Streamlined detection of Nipah virus antibodies using a split NanoLuc biosensor.

Nipah virus (NiV) is an emerging zoonotic RNA virus that can cause fatal respiratory and neurological diseases in animals and humans. Accurate NiV diagnostics and surveillance tools are crucial for the identification of acute and resolved infections and to improve our understanding of NiV transmission and circulation. Here, we have developed and validated a split NanoLuc luciferase NiV glycoprotein (G) biosensor for detecting antibodies in clinical and animal samples. This assay is performed by simply mixing reagents and measuring luminescence, which depends on the complementation of the split NanoLuc luciferase G biosensor following its binding to antibodies. This anti-NiV-G "mix-and-read" assay was validated using the WHO's first international standard for anti-NiV antibodies and more than 700 serum samples from the NiV-endemic country of Bangladesh. Anti-NiV antibodies from survivors persisted for at least 8 years according to both ⍺NiV-G mix-and-read and NiV neutralization assays. The ⍺NiV-G mix-and-read assay sensitivity (98.6%) and specificity (100%) were comparable to anti-NiV IgG ELISA performance but failed to detect anti-NiV antibodies in samples collected less than a week following the appearance of symptoms. Overall, the anti-NiV-G biosensor represents a simple, fast, and reliable tool that could support the expansion of NiV surveillance and retrospective outbreak investigations.

RNA editing sites and triplet usage in exomes of bat RNA virus genomes of the family Paramyxoviridae.

Bats contain a diverse spectrum of viral species in their bodies. The RNA virus family Paramyxoviridae tends to infect several vertebrate species, which are accountable for a variety of devastating infections in both humans and animals. Viruses of this kind include measles, mumps, and Hendra. Some synonymous codons are favoured over others in mRNAs during gene-to-protein synthesis process. Such phenomenon is termed as codon usage bias (CUB). Our research emphasized many aspects that shape the CUB of genes in the Paramyxoviridae family found in bats. Here, the nitrogenous base A occurred the most. AT was found to be abundant in the coding sequences of the Paramyxoviridae family. RSCU data revealed that A or T ending codons occurred more frequently than predicted. Furthermore, 3 overrepresented codons (CAT, AGA, and GCA) and 7 underrepresented codons (CCG, TCG, CGC, CGG, CGT, GCG and ACG) were detected in the viral genomes. Correspondence analysis, neutrality plot, and parity plots highlight the combined impact of mutational pressure and natural selection on CUB. The neutrality plot of GC12 against GC3 yielded a regression coefficient value of 0.366, indicating that natural selection had a significant (63.4 %) impact. Moreover, RNA editing analysis was done, which revealed the highest frequency of C to T mutations. The results of our research revealed the pattern of codon usage and RNA editing sites in Paramyxoviridae genomes.

Genetic diversity accelerates canine distemper virus adaptation to ferrets.

RNA viruses adapt rapidly to new host environments by generating highly diverse genome sets, so-called "quasispecies." Minor genetic variants promote their rapid adaptation, allowing for the emergence of drug-resistance or immune-escape mutants. Understanding these adaptation processes is highly relevant to assessing the risk of cross-species transmission and the safety and efficacy of vaccines and antivirals. We hypothesized that genetic memory within a viral genome population facilitates rapid adaptation. To test this, we investigated the adaptation of the Morbillivirus canine distemper virus to ferrets and compared an attenuated, Vero cell-adapted virus isolate with its recombinant derivative over consecutive ferret passages. Although both viruses adapted to the new host, the reduced initial genetic diversity of the recombinant virus resulted in delayed disease onset. The non-recombinant virus gradually increased the frequencies of beneficial mutations already present at very low frequencies in the input virus. In contrast, the recombinant virus first evolved de novo mutations to compensate for the initial fitness impairments. Importantly, while both viruses evolved different sets of mutations, most mutations found in the adapted non-recombinant virus were identical to those found in a previous ferret adaptation experiment with the same isolate, indicating that mutations present at low frequency in the original virus stock serve as genetic memory. An arginine residue at position 519 in the carboxy terminus of the nucleoprotein shared by all adapted viruses was found to contribute to pathogenesis in ferrets. Our work illustrates the importance of genetic diversity for adaptation to new environments and identifies regions with functional relevance.IMPORTANCEWhen viruses encounter a new host, they can rapidly adapt to this host and cause disease. How these adaptation processes occur remains understudied. Morbilliviruses have high clinical and veterinary relevance and are attractive model systems to study these adaptation processes. The canine distemper virus is of particular interest, as it exhibits a broader host range than other morbilliviruses and frequently crosses species barriers. Here, we compared the adaptation of an attenuated virus and its recombinant derivative to that of ferrets. Pre-existing mutations present at low frequency allowed faster adaptation of the non-recombinant virus compared to the recombinant virus. We identified a common point mutation in the nucleoprotein that affected the pathogenesis of both viruses. Our study shows that genetic memory facilitates environmental adaptation and that erasing this genetic memory by genetic engineering results in delayed and different adaptation to new environments, providing an important safety aspect for the generation of live-attenuated vaccines.

