A Novel Jeilongvirus from Florida, USA, Has a Broad Host Cell Tropism Including Human and Non-Human Primate Cells.

A novel jeilongvirus was identified through next-generation sequencing in cell cultures inoculated with spleen and kidney extracts. The spleen and kidney were obtained from a Peromyscus gossypinus rodent (cotton mouse) found dead in the city of Gainesville, in North-Central Florida, USA. Jeilongviruses are paramyxoviruses of the subfamily Orthoparamyxovirinae that have been found in bats, cats, and rodents. We designated the virus we discovered as Gainesville rodent jeilong virus 1 (GRJV1). Preliminary results indicate that GRJV1 can complete its life cycle in various human, non-human primate, and rodent cell lines, suggesting that the virus has a generalist nature with the potential for a spillover event. The early detection of endemic viruses circulating within hosts in North-Central Florida can significantly enhance surveillance efforts, thereby bolstering our ability to monitor and respond to potential outbreaks effectively.

Pongamia pinnata L. seed-derived karanjin as prominent antiviral agent against Newcastle disease virus.

Current study aim to explore Karanjin (Kar) isolated from the seeds of Pongamia pinnata as a potential antiviral polyphenol against Newcastle disease virus (NDV) through comprehensive investigations. For the in vitro study, plaque assays, gene and protein expression were used to analyse the inhibitory impact of Kar on NDV, where it reduced NDV replication as shown by a 13-fold suppression of the hemagglutinin-neuraminidase (HN) gene and decrease about 60% in virus activity. In ovo study showed that Kar mitigates NDV effects in chicken embryos. In silico studies involving molecular docking and molecular dynamics simulations showed strong binding between Kar and HN protein of NDV. Kar also influenced glucose metabolism, enhancing antiviral responses, as showed by the upregulation of GLUT1 and HEX genes through RT-qPCR and HPLC analyses. The study contributes valuable insights for future investigations into therapeutic applications of Kar against viral infections.

Epidemiology of human metapneumovirus in Taiwan from 2013 to 2023.

Human metapneumovirus (HMPV) is a member of the genus Metapneumovirus in the family Pneumoviridae of the order Mononegavirales that can cause upper and lower respiratory tract disease. This retrospective study describes the epidemiology of hMPV based on community viral surveillance results from sentinel sites across Taiwan from 2013 to 2023. A total of 114 hMPV strains were isolated and analyzed to assess viral evolution through sequencing of their fusion protein genes. This study revealed that hMPV cases occur almost year-round in Taiwan, with a peak occurring during spring (March to May). Of the 114 infected patients, 68.4% were children under 4 years old. The geographical distribution of hMPV positivity was highest in Penghu County, followed by Changhua County and Hsinchu County. The clinical symptoms of hMPV infection are nonspecific, with fever (56.1%), cough (44.7%), rhinorrhea (21.1%), and sore throat (14.9%) being the most common. However, a few patients also developed severe central nervous system symptoms (1.8%) or dyspnea (0.9%). Phylogenetic analysis revealed genetic diversity among the 114 isolated hMPV strains, with the A2 lineage (57.9%) being the most frequently observed, followed by the B2 lineage (33.3%), in the Taiwanese community from 2013 to 2023. In conclusion, hMPV causes a serious acute respiratory disease in Taiwan that should not be neglected. Further epidemiological surveillance and investigations of the clinical characteristics of hMPV should be performed continually for prevention and control of this virus.

Immune Responses in the Harderian Gland After Newcastle Disease Vaccination in Chickens with Maternal Antibodies.

