Levels Of Viral Replication Research Articles

Biological Significance of Dual Mutations A494D and E495K of the Genotype III Newcastle Disease Virus Hemagglutinin-Neuraminidase In Vitro and In Vivo.

As a multifunctional protein, the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is involved in various biological functions. A velogenic genotype III NDV JS/7/05/Ch evolving from the mesogenic vaccine strain Mukteswar showed major amino acid (aa) mutations in the HN protein. However, the precise biological significance of the mutant HN protein remains unclear. This study sought to investigate the effects of the mutant HN protein on biological activities in vitro and in vivo. The mutant HN protein (JS/7/05/Ch-type HN) significantly enhanced the hemadsorption (HAd) and fusion promotion activities but impaired the neuraminidase (NA) activity compared with the original HN protein (Mukteswar-type HN). Notably, A494D and E495K in HN exhibited a synergistic role in regulating biological activities. Moreover, the mutant HN protein, especially A494D and E495K in HN, enhanced the F protein cleavage level, which can contribute to the activation of the F protein. In vitro infection assays further showed that NDVs bearing A494D and E495K in HN markedly impaired the cell viability. Simultaneously, A494D and E495K in HN enhanced virus replication levels at the early stage of infection but weakened later in infection, which might be associated with the attenuated NA activity and cell viability. Furthermore, the animal experiments showed that A494D and E495K in HN enhanced case fatality rates, virus shedding, virus circulation, and histopathological damages in NDV-infected chickens. Overall, these findings highlight the importance of crucial aa mutations in HN in regulating biological activities of NDV and expand the understanding of the enhanced pathogenicity of the genotype III NDV.

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.

Effects of Caspase-3 on HIV-1 Latency in a 3.01 Cells

Latent infection is a major barrier for cure of HIV-1/AIDS. HIV-1 is capable of establishing latency and the components of the apoptotic pathways may affect viral latency. However, it is not well-known how anti/pro-apoptotic components modulate HIV-1 replication and latency. Using the susceptible A3.01 cell line, we investigated some long-term effects of caspase-3 activities on HIV-1 DNA levels using a sensitive real-time PCR assay. Here we report that viral DNA levels increased upon treatment with caspase-3 inhibitor, Z-DEVD and decreased with caspase-3 activator, PAC1. We also simultaneously measured viral RNA from supernatants of these cell cultures and found that the degree of HIV-1 latency is inversely proportional to levels of viral replication. Furthermore, we demonstrated that inhibition of caspase-3 activities promoted viral latency and inhibited viral replication in several ways, which may include: 1) inhibition of viral RNA un coating with increased Trim5a expression; 2) deleterious mutations in the viral genome with increased APOBEC3G; 3) transcriptional interference with decreased levels of the host factors, NF-κB p65, Ap-1, Sp-1, NFAT, STAT1/3/5, IRF3/7, inactivated YB-1 and MAPK, Erk1/2 and p38, and inhibition of full-length of HIV-1 mRNA and P-TEF b signaling; 4) epigenetic silencing with decreased PCAF; 5) blocking trafficking of the components of viral particle and budding with decreased Tag101 and Alix. These data suggest that HIV-1 infection can employ or even manipulate the cellular apoptotic status to favor viral survival and escape monitoring and destruction by the host immune system.

Genotypic Resistance Testing of HIV-1 DNA in Peripheral Blood Mononuclear Cells.

HIV-1 DNA exists in nonintegrated linear and circular episomal forms and as integrated proviruses. In patients with plasma viremia, most peripheral blood mononuclear cell (PBMC) HIV-1 DNA consists of recently produced nonintegrated virus DNA while in patients with prolonged virological suppression (VS) on antiretroviral therapy (ART), most PBMC HIV-1 DNA consists of proviral DNA produced months to years earlier. Drug-resistance mutations (DRMs) in PBMCs are more likely to coexist with ancestral wild-type virus populations than they are in plasma, explaining why next-generation sequencing is particularly useful for the detection of PBMC-associated DRMs. In patients with ongoing high levels of active virus replication, the DRMs detected in PBMCs and in plasma are usually highly concordant. However, in patients with lower levels of virus replication, it may take several months for plasma virus DRMs to reach detectable levels in PBMCs. This time lag explains why, in patients with VS, PBMC genotypic resistance testing (GRT) is less sensitive than historical plasma virus GRT, if previous episodes of virological failure and emergent DRMs were either not prolonged or not associated with high levels of plasma viremia. Despite the increasing use of PBMC GRT in patients with VS, few studies have examined the predictive value of DRMs on the response to a simplified ART regimen. In this review, we summarize what is known about PBMC HIV-1 DNA dynamics, particularly in patients with suppressed plasma viremia, the methods used for PBMC HIV-1 GRT, and the scenarios in which PBMC GRT has been used clinically.