A temperature-sensitive and interferon-silent Sendai virus vector for CRISPR-Cas9 delivery and gene editing in primary human cells.

The transformative potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and interferon-silent Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types without inducing IFN responses. ts SeV demonstrates unprecedented transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhigh stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help to further expand the possibilities in personalized medicine and the treatment of genetic disorders.

Molecular Analysis of Copy-Back Defective Interfering RNAs of Morbilliviruses.

Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid molecules of positive sense antigenome and negative sense genome. They possess perfectly complementary ends allowing the formation of extremely stable double-stranded RNA panhandle structures. The presence of the 3'-terminal promoter allows replication of these molecules by the viral polymerase. They thereby negatively interfere with replication of standard genomes. In addition, the double-stranded RNA stem structures are highly immunostimulatory and activate antiviral cell-intrinsic innate immune responses. Thus, copy-back defective interfering RNAs severely affect the virulence and pathogenesis of morbillivirus stocks. We describe two biochemical methods to analyze copy-back defective interfering RNAs in virus-infected samples, or purified viral RNA. First, we present our Northern blotting protocol that allows accurate size determination of defective interfering RNA molecules and estimation of the relative contamination level of virus preparations. Second, we describe a PCR approach to amplify defective interfering RNAs specifically, which allows detailed sequence analysis.

Identification of Host Factors of Paramyxoviruses by siRNA Genome-Wide Screens.

Measles is a highly infectious disease that continues to spread mainly in developing countries, often resulting in child mortality. Despite the existence of effective vaccines, no specific antivirals are available as targeted therapy to combat measles virus (MeV). The implementation of genome-wide siRNA screens can provide a powerful platform to discover host factors that mediate MeV infection and replication, which could be essential to develop novel therapeutic strategies against this disease. Here, we describe a human genome-wide siRNA screen for MeV.

Canine Distemper Virus Pathogenesis in the Ferret Model.

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

Identification of Viruses in Molossus Bats from the Brazilian Amazon: A Descriptive Metagenomic Analysis.

Bats are widely distributed in Brazil, including the Amazon region, and their association with viral pathogens is well-known. This work aimed to evaluate the metavirome in samples of Molossus sp. bats captured in the Brazilian Amazon from 2019 to 2021. Lung samples from 58 bats were divided into 13 pools for RNA isolation and sequencing followed by bioinformatic analysis. The Retroviridae family showed the highest abundance of viral reads. Although no complete genome could be recovered, the Paramyxoviridae and Dicistroviridae families showed the formation of contigs with satisfactory identity and size characteristics for further analysis. One contig of the Paramyxoviridae family was characterized as belonging to the genus Morbillivirus, being grouped most closely phylogenetically to Porcine morbillivirus. The contig related to the Dicistroviridae family was identified within the Cripavirus genus, with 94%, 91%, and 42% amino acid identity with Culex dicistrovirus 2, Rhopalosiphum padi, and Aphid lethal paralysis, respectively. The presence of viruses in bats needs constant updating since the study was able to identify viral sequences related to families or genera still poorly described in the literature in association with bats.

