Affect Virus Replication Research Articles

Multigenic family 110 (1L-5-6L) of African swine fever virus modulate cytokine genes expression in vitro.

African swine fever (ASF) is a viral disease that affects pigs and wild boars providing economic burden in swine industry. In this study, we investigated the effect of deleting the ASFV multigene family 110 (MGF110) fragment (1L-5-6L) on apoptosis modulation and the expression of proinflammatory cytokines. Gene expression in swine peripheral blood macrophages infected with either the parental "Volgograd/14c" strain or the gene-deleted "Volgograd/D(1L-5-6L) MGF110" strain was analyzed. Caspase-3 activity was 1.15 times higher in macrophages infected with the parental ASFV strain compared to the gene-deleted strain. Gene expression analysis of Caspase-3 (Cas-3), Interferon-A (IFN-A), Tumor Necrosis Factor A (TNF-A), B-cell Lymphoma-2 (Bcl-2), Nuclear Factor Kappa B (NF-kB), Interleukin-12 (IL-12), and Heat Shock Protein-70 (HSP-70) using RT-qPCR at various time points after infection revealed significant differences in expression profiles between the strains. The peak expression of cytokines (except NF-kB) occurred at 24h post-infection with the "Volgograd/D(1L-5-6L) MGF110" strain. In samples infected with the ASFV "Volgograd/14c" strain, the most intense expression was observed at 72 and 96h, except for Bcl-2 and NF-kB, which peaked at 6h post-infection. The cytokine expression trend for the "Volgograd/D(1L-5-6L) MGF110" strain was more stable with higher expression values. The expression trend for the parental strain increased over time, reaching maximum values at 72 and 96h post-infection, but the overall expression level was lower than that of the gene-deleted strain. These findings suggest that deleting the multigene family 110 members (1L-5-6L) contributes to ASFV attenuation without affecting virus replication kinetics.

Telomeric repeats in the commercial SB-1 vaccine facilitate viral integration and contribute to vaccine efficacy

Marek’s disease virus (MDV) integrates its genome into the telomeres of host chromosomes and causes fatal lymphomas in chickens. This integration is facilitated by telomeric repeat sequences (TMRs) at the ends of the viral genome, and is crucial for MDV-induced lymphomagenesis. The SB-1 vaccine virus is commonly used in commercial bivalent vaccines against MDV and also contains TMRs at its ends. Here, we demonstrate that SB-1 efficiently integrates its genome into the chromosomes of latently infected T cells. Deletion of the TMRs from the SB-1 genome did not affect virus replication, but severely impaired virus integration and genome maintenance in latently infected T cells and in chickens. Strikingly, the reduced integration and maintenance of latent SB-1 significantly impaired vaccine protection. Taken together, our data revealed that the TMRs facilitate SB-1 integration and that integration and/or maintenance of the latent viral genome is critical for vaccine protection.

HnRNPs in antiviral innate immunity.

During virus infection, many host proteins are redirected from their normal cellular roles to restrict and terminate infection. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are cellular RNA-binding proteins critical to host nucleic acid homeostasis, but can also be involved in the viral infection process, affecting virus replication, assembly and propagation. It has become evident that hnRNPs play important roles in modulation of host innate immunity, which provides critical initial protection against infection. These novel findings can potentially lead to the leveraging of hnRNPs in antiviral therapies. We review hnRNP involvement in antiviral innate immunity, in humans, mice and other animals, and discuss hnRNP targeting as a potential novel antiviral therapeutic.

Recruitment of multi-segment genomic RNAs by Bluetongue virus requires a preformed RNA network.

How do segmented RNA viruses correctly recruit their genome has yet to be clarified. Bluetongue virus is a double-stranded RNA virus with 10 segments of different sizes, but it assembles its genome in single-stranded form through a series of specific RNA-RNA interactions prior to packaging. In this study, we determined the structure of each BTV transcript, individually and in different combinations, using 2'-hydroxyl acylation analysed by primer extension and mutational profiling (SHAPE-MaP). SHAPE-MaP identified RNA structural changes during complex formation and putative RNA-RNA interaction sites. Our data also revealed a core RNA-complex of smaller segments which serves as the foundation ('anchor') for the assembly of a complete network composed of ten ssRNA segments. The same order of core RNA complex formation was identified in cells transfected with viral RNAs. No viral protein was required for these assembly reactions. Further, substitution mutations in the interacting bases within the core assemblies, altered subsequent segment addition and affected virus replication. These data identify a wholly RNA driven reaction that may offer novel opportunities for designed attenuation or antiviral therapeutics.