Development and characterization of type I interferon receptor knockout sheep: A model for viral immunology and reproductive signaling.

Type I interferons (IFNs) initiate immune responses to viral infections. Their effects are mediated by the type I IFN receptor, IFNAR, comprised of two subunits: IFNAR1 and IFNAR2. One or both chains of the sheep IFNAR were disrupted in fetal fibroblast lines using CRISPR/Cas9 and 12 lambs were produced by somatic cell nuclear transfer (SCNT). Quantitative reverse transcription-polymerase chain reaction for IFN-stimulated gene expression showed that IFNAR deficient sheep fail to respond to IFN-alpha. Furthermore, fibroblast cells from an IFNAR2 −/− fetus supported significantly higher levels of Zika virus (ZIKV) replication than wild-type fetal fibroblast cells. Although many lambs have died from SCNT related problems or infections, one fertile IFNAR2 −/− ram lived to over 4 years of age, remained healthy, and produced more than 80 offspring. Interestingly, ZIKV infection studies failed to demonstrate a high level of susceptibility. Presumably, these sheep compensated for a lack of type I IFN signaling using the type II, IFN-gamma and type III, IFN-lambda pathways. These sheep constitute a unique model for studying the pathogenesis of viral infection. Historical data supports the concept that ruminants utilize a novel type I IFN, IFN-tau, for pregnancy recognition. Consequently, IFNAR deficient ewes are likely to be infertile, making IFNAR knockout sheep a valuable model for studying pregnancy recognition. A breeding herd of 32 IFNAR2 +/− ewes, which are fertile, has been developed for production of IFNAR2 −/− sheep for both infection and reproduction studies.

Bimodal distribution pattern associated with the PCR cycle threshold (Ct) and implications in COVID-19 infections

SARS-CoV-2 is notable for its extremely high level of viral replication in respiratory epithelial cells, relative to other cell types. This may partially explain the high transmissibility and rapid global dissemination observed during the COVID-19 pandemic. Polymerase chain reaction (PCR) cycle threshold (Ct) number has been widely used as a proxy for viral load based on the inverse relationship between Ct number and amplifiable genome copies present in a sample. We examined two PCR platforms (Centers for Disease Control and Prevention 2019-nCoV Real-time RT-PCR, Integrated DNA Technologies; and TaqPath COVID-19 multi-plex combination kit, ThermoFisher Scientific) for their performance characteristics and Ct distribution patterns based on results generated from 208,947 clinical samples obtained between October 2020 and September 2021. From 14,231 positive tests, Ct values ranged from 8 to 39 and displayed a pronounced bimodal distribution. The bimodal distribution persisted when stratified by gender, age, and time period of sample collection during which different viral variants circulated. This finding may be a result of heterogeneity in disease progression or host response to infection irrespective of age, gender, or viral variants. Quantification of respiratory mucosal viral load may provide additional insight into transmission and clinical indicators helpful for infection control.

Acteoside attenuates RSV-induced lung injury by suppressing necroptosis and regulating metabolism