Newcastle disease virus: the past and current situation in Indonesia.

The Newcastle disease virus (NDV) outbreak was first reported in Java Island, Indonesia, in 1926, which was then reported further in Newcastle-upon-Tyne, England. Nevertheless, the NDV is still endemic in Indonesia, with outbreaks occurring in free-range and commercial chicken farms. The dynamic evolution of the NDV has led to the further development of vaccines and diagnostic tools for more effective control of this virus. This paper discusses the history of the NDV occurrence, vaccines, the development of diagnostic tools, and the epidemiological condition of the NDV in Indonesia. Indonesia, which has the largest poultry population in the world after China, has challenges in preventing and controlling this virus that causes economic losses to the farmers and has an impact on the welfare of the poultry farming community in Indonesia.

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.

Dangerous Risk Group-4 (RG-4) emergent viruses.

World Health Organization (WHO) Risk Group-4 (RG-4) pathogens are among the most dangerous of the emergent and re-emergent viruses. International health agencies, working in concert, bridge the gaps in health care for populations at risk for RG-4 viral pathogen exposure. RG-4 virus research incorporates Biodefense Program and Biosafety Laboratory (BSL)-4 technologies. RG-4 viruses include Arena-viridae, Filo-viridae, Flavi-viridae, Herpes-viridae, Nairo-viridae, Paramyxo-viridae, and Pox-viridae.

Interferon-independent processes constrain measles virus cell-to-cell spread in primary human airway epithelial cells.

Amplification of measles virus (MeV) in human airway epithelia may contribute to its extremely high contagious nature. We use well-differentiated primary cultures of human airway epithelial cells (HAE) to model ex vivo how MeV spreads in human airways. In HAE, MeV spreads cell-to-cell for 3-5 days, but then, infectious center growth is arrested. What stops MeV spread in HAE is not understood, but interferon (IFN) is known to slow MeV spread in other in vitro and in vivo models. Here, we assessed the role of type I and type III IFN in arresting MeV spread in HAE. The addition of IFN-ß or IFN-λ1 to the medium of infected HAE slowed MeV infectious center growth, but when IFN receptor signaling was blocked, infectious center size was not affected. In contrast, blocking type-I IFN receptor signaling enhanced respiratory syncytial virus spread. HAE were also infected with MeV mutants defective for the V protein. The V protein has been demonstrated to interact with both MDA5 and STAT2 to inhibit activation of innate immunity; however, innate immune reactions were unexpectedly muted against the V-defective MeV in HAE. Minimal innate immunity activation was confirmed by deep sequencing, quantitative RT-PCR, and single-cell RNA-seq analyses of the transcription of IFN and IFN-stimulated genes. We conclude that in HAE, IFN-signaling can contribute to slowing infectious center growth; however, IFN-independent processes are most important for limiting cell-to-cell spread. IMPORTANCE Fundamental biological questions remain about the highly contagious measles virus (MeV). MeV amplifies within airway epithelial cells before spreading to the next host. This final step likely contributes to the ability of MeV to spread host-to-host. Over the course of 3-5 days post-infection of airway epithelial cells, MeV spreads directly cell-to-cell and forms infectious centers. Infectious center formation is unique to MeV. In this study, we show that interferon (IFN) signaling does not explain why MeV cell-to-cell spread is ultimately impeded within the cell layer. The ability of MeV to spread cell-to-cell in airway cells without appreciable IFN induction may contribute to its highly contagious nature. This study contributes to the understanding of a significant global health concern by demonstrating that infectious center formation occurs independent of the simplest explanation for limiting viral transmission within a host.

Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria: kingdom Orthornavirae: phylum Negarnaviricota).

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Langya virus outbreak: current challenges and lesson learned from previous henipavirus outbreaks in China, Australia, and Southeast Asia.