Immune responses in the Harderian gland (HG) were characterized after Newcastle disease virus (NDV) LaSota ocular vaccination in antibody-naïve specific-pathogen-free (SPF) chickens and in chickens of commercial origin with NDV maternally derived antibodies (MDA). Ocular LaSota vaccination of 13-day-old white leghorn SPF chickens elicited serum antibody levels that consistently increased 15 days postvaccination, while the specific IgA response in lacrimal fluids was already detectable 10 days after vaccination. Eleven days postvaccination, the relative abundance of B cells, as well as T-helper cells (CD4+) and cytotoxic T cells (CD8+), in HGs was significantly increased, achieving maximum frequencies 16 days postvaccination. In a second experiment, chickens with MDA originating from NDV-vaccinated commercial white leghorn layer breeders, as well as white leghorn SPF chickens, were vaccinated with NDV LaSota. The LaSota virus successfully replicated in periocular tissues and in the trachea both in commercial and control SPF chickens after vaccination at 2 or 15 days of age (DOA). Vaccination at 2 DOA did not induce a serum NDV antibody response in chickens of commercial origin. In contrast, seroconversion was elicited in commercial chickens upon vaccination at 15 DOA, likely associated with waning of MDA. Unlike systemic IgG responses, vaccination at 2 or 15 DOA elicited strong specific IgA responses in lacrimal fluids in commercial chickens. The IgA response was highest 9 days after vaccination and showed a tendency to decline 15 days postvaccination. Commercial chickens vaccinated at 2 DOA showed increased B cells in HG 10 and 16 days postvaccination. The expansion of B cells in the HG in these chickens is consistent with increased IgA levels detected in lacrimal fluids. In contrast, control SPF chickens showed a more limited B-cell expansion in HG and lower IgA levels. Vaccination at 15 DOA also triggered a greater increase of B cells in HGs in commercial chickens than in control SPF chickens. The B-cell response was accompanied by T-helper (CD4+) cell expansion, occurring both in commercial and control SPF chickens. These cells expanded to a lesser extent when vaccination was performed at 2 DOA compared with vaccination at 15 DOA. CD8+ showed significant expansion irrespective of vaccination day and without differences detected between control SPF chickens and chickens with MDA. We conclude that NDV LaSota elicits vigorous humoral and cell immune responses in the HG. Furthermore, unlike the interference shown by MDA on vaccine-induced serum antibody responses, MDA do not interfere with the mucosal immune response of the HG.

