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Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we amplified regions of the SARS-CoV-2 Spike protein gene from RNA acquired from all 14 NYC wastewater treatment plants (WWTPs) and ascertained the diversity of lineages from these samples using high throughput sequencing. Here we report the detection and increasing frequencies of novel SARS-CoV-2 lineages not recognized in GISAID’s EpiCoV database. These lineages contain mutations rarely observed in clinical samples, including Q493K, Q498Y, H519N and T572N. Many of these mutations were found to expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. In addition, pseudoviruses containing the Spike amino acid sequence of these lineages were found to be resistant to many different classes of receptor binding domain (RBD) binding neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these mutations, including the possibility of a non-human animal reservoir. Although wastewater sampling cannot provide direct inference of SARS-CoV-2 clinical sequences, our research revealed several lineages that could be relevant to public health and they would not have been discovered if not for wastewater surveillance.

The safety and high efficiency of adeno-associated virus (AAV) vectors has facilitated their wide-scale use to deliver therapeutic genes for experimental and clinical purposes in diseases affecting the central nervous system (CNS). AAV1, 2, 5, 8, 9, and rh10 are the most commonly used serotypes for CNS applications. Most AAVs are known to transduce genes predominantly into neurons. However, the precise tropism of AAVs in the dentate gyrus (DG), the region where persistent neurogenesis occurs in the adult brain, is not fully understood. We stereotaxically injected 1.5 × 10

Purpose: Despite numerous recent advances in retinal gene therapy using adeno-associated viruses (AAVs) as delivery vectors, there remains a crucial need to identify viral vectors with the ability to transduce specific retinal cell types and that have a larger carrying capacity than AAV. In this study, we evaluate the retinal tropism of 2 chimeric helper-dependent adenoviruses (HDAds), helper-dependent adenovirus serotype 5 (HDAd5)/3 and HDAd5/35, both ex vivo using human retinal explants and in vivo using rats. Methods: We transduced cultured human retinal explants with HDAd5/3 and HDAd5/35 carrying an eGFP vector and evaluated tropism and transduction efficiency using immunohistochemistry. To assess in vivo transduction efficiency, subretinal injections were performed in wild-type Sprague-Dawley rats. For both explants and subretinal injections, we delivered 10 μL (1 × 10(6) vector genomes/mL) and assessed tropism at 7- and 14-days post-transduction, respectively. Results: HDAd5/3 and HDAd5/35 both transduced human retinal ganglion cells (RGCs) and Müller cells, but not photoreceptors, in human retinal explants. However, subretinal injections in albino rats resulted in transduction of the retinal pigmented epithelium only, highlighting species-specific differences in retinal tropism and the value of a human explant model when testing vectors for eventual human gene therapy. Conclusions: Chimeric HDAds are promising candidates for the delivery of large genes, multiple genes, or neuroprotective factors to Müller cells and RGCs. These vectors may have utility for targeted therapy of neurodegenerative diseases primarily involving retinal ganglion or Müller cell types, such as glaucoma or macular telangiectasia type 2.

International audience; Influenza A viruses cause important diseases in both human and animal. The PB1-F2 protein is a virulence factor expressed by some influenza viruses. Its deleterious action for the infected host is mostly described in mammals, while the available information is scarce in avian hosts. In this work, we compared the effects of PB1-F2 in avian and mammalian hosts by taking advantage of the zoonotic capabilities of an avian H7N1 virus. In vitro, the H7N1 virus did not behave differently when PB1-F2 was deficient while a H3N2 virus devoid of PB1-F2 was clearly less inflammatory. Likewise, when performing in vivo challenges of either chickens or embryonated eggs, with the wild-type or the PB1-F2 deficient virus, no difference could be observed in terms of mortality, host response or tropism. PB1-F2 therefore does not appear to play a major role as a virulence factor in the avian host. However, when infecting NF-κB-luciferase reporter mice with the H7N1 viruses, a massive PB1-F2-dependent inflammation was quantified, highlighting the host specificity of PB1-F2 virulence. Surprisingly, a chimeric 7:1 H3N2 virus harboring an H7N1-origin segment 2 (i.e. expressing the avian PB1-F2) induced a milder inflammatory response than its PB1-F2-deficient counterpart. This result shows that the pro-inflammatory activity of PB1-F2 is governed by complex mechanisms involving components from both the virus and its infected host. Thus, a mere exchange of segment 2 between strains is not sufficient to transmit the deleterious character of PB1-F2.

Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains—a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD- Prkdcem26Cd52Il2rgem26Cd22 /NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

Adenoviruses (Ads), common self-limiting pathogens in humans and animals, usually cause conjunctivitis, mild upper respiratory tract infection or gastroenteritis in humans and hepatotoxicity syndrome in chickens and dogs, posing great threats to public health and livestock husbandry. Artificially modified Ads, which wipe out virulence-determining genes, are the most frequently used viral vectors in gene therapy, and some Ad vector (AdV)-related medicines and vaccines have been licensed and applied. Inherent liver tropism enables AdVs to specifically deliver drugs/genes to the liver; however, AdVs are closely associated with acute hepatotoxicity in immunocompromised individuals, and the side effects of AdVs, which stimulate a strong inflammatory reaction in the liver and cause acute hepatotoxicity, have largely limited clinical application. Therefore, this review systematically elucidates the intimate relationship between AdVs and hepatotoxicity in terms of virus and host and precisely illustrates the accumulated understanding in this field over the past decades. This review demonstrates the liver tropism of AdVs and molecular mechanism of AdV-induced hepatotoxicity and looks at the studies on AdV-mediated animal hepatotoxicity, which will undoubtedly deepen the understanding of AdV-caused liver injury and be of benefit in the further safe development of AdVs.

Summary Beginning in the summer of 2020, a variant of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the United Kingdom (UK). This B.1.1.7 variant increased rapidly in prevalence among sequenced strains, attributed to an increase in infection and/or transmission efficiency. The UK variant has 19 nonsynonymous mutations across its viral genome including 8 substitutions or deletions in the spike protein, which interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that, of the 8 individual spike protein substitutions, only N501Y exhibited consistent fitness gains for replication in the upper airway in the hamster model as well as primary human airway epithelial cells. The N501Y substitution recapitulated the phenotype of enhanced viral transmission seen with the combined 8 UK spike mutations, suggesting it is a major determinant responsible for increased transmission of this variant. Mechanistically, the N501Y substitution improved the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil and South Africa, our results indicate that N501Y substitution is a major adaptive spike mutation of major concern.

Chlamydia muridarum actively grows in murine mucosae and is a representative model of human chlamydial genital tract disease. In contrast, C. trachomatis infections in mice are limited and rarely cause disease. The factors that contribute to these differences in host adaptation and specificity remain elusive. Overall genomic similarity leads to challenges in the understanding of these significant differences in tropism. A region of major genetic divergence termed the plasticity zone (PZ) has been hypothesized to contribute to the host specificity. To evaluate this hypothesis, lateral gene transfer was used to generate multiple hetero-genomic strains that are predominately C. trachomatis but have replaced regions of the PZ with those from C. muridarum. In vitro analysis of these chimeras revealed C. trachomatis-like growth as well as poor mouse infection capabilities. Growth-independent cytotoxicity phenotypes have been ascribed to three large putative cytotoxins (LCT) encoded in the C. muridarum PZ. However, analysis of PZ chimeras supported that gene products other than the LCTs are responsible for cytopathic and cytotoxic phenotypes. Growth analysis of associated chimeras also led to the discovery of an inclusion protein, CTL0402 (CT147), and homolog TC0424, which was critical for the integrity of the inclusion and preventing apoptosis.