Background: Necroptosis and inflammation are closely related to the pathogenesis of respiratory syncytial virus (RSV). Acteoside (AC), a natural phenylpropanoid glycoside from Kuding Tea, has significant anti-RSV effect. However, the roles of AC on RSV-induced lung necroptosis and inflammation are yet to be elucidated. Methods: The effects of AC were investigated in BALB/c mice and A549 cells. Lung histopathology was observed through H&E staining. The viral titer was assessed via plaque assay. The RSV-F expression was determined by RT-qPCR and immunohistochemistry assay. The levels of cytokines were detected by ELISA and RT-qPCR. The necroptosis rate and mitochondrial membrane potential were evaluated via flow cytometry. The expressions of HMGB1/NF-κB and RIP1/RIP3/MLKL/PGAM5/DRP1 were detected by western blot. Additionally, untargeted metabolomics was conducted to investigate the metabolic profiles and related metabolic pathways via Gas Chromatography-Mass Spectrometry. Results: The results showed that compared with the RSV-infected group, AC treatment significantly attenuated lung pathological damage, virus replication, and cytokines levels. AC also alleviated RSV-induced necroptosis and mitochondrial dysfunction in vitro and in vivo. Moreover, AC treatment down-regulated the expression of HMGB1, p-Iκbα/Iκbα, p-p65/p65, RIP1, RIP3, MLKL, PGAM5, and DRP1. Furthermore, metabolomic analyses suggested that the perturbations in major metabolites of AC therapy were related to variations in amino acid and energy metabolism. Conclusion: Our findings validated the beneficial effects of AC in suppressing necroptosis and regulating metabolism, suggesting AC may be a new drug candidate for RSV infection.

Antiviral activity of extracellular vesicles derived from respiratory syncytial virus-infected airway epithelial cells.

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in children and elderly. No vaccine or effective treatment is currently available for RSV. Extracellular vesicles (EVs) are microvesicles known to carry biologically active molecules, including RNA, DNA and proteins (i.e. cargo). Viral infections can induce profound changes in EV cargo, and the cargo can modulate cellular responses of recipient cells. We have recently shown that EVs isolated from RSV-infected cells were able to activate innate immune response by inducing cytokine and chemokine release from human monocytes and airway epithelial cells, however, we did not investigate the potential antiviral contribution of EVs to a subsequent infection. The objective of this study was to assess the presence of innate immune mediators, including type I and III interferons (IFNs) in EVs released from airway epithelial cells infected with RSV, and their potential role in modulating viral replication in recipient cells. EV-derived from cells infected with RSV were associated with significant amounts of cytokine and chemokines, as well as IFN-β and -λ, compared to EVs isolated from mock-infected cells. Cells treated with RSV-EVs showed significantly lower levels of viral replication compared to untreated or mock-EV-treated RSV infected cells. Cellular pretreatment with Cerdulatinib, an IFN receptor signaling inhibitor, inhibited the antiviral activity of RSV-EVs in recipient airway epithelial cells. Furthermore, treatment of A549 cells with RSV-EVs induced the expression of IFN-dependent antiviral genes, supporting the idea that RSV-EVs exerts their antiviral activity through an interferon-dependent mechanism. Finally, we determined the concentrations of soluble and EV-associated IFN-β and IFN-λ in five nasopharyngeal secretions (NPS) of children with viral infections. There were significant levels of IFN-λ in NPS and NPS-derived EVs, while IFN-β was not detected in either of the two types of samples. EVs released from RSV-infected cells could represent a potential therapeutic approach for modulating RSV replication in the airways.

Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

Oncolytic adenoviruses (OAd) can be employed to efficiently eliminate cancer cells through multiple mechanisms of action including cell lysis and immune activation. Our OAds, AdΔΔ and Ad-3∆-A20T, selectively infect, replicate in, and kill adenocarcinoma cells with the added benefit of re-sensitising drug-resistant cells in preclinical models. Further modifications are required to enable systemic delivery in patients due to the rapid hepatic elimination and neutralisation by blood factors and antibodies. Here, we show data that support the use of coating OAds with gold nanoparticles (AuNPs) as a possible new method of virus modification to help augment tumour uptake. The pre-incubation of cationic AuNPs with AdΔΔ, Ad-3∆-A20T and wild type adenovirus (Ad5wt) was performed prior to infection of prostate/pancreatic cancer cell lines (22Rv, PC3, Panc04.03, PT45) and a pancreatic stellate cell line (PS1). Levels of viral infection, replication and cell viability were quantified 24–72 h post-infection in the presence and absence of AuNPs. Viral spread was assessed in organotypic cultures. The presence of AuNPs significantly increased the uptake of Ad∆∆, Ad-3∆-A20T and Ad5wt in all the cell lines tested (ranging from 1.5-fold to 40-fold), compared to virus alone, with the greatest uptake observed in PS1, a usually adenovirus-resistant cell line. Pre-coating the AdΔΔ and Ad-3∆-A20T with AuNPs also increased viral replication, leading to enhanced cell killing, with maximal effect in the most virus-insensitive cells (from 1.4-fold to 5-fold). To conclude, the electrostatic association of virus with cationic agents provides a new avenue to increase the dose in tumour lesions and potentially protect the virus from detrimental blood factor binding. Such an approach warrants further investigation for clinical translation.