Respuestas inmunes en la glándula de Harder después de la vacunación contra la enfermedad de Newcastle en pollos con anticuerpos maternos. Se caracterizaron las respuestas inmunes en la glándula de Harder (HG) después de la vacunación ocular con la cepa LaSota contra el virus de la enfermedad de Newcastle (NDV) en pollos libres de patógenos espec'ificos (SPF) sin anticuerpos y en pollos de origen comercial con anticuerpos maternos (MDA) contra el virus de Newcastle. La vacunación ocular con cepa LaSota en aves White Leghorn libres de patógenos espec'ificos de 13 d'ias de edad indujo niveles de anticuerpos séricos que aumentaron consistentemente 15 d'ias después de la vacunación, mientras que la respuesta espec'ifica de IgA en los fluidos lagrimales ya era detectable a los 10 d'ias después de la vacunación. Once d'ias después de la vacunación, la abundancia relativa de células B, as'i como de células T colaboradoras (CD4+) y células T citotóxicas (CD8+), en la glándula de Harder aumentó significativamente, alcanzando frecuencias máximas 16 d'ias después de la vacunación. En un segundo experimento, se vacunaron con la cepa LaSota de Newcastle pollos con anticuerpos maternales procedentes de reproductoras comerciales de aves de postura White Leghorn vacunadas con el NDV, as'i como gallinas White Leghorn libres de patógenos espec'ificos. El virus LaSota se replicó con éxito en los tejidos perioculares y en la tráquea tanto de pollos comerciales como en las aves controles libres de patógenos espec'ificos después de la vacunación a los 2 o 15 d'ias de edad. La vacunación a los dos d'ias de edad no indujo una respuesta de anticuerpos séricos contra el virus de Newcastle en pollos de origen comercial. Por el contrario, se indujo seroconversión en pollos comerciales tras la vacunación a los 15 d'ias de edad, probablemente asociada con una disminución de los anticuerpos maternos. A diferencia de las respuestas sistémicas de IgG, la vacunación a los dos o 15 d'ias de edad provocó fuertes respuestas de IgA espec'ificas en los fluidos lagrimales en pollos comerciales. La respuesta de IgA fue máxima nueve d'ias después de la vacunación y mostró una tendencia a disminuir 15 d'ias después de la vacunación. Los pollos comerciales vacunados con dos d'ias de edad mostraron un aumento de células B en la glándula de Harder 10 y 16 d'ias después de la vacunación. La expansión de las células B en la glándula de Harder en estos pollos es consistente con el aumento de los niveles de IgA detectados en los fluidos lagrimales. Por el contrario, los pollos controles libres de patógenos espec'ificos mostraron una expansión de células B más limitada en la glándula de Harder y niveles más bajos de IgA. La vacunación con 15 d'ias de edad también provocó un aumento importante de células B en la glándula de Harder en pollos comerciales en comparación con los pollos controles libres de patógenos espec'ificos. La respuesta de las células B estuvo acompañada por la expansión de las células T colaboradoras (CD4+), que ocurrió tanto en pollos comerciales como en los controles libres de patógenos espec'ificos. Estas células se expandieron en menor medida cuando la vacunación se realizó a los dos d'ias de edad en comparación con la vacunación a los 15 d'ias de edad. Las células CD8+ mostraron una expansión significativa independientemente del d'ia de vacunación y sin diferencias detectadas entre los pollos control libres de patógenos espec'ificos y los pollos con anticuerpos maternales. Se concluye que la cepa de Newcastle LaSota provoca robustas respuestas inmunes humorales y celulares en la glándula de Harder. Además, a diferencia de la interferencia mostrada por los anticuerpos maternos en las respuestas de anticuerpos séricos inducidas por la vacuna, los anticuerpos maternos no interfieren con la respuesta inmune de la mucosa de la glándula de Harder.

Assessment of PPMV-1 Genotype VI Virulence in Pigeons and Chickens and Protective Effectiveness of Paramyxovirus Vaccines in Pigeons.

Pigeon paramyxovirus serotype 1 (PPMV-1), an antigenic and host variant of avian paramyxovirus Newcastle disease virus (NDV), primarily originating from racing pigeons, has become a global panzootic. Egypt uses both inactivated PPMV-1 and conventional NDV vaccines to protect pigeons from disease and mortality. However, the impact of prevalent strains and the effectiveness of available vaccines in pigeons in Egypt are unclear. This study investigates the virulence of PPMV-1 (Pigeon/Egypt/Sharkia-19/2015/KX580988) and evaluates available paramyxovirus vaccines in protecting pigeons against a PPMV-1 challenge. Ten-day-old specific-pathogen-free (SPF) embryonated chicken eggs infected with this strain exhibited a mean death time (MDT) of 86.4 ± 5.88 h. The intracerebral pathogenicity index (ICPI) in day-old chickens was 0.8, while pigeons experienced an ICPI of 0.96 and an intravenous pathogenicity index (IVPI) of 2.11. These findings classify the strain as virulent and velogenic. Experimental infection of pigeons with this PPMV-1 strain at 106 EID50/0.1 mL resulted in a 62.5% mortality rate, displaying nervous and enteric distress. The virus caused extensive lesions in visceral organs, with strong immunohistochemistry signals in all examined organs, indicating the systemic spread of the virus concurrent to its neurotropic and viscerotropic tropism. Furthermore, vaccination using an inactivated PPMV-1 and live NDV LaSota vaccine regimen protected 100% of pigeons against mortality, while with a single NDV LaSota vaccine, it was 62.5%. The PPMV alone or combined with NDV LaSota induced protective levels of haemagglutination inhibition (HI) antibody titres and reduced virus shedding from buccal and cloacal cavities. Based on generalised linear gamma model analysis, both PPMV-1 and NDV LaSota are antigenically comparable by HI. These findings suggest that using both inactivated PPMV-1 (G-VI) and live attenuated NDV (LaSota) vaccines is an effective prophylactic regimen for preventing and controlling PPMV-1 and NDV in pigeons, thereby reducing the risk of interspecies transmission.