Introduction The outbreak of the newly discovered human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has disrupted the normal life of almost every civilization worldwide. Studies have shown that the coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 can affect multiple human organs and physiological systems, but the respiratory system remains the primary location for viral infection. Areas covered We summarize how omics technologies are used in SARS-CoV-2 research and specifically review the current knowledge of COVID-19 from the aspect of human bronchial-pulmonary proteomics. Also, knowledge gaps in COVID-19 that can be fulfilled by proteomics are discussed. Expert opinion Overall, human bronchial-pulmonary proteomics plays an important role in revealing the dynamics, functions, tropism, and pathogenicity of SARS-CoV-2, which is crucial for COVID-19 biomarker and therapeutic target discoveries. To more fully understand the impact of COVID-19, research from various angles using multi-omics approaches should also be conducted on the lungs as well as other organs.

Due to redundancy with other tyrosine phosphatases, the ubiquitously expressed tyrosine phosphatase SHP-2 (encoded by Ptpn11) is not required for T cell development. However, Ptpn11 gene deletion driven by CD4 Cre recombinase leads to cartilage tumors in the wrist. Using a fate mapping system, we demonstrate that wrist tumor development correlates with increased frequency and numbers of non-hematopoietic lineage negative CD45 negative cells with a bone chondrocyte stromal cell precursor cell (BCSP) phenotype. Importantly, the BCSP subset has a history of CD4 expression and a marked wrist location tropism, explaining why the wrist is the main site of tumor development. Mechanistically, we found that in SHP-2 absence, SOX-9 is no longer regulated, leading to an uncontrolled proliferation of the BCSP subset. Altogether, these results identify a unique subset of chondrocyte precursors tightly regulated by SHP-2. These findings underscore the need for the development of methods to therapeutically target this subset of cells, which could potentially have an impact on treatment of SHP-2 dysfunction linked debilitating diseases.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease that affects 1:5,000 live male births and is characterized by muscle wasting. By the age of 13 years, affected individuals are often wheelchair bound and suffer from respiratory and cardiac failure, which results in premature death. Although the administration of corticosteroids and ventilation can relieve the symptoms and extend the patients' lifespan, currently no cure exists for DMD. Among the different approaches under preclinical and clinical testing, gene therapy, using adeno-associated viral (AAV) vectors, is one of the most promising. In this study, we delivered intravenously AAV9 vectors expressing the microdystrophin MD1 (ΔR4-R23/ΔCT) under control of the synthetic muscle-specific promoter Spc5-12 and assessed the effect of adding a cardiac-specific cis-regulatory module (designated as CS-CRM4) on its expression profile in skeletal and cardiac muscles. Results show that Spc5-12 promoter, in combination with an AAV serotype that has high tropism for the heart, drives high MD1 expression levels in cardiac muscle in mdx mice. The additional regulatory element CS-CRM4 can further improve MD1 expression in cardiac muscles, but its effect is dose dependent and enhancement becomes evident only at lower vector doses.

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease.