Heterogeneous nuclear ribonucleoprotein A3 binds to the internal ribosomal entry site of enterovirus A71 and affects virus replication in neural cells.

Enterovirus A71 (EV-A71) belongs to the genus Enterovirus of the Picornaviridae family and often causes outbreaks in Asia. EV-A71 infection usually causes hand, foot, and mouth disease and can even affect the central nervous system, causing neurological complications or death. The 5'-untranslated region (5'-UTR) of EV-A71 contains an internal ribosome entry site (IRES) that is responsible for the translation of viral proteins. IRES-transacting factors can interact with the EV-A71 5'-UTR to regulate IRES activity. Heterogeneous nuclear ribonucleoprotein (hnRNP) A3 is a member of the hnRNP A/B protein family of RNA-binding proteins and is involved in RNA transport and modification. We found that hnRNP A3 knockdown promoted the replication of EV-A71 in neural calls. Conversely, increasing the expression of hnRNP A3 within cells inhibits the growth of EV-A71. HnRNP A3 can bind to the EV-A71 5'-UTR, and knockdown of hnRNP A3 enhances the luciferase activity of the EV-A71 5'-UTR IRES. The localization of hnRNP A3 shifts from the nucleus to the cytoplasm of infected cells during viral infection. Additionally, EV-A71 infection can increase the protein expression of hnRNP A3, and the protein level is correlated with efficient viral growth. Based on these findings, we concluded that hnRNP A3 plays a negative regulatory role in EV-A71 replication within neural cells.

Replication of Human Norovirus in Human Intestinal Enteroids Is Affected by Fecal Sample Processing.

Human intestinal enteroids (HIEs) culture is an emerging model for assessing the infectivity of human noroviruses (HuNoVs). The model is based on detecting an increase in HuNoV RNA post-infection of HIEs. HuNoV fecal samples used for HIE infection are traditionally processed by serial filtration. Recently, processing HuNoV fecal samples by serial centrifugation was shown to retain vesicles containing HuNoV. The objective of this study was to investigate whether serially centrifuged fecal samples, RNA extraction kit (QIAamp versus MagMaX) and HIE age (newer versus older) affect HuNoV RNA fold increase in HIE. HuNoV GII.1, GII.4 and GII.6 fecal samples were prepared by serial centrifugation and filtration and the viral RNA in HIE was quantified at 1 and 72 h post-infection (hpi) following RNA extraction and RT-qPCR. The serially filtered GII.1, GII.4 and GII.6 showed successful replication in HIE, resulting in mean log increases of 2.2, 2 and 1.2, respectively, at 72 vs. 1 hpi. In contrast, only serially centrifuged GII.1 showed consistently successful replication. However, using newer HIE passages and the MagMAX kit resulted in mean log fold increases for serially centrifuged GII.1, GII.4 and GII.6 (1.6, 2.3 and 1.8 log, respectively) that were similar to serially filtered samples. Therefore, HuNoV fecal sample processing and HIE age can affect virus replication in the HIE model.

Cold atmospheric plasma can effectively disinfect SARS‐CoV‐2 in the wastewater

AbstractCOVID‐19 is currently pandemic and the detection of SARS‐CoV‐2 variants in wastewater is causing widespread concern. Herein, cold atmospheric plasma (CAP) is proposed as a novel wastewater disinfection technology that effectively inactivates SARS‐CoV‐2 transcription‐ and replication‐competent virus‐like particles, coronavirus GX_P2V, pseudotyped SARS‐CoV‐2 variants, and porcine epidemic diarrhoea virus in a large volume of water within 180 s (inhibition rate > 99%). Further, CAP disinfection did not adversely affect the viability of various human cell lines. It is identified that CAP produced peroxynitrite (ONOO−), ozone (O3), superoxide anion radicals (O2−), and hydrogen peroxide (H2O2) as the major active substances for coronavirus disinfection. Investigation of the mechanism showed that active substances not only reacted with the coronavirus spike protein and affected its infectivity, but also destroyed the nucleocapsid protein and genome, thus affecting virus replication. This method provides an efficient and environmentally friendly strategy for the elimination of SARS‐CoV‐2 and other coronaviruses from wastewater.