Complete genome sequence of the avian paramyxovirus serotype 9 strain duck/Miyazaki/128/2021.

Here, we report the complete genome sequence of the avian paramyxovirus serotype 9 strain duck/Miyazaki/128/2021, which was determined using the Illumina MiSeq platform. The position of the hemagglutinin-neuraminidase stop codon differed from that of the only other available completely sequenced prototype strain, duck/New York/22/1977.

Human parainfluenza virus 3 field strains undergo extracellular fusion protein cleavage to activate entry.

Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F in vivo were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation in vivo, reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread. IMPORTANCE: Enveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.

The first emergence of paramyxovirus type 12 in wild birds in mainland, China.

Avian paramyxoviruses (APMV) belong to the subfamily Avulavirinae of the family Paramyxoviridae and include 22 distinct subtypes or serotypes (1-22). Avian paramyxovirus serotype 12 (APMV-12) is found sporadically in wild birds worldwide, and reports from only Italy and Taiwan have been published to date; information on its genetic variation and biological characteristics is still limited. In this study, 3 APMV-12 strains, designated WB19, LY9, and LY11, were isolated from 8643 wild bird faecal samples during the annual influenza virus surveillance of wild birds in Guangdong, China between 2018 and 2024, which is first reported in mainland China. The complete genomes of the 3 viruses with 6 gene segments, 3'-N-P-M-F-HN-L-5', were 15,231 nt in length. Phylogenetic analysis based on the whole genome showed that the 3 APMV-12 strains had the highest homology with an APMV-12 strain isolated from Taiwan in 2015, followed by the prototype APMV-12 strains isolated from mallard ducks in Italy in 2005. Genetic analysis of the whole gene of each of them indicated that they were derived from a Eurasian lineage. This study provides additional evidence that wild birds transmit viruses between countries, and this should be monitored to understand APMV transmission, evolution and epidemiology.

Genomic Diversity and Evolutionary Insights of Avian Paramyxovirus-1 in Avian Populations in Pakistan.

The virulent form of Avian paramyxovirus-1 (APMV-1), commonly known as Newcastle Disease Virus (NDV), is a pathogen with global implications for avian health, affecting both wild and domestic bird populations. In Pakistan, recurrent Newcastle Disease (caused by NDV) outbreaks have posed significant challenges to the poultry industry. Extensive surveillance in Pakistan over 20 years has demonstrated a dynamic genetic diversity among circulating APMV-1 strains, emphasizing the potential necessity for customized vaccination strategies and continuous surveillance. In this study, 13 APMV-1-positive isolates harboring four different APMV-1 genotypes circulating throughout Pakistan were identified. These included the highly virulent genotypes VII and XIII, genotype XXI, commonly associated with Columbiformes, and genotype II, hypothesized to have been detected following vaccination. These findings underscore the intricate interplay of mutational events and host-immune interactions shaping the evolving NDV landscape. This study advances our understanding of the evolutionary dynamics of APMV-1 in Pakistan, highlighting the need for tailored vaccination strategies and continuous surveillance to enable effective APMV-1 management in avian populations, further emphasizing the importance of globally coordinated strategies to tackle APMV-1, given its profound impact on wild and domestic birds.

Paramyxovirus matrix proteins modulate host cell translation via exon-junction complex interactions in the cytoplasm.