Emerging mutations in SARS-CoV-2 cause several waves of COVID-19 pandemic. Here we investigate the infectivity and antigenicity of ten emerging SARS-CoV-2 variants—B.1.1.298, B.1.1.7(Alpha), B.1.351(Beta), P.1(Gamma), P.2(Zeta), B.1.429(Epsilon), B.1.525(Eta), B.1.526-1(Iota), B.1.526-2(Iota), B.1.1.318—and seven corresponding single amino acid mutations in the receptor-binding domain using SARS-CoV-2 pseudovirus. The results indicate that the pseudovirus of most of the SARS-CoV-2 variants (except B.1.1.298) display slightly increased infectivity in human and monkey cell lines, especially B.1.351, B.1.525 and B.1.526 in Calu-3 cells. The K417N/T, N501Y, or E484K-carrying variants exhibit significantly increased abilities to infect mouse ACE2-overexpressing cells. The activities of furin, TMPRSS2, and cathepsin L are increased against most of the variants. RBD amino acid mutations comprising K417T/N, L452R, Y453F, S477N, E484K, and N501Y cause significant immune escape from 11 of 13 monoclonal antibodies. However, the resistance to neutralization by convalescent serum or vaccines elicited serum is mainly caused by the E484K mutation. The convalescent serum from B.1.1.7- and B.1.351-infected patients neutralized the variants themselves better than other SARS-CoV-2 variants. Our study provides insights regarding therapeutic antibodies and vaccines, and highlights the importance of E484K mutation., Li Zhang, Zhimin Cui, and Qianqian Li et al. compare the infectivity, host tropism, and antigenicity of 10 SARS-CoV-2 variants using a VSV-based pseudovirus system. Their results suggest that variants carrying E484K display the most significant reduction in sensitivity to neutralization, and may provide further insight into the development of relevant therapeutics for SARS-CoV-2 infection.

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi parasites. During mammalian infection, T. cruzi alternates between an intracellular stage and extracellular stage. T. cruzi adapts its metabolism to this lifestyle, while also reshaping host metabolic pathways. Such host metabolic adaptations compensate for parasite-induced stress, but may promote parasite survival and proliferation. Recent work has demonstrated that metabolism controls parasite tropism and location of Chagas disease symptoms, and regulates whether infection is mild or severe. Such findings have important translational applications with regards to treatment and diagnostic test development, though further research is needed with regards to in vivo parasite metabolic gene expression, relationship between magnitude of local metabolic perturbation, parasite strain and disease location, and host-parasite-microbiota co-metabolism.

Coxsackievirus B1 (CVB1) is a leading causative agent of severe infectious diseases in humans and has been reported to be associated with outbreaks of aseptic meningitis, myocarditis, and the development of chronic diseases such as type 1 diabetes mellitus (T1DM). There is no approved vaccine or effective antiviral therapy to treat CBV1 infection. And animal models to assess the effects of antiviral agents and vaccine remain limited. In this study, we established a neonatal mouse model of CVB1 using a clinically isolated strain to characterize the pathological manifestations of virus infection and to promote the development of vaccines and antiviral drugs against CVB1. One-day-old BALB/c mice were susceptible to CVB1 infection by intraperitoneal injection. Mice challenged with CVB1 at a low dose [10 median tissue culture infective dose (TCID50)] exhibited a series of clinical symptoms, such as inactivity, emaciation, limb weakness, hair thinning, hunching and even death. Pathological examination and tissue viral load analysis showed that positive signals of CVB1 were detected in the heart, spinal cord, limb muscle and kidney without pathological damage. Particularly, CVB1 had a strong tropism towards the pancreas, causing severe cellular necrosis with inflammatory infiltration, and was spread by viraemia. Notably, the monoclonal antibody (mAb) 6H5 and antisera elicited from CVB1-vaccinated mice effectively protected the mice from CVB1 infection in the mouse model. In summary, the established neonatal mouse model is an effective tool for evaluating the efficacy of CVB1 antiviral reagents and vaccines. Supplementary Information The online version contains supplementary material available at 10.1007/s12250-021-00444-1.

Arboviruses and insect-specific viruses (ISVs) are two major types of viruses harbored by mosquitoes that are distinguished by the involvement of vertebrate hosts in their transmission cycles. While intensive studies have focused on the transmission, tissue tropism, and evolution of arboviruses, these characteristics are poorly investigated in ISVs, which dominate the mosquito virome. Therefore, in this study, we collected two mosquito species, Anopheles sinensis and Culex quinquefasciatus, in the field and used a metatranscriptomics approach to characterize their RNA viromes in different tissues, such as the midgut, legs, salivary gland, eggs, and the remainder of the carcass. Blood-engorged individuals of these species were captured in 3 locations, and 60 mosquitoes were pooled from each species and location. A total of 40 viral species from diverse viral taxa associated with all viral RNA genome types were identified, among which 19 were newly identified in this study. According to the current viral taxonomy, some of these viruses, such as Yancheng

