Marek's disease virus (MDV) causes lymphomas and neurological disorders in chickens. Although vaccination largely controls outbreaks, highly virulent strains continue to emerge. The major MDV-encoded oncoprotein is Meq, functions as a transcription factor. Amino acid polymorphisms in Meq have been reported to influence virulence. Despite routine vaccination, MD still occurs sporadically in Japan. Japanese isolates harbour characteristic Meq polymorphisms, but their impact on MDV virulence remains unclear. We investigated the transcriptional regulation by Meq from Japanese isolates and evaluated the pathogenicity of recombinant MDV (rMDV) encoding Meq from the Japanese isolate Nr-c1. Nr-c1-Meq exhibited reduced transrepression and transactivation on viral gene promoters. An rMDV encoding Nr-c1-Meq (vNr-c1-Meq) induced lower mortality and tumourigenicity than an rMDV encoding Meq from the parental very virulent RB-1B strain (vRB-1B). vNr-c1-Meq did not cause visceral tumours or neurological disorders but resulted in distinct clinical signs, including open-mouth breathing. In lymphoid tissues from vNr-c1-Meq-infected chickens, a lower proportion of CD4+ T cells, the targets of MDV transformation, and lower viral loads were observed than those in vRB-1B-infected chickens. Histopathological examination revealed increased lymphocyte infiltration in bronchus-associated lymphoid tissues (BALT) in the vNr-c1-Meq group. Additionally, flow cytometric analysis showed elevated γδ T-cell proportions, which positively correlated with IFN-γ expression in vNr-c1-Meq-infected chickens and were linked to BALT hyperplasia. In comparison to the vRB-1B, vNr-c1-Meq infection resulted in attenuated disease progression and altered clinical signs. These findings suggest that Meq polymorphisms not only influence MD virulence but also clinical presentation.

Marek’s disease virus (MDV) causes Marek’s disease (MD) in chickens, which is characterized by malignant lymphomas and neurological disorders. Although MD is currently controlled using live vaccines, the virulence of field strains has continuously increased in recent decades. Polymorphisms in the MDV-encoded oncoprotein Meq are shared among field strains according to their virulence. In particular, very virulent MDV strains harbor characteristic amino acid changes in the basic region of Meq at positions 77 and 80; however, the contribution of these polymorphisms to virulence remains unclear. To assess the impact of these polymorphisms on MDV virulence, we generated recombinant MDV (rMDV) based on the very virulent RB-1B strain, harboring K77E and D80Y substitutions in Meq found in low-virulent strains (rRB-1B_Meq77/80). Chickens were challenged with rMDVs, and survival rates and tumor incidence were evaluated. Viral loads in major organs were quantified by quantitative PCR, and the dynamics of MDV-infected cells and T cells were analyzed using flow cytometry. In addition, histopathological analysis was performed to further examine differences in pathogenesis in detail. To elucidate the mechanisms underlying pathogenesis, we conducted reporter assays to assess the effect of these polymorphisms in the basic region on its transcriptional regulatory activity. rRB-1B_Meq77/80 exhibited a reduced virulence but unexpectedly caused other clinical signs, including open-mouth breathing, in infected chickens. Quantitative PCR analysis showed consistently lower viral loads across all examined organs in rRB-1B_Meq77/80-infected chickens. Flow cytometric analysis revealed a reduction in MDV-infected cells, accompanied by a notable increase in CD8⁺ T cell populations. Histopathological analysis showed bronchus-associated lymphoid tissue hyperplasia in the lungs. Reporter assays revealed that most amino acid substitutions in the basic region in low-virulence strains reduced transcriptional regulatory activity. Our data indicate that polymorphisms at positions 77 and 80 in the Meq of low-virulence strains reduce MDV virulence and Meq-mediated transcription and possibly alter pathogenesis. This study improves our understanding of the mechanisms underlying MDV virulence.