Viruses have evolved myriad strategies to exploit the translation machinery of host cells to potentiate their replication. However, how paramyxovirus (PMVs) modulate cellular translation for their own benefit has not been systematically examined. Utilizing puromycylation labeling, overexpression of individual viral genes, and infection with wild-type virus versus its gene-deleted counterpart, we found that PMVs significantly inhibit host cells' nascent peptide synthesis during infection, with the viral matrix being the primary contributor to this effect. Using the rNiV-NPL replicon system, we discovered that the viral matrix enhances viral protein translation without affecting viral mRNA transcription and suppresses host protein expression at the translational level. Polysome profile analysis revealed that the HPIV3 matrix promotes the association of viral mRNAs with ribosomes, thereby enhancing their translation efficiency during infection. Intriguingly, our NiV-Matrix interactome identified the core exon-junction complex (cEJC), critical for mRNA biogenesis, as a significant component that interacts with the paramyxoviral matrix predominantly in the cytoplasm. siRNA knockdown of eIF4AIII simulated the restriction of cellular functions by the viral matrix, leading to enhanced viral gene translation and a reduction in host protein synthesis. Moreover, siRNA depletion of cEJC resulted in a 2-3 log enhancement in infectious virus titer for various PMVs but not SARS-CoV-2, enterovirus D68, or influenza virus. Our findings characterize a host translational interference mechanism mediated by viral matrix and host cEJC interactions. We propose that the PMV matrix redirects ribosomes to translate viral mRNAs at the expense of host cell transcripts, enhancing viral replication, and thereby enhancing viral replication. These insights provide a deeper understanding of the molecular interactions between paramyxoviruses and host cells, highlighting potential targets for antiviral strategies.

Tetracistronic minigenomes elucidate a functional promoter for Ghana virus and unveils Cedar virus replicase promiscuity for all henipaviruses.

Batborne henipaviruses, such as Nipah and Hendra viruses, represent a major threat to global health due to their propensity for spillover, severe pathogenicity, and high mortality rate in human hosts. Coupled with the absence of approved vaccines or therapeutics, work with the prototypical species and uncharacterized, emergent species is restricted to high biocontainment facilities. There is a scarcity of such specialized spaces for research, and often, the scope and capacity of research, which can be conducted at BSL-4, is limited. Therefore, there is a pressing need for innovative life-cycle modeling systems to enable comprehensive research within lower biocontainment settings. This work showcases tetracistronic, transcription, and replication-competent minigenomes for the Nipah, Hendra, and Cedar viruses, which encode viral proteins facilitating budding, fusion, and receptor binding. We validate the functionality of all encoded viral proteins and demonstrate a variety of applications to interrogate the viral life cycle. Notably, we found that the Cedar virus replicase exhibits remarkable promiscuity, efficiently driving replication and transcription of minigenomes from all tested henipaviruses. We also apply this technology to Ghana virus (GhV), an emergent species that has so far not been isolated in culture. We demonstrate that the reported sequence of GhV is incomplete, but that this missing sequence can be substituted with analogous sequences from other henipaviruses. The use of our GhV system establishes the functionality of the GhV replicase and identifies two antivirals that are highly efficacious against the GhV polymerase. IMPORTANCE: Henipaviruses are recognized as significant global health threats due to their high mortality rates and lack of effective vaccines or therapeutics. Due to the requirement for high biocontainment facilities, the scope of research which may be conducted on henipaviruses is limited. To address this challenge, we developed innovative tetracistronic, transcription, and replication-competent minigenomes. We demonstrate that these systems replicate key aspects of the viral life cycle, such as budding, fusion, and receptor binding, and are safe for use in lower biocontainment settings. Importantly, the application of this system to the Ghana virus revealed that its known sequence is incomplete; however, substituting the missing sequences with those from other henipaviruses allowed us to overcome this challenge. We demonstrate that the Ghana virus replicative machinery is functional and can identify two orally efficacious antivirals effective against it. Our research offers a versatile system for life-cycle modeling of highly pathogenic henipaviruses at low biocontainment.