Evaluation of the Deletion of the African Swine Fever Virus Gene O174L from the Genome of the Georgia Isolate.

African swine fever virus (ASFV) is a structurally complex, double-stranded DNA virus, which causes African swine fever (ASF), a contagious disease affecting swine. ASF is currently affecting pork production in a large geographical region, including Eurasia and the Caribbean. ASFV has a large genome, which harbors more than 160 genes, but most of these genes' functions have not been experimentally characterized. One of these genes is the O174L gene which has been experimentally shown to function as a small DNA polymerase. Here, we demonstrate that the deletion of the O174L gene from the genome of the virulent strain ASFV Georgia2010 (ASFV-G) does not significantly affect virus replication in vitro or in vivo. A recombinant virus, having deleted the O174L gene, ASFV-G-∆O174L, was developed to study the effect of the O174L protein in replication in swine macrophages cultures in vitro and disease production when inoculated in pigs. The results demonstrated that ASFV-G-∆O174L has similar replication kinetics to parental ASFV-G in swine macrophage cultures. In addition, animals intramuscularly inoculated with 102 HAD50 of ASFV-G-∆O174L presented a clinical form of the disease that is indistinguishable from that induced by the parental virulent strain ASFV-G. All animals developed a lethal disease, being euthanized around day 7 post-infection. Therefore, although O174L is a well-characterized DNA polymerase, its function is apparently not critical for the process of virus replication, both in vitro and in vivo, or for disease production in domestic pigs.

Investigation of the individual genetic evolution of SARS-CoV-2 in a small cluster during the rapid spread of the BF.5 lineage in Tokyo, Japan.

There has been a decreasing trend in new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases and fatalities worldwide. The virus has been evolving, indicating the potential emergence of new variants and uncertainties. These challenges necessitate continued efforts in disease control and mitigation strategies. We investigated a small cluster of SARS-CoV-2 Omicron variant infections containing a common set of genomic mutations, which provided a valuable model for investigating the transmission mechanism of genetic alterations. We conducted a study at a medical center in Japan during the Omicron surge (sub-lineage BA.5), sequencing the entire SARS-CoV-2 genomes from infected individuals and evaluating the phylogenetic tree and haplotype network among the variants. We compared the mutations present in each strain within the BA.5 strain, TKYnat2317, which was first identified in Tokyo, Japan. From June 29th to July 4th 2022, nine healthcare workers (HCWs) tested positive for SARS-CoV-2 by real-time PCR. During the same period, five patients also tested positive by real-time PCR. Whole genome sequencing revealed that the infected patients belonged to either the isolated BA.2 or BA.5 sub-lineage, while the healthcare worker infections were classified as BF.5. The phylogenetic tree and haplotype network clearly showed the specificity and similarity of the HCW cluster. We identified 12 common mutations in the cluster, including I110V in nonstructural protein 4 (nsp4), A1020S in the Spike protein, and H47Y in ORF7a, compared to the BA.5 reference. Additionally, one case had the extra nucleotide-deletion mutation I27* in ORF10, and low frequencies of genetic alterations were also found in certain instances. The results of genome sequencing showed that the nine HCWs shared a set of genetic mutations, indicating transmission within the cluster. Minor mutations observed in five HCW individuals suggested the emergence of new virus variants. Five amino acid substitutions occurred in nsp3, which could potentially affect virus replication or immune escape. Intra-host evolution also generated additional mutations. The cluster exhibited a mild disease course, with individuals in this case, recovering without requiring any medical treatments. Further investigation is needed to understand the relationship between the genetic evolution of the virus and the symptoms.

Higher Infectivity of the Human Immunodeficiency Virus in Sensitive Cells with a Modification of the CCR5 Gene.