Background: Marek’s disease (MD) is a pathology affecting chickens caused by Marek’s disease virus (MDV), an acute transforming alphaherpesvirus of the genus Mardivirus. MD is characterized by paralysis, immune suppression, and the rapid formation of T-cell (primarily CD4+) lymphomas. Over the last 50 years, losses due to MDV infection have been controlled worldwide through vaccination; however, these live-attenuated vaccines are non-sterilizing and potentially contributed to the virulence evolution of MDV field strains. Mutations common to field strains that can overcome vaccine protection were identified in the C-terminal proline-rich repeats of the oncoprotein Meq (Marek’s EcoRI-Q-encoded protein). These mutations in meq have been found to be distinct to their region of origin, with high virulence strains obtained in Europe differing from those having evolved in the US. The present work reports on meq mutations identified in MDV field strains in Nigeria, arising at farms employing different vaccination practices. Materials and Methods: DNA was isolated from FTA cards obtained at 12 farms affected by increased MD in the Plateau State, Nigeria. These sequences included partial whole genomes as well as targeted sequences of the meq oncogenes from these strains. Several of the meq genes were cloned for expression and their localization ability to interact with the chicken NF-IL3 protein, a putative Meq dimerization partner, were assessed. Results: Sequence analysis of the meq genes from these Nigerian field strains revealed an RB1B-like lineage co-circulating with a European Polen5-like lineage, as well as recombinants harboring a combination of these mutations. In a number of these isolates, Meq mutations accumulated in both N-terminal and C-terminal domains. Discussion: Our data, suggest a direct effect of the vaccine strategy on the selection of Meq mutations. Moreover, we posit the evolution of the next higher level of virulence MDVs, a very virulent plus plus pathotype (vv++).

RNA viruses continue to remain a clear and present threat for potential pandemics due to their rapid evolution. To mitigate their impact, we urgently require antiviral agents that can inhibit multiple families of disease-causing viruses, such as arthropod-borne and respiratory pathogens. Potentiating host antiviral pathways can prevent or limit viral infections before escalating into a major outbreak. Therefore, it is critical to identify broad-spectrum antiviral agents. We have tested a small library of innate immune agonists targeting pathogen recognition receptors, including TLRs, STING, NOD, Dectin and cytosolic DNA or RNA sensors. We observed that TLR3, STING, TLR8 and Dectin-1 ligands inhibited arboviruses, Chikungunya virus (CHIKV), West Nile virus (WNV) and Zika virus, to varying degrees. Cyclic dinucleotide (CDN) STING agonists, such as cAIMP, diABZI, and 2',3'-cGAMP, and Dectin-1 agonist scleroglucan, demonstrated the most potent, broad-spectrum antiviral function. Comparative transcriptome analysis revealed that CHIKV-infected cells had larger number of differentially expressed genes than of WNV and ZIKV. Furthermore, gene expression analysis showed that cAIMP treatment rescued cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provided protection against CHIKV in a CHIKV-arthritis mouse model. Cardioprotective effects of synthetic STING ligands against CHIKV, WNV, SARS-CoV-2 and enterovirus D68 (EV-D68) infections were demonstrated using human cardiomyocytes. Interestingly, the direct-acting antiviral drug remdesivir, a nucleoside analogue, was not effective against CHIKV and WNV, but exhibited potent antiviral effects against SARS-CoV-2, RSV (respiratory syncytial virus), and EV-D68. Our study identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses, which can be rapidly deployed to prevent or mitigate future pandemics.

Marek’s disease virus (MDV), an Alphaherpesvirus belonging to the genus Mardivirus, causes T cell lymphomas in chickens and remains one of the greatest threats to poultry production worldwide [...]

RNA viruses continue to remain a threat for potential pandemics due to their rapid evolution. Potentiating host antiviral pathways to prevent or limit viral infections is a promising strategy. Thus, by testing a library of innate immune agonists targeting pathogen recognition receptors, we observe that Toll-like receptor 3 (TLR3), stimulator of interferon genes (STING), TLR8, and Dectin-1 ligands inhibit arboviruses, Chikungunya virus (CHIKV), West Nile virus, and Zika virus to varying degrees. STING agonists (cAIMP, diABZI, and 2′,3′-cGAMP) and Dectin-1 agonist scleroglucan demonstrate the most potent, broad-spectrum antiviral function. Furthermore, STING agonists inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enterovirus-D68 (EV-D68) infection in cardiomyocytes. Transcriptome analysis reveals that cAIMP treatment rescue cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provides protection against CHIKV in a chronic CHIKV-arthritis mouse model. Our study describes innate immune signaling circuits crucial for RNA virus replication and identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses.