Background: Primary vitreoretinal lymphoma (PVRL), a rare malignancy of the eye, is strongly related to primary central nervous system lymphoma (PCNSL). We hypothesized that lymphoma cells disseminate to the CNS and eye tissue via distinct homing receptors. The objective of this study was to test expression of CXCR4, CXCR5, CXCR7 and CD44 homing receptors on CD20 positive B-lymphoma cells on enucleated eyes using a PCNSL xenograft mouse model. Methods: We used indirect immunofluorescence double staining for CD20/CXCR4, CD20/CXCR5, CD20/CXCR7 and CD20/CD44 on enucleated eyes of a PCNSL xenograft mouse model with PVRL phenotype (PCNSL group) in comparison to a secondary CNS lymphoma xenograft mouse model (SCNSL group). Lymphoma infiltration was evaluated with an immunoreactive score (IRS). Results: 11/13 paired eyes of the PCNSL but none of the SCNSL group were infiltrated by CD20-positive cells. Particularly the choroid and to a lesser extent the retina of the PCNSL group were infiltrated by CD20+/CXCR4+, CD20+/CXCR5+, few CD20+/CD44+ but no CD20+/CXCR7+ cells. Expression of CXCR4 (p = 0.0205), CXCR5 (p = 0.0004) and CD44 (p < 0.0001) was significantly increased in the PCNSL compared to the SCNSL group. Conclusions: CD20+ PCNSL lymphoma cells infiltrating the eye co-express distinct homing receptors such as CXCR4 and CXCR5 in a PVRL homing mouse model. These receptors may be involved in PVRL homing into the eye.

Hand, foot, and mouth disease (HFMD) is a contagious disease that threatens the health of children under 5 years of age. Coxsackievirus A10 (CV-A10) is one of the main pathogens of HFMD. Currently, preventive vaccines and specific therapeutic drugs are not available for CV-A10. In this study, a total of 327 stool specimens were collected from pediatric patients from 2009 to 2017 during HFMD surveillance, among which 14 CV-A10 strains could only be isolated from rhabdomyosarcoma cells, but not from KMB17 and Vero cells. Through adaptive culture, 2 and 11 CV-A10 strains were recovered from Vero and KMB17 cell cultures, respectively. The growth of CV-A10 strains in Vero cells was better than that in KMB17 cells. The 14 CV-A10 strains belonged to the F genotype, and the nucleotides and amino acids of their complete genomes shared 92.6%-96.3% and 98.4%-98.9% identities, respectively. The different CV-A10 strains exhibited varying virulence in vivo, but had similar effects on tissue injury, with the hind limb muscles, kidneys, and lungs being severely affected. Additionally, the hind limb muscles had the highest viral loads. CV-A10 was found to exhibit a strong tropism to muscle tissue. The results of this study are critical to developing vaccines against CV-A10 infections.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious disease with high morbidity and mortality in domestic pigs. Although adaptation of ASFV to Vero cells has been investigated, the phenotypic changes and the corresponding genomic variations during adaptation of ASFV to other cell lines remain unclear. To obtain a cell-adapted ASFV strain, different cell lines were tested to determine whether they support ASFV infection. Interestingly, the ASFV wild-type strain ASFV-HLJ/18 can infect HEK293T cells and replicate at a low level. After continuous passaging, the adapted ASFV strain can replicate efficiently in both HEK293T and Vero cells. However, the adapted ASFV strain displayed reduced infectivity in primary porcine alveolar macrophages compared to the corresponding wild-type strain. Furthermore, stepwise losses at the left variable end of the MGF genes and accumulative mutations were identified during passaging, indicating that the ASFV strain gradually adapted to HEK293T cells. Comparison of MGF deletions in other cell culture-adapted ASFV strains revealed that the deletions of MGF300 (1L, 2R and 4L) and MGF360 genes (8L, 9L, 10L and 11L) play an important role for the adaptation of ASFV to HEK293T cells at the early stage. The biological functions of the deletions and mutants associated with ASFV infection in HEK293T cells and pigs warrant further study. Overall, our findings provide new targets to elucidate the molecular mechanism of adaptation of ASFV to cell lines.