A three-way interface of the Nipah virus phosphoprotein X-domain coordinates polymerase movement along the viral genome.

Nipah virus (NiV) is a highly pathogenic paramyxovirus causing frequently lethal encephalitis in humans. The NiV genome is encapsidated by the nucleocapsid (N) protein. RNA synthesis is mediated by the viral RNA-dependent RNA polymerase (RdRP), consisting of the polymerase (L) protein complexed with the homo-tetrameric phosphoprotein (P). The advance of the polymerase along its template requires iterative dissolution and reformation of transient interactions between P and N protomers in a highly regulated process that remains poorly understood. This study applied functional and biochemical NiV polymerase assays to the problem. We mapped three distinct protein interfaces on the C-terminal P-X domain (P-XD), which form a triangular prism and engage L, the C-terminal N tail, and the globular N core, respectively. Transcomplementation assays using NiV L and N-tail binding-deficient mutants revealed that only one XD of a P tetramer binds to L, whereas three must be available for N-binding for efficient polymerase activity. The dissolution of the N-tail complex with P-XD was coordinated by a transient interaction between N-core and the α-1/2 face of this XD but not unoccupied XDs of the P tetramer, creating a timer for coordinated polymerase advance. IMPORTANCE: Mononegaviruses comprise major human pathogens such as the Ebola virus, rabies virus, respiratory syncytial virus, measles virus, and Nipah virus (NiV). For replication and transcription, their polymerase complexes must negotiate a protein-encapsidated RNA genome, which requires the highly coordinated continuous formation and resolution of protein-protein interfaces as the polymerase advances along the template. The viral P protein assumes a central role in this process, but the molecular mechanism of ensuring polymerase mobility is poorly understood. Studying NiV polymerase complexes, we applied functional and biochemical assays to map three distinct interfaces in the NiV P XD and identified transient interactions between XD and the nucleocapsid core as instrumental in coordinating polymerase advance. These results define a conserved molecular principle regulating paramyxovirus polymerase dynamics and illuminate a promising druggable target for the structure-guided development of broad-spectrum polymerase inhibitors.

Small hydrophobic (SH) proteins of Pneumoviridae and Paramyxoviridae: small but mighty.

Small hydrophobic (SH) proteins are a class of viral accessory proteins expressed by many members of the negative-stranded RNA viral families Paramyxoviridae and Pneumoviridae. Identified SH proteins are type I or II transmembrane (TM) proteins with a single-pass TM domain. Little is known about the functions of SH proteins; however, several possess viroporin activity, enhancing membrane permeability of infected cells or those expressing SH protein. Moreover, several SH proteins inhibit apoptosis and immune signaling pathways within infected cells, including TNF and interferon signaling, or activate inflammasomes. SH proteins are generally nonessential for viral replication in vitro, but loss of SH is often associated with reduced replication in vivo, suggesting a role in enhancing viral replication or evading host immunity. Analogous proteins are expressed by a variety of pathogens of public health importance; thus, understanding the functional importance and mechanisms of SH proteins provides insight into the pathogenesis and replication of negative-sense RNA viruses.

Structural basis of paramyxo- and pneumovirus polymerase inhibition by non-nucleoside small-molecule antivirals.

Small-molecule antivirals can be used as chemical probes to stabilize transitory conformational stages of viral target proteins, facilitating structural analyses. Here, we evaluate allosteric pneumo- and paramyxovirus polymerase inhibitors that have the potential to serve as chemical probes and aid the structural characterization of short-lived intermediate conformations of the polymerase complex. Of multiple inhibitor classes evaluated, we discuss in-depth distinct scaffolds that were selected based on well-understood structure-activity relationships, insight into resistance profiles, biochemical characterization of the mechanism of action, and photoaffinity-based target mapping. Each class is thought to block structural rearrangements of polymerase domains albeit target sites and docking poses are distinct. This review highlights validated druggable targets in the paramyxo- and pneumovirus polymerase proteins and discusses discrete structural stages of the polymerase complexes required for bioactivity.

