. IMPORTANCE Bats are a reservoir of large numbers of viruses. Among bat-borne zoonotic viruses, members of Coronaviridae and Paramyxoviridae have had the largest impact on human health. The repeated spillover of bat viruses to humans, often with devastating results, has led to increased surveillance and virus discovery efforts in hot spots for virus emergence, largely Asia and Africa. Apart from rabies virus, little surveillance of viruses in bats is performed in North America. Here, viral metagenomic sequencing identified a close relative to HPIV4 in a big brown bat found in a motel room in South Dakota. The virus, EfORV, was 72% identical to HPIV4, which causes clinically significant respiratory disease, mainly in children; it represents the first bat paramyxovirus identified in North America. Close genetic relationships between bat and mammalian orthorubulaviruses underscore the importance of bats as a reservoir for zoonotic viruses.

Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions.

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

Paramyxovirus-Like Particles as Protein Delivery Vehicles.

We have developed a flexible platform for delivery of proteins to target cell interiors using paramyxovirus-like particles. The key enabling feature is an appendage, 15 to 30 amino acid residues in length, that is added to cargo proteins and that induces them to bind to the viral matrix (M) protein during virus-like particle (VLP) assembly. The cargo is then incorporated within the VLPs as they bud, using the same interactions that normally direct viral genome packaging. The appendage can also serve as an epitope tag for cargo detection using a nucleocapsid (NP) protein-specific monoclonal antibody. Using this approach, we generated Renilla luciferase-loaded VLPs, green fluorescent protein-loaded VLPs, superoxide dismutase-loaded VLPs, and Cre recombinase-loaded VLPs. In each case, the VLPs could efficiently deliver their functional cargos to target cells and, in the case of Cre recombinase, to target cell nuclei. The strategy was employed using two different VLP production platforms, one based on parainfluenza virus 5 (PIV5) and the other based on Nipah virus, and in both cases efficient cargo packaging and delivery could be achieved. These findings provide a foundation for development of paramyxovirus-like particles as tools for safe and efficient delivery of therapeutic proteins to cells and tissues. IMPORTANCE Therapeutic proteins including transcription factors and genome editors have enormous clinical potential but are currently limited in part due to the challenges of safely and efficiently delivering these proteins to the interiors of target cells. Here, we have developed a new strategy for protein delivery based on manipulation of paramyxovirus genome packaging interactions.

Novel Paju Apodemus paramyxovirus 1 and 2, harbored by Apodemus agrarius in the Republic of Korea.

Paramyxoviruses harbored by multiple natural reservoirs pose a potential threat to public health. Jeilongvirus has been proposed as a novel paramyxovirus genus found in rodents, bats, and cats. Paramyxovirus RNA was detected in 108/824 (13.1%) Apodemus agrarius captured at 14 trapping sites in the Republic of Korea. We first present two genetically distinct novel paramyxoviruses, Paju Apodemus paramyxovirus 1 (PAPV-1) and 2 (PAPV-2). The disparity between PAPV-1 (19,716 nucleotides) and -2 (17,475 nucleotides) revealed the presence of the SH gene and length of the G gene in the genome organization. The phylogeny of PAPV-1 and -2 belonged to distinct genetic lineages of Jeilongvirus, respectively, even though these viruses were originated from A. agrarius. PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III interferons, interferon-stimulated genes, and pro-inflammatory cytokines. Therefore, this study provides insights into the molecular epidemiology, genetic diversity, and virus-host interactions of novel rodent-borne paramyxoviruses.

Epizootic reptilian ferlavirus infection in individual and multiple snake colonies with additional evidence of the virus in the male genital tract.

Reptilian ferlavirus, a pathogen of serious concern in snakes, has been reported in Western countries, but little is known about its prevalence in Thailand, where many snake breeding farms are located. In this study, we investigated the reptilian ferlavirus via swab samples derived from 49 diseased snakes and 77 healthy snakes as well as tissue samples taken from nine dead snakes from five independent snake farms. Using molecular detection, we found the ferlavirus in 8.16% of diseased snakes, but not in healthy snakes. Out of nine farmed snakes, eight snakes derived from four farms were found to be positive. Four complete genome sequences of the ferlavirus were successfully obtained and phylogenetically clustered to the highly pathogenic ferlavirus. Tissue tropism of the ferlavirus was identified in various epithelial cell types using the in situ hybridization technique. Interestingly, the hybridization signals were strongly labeled in the male genital tract. Transmission electron microscopy was used to support the ferlaviral localization in the male genital tract. This study provides the first evidence of ferlavirus localization in the male genital tract and contributes to the knowledge about ferlavirus epidemiology, indicating that there needs to be further awareness and elucidation regarding vertical transmission of reptilian ferlavirus.