As a natural mutation of the human ccr5 gene has been shown to confer resistance to human immunodeficiency virus type 1 (HIV-1) infection, a new avenue has opened in the development of alternative treatment approaches through genome editing. One of the two chemokine co-receptors of the plasma membrane is utilized by HIV-1 to infect CD4+ cells. HIV-1 strains that utilize CCR5 circulate in early infection, and strains that utilize CXCR4 circulate at advanced stages. A complex relationship may exist in the expression regulation of the receptors and may affect virus replication in cells that normally do not express CCR5 on the membrane, such as the MT-4 cell line. MT-4 cells were used to study the effect of ccr5 modification HIV-1 replication in vitro. Genetic modification of ccr5 in MT-4 cells was shown to increase the activities of HIV-1 strains, especially in homozygote. The results indicate that genome editing should be performed with caution in human cells and that the issue needs comprehensive investigation.

The Small GTPase Rab14 Regulates the Trafficking of Ceramide from Endoplasmic Reticulum to Golgi Apparatus and Facilitates Classical Swine Fever Virus Assembly.

Classical swine fever virus (CSFV) is a highly pathogenic RNA virus belonging to the Flaviviridae family that can cause deadly classical swine fever (CSF) in pigs. However, the molecular details of virus replication in the host are still unclear. Our previous studies have reported that several Rab proteins mediate CSFV entry into host cells, but it is unknown whether CSFV hijacks other Rab proteins for effective viral infection. Here, we systematically studied the role of Rab14 protein in regulating lipid metabolism for promoting viral assembly. First, Rab14 knockdown and overexpression significantly affected CSFV replication, indicating the essential role of Rab14 in CSFV infection. Interestingly, Rab14 could significantly affect virus replication in the late stage of infection. Mechanistically, CSFV NS5A recruited Rab14 to the ER, followed by ceramide transportation to the Golgi apparatus, where sphingomyelin was synthesized. The experimental data of small molecule inhibitors, RNA interference, and replenishment assay showed that the phosphatidylinositol-3-kinase (PI3K)/AKT/AS160 signaling pathway regulated the function of Rab14 to affect the transport of ceramide. More importantly, sphingomyelin on the Golgi apparatus contributed to the assembly of viral particles. Blockage of the Rab14 regulatory pathway induced the reduction of the content of sphingomyelin on the Golgi apparatus, impairing the assembly of virus particles. Our study clarifies that Rab14 regulates lipid metabolism and promotes CSFV replication, which provides insight into a novel function of Rab14 in regulating vesicles to transport lipids to the viral assembly factory. IMPORTANCE The Rab protein family members participate in the viral replication of multiple viruses and play important roles in the virus infection cycle. Our previous research focused on Rab5/7/11, which regulated the trafficking of vesicles in the early stage of CSFV infection, especially in viral endocytosis. However, the role of other Rab proteins in CSFV replication is unclear and needs further clarification. Strikingly, we screened some Rabs and found the important role of Rab14 in CSFV infection. Virus infection mobilized Rab14 to regulate the vesicle to transport ceramide from the ER to the Golgi apparatus, further promoting the synthesis of sphingomyelin and facilitating virus assembly. The treatment of inhibitors showed that the lipid transport mediated by Rab14 was regulated by the PI3K/AKT/AS160 signaling pathway. Knockdown of Rab14 or the treatment with PI3K/AKT/AS160 inhibitors reduced the ceramide content in infected cells and hindered virus assembly. Our study is the first to explain that vesicular lipid transport regulated by Rab promotes CSFV assembly, which is conducive to the development of antiviral drugs.

In SARS-CoV-2 delta variants, Spike-P681R and D950N promote membrane fusion, Spike-P681R enhances spike cleavage, but neither substitution affects pathogenicity in hamsters

The SARS-CoV-2 delta (B.1.617.2 lineage) variant was first identified at the end of 2020 and possessed two unique amino acid substitutions in its spike protein: S-P681R, at the S1/S2 cleavage site, and S-D950N, in the HR1 of the S2 subunit. However, the roles of these substitutions in virus phenotypes have not been fully characterized. We used reverse genetics to generate Wuhan-D614G viruses with these substitutions and delta viruses lacking these substitutions and explored how these changes affected their viral characteristics invitro and invivo. S-P681R enhanced spike cleavage and membrane fusion, whereas S-D950N slightly promoted membrane fusion. Although S-681R reduced the virus replicative ability especially in VeroE6 cells, neither substitution affected virus replication in Calu-3cells and hamsters. The pathogenicity of all recombinant viruses tested in hamsters was slightly but not significantly affected. Our observations suggest that the S-P681R and S-D950N substitutions alone do not increase virus pathogenicity, despite of their enhancement of spike cleavage or fusogenicity. A full list of funding bodies that contributed to this study can be found under Acknowledgments.