Background and aims: A new novel virus, Langya virus (LayV), was detected in China in August 2022, 3 years after the COVID-19 pandemic. LayV is similar to the previously discovered Mojiang henipavirus. Other zoonotic henipaviruses include the Hendra and Nipah viruses. The emergence of the zoonotic Langya virus is attributed to climate change and wildlife encroachment, as LayV is detected in shrews. Those who are infected in China showed various symptoms, but no deaths have been recorded yet. This review aims to shed light on the current state of Langya virus outbreak, its infection control efforts, and the remaining challenges that need to be addressed to curb the outbreak. Methods: We utilized online publication databases such as PubMed, Google Scholar, and Scopus in writing this review article. Results: A surveillance study on thirty-five febrile patients in Eastern China identified the Langya virus outbreak. The current efforts from the Chinese government and health authorities to reduce the transmission and spread of Langya virus such as isolation and characterization of LayV, challenges associated with the increase in cases of LayV, and trackable recommendations such as strengthening the healthcare system in China, sensitization of people about risks associated with Langya virus outbreaks, creating an intensive surveillance system network, etc. were discussed. Conclusion: It is germane and pertinent that the Chinese government and health authorities continue to intensify efforts against Langya virus and address the challenges to effectively reduce transmission.

Respiratory Syncytial Virus: A Comprehensive Review of Transmission, Pathophysiology, and Manifestation.

With an increasing global incidence in children younger than the age of five, respiratory syncytial virus (RSV) is one of the most common viral respiratory infections worldwide. Despite the increasing number of cases among infants and young children, RSV can infect any age group; however, some individuals are more high risk than others. Premature infants, young children, elderly, and immunocompromised individuals are the most likely to suffer a more severe presentation of RSV in comparison to healthy adults. RSV is transmitted through respiratory droplets via direct contact with an infected individual or with contaminated surfaces. The viral genome of RSV consists of 11 proteins. Out of these 11, two proteins allow for the attachment of the virus to the respiratory epithelial cells and fusion with host cells. Upon fusion, the viral material transfers to the host cell, where viral replication occurs. It is important to acknowledge that an individual is considered infectious and can transmit the virus even before the symptomatic presentation of RSV begins. As long as the individual is shedding the virus, he or she is considered infectious. The length of viral shedding also differs depending on the severity of the infection, who is infected, and the underlying immune status of an individual. Currently, there is no definitive treatment for RSV; however, supportive therapy is considered the mainstay treatment. Some pharmaceutical treatments such as ribavirin have been FDA-approved; however, the administration is typically limited to children and infants. Palivizumab is also administered as an immune prophylaxis; however, both therapies are constantly at the end of a cost-effective debate due to their extensively expensive nature and questionable adverse effect profiles. Supportive therapy includes hydration, supplemental oxygen, and mechanical ventilation in hospitalized cases; however, most RSV cases can be treated as outpatient cases. Prevention techniques such as hand washing and maintaining social distancing are imperative to minimize the transmission of the virus as much as remotely possible.

Computational identification of drug-like marine natural products as potential RNA polymerase inhibitors against Nipah virus.

Nipah virus (NiV) has been an alarming threat to human populations in southern Asia for more than a decade. It is one of the most deadly viruses in the Mononegavirales order. Despite its high mortality rate and virulence, no chemotherapeutic agent or vaccine is publicly available. Hence, this work was conducted to computationally screen marine natural products database for drug-like potential inhibitors for the viral RNA-dependent RNA polymerase (RdRp). The structural model was subjected to molecular dynamics (MD) simulation to obtain the native ensemble of the protein. The CMNPDB dataset of marine natural products was filtered to retain only compounds following Lipinski's five rules. The molecules were energy minimized and docked into different conformers of the RdRp using AutoDock Vina. The best 35 molecules were rescored by GNINA, a deep learning-based docking software. The resulting nine compounds were evaluated for their pharmacokinetic profiles and medicinal chemistry properties. The best five compounds were subjected to MD simulation for 100 ns, followed by binding free energy estimation via Molecular Mechanics/ Generalized Born Surface Area (MM/GBSA) calculations. The results showed remarkable behavior of five hits as inferred by stable binding pose and orientation to block the exit channel of RNA synthesis products in the RdRp cavity. These hits are promising starting materials for in vitro validation and structural modifications to enhance the pharmacokinetic and medicinal chemistry properties for developing antiviral lead compounds.