Marek's disease virus (MDV) causes malignant lymphoma in chickens (Marek's disease, MD). Although MD is currently controlled by vaccination, MDV strains have continuously increased in virulence over the recent decades. Polymorphisms in Meq, an MDV-encoded oncoprotein that serves as a transcription factor, have been associated with the enhanced virulence of the virus. In addition, insertions and deletions in Meq have been observed in MDV strains of higher virulence, but their contribution to said virulence remains elusive. In this study, we investigated the contribution of an insertion (L-Meq) and a deletion in the Meq gene (S-Meq) to its functions and MDV pathogenicity. Reporter assays revealed that both insertion and deletion enhanced the transactivation potential of Meq. Additionally, we generated RB-1B-based recombinant MDVs (rMDVs) encoding each Meq isoform and analyzed their pathogenic potential. rMDV encoding L-Meq indueced the highest mortality and tumor incidence in infected animals, whereas the rMDV encoding S-Meq exhibited the lowest pathogenicity. Thus, insertion enhanced the transactivation activity of Meq and MDV pathogenicity, whereas deletion reduced pathogenicity despite having increased transactivation activity. These data suggest that other functions of Meq affect MDV virulence. These data improve our understanding of the mechanisms underlying the evolution of MDV virulence.

Zika virus (ZIKV) is a mosquito-borne flavivirus that became widely recognized due to the epidemic in Brazil in 2015. Since then, there has been nearly a 20-fold increase in the incidence of microcephaly and birth defects seen among women giving birth in Brazil, leading the Centers for Disease Control and Prevention (CDC) to officially declare a causal link between prenatal ZIKV infection and the serious brain abnormalities seen in affected infants. Here, we used a unique rat model of prenatal ZIKV infection to study three possible long-term outcomes of congenital ZIKV infection: (1) behavior, (2) cell proliferation, survival, and differentiation in the brain, and (3) immune responses later in life. Adult offspring that were prenatally infected with ZIKV exhibited motor deficits in a sex-specific manner, and failed to mount a normal interferon response to a viral immune challenge later in life. Despite undetectable levels of ZIKV in the brain and serum in these offspring at P2, P24, or P60, these results suggest that prenatal exposure to ZIKV results in lasting consequences that could significantly impact the health of the offspring. To help individuals already exposed to ZIKV, as well as be prepared for future outbreaks, we need to understand the full spectrum of neurological and immunological consequences that could arise following prenatal ZIKV infection.

All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD).

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

Three new coordination compounds containing 5‑diethylamino‑2‑((4‑(phenyldiazenyl) phenylimino) methyl) phenol ligand have been synthesized and fully characterized. Single crystal structure showed that the ligand shows multiple coordination mode and the phenyl group next to azo group could rotate during the coordination process. In Mn(HDAPPP)2Cl2·CH3CN (1), the HDAPPP behaved as a mono ligand because of the N of the imine group is protonated in the keto form and does not participate in coordination. While the Cu(DAPPP)2 (2) and Ni(DAPPP)2 (3) are classic complexes containing azo-Schiff base ligand. The fluorescence emission peaks of compounds indicated that the fluorescence of the compounds are mainly derived from the π-π* charge transition of the ligand and affected by the rotation of ligand. The UV–Vis data revealed that the compound 1–3 with the maximum absorption peak at 425 nm, 440 nm and 338 nm, respectively. This clearly confirmed that the compound 1 is mainly based on the keto configuration while the compound 3 is mainly based on the enol configuration. Z-scan experiments showed that all compounds exhibited reverse saturable absorption effect and self-defocusing effect for nonlinear optical materials. The nonlinear absorption coefficients (β) of compound 1–3 are evaluated to be 6 × 10−11 m·W−1, 4.4 × 10−11 m·W−1, and 1.7 × 10−11 m·W−1 respectively, and the nonlinear refraction coefficients (n2) are −1.4 × 10−17 m2·W−1, −1.6 × 10−17 m2·W−1, −7.0 × 10−18 m2·W−1 respectively. The nonlinear optical property of compound 1 and 2 are better than compound 3, which are influenced by ligand rotation after coordination.