Advances in deciphering the interactions between viral proteins of influenza A virus and host cellular proteins.

Influenza A virus (IAV) poses a severe threat to the health of animals and humans. The genome of IAV consists of eight single-stranded negative-sense RNA segments, encoding ten essential proteins as well as certain accessory proteins. In the process of virus replication, amino acid substitutions continuously accumulate, and genetic reassortment between virus strains readily occurs. Due to this high genetic variability, new viruses that threaten animal and human health can emerge at any time. Therefore, the study on IAV has always been a focus of veterinary medicine and public health. The replication, pathogenesis, and transmission of IAV involve intricate interplay between the virus and host. On one hand, the entire replication cycle of IAV relies on numerous proviral host proteins that effectively allow the virus to adapt to its host and support its replication. On the other hand, some host proteins play restricting roles at different stages of the viral replication cycle. The mechanisms of interaction between viral proteins and host cellular proteins are currently receiving particular interest in IAV research. In this review, we briefly summarize the current advances in our understanding of the mechanisms by which host proteins affect virus replication, pathogenesis, or transmission by interacting with viral proteins. Such information about the interplay between IAV and host proteins could provide insights into how IAV causes disease and spreads, and might help support the development of antiviral drugs or therapeutic approaches.

WY14643 Increases Herpesvirus Replication and Inhibits IFNβ Production Independently of PPARα Expression.

Peroxisome proliferator activated receptor (PPAR) agonists are commonly used to treat metabolic disorders in humans because they regulate fatty acid oxidation and cholesterol metabolism. In addition to their roles in controlling metabolism, PPAR agonists also regulate inflammation and are immunosuppressive in models of autoimmunity. We aimed to test whether activation of PPARα with clinically relevant ligands could impact gammaherpesvirus infection using murine gammaherpesvirus-68 (MHV68, MuHV-4). We found that PPAR agonists WY14643 and fenofibrate increased herpesvirus replication in vitro. In vivo, WY14643 increased viral replication and caused lethality in mice. Unexpectedly, these effects proved independent of PPARα. We found that WY14643 suppressed production of type I interferon after MHV68 infection in vitro and in vivo. Taken together, our data indicate that caution should be employed when using PPARα agonists in immuno-metabolic studies, as they can have off-target effects on viral replication through the inhibition of type I interferon production. IMPORTANCE PPAR agonists are used clinically to treat both metabolic and inflammatory disorders. Because viruses are known to rewire host metabolism to their own benefit, the intersection of immunity, metabolism, and virology is an important research area. Our article is an important contribution to this field for two reasons. First, it shows a role for PPARα agonists in altering virus replication. Second, it shows that PPARα agonists can affect virus replication in a manner independent of their predicted target. This knowledge is valuable for anyone seeking to use PPARα agonists as a research tool.

RIG-I and MDA5 are modulated by bone morphogenetic protein (BMP6) and are essential for restricting Zika virus infection in human Sertoli cells.