The SARS-CoV-2 spike protein Q677P/H mutation and furin cleavage site (FCS) have been shown to affect cell tropism and virus transmissibility. Here, we analyzed the frequency of Q677P/H and FCS point mutations in 1,144,793 human and 1042 animal spike protein sequences and from those of the emergent variants B.1.1.7, B.1.351, P.1, B.1.429 + B.1.427, and B.1.525, which were deposited in the database of the GISAID Initiative. Different genetic polymorphisms, particularly P681H and A688V, were detected in the FCS, mainly in human isolates, and otherwise, only pangolin and bat sequences had these mutations. Multiple FCS amino acid deletions such as Δ680SPRRA684 and Δ685RSVA688 were only detected in eight and four human isolates, respectively. Surprisingly, deletion of the entire FCS motif as Δ680SPRRARSVA688 and Δ680SPRRARSVAS689 was detected only in three human isolates. On the other hand, analysis of FCS from emergent variants showed no deletions in the FCS except for spike P681del, which was detected in seven B.1.1.7 isolates from the USA. Spike Q677P was detected only once in variant, B.1.1.7, whereas Q677H was detected in all variants, i.e., B.1.1.7 (n = 1938), B.1.351 (n = 28), P.1 (n = 9), B.1.429 + B.1.427 (n = 132), and B.1.525 (n = 1584). Structural modeling predicted that mutations or deletions at or near the FCS significantly alter the cleavage loop structure and would presumably affect furin binding. Taken together, our results show that Q677H and FCS point mutations are prevalent and may have various biological effects on the circulating variants. Therefore, we recommend urgent monitoring and surveillance of the investigated mutations, as well as laboratory assessment of their pathogenicity and transmissibility.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by the host furin protease. Proteolytic cleavage activates the spike protein, thereby affecting both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogenesis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited low growth properties. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, thereby indicating the attenuated variant nature of S gene mutants. This transient infection was sufficient for inducing protective neutralizing antibodies that cross-react with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Taken together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of hamsters and highlighted the potential benefits of S gene mutants as potential immunogens. IMPORTANCE SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. In this study, we employed SARS-CoV-2 mutants lacking the S protein cleavage site and characterized their growth and pathogenicity using hamsters, a laboratory animal model for SARS-CoV-2 infection. These mutants exerted low pathogenicity but induced sufficient levels of neutralizing antibodies in hamsters, which protected hamsters from rechallenge with pathogenic clinical SARS-CoV-2 strains. These virus mutants may be used as protective immunogens against SARS-CoV-2 infection.

Treatment of castration-resistant prostate cancer remains a challenging clinical problem. Despite the promising effects of immunotherapy in other solid cancers, prostate cancer has remained largely unresponsive. Oncolytic viruses represent a promising therapeutic avenue, as oncolytic virus treatment combines tumour cell lysis with activation of the immune system and mounting of effective anti-tumour responses. Mammalian Orthoreoviruses are non-pathogenic human viruses with a preference of lytic replication in human tumour cells. In this study, we evaluated the oncolytic efficacy of the bioselected oncolytic reovirus mutant jin-3 in multiple human prostate cancer models. The jin-3 reovirus displayed efficient infection, replication, and anti-cancer responses in 2D and 3D prostate cancer models, as well as in ex vivo cultured human tumour slices. In addition, the jin-3 reovirus markedly reduced the viability and growth of human cancer cell lines and patient-derived xenografts. The infection induced the expression of mediators of immunogenic cell death, interferon-stimulated genes, and inflammatory cytokines. Taken together, our data demonstrate that the reovirus mutant jin-3 displays tumour tropism, and induces potent oncolytic and immunomodulatory responses in human prostate cancer models. Therefore, jin-3 reovirus represents an attractive candidate for further development as oncolytic agent for treatment of patients with aggressive localised or advanced prostate cancer.