Clinicopathological and Molecular Investigation of Newcastle Disease Outbreaks in Vaccinated and Non-Vaccinated Broiler Chicken Flocks in Nepal.

Newcastle disease (ND) is a highly contagious viral disease caused by the paramyxovirus, which is a single-stranded ribonucleic acid (RNA) virus. This study was conducted to investigate ND outbreaks in 10 vaccinated or non-vaccinated broiler farms, collectively housing 9840 birds of various ages in the Chitwan and Nawalpur districts of Nepal from July to December 2021. Clinically, the affected birds exhibited symptoms such as limb paralysis, greenish diarrhea (seven out of ten flocks), torticollis (two out of ten flocks), inappetence, and drowsiness (ten out of ten flocks). Birds that succumbed during the clinical course underwent a necropsy for gross pathology and samples were collected for the histopathology and molecular diagnosis. The gross and microscopic examination revealed hemorrhages in the proventriculus, erosions and ulcers in the small intestine, congestion, as well as sero-mucosal hemorrhages in the trachea of affected birds, which are typical of ND. Rapid test kits further confirmed the presence of the ND virus antigen while excluding the avian influenza virus. Furthermore, M gene-based real time polymerase chain reaction (RT-PCR) was performed in the pooled samples from the affected birds and the presence of a velogenic strain of the ND virus was identified. The phylogenetic analysis of the RT-PCR positive strain based on the partial F gene nucleotide sequence revealed these strains as genotype VII.2 (formerly VIIi). The findings highlight the occurrence of clinical ND outbreaks in farms despite adherence to recommended vaccination protocols in broiler flocks, underscoring the need for a regular comprehensive investigation involving in-depth examinations of available vaccines and genetic analyses.

Evaluation of the sensitivity of a measles diagnostic real-time RT-PCR assay incorporating recently observed priming mismatch variants, 2024.

We investigated a variant of measles virus that encodes three mismatches to the reverse priming site for a widely used diagnostic real-time RT-PCR assay; reduction of sensitivity was hypothesised. We examined performance of the assay in context of the variant using in silico data, synthetic RNA templates and clinical specimens. Sensitivity was reduced observed at low copy numbers for templates encoding the variant sequence. We designed and tested an alternate priming strategy, rescuing the sensitivity of the assay.

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.

Integrated cryoEM structure of a spumaretrovirus reveals cross-kingdom evolutionary relationships and the molecular basis for assembly and virus entry.

Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle.

Nipah virus-associated neuropathology in African green monkeys during acute disease and convalescence.

BACKGROUND: Nipah virus is an emerging zoonotic virus that causes severe respiratory disease and meningoencephalitis. The pathophysiology of Nipah virus meningoencephalitis is poorly understood. METHODS: We have collected the brains of African green monkeys during multiple Nipah virus, Bangladesh studies, resulting in 14 brains with Nipah virus-associated lesions. RESULTS: The lesions seen in the brain of African green monkeys infected with Nipah virus, Bangladesh were very similar to those observed in humans with Nipah virus, Malaysia infection. We observed viral RNA and antigen within neurons and endothelial cells, within encephalitis foci and in uninflamed portions of the CNS. CD8+ T cells had a consistently high prevalence in CNS lesions. We developed a UNet model for quantifying and visualizing inflammation in the brain in a high-throughput and unbiased manner. While CD8+ T cells had a consistently high prevalence in CNS lesions, the model revealed that CD68+ cells were numerically the immune cell with the highest prevalence in the CNS of NiV-infected animals. CONCLUSION: Our study provides an in-depth analysis on Nipah virus infection in the brains of primates, and similarities between lesions in patients and the animals in our study validate this model.