Sexual transmission of Zika virus (ZIKV) is associated with virus persistence in the testes and shedding in the seminal fluid for months after recovery. We previously demonstrated that ZIKV can establish long-term replication without causing cytotoxicity in human Sertoli cells (SC), responsible for maintaining the immune privileged compartment of seminiferous tubules. Functional gene expression analyses also predicted activation of multiple virus sensing pathways including TLR3, RIG-I, and MDA5. Here, we elucidated which of the RNA virus sensing receptors play a decisive role in restricting ZIKV replication. We show that both poly I:C and IFN-β treatment induced a robust antiviral state and reduced ZIKV replication significantly, suggesting that virus sensing and antiviral signaling are functional in SC. Silencing of TLR3, 7, and 9 did not affect virus replication kinetics; however, both RIG-I and MDA5 played a synergistic role in inducing an anti-ZIKV response. Further, the impact of SC-specific immunosuppressive pathways that collectively regulate SC function, specifically the TGF-β superfamily members, TGF-β, Activin A, and BMP6, on ZIKV replication was investigated. While ZIKV did not modulate the expression of TGF-β and Activin A, BMP6 signaling was suppressed at later stages of infection. Notably, treatment with BMP6 increased IFN-β, p-IRF3, and p-STAT1 levels, and expression of key interferon-stimulated genes including MDA5, suggesting that BMP6 enhances antiviral response in SC. Collectively, this study further delineates the key role of the RIG-I-like receptors in sensing ZIKV in SC, and reveals a novel role of BMP6 in modulating innate immune and antiviral response in the testes.

АНАЛИЗ ГЕНОМНОГО РАЗНООБРАЗИЯ ИЗОЛЯТОВ И ШТАММОВ ВИРУСА АФРИКАНСКОЙ ЧУМЫ СВИНЕЙ И ФУНКЦИИ MGF360 И MGF505

The review provides information on the functions and genomic diversity of African swine fever virus isolates and strains of multigene families 360 and 505. A feature of the ASF virus genome is the presence of a large number of multigene families (MGFs) in it. The genome of the ASF virus widely contains genes of the multigene families 360 and 530, which affect virus replication in macrophages and virulence. These families of ASF virus are known to play an important role in suppressing the type I interferon response and increasing virulence in domestic pigs. The mechanism by which these genes influence virus-host interactions is still unknown. Gain or loss of fragments of multigene families results in variations in genomic DNA length observed between different ASFV isolates. There is evidence that isolates and strains with deletions in the MGS 360 and 505 region are able to reduce the virulence of the ASF virus. Studies by many researchers show that multiple deletions in several genes of the African swine fever virus can reduce virulence and actively contribute to the formation of cellular immunity.

RSV Replication, Transmission, and Disease Are Influenced by the RSV G Protein.

It is important to understand the features affecting virus replication, fitness, and transmissibility as they contribute to the outcome of infection and affect disease intervention approaches. Respiratory syncytial virus (RSV) is a major contributor to respiratory disease, particularly in the infant and elderly populations. Although first described over 60 years ago, there are no approved vaccines and there are limited specific antiviral treatments due in part to our incomplete understanding of the features affecting RSV replication, immunity, and disease. RSV studies have typically focused on using continuous cell lines and conventional RSV strains to establish vaccine development and various antiviral countermeasures. This review outlines how the RSV G protein influences viral features, including replication, transmission, and disease, and how understanding the role of the G protein can improve the understanding of preclinical studies.

RNA-seq and LC-MS/MS analysis of antiviral effects mediated by cold stress and stress hormone corticosterone in chicken DF-1 cells

Cold stress in poultry is a global problem that causes huge economic losses and threatens the health and welfare of poultry. However, knowledge of chicken responses to virus infection under cold stress is limited. The purpose of this research was to investigate the effects of cold stress on gene expression and viral replication in chicken DF-1 cells in hypothermia. In addition, the characterization of circulating steroid hormone profiles in the plasma of chickens under cold stress was analyzed by liquid chromatography-tandem mass spectrometry. Herein, we performed RNA sequencing to obtain DF-1 cell transcriptional profiles under cold stress. A total of 9499 differentially expressed genes (DEGs) were identified in DF-1 cells. Overexpressed DEGs were related to the proteasome, cell cycle, spliceosome, ribosome biogenesis, and mammalian target of rapamycin (mTOR). Down-regulated DEGs were related to ribosomes, oxidative phosphorylation, apoptosis, and the p53 signaling pathway. Gene set enrichment analysis showed that the DEGs mainly affect host ribosome translation and mitochondrial respiratory electron transport. The principal steroid hormone alterations in chickens subjected to cold stress included dihydrotestosterone, testosterone, β-sitosterol, androstenedione, 7a,27-dihydroxycholesterol,7-ketocholesterol, and desmosterol, which are associated with endocrine resistance, ovarian steroidogenesis, and steroid hormone biosynthesis. In addition, Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and Influenza A (H9N2) Virus replication in DF-1 cells is significantly inhibited by cold stress. Moreover, the plasma concentrations of corticosterone, an important stress hormone in poultry, were significantly elevated in chickens subjected to cold stress, and we found that IBV and vesicular stomatitis virus (VSV) replication were strongly inhibited in DF-1 cells pretreated with CORT, but NDV and H9N2 replication were unaffected. In conclusion, in response to cold stress, the translation efficiency and mitochondrial respiratory chain are temporarily weakened in DF-1 cells, which affects virus replication. Chickens may regulate aromatase deficiency, androstenedione metabolism, androgen and estrogen metabolism, and 17-beta hydroxysteroid dehydrogenase III deficiency through steroid hormones in response to cold stress. This study provides valuable insights into the molecular regulatory mechanisms of poultry under cold stress and may support further research on the intrinsic link between steroid hormones and virus replication under stress.