Orthoparamyxovirinae C Proteins Have a Common Origin and a Common Structural Organization.

The protein C is a small viral protein encoded in an overlapping frame of the P gene in the subfamily Orthoparamyxovirinae. This protein, expressed by alternative translation initiation, is a virulence factor that regulates viral transcription, replication, and production of defective interfering RNA, interferes with the host-cell innate immunity systems and supports the assembly of viral particles and budding. We expressed and purified full-length and an N-terminally truncated C protein from Tupaia paramyxovirus (TupV) C protein (genus Narmovirus). We solved the crystal structure of the C-terminal part of TupV C protein at a resolution of 2.4 Å and found that it is structurally similar to Sendai virus C protein, suggesting that despite undetectable sequence conservation, these proteins are homologous. We characterized both truncated and full-length proteins by SEC-MALLS and SEC-SAXS and described their solution structures by ensemble models. We established a mini-replicon assay for the related Nipah virus (NiV) and showed that TupV C inhibited the expression of NiV minigenome in a concentration-dependent manner as efficiently as the NiV C protein. A previous study found that the Orthoparamyxovirinae C proteins form two clusters without detectable sequence similarity, raising the question of whether they were homologous or instead had originated independently. Since TupV C and SeV C are representatives of these two clusters, our discovery that they have a similar structure indicates that all Orthoparamyxovirine C proteins are homologous. Our results also imply that, strikingly, a STAT1-binding site is encoded by exactly the same RNA region of the P/C gene across Paramyxovirinae, but in different reading frames (P or C), depending on which cluster they belong to.

In silico structural elucidation of Nipah virus L protein and targeting RNA-dependent RNA polymerase domain by nucleoside analogs.

The large (L) protein of Mononegavirales is a multi-domain protein that performs transcription and genome replication. One of the important domains in L is the RNA-dependent RNA polymerase (RdRp), a promising target for antiviral drugs. In this work, we employed rigorous computational comparative modeling to predict the structure of L protein of Nipah virus (NiV). The RdRp domain was targeted by a panel of nucleotide analogs, previously reported to inhibit different viral RNA polymerases, using molecular docking. Best binder compounds were subjected to molecular dynamics simulation to validate their binding. Molecular mechanics/generalized-born surface area (MM/GBSA) calculations estimated the binding free energy. The predicted model of NiV L has an excellent quality as judged by physics- and knowledge-based validation tests. Galidesivir, AT-9010 and Norov-29 scored the top nucleotide analogs to bind to the RdRp. Their binding free energies obtained by MM/GBSA (-31.01 ± 3.9 to -38.37 ± 4.8 kcal/mol) ranked Norov-29 as the best potential inhibitor. Purine nucleotide analogs are expected to harbor the scaffold for an effective drug against NiV. Finally, this study is expected to provide a start point for medicinal chemistry and drug discovery campaigns toward identification of effective chemotherapeutic agent(s) against NiV.Communicated by Ramaswamy H. Sarma.

A novel and highly divergent Canine Distemper Virus lineage causing distemper in ferrets in Australia.

Canine distemper virus (CDV) causes a highly contagious systemic infection in an array of animal species. In this study we report an outbreak of distemper in ferrets in two research facilities in Australia, caused by a novel lineage of CDV. While the CDV strain caused mainly mild symptoms in ferrets, histopathology results presented a typical profile of distemper pathology, with multi-system virus replication. Through the development of a discriminatory PCR, paired with full genome sequencing, we revealed that the outbreak was caused by a novel lineage of CDV. The novel CDV lineage was highly divergent, with less than 93% similarity across the H gene to other described lineages, including the vaccine strain, and diverged approximately 140-400 years ago. Enhanced surveillance to determine the prevalence of CDV in ferrets, dogs and other at-risk species is critical to better understand the presence and diversity of CDV in Australia currently.