Recapitulating infection, thermal sensitivity and antiviral treatment of seasonal coronaviruses in human airway organoids

SummaryBackgroundHuman seasonal coronaviruses usually cause mild upper-respiratory tract infection, but severe complications can occur in specific populations. Research into seasonal coronaviruses is limited and robust experimental models are largely lacking. This study aims to establish human airway organoids (hAOs)-based systems for seasonal coronavirus infection and to demonstrate their applications in studying virus-host interactions and therapeutic development.MethodsThe infections of seasonal coronaviruses 229E, OC43 and NL63 in 3D cultured hAOs with undifferentiated or differentiated phenotypes were tested. The kinetics of virus replication and production was profiled at 33 °C and 37 °C. Genome-wide transcriptome analysis by RNA sequencing was performed in hAOs under various conditions. The antiviral activity of molnupiravir and remdesivir, two approved medications for treating COVID19, was tested.FindingsHAOs efficiently support the replication and infectious virus production of seasonal coronaviruses 229E, OC43 and NL63. Interestingly, seasonal coronaviruses replicate much more efficiently at 33 °C compared to 37 °C, resulting in over 10-fold higher levels of viral replication. Genome-wide transcriptomic analyses revealed distinct patterns of infection-triggered host responses at 33 °C compared to 37 °C temperature. Treatment of molnupiravir and remdesivir dose-dependently inhibited the replication of 229E, OC43 and NL63 in hAOs.InterpretationHAOs are capable of modeling 229E, OC43 and NL63 infections. The intriguing finding that lower temperature resembling that in the upper respiratory tract favors viral replication may help to better understand the pathogenesis and transmissibility of seasonal coronaviruses. HAOs-based innovative models shall facilitate the research and therapeutic development against seasonal coronavirus infections.FundingThis research is supported by funding of a VIDI grant (No. 91719300) from the Netherlands Organization for Scientific Research and the Dutch Cancer Society Young Investigator Grant (10140) to Q.P., and the ZonMw COVID project (114025011) from the Netherlands Organization for Health Research and Development to R.R.

Immature Brain Cortical Neurons Have Low Transcriptional Competence to Activate Antiviral Defences and Control RNA Virus Infections

Virus infections of the central nervous system (CNS) cause important diseases of humans and animals. As in other tissues, innate antiviral responses mediated by type I interferons (IFNs) are crucially important in controlling CNS virus infections. The maturity of neuronal populations is an established critical factor determining the outcome of CNS virus infection. Using primary cultures of mouse cortical neurons, we investigated the relationships between neuronal maturation, type I IFN responses, and the outcome of Semliki Forest virus infection. The virus replicated better, infected more cells, and produced higher titres of infectious viruses in immature neurons. Complete transcriptome analysis demonstrated that resting immature neurons have low transcriptional competence to mount antiviral responses. They had no detectable transcription of the genes Ddx58 and Ifih1, which encode key RNA virus cytoplasmic sensors RIG-I and MDA5, and very low expression of genes encoding key regulators of associated signalling pathways. Upon infection, immature neurons failed to mount an antiviral response as evidenced by their failure to produce chemokines, IFNs, and other cytokines. Treatment of immature neurons with exogenous IFNβ prior to infection resulted in antiviral responses and lower levels of virus replication and infectious virus production. In contrast, resting mature neurons generated a robust antiviral response. This was augmented by pretreatment with IFNβ. Infection of mature neurons derived from IFNAR−/− mice did not make an antiviral response and replicated virus to high levels.

A 77 Amino Acid Region in the N-Terminal Half of the HSV-1 E3 Ubiquitin Ligase ICP0 Contributes to Counteracting an Established Type 1 Interferon Response.