A Deletion Encompassing the Furin Cleavage Site in the Spike Encoding Gene Does Not Alter SARS-CoV-2 Replication in Lung Tissues of Mink and Neutralization by Convalescent Human Serum Samples.

SARS-CoV-2 has been shown to lose the furin polybasic cleavage site (FCS) following adaptation on cell culture. Deletion occurring in this region, which may include also the FCS flanking regions, seem not to affect virus replication in vitro; however, a chimeric SARS-CoV-2 virus without the sole FCS motif has been associated with lower virulence in mice and lower neutralization values. Moreover, SARS-CoV-2 virus lacking the FCS was shed to lower titers from experimentally infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. In this study, we investigated the replication kinetics and cellular tropism of a SARS-CoV-2 isolate carrying a 10-amino acid deletion in the spike protein spanning the FCS in lung ex vivo organ cultures of mink. Furthermore, we tested the neutralization capabilities of human convalescent SARS-CoV-2 positive serum samples against this virus. We showed that this deletion did not significantly hamper neither ex vivo replication nor neutralization activity by convalescent serum samples. This study highlights the importance of the preliminary phenotypic characterization of emerging viruses in ex vivo models and demonstrates that mink lung tissues are permissive to the replication of a mutant form of SARS-CoV-2 showing a deletion spanning the FCS. Notably, we also highlight the need for sequencing viral stocks before any infection study as large deletions may occur leading to the misinterpretation of results.

Accumulation of copy-back viral genomes during respiratory syncytial virus infection is preceded by diversification of the copy-back viral genome population followed by selection.

RNA viruses generate nonstandard viral genomes during their replication, including viral genomes of the copy-back (cbVGs) type that cannot replicate in the absence of a standard virus. cbVGs play a crucial role in shaping virus infection outcomes due to their ability to interfere with virus replication and induce strong immune responses. However, despite their critical role during infection, the principles that drive the selection and evolution of cbVGs within a virus population are poorly understood. As cbVGs are dependent on the virus replication machinery to be generated and replicated, we hypothesized that host factors that affect virus replication exert selective pressure on cbVGs and drive their evolution within a virus population. To test this hypothesis, we used respiratory syncytial virus (RSV) as a model and took an experimental evolution approach by serially passaging RSV in immune-competent human lung adenocarcinoma A549 control and immune-deficient A549 Signal transducer and activator of transcription 1 (STAT1) KO cells, which allow higher levels of virus replication. As predicted, we observed that virus populations accumulated higher amounts of cbVGs in the more permissive A549 STAT1 KO cells over time; however, unexpectedly, the predominant cbVG species after passages in the two conditions were different. While A549 STAT1 KO cells accumulated relatively short cbVGs, A549 control cells mainly contained cbVGs of much longer predicted size, which have not been described previously. These long cbVGs were predominant at first in both cell lines in vitro and the predominant ones observed in samples from RSV-infected patients. Although sustained high replication levels are associated with cbVG generation and accumulation, our data show that sustained high levels of virus replication are critical for cbVG population diversification, a process that precedes the generation of shorter cbVGs that selectively accumulate over time. Taken together, we show that selection and evolution of cbVGs within a virus population are shaped by how resistant or permissive a host is to RSV.