Since Zika virus (ZIKV) first emerged as a public health concern in 2015, our ability to identify and track the long-term neurological sequelae of prenatal Zika virus (ZIKV) infection in humans has been limited. Our lab has developed a rat model of maternal ZIKV infection with associated vertical transmission to the fetus that results in significant brain malformations in the neonatal offspring. Here, we use this model in conjunction with longitudinal magnetic resonance imaging (MRI) to expand our understanding of the long-term neurological consequences of prenatal ZIKV infection in order to identify characteristic neurodevelopmental changes and track them across time. We exploited both manual and automated atlas-based segmentation of MR images in order to identify long-term structural changes within the developing rat brain following inoculation. The paradigm involved scanning three cohorts of male and female rats that were prenatally inoculated with 107 PFU ZIKV, 107 UV-inactivated ZIKV (iZIKV), or diluent medium (mock), at 4 different postnatal day (P) age points: P2, P16, P24, and P60. Analysis of tracked brain structures revealed significantly altered development in both the ZIKV and iZIKV rats. Moreover, we demonstrate that prenatal ZIKV infection alters the growth of brain regions throughout the neonatal and juvenile ages. Our findings also suggest that maternal immune activation caused by inactive viral proteins may play a role in altered brain growth throughout development. For the very first time, we introduce manual and automated atlas-based segmentation of neonatal and juvenile rat brains longitudinally. Experimental results demonstrate the effectiveness of our novel approach for detecting significant changes in neurodevelopment in models of early-life infections.

Marek's disease (MD) is an alphaherpesvirus (Marek's disease virus, MDV)-induced pathology of chickens associated with paralysis, immunosuppression, neurological signs, and T-cell lymphomas. MD is controlled in poultry production via live attenuated vaccines. The purpose of the current study was to compare methods for precipitating exosomes from vaccinated and protected chicken sera (VEX) and tumor-bearing chicken sera (TEX) for biomarker analysis of vaccine-induced protection and MD lymphomas respectively. A standard polyethylene glycol (PEG, 8%) method was compared to a commercial reagent (total exosome isolation reagent, TEI) for exosome yield and RNA content. Although exosomes purified by PEG or TEI were comparable in size and morphology, TEI-reagent yielded 3-4-fold greater concentration. Relative expression of 8 out of 10 G. gallus- and MDV1-encoded miRNAs examined displayed significant difference depending upon the precipitation method used. Standard PEG yields comparable, albeit lower amounts of exosomes than the TEI-reagent and a distinctive miRNA composition.

SUMMARYThe SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates entry of SARS-CoV-2 into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. Antibodies to the RBD domain of SARS-CoV (SARS-CoV-1), a closely related coronavirus which emerged in 2002-2003, have been shown to potently neutralize SARS-CoV-1 S-protein-mediated entry, and the presence of anti-RBD antibodies correlates with neutralization in SARS-CoV-2 convalescent sera. Here we show that immunization with the SARS-CoV-2 RBD elicits a robust neutralizing antibody response in rodents, comparable to 100 µg/ml of ACE2-Ig, a potent SARS-CoV-2 entry inhibitor. Importantly, anti-sera from immunized animals did not mediate antibody-dependent enhancement (ADE) of S-protein-mediated entry under conditions in which Zika virus ADE was readily observed. These data suggest that an RBD-based vaccine for SARS-CoV-2 could be safe and effective.

Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek’s disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection.