Background The crucial role of type I interferon (IFN-I, IFN-α/β) is well known to control central nervous system (CNS) neuroinflammation caused by neurotrophic flaviviruses such as Japanese encephalitis virus (JEV) and West Nile virus. However, an in-depth analysis of IFN-I signal-dependent cellular factors that govern CNS-restricted tropism in JEV infection in vivo remains to be elucidated. Methods Viral dissemination, tissue tropism, and cytokine production were examined in IFN-I signal-competent and -incompetent mice after JEV inoculation in tissues distal from the CNS such as the footpad. Bone marrow (BM) chimeric models were used for defining hematopoietic and tissue-resident cells in viral dissemination and tissue tropism. Results The paradoxical and interesting finding was that IFN-I signaling was essentially required for CNS neuroinflammation following JEV inoculation in distal footpad tissue. IFN-I signal-competent mice died after a prolonged neurological illness, but IFN-I signal-incompetent mice all succumbed without neurological signs. Rather, IFN-I signal-incompetent mice developed hemorrhage-like disease as evidenced by thrombocytopenia, functional injury of the liver and kidney, increased vascular leakage, and excessive cytokine production. This hemorrhage-like disease was closely associated with quick viral dissemination and impaired IFN-I innate responses before invasion of JEV into the CNS. Using bone marrow (BM) chimeric models, we found that intrinsic IFN-I signaling in tissue-resident cells in peripheral organs played a major role in inducing the hemorrhage-like disease because IFN-I signal-incompetent recipients of BM cells from IFN-I signal-competent mice showed enhanced viral dissemination, uncontrolled cytokine production, and increased vascular leakage. IFN-I signal-deficient hepatocytes and enterocytes were permissive to JEV replication with impaired induction of antiviral IFN-stimulated genes, and neuron cells derived from both IFN-I signal-competent and -incompetent mice were vulnerable to JEV replication. Finally, circulating CD11b+Ly-6C+ monocytes infiltrated into the distal tissues inoculated by JEV participated in quick viral dissemination to peripheral organs of IFN-I signal-incompetent mice at an early stage. Conclusion An IFN-I signal-dependent model is proposed to demonstrate how CD11b+Ly-6C+ monocytes are involved in restricting the tissue tropism of JEV to the CNS.

Crane-associated adenovirus 1 (CrAdV-1) is a proposed novel virus in the genus Aviadenovirus, first detected in faecal samples from hooded cranes (a vulnerable crane species) on the Izumi plain, a major overwintering site for migratory cranes in Japan. CrAdV-1 was genetically characterized in that study; however, its virological characteristics remain largely unclear. To investigate the prevalence and organ tropism of CrAdV-1, we collected swab and organ samples from dead or debilitated cranes on the Izumi plain. CrAdV-1 gene was detected in 47% (45/95) of tested cranes, comprising mainly hooded cranes but also white-naped and sandhill cranes. These results indicate that CrAdV-1 shedding is widespread among cranes overwintering on the Izumi plain. Phylogenetic analyses revealed that the 68 nucleotide sequences determined from the positive swabs formed a single cluster, suggesting phylogenetic differences between CrAdV-1 and other aviadenoviruses. CrAdV-1 prevalence showed a significant linear increase with time through the overwintering period (November to February), especially among juveniles. These findings indicate that CrAdV-1 spreads mainly by transmission between juveniles progressively through the overwintering period. The CrAdV-1 gene-positive rate was significantly higher in cloacal swabs than conjunctival or tracheal swabs. Copy numbers for the partial CrAdV-1 gene sequence were markedly high in the colon samples from three of the four cranes investigated for organ tropism. We also detected relatively high copy numbers in the cerebrum, trachea, lung and heart, suggesting that CrAdV-1 mainly targets these four organs and transmitted via the faecal-oral route and airborne transmission. These results contribute to further understanding of the virological characteristics of CrAdV-1.