ABSTRACTHerpes simplex virus 1 (HSV-1) is a human pathogen capable of establishing lifelong latent infections that can reactivate under stress conditions. A viral immediate early protein that plays important roles in the HSV-1 lytic and latent infections is the viral E3 ubiquitin ligase, ICP0. ICP0 transactivates all temporal classes of HSV-1 genes and facilitates viral gene expression. ICP0 also impairs the antiviral effects of interferon (IFN)-β, a component of host innate defenses known to limit viral replication. To begin to understand how ICP0 allows HSV-1 to disarm the IFN-β response, we performed genetic analyses using a series of ICP0 truncation mutants in the absence and presence of IFN-β in cell culture. We observed that IFN-β pretreatment of cells significantly impaired the replication of the ICP0 truncation mutants, n212 and n312, which code for the first 211 and 311 amino acids of ICP0, respectively; this effect of IFN-β correlated with decreased HSV-1 early and late gene expression. This increased sensitivity to IFN-β was not as apparent with the ICP0 mutant, n389. Our mapping studies indicate that loss of 77 amino acids from residues 312 to 388 in the N-terminal half of ICP0 resulted in a virus that was significantly more sensitive to cells pre-exposed to IFN-β. This 77 amino acid region contains a phospho-SUMO-interacting motif or -SIM, which we propose participates in ICP0’s ability to counteract the antiviral response established by IFN-β.IMPORTANCE Interferons (IFNs) are secreted cellular factors that are induced by viral infection and limit replication. HSV-1 is largely refractory to the antiviral effects of type 1 IFNs, which are synthesized shortly after viral infection, in part through the activities of the viral regulatory protein, ICP0. To understand how ICP0 impedes the antiviral effects of type 1 IFNs, we used a series of HSV-1 ICP0 mutants and examined their viral replication and gene expression levels in cells stimulated with IFN-β (a type 1 IFN). Our mapping data identifies a discrete 77 amino acid region in the N-terminal half of ICP0 that facilitates HSV-1 resistance to IFN-β. This region of ICP0 is modified by phosphorylation and binds to the posttranslational modification SUMO, suggesting that HSV, and potentially other viruses, may counteract type 1 IFN signaling by altering SUMO and/or SUMO modified cellular proteins.

Lineage 1 Porcine Reproductive and Respiratory Syndrome Virus Attenuated Live Vaccine Provides Broad Cross-Protection against Homologous and Heterologous NADC30-Like Virus Challenge in Piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that endangers the swine industry worldwide. Recently, lineage 1 PRRSVs, especially NADC30-like PRRSVs, have become the major endemic strains in many pig-breeding countries. Since 2016, NADC30-like PRRSV has become the predominant strain in China. Unfortunately, current commercial vaccines cannot provide sufficient protection against this strain. Here, an attenuated lineage 1 PRRSV strain, named SD-R, was obtained by passaging an NADC30-like PRRSV strain SD in Marc-145 cells for 125 passages. Four-week-old PRRSV-free piglets were vaccinated intramuscularly with 105.0TCID50 SD-R and then challenged intramuscularly (2 mL) and intranasally (2 mL) with homologous NADC30-like PRRSV SD (1 × 105.0TCID50/mL) and heterologous NADC30-like PRRSV HLJWK108-1711 (1 × 105.0TCID50/mL). The results showed that antibodies against specific PRRSVs in 5 of 5 immunized piglets were positive after a 14-day post-vaccination and did not develop fever or clinical diseases after NADC30-like PRRSV challenges. Additionally, compared with challenge control piglets, vaccinated piglets gained significantly more weight and showed much milder pathological lesions. Furthermore, the viral replication levels of the immunized group were significantly lower than those of the challenge control group. These results demonstrate that lineage 1 PRRSV SD-R is a good candidate for an efficacious vaccine, providing complete clinical protection for piglets against NADC30-like PRRSVs.

Hotspot siRNA Confers Plant Resistance against Viral Infection.