Marek's disease virus (MDV) is the causative agent of Marek's disease (MD), a complex pathology of chickens characterized by paralysis, immunosuppression, and T-cell lymphomagenesis. MD is controlled in poultry production via vaccines administered in ovo or at hatch, and these confer protection against lymphoma formation, but not superinfection by MDV field strains. Despite vaccine-induced humoral and cell-mediated immune responses, mechanisms eliciting systemic protection remain unclear. Here we report the contents of serum exosomes to assess their possible roles as indicators of systemic immunity, and alternatively, tumor formation. We examined the RNA and protein content of serum exosomes from CVI988 (Rispens)-vaccinated and protected chickens (VEX), and unvaccinated tumor-bearing chickens (TEX), via deep-sequencing and mass spectrometry, respectively. Bioinformatic analyses of microRNAs (miRNAs) and predicted miRNA targets indicated a greater abundance of tumor suppressor miRNAs in VEX compared to TEX. Conversely, oncomiRs originating from cellular (miRs 106a-363) and MDV miRNA clusters were more abundant in TEX compared to VEX. Most notably, mRNAs mapping to the entire MDV genome were identified in VEX, while mRNAs mapping to the repeats flanking the unique long (IRL/TRL) were identified in TEX. These data suggest that long-term systemic vaccine-induced immune responses may be mediated at the level of VEX which transfer viral mRNAs to antigen presenting cells systemically. Proteomic analyses of these exosomes suggested potential biomarkers for VEX and TEX. These data provide important putative insight into MDV-mediated immune suppression and vaccine responses, as well as potential serum biomarkers for MD protection and susceptibility.

Zika virus (ZIKV), a mosquito-borne flavivirus, has been associated with microcephaly and other neurological disorders in infants born to infected mothers. Despite being declared an international emergency by the World Health Organization, comparatively very little is known about the pathogenesis, mechanisms, or behavioral consequences of maternal ZIKV infection in the offspring. Our lab is interested in developing a working animal model to answer some of these questions. Here, we use a rat model of prenatal ZIKV infection to measure the level of infectivity, as well as the rate of viral clearance in both the mother and her pups. We examine various aspects of brain development in pups, including cortical thickness, microglia morphology, apoptosis, and neurogenesis. Given that pregnancy is associated with significant immunomodulation, we are also interested in the role that pregnancy has on the impact of ZIKV infection, therefore we compare viral infectivity between both pregnant and non-pregnant female rats. In this study, we show that prenatal ZIKV infection results in an increase in cell death and reduces hippocampal and cortical volumes in the neonatal rat brain. For the first time, we demonstrate the efficacy and validity of a rat model for maternal ZIKV infection with vertical transmission to the fetus.

Annual changes in day length enhance or suppress diverse aspects of immune function, giving rise to seasonal cycles of illness and mortality. The daily light–dark cycle also entrains circadian rhythms in immunity. Most published reports on immunological seasonality rely on measurements or interventions performed only at one point in the day. Because there can be no perfect matching of circadian phase across photoperiods of different duration, the manner in which these timescales interact to affect immunity is not understood. We examined whether photoperiodic changes in immune function reflect phenotypic changes that persist throughout the daily cycle, or merely reflect photoperiodic shifts in the circadian phase alignment of immunological rhythms. Diurnal rhythms in blood leukocyte trafficking, infection induced sickness responses, and delayed-type hypersensitivity skin inflammatory responses were examined at high-frequency sampling intervals (every 3 h) in Siberian hamsters (Phodopus sungorus) following immunological adaptation to summer or winter photoperiods. Photoperiod profoundly enhanced or suppressed immune function, in a trait-specific manner, and we were unable to identify a phase alignment of diurnal waveforms which eliminated these enhancing and suppressing effects of photoperiod. These results support the hypothesis that seasonal timescales affect immunity via mechanisms independent of circadian entrainment of the immunological circadian waveform.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with microcephaly and other neurological disorders in infants born to infected mothers. Despite being declared an international emergency by the World Health Organization, very little is known about the mechanisms of ZIKV pathogenesis or the long-term consequences of maternal ZIKV infection in the affected offspring, largely due to the lack of appropriate rodent models. To address this issue, our lab has developed a working model of prenatal ZIKV infection in rats. In this study, we infected immune competent pregnant female rats with 105-107 PFU of ZIKV (PRVABC59, Puerto Rico/Human/Dec 2015) in order to examine its pathogenesis in the dams and pups. We examined the febrile response and sickness behavior in the dams, in addition to neonatal mortality, microglia morphology, cortical organization, apoptosis, and brain region-specific volumes in the offspring. Here, we demonstrate that pregnant and non-pregnant female rats have a distinct febrile response to ZIKV infection. Moreover, prenatal ZIKV infection increased cell death and reduced tissue volume in the hippocampus and cortex in the neonatal offspring. For the first time, we demonstrate the efficacy and validity of an immunocompetent rat model for maternal ZIKV infection that results in significant brain malformations in the neonatal offspring.