While the viromes and immune systems of bats and rodents have been extensively studied, comprehensive data are lacking for insectivores (order Eulipotyphla) despite their wide geographic distribution. Anthropogenic land use and outdoor recreational activities, as well as changes in the range of shrews, may lead to an expansion of the human-shrew interface with the risk of spillover infections, as reported for Borna disease virus 1. We investigated the virome of 45 individuals of 4 white-toothed shrew species present in Europe, using metagenomic RNA sequencing of tissue and intestine pools. Moderate to high abundances of sequences related to the families Paramyxoviridae, Nairoviridae, Hepeviridae and Bornaviridae were detected. Whole genomes were determined for novel orthoparamyxoviruses (n=3), orthonairoviruses (n=2) and an orthohepevirus. The novel paramyxovirus, tentatively named Hasua virus, was phylogenetically related to the zoonotic Langya virus and Mòjiāng virus. The novel orthonairoviruses, along with the potentially zoonotic Erve virus, fall within the shrew-borne Thiafora virus genogroup. The highest viral RNA loads of orthoparamyxoviruses were detected in the kidneys, in well-perfused organs for orthonairoviruses and in the liver and intestine for orthohepevirus, indicating potential transmission routes. Notably, several shrews were found to be coinfected with viruses from different families. Our study highlights the virus diversity present in shrews, not only in biodiversity-rich regions but also in areas influenced by human activity. This study warrants further research to characterize and assess the clinical implications and risk of these viruses and the importance of shrews as reservoirs in European ecosystems.

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.

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.

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.

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.

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.

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.

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.
OBJECTIVE: 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.

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.

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.