Simple SummaryA hallmark of antiviral RNAi is the production of viral siRNA (vsiRNA). Profiling of vsiRNAs indicates that certain hotspot regions of viral genome or transcribed viral RNAs are more prone to RNAi-mediated cleavage. However, the biological relevance of hotspot vsiRNAs to the host innate defence remains to be elucidated. Here, we show that direct targeting a hotspot by synthetic vsiRNA confers plant resistance to virus infection. Hotspot and coldspot vsiRNAs, based on vsiRNA profile of the African cassava mosaic virus (ACMV), were synthesised. However, only the double-stranded hotspot vsiRNA protected plants from ACMV infection with undetectable levels of viral DNA replication and viral mRNA. We further demonstrated that the hotspot vsiRNA-mediated virus resistance had a threshold effect and required an active RDR6. These data show that hotspot vsiRNAs bear a functional significance on antiviral RNAi, suggesting that they may have the potential as exogenous protection agents for controlling destructive plant viral diseases.A hallmark of antiviral RNA interference (RNAi) is the production of viral small interfering RNA (vsiRNA). Profiling of vsiRNAs indicates that certain regions of viral RNA genome or transcribed viral RNA, dubbed vsiRNA hotspots, are more prone to RNAi-mediated cleavage for vsiRNA biogenesis. However, the biological relevance of hotspot vsiRNAs to the host innate defence against pathogens remains to be elucidated. Here, we show that direct targeting a hotspot by a synthetic vsiRNA confers host resistance to virus infection. Using Northern blotting and RNAseq, we obtained a profile of vsiRNAs of the African cassava mosaic virus (ACMV), a single-stranded DNA virus. Sense and anti-sense strands of small RNAs corresponding to a hotspot and a coldspot vsiRNA were synthesised. Co-inoculation of Nicotiana benthamiana with the double-stranded hotspot siRNA protected plants from ACMV infection, where viral DNA replication and accumulation of viral mRNA were undetectable. The sense or anti-sense strand of this hotspot vsiRNA, and the coldspot vsiRNA in both double-stranded and single-stranded formats possessed no activity in viral protection. We further demonstrated that the hotspot vsiRNA-mediated virus resistance had a threshold effect and required an active RDR6. These data show that hotspot vsiRNAs bear a functional significance on antiviral RNAi, suggesting that they may have the potential as an exogenous protection agent for controlling destructive viral diseases in plants.

Avian Reoviruses From Wild Birds Exhibit Pathogenicity to Specific Pathogen Free Chickens by Footpad Route.

Avian reoviruses (ARVs) are ubiquitous in domestic poultry with 80% of them being non-pathogenic and they are frequently found in clinically healthy birds. ARVs have also been known to be the etiological agents of viral arthritis (VA), tenosynovitis, myocarditis, runting-stunting syndrome (RSS), and respiratory and enteric disease in chickens. Significant economic losses during the process of poultry husbandry are due, in part, to unmitigated ARV infections throughout the poultry industry. Recently, many isolates shared genetic similarities between those recovered from wild birds and those recovered from poultry. One explanation may be that there is a degree of spillover and spillback of ARVs between the two groups. However, studies on the role of wild birds in the epidemiology and pathogenicity of ARVs are insufficient. Here, we describe the pathogenicity in specific pathogen-free (SPF) chickens of ARV originating from wild birds. The challenge experiment was conducted in six groups including a negative control group, a positive control group (reference strain of S1133), and four groups (A15-157, A18-13, A18-205, A19-106) infected with ARVs from wild birds. The 7-day-old SPF chickens were inoculated with 106TCID50 ARV to evaluate the clinical signs, changes in weight gain, gross lesions, histological changes, virus replication, and serum antibody levels. The peak of clinical signs was from 3 to 5 days post infection (dpi). In addition, the death of one chicken was found in the group infected with the A18-13 isolate. Reduced body weight was also found in chickens infected with ARVs from wild birds compared to the negative control group. All the ARVs infection groups showed noticeable swelling of the footpad. In addition, ARVs were detected in the bursa, tendon, and hock joint by reverse transcription-polymerase chain reaction (RT-PCR) in all infected groups at 5 and 15 dpi. Histopathological observations revealed acute inflammatory responses on the synovium covering the joint surfaces (arthritis) and tendon sheaths (tenosynovitis), as well as bursa atrophy and lymphocyte depletion. The analysis of the humoral response was performed by ELISA assay, and chickens infected with ARVs showed seroconverted. In conclusion, this study described the typical severe disease of acute VA and tenosynovitis in SPF chickens infected with ARVs derived from wild birds. This study confirmed the pathogenicity of ARVs infection in SPF chickens for the first time, and these results enrich our understanding of the pathogenicity of ARVs derived from wild birds.

Human microglial models to study HIV infection and neuropathogenesis: a literature overview and comparative analyses.

HIV persistence in the CNS despite antiretroviral therapy may cause neurological disorders and poses a critical challenge for HIV cure. Understanding the pathobiology of HIV-infected microglia, the main viral CNS reservoir, is imperative. Here, we provide a comprehensive comparison of human microglial culture models: cultured primary microglia (pMG), microglial cell lines, monocyte-derived microglia (MDMi), stem cell–derived microglia (iPSC-MG), and microglia grown in 3D cerebral organoids (oMG) as potential model systems to advance HIV research on microglia. Functional characterization revealed phagocytic capabilities and responsiveness to LPS across all models. Microglial transcriptome profiles of uncultured pMG showed the highest similarity to cultured pMG and oMG, followed by iPSC-MG and then MDMi. Direct comparison of HIV infection showed a striking difference, with high levels of viral replication in cultured pMG and MDMi and relatively low levels in oMG resembling HIV infection observed in post-mortem biopsies, while the SV40 and HMC3 cell lines did not support HIV infection. Altogether, based on transcriptional similarities to uncultured pMG and susceptibility to HIV infection, MDMi may serve as a first screening tool, whereas oMG, cultured pMG, and iPSC-MG provide more representative microglial culture models for HIV research. The use of current human microglial cell lines (SV40, HMC3) is not recommended.Supplementary informationThe online version contains supplementary material available at 10.1007/s13365-021-01049-w.

Incursion of H5N8 high pathogenicity avian influenza virus (HPAIV) into gamebirds in England.

The 2016-17 European outbreak of H5N8 HPAIV (Clade 2.3.4.4b) affected a wider range of avian species than the previous H5N8 outbreak (2014-15), including an incursion of H5N8 HPAIV into gamebirds in England. Natural infection of captive-reared pheasants (Phasianus colchicus) led to variable disease presentation; clinical signs included ruffled feathers, reluctance to move, bright green faeces, and/or sudden mortality. Several birds exhibited neurological signs (nystagmus, torticollis, ataxia). Birds exhibiting even mild clinical signs maintained substantial levels of virus replication and shedding, with preferential shedding via the oropharyngeal route. Gross pathology was consistent with HPAIV, in gallinaceous species but diphtheroid plaques in oropharyngeal mucosa associated with necrotising stomatitis were novel but consistent findings. However, minimal or modest microscopic pathological lesions were detected despite the systemic dissemination of the virus. Serology results indicated differences in the timeframe of exposure for each case (n = 3). This supported epidemiological conclusions confirming that the movement of birds between sites and other standard husbandry practices with limited hygiene involved in pheasant rearing (including several fomite pathways) contributed to virus spread between premises.

Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models—the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.

Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of IFN-mediated innate immune defenses.

The pandemic spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19), represents an ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 replication. Utilizing a three-dimensional (3D) air-liquid interface (ALI) model that closely mimics the natural tissue physiology of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. Respiratory tissue incubated at 40°C remained permissive to SARS-CoV-2 entry but refractory to viral transcription, leading to significantly reduced levels of viral RNA replication and apical shedding of infectious virus. We identify tissue temperature to play an important role in the differential regulation of epithelial host responses to SARS-CoV-2 infection that impact upon multiple pathways, including intracellular immune regulation, without disruption to general transcription or epithelium integrity. We present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication in respiratory epithelia. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 independently of canonical interferon (IFN)-mediated antiviral immune defenses.

Use of Micro-Computed Tomography to Visualize and Quantify COVID-19 Vaccine Efficiency in Free-Breathing Hamsters.

The SARS-CoV-2 pandemic has impacted the health of humanity after the outbreak in Hubei, China in late December 2019. Ever since, it has taken unprecedented proportions and rapidity causing over a million fatal cases. Recently, a robust Syrian golden hamster model recapitulating COVID-19 was developed in search for effective therapeutics and vaccine candidates. However, overt clinical disease symptoms were largely absent despite high levels of virus replication and associated pathology in the respiratory tract. Therefore, we used micro-computed tomography (μCT) to longitudinally visualize lung pathology and to preclinically assess candidate vaccines. μCT proved to be crucial to quantify and noninvasively monitor disease progression, to evaluate candidate vaccine efficacy, and to improve screening efforts by allowing longitudinal data without harming live animals. Here, we give a comprehensive guide on how to use low-dose high-resolution μCT to follow-up SARS-CoV-2-induced disease and test the efficacy of COVID-19 vaccine candidates in hamsters. Our approach can likewise be applied for the preclinical assessment of antiviral and anti-inflammatory drug treatments in vivo.