The strain 68-1 rhesus cytomegalovirus (RhCMV)–based vaccine for simian immunodeficiency virus (SIV) can stringently protect rhesus macaques (RMs) from SIV challenge by arresting viral replication early in primary infection. This vaccine elicits unconventional SIV-specific CD8 + T cells that recognize epitopes presented by major histocompatibility complex (MHC)–II and MHC-E instead of MHC-Ia. Although RhCMV/SIV vaccines based on strains that only elicit MHC-II– and/or MHC-Ia–restricted CD8 + T cells do not protect against SIV, it remains unclear whether MHC-E–restricted T cells are directly responsible for protection and whether these responses can be separated from the MHC-II–restricted component. Using host microRNA (miR)–mediated vector tropism restriction, we show that the priming of MHC-II and MHC-E epitope–targeted responses depended on vector infection of different nonoverlapping cell types in RMs. Selective inhibition of RhCMV infection in myeloid cells with miR-142–mediated tropism restriction eliminated MHC-E epitope–targeted CD8 + T cell priming, yielding an exclusively MHC-II epitope–targeted response. Inhibition with the endothelial cell–selective miR-126 eliminated MHC-II epitope–targeted CD8 + T cell priming, yielding an exclusively MHC-E epitope–targeted response. Dual miR-142 + miR-126–mediated tropism restriction reverted CD8 + T cell responses back to conventional MHC-Ia epitope targeting. Although the magnitude and differentiation state of these CD8 + T cell responses were generally similar, only the vectors programmed to elicit MHC-E–restricted CD8 + T cell responses provided protection against SIV challenge, directly demonstrating the essential role of these responses in RhCMV/SIV vaccine efficacy.

A serious limitation of current adeno-associated viral (AAV) capsids employed for subretinal delivery is achieving adequate lateral spread beyond the injection site, required for the efficient delivery of gene therapy to the outer retina and/or RPE. AAVBR1 is a unique AAV with exceptional tropism for CNS microvasculature following systemic delivery. Here, we used in vivo and ex vivo analysis to show that subretinal delivery of AAVBR1.GFP in mice achieves superior tropism to RPE and outer retina than either AAV2.GFP or AAV8.GFP, two of the most common capsids used for subretinal delivery. At a low (5 × 108 vg) subretinal dose, the AAVBR1.GFP signal was visible by 48 h and significantly surpassed peak fluorescence of other AAVs in retina and RPE. The co-injection of AAVBR1.GFP with the AAVBR1-specific heptapeptide, NRGTEWD, significantly blocked the AAVBR1.GFP signal, but had no effect on AAV2.GFP fluorescence, confirming that AAVBR1’s enhanced tropism for RPE and outer retina derives from this 7AA modification within the capsid-binding motif. Enhanced dispersal and consequent transduction suggest that AAVBR1 can be employed at a lower dosage than the standard AAV2 capsid to achieve equivalent expression for gene therapy, warranting further evaluation of its utility as a therapeutic vehicle for subretinal delivery.

Chagas disease (CD) is a parasitic, systemic, chronic, and often fatal illness caused by infection with the protozoan Trypanosoma cruzi. The World Health Organization classifies CD as the most prevalent of povertypromoting neglected tropical diseases, the most important parasitic one, and the third most infectious disease in Latin America. Currently, CD is a global public health issue that affects 6−8 million people. However, the current approved treatments are limited to two nitroheterocyclic drugs developed more than 50 years ago. Many efforts have been made in recent decades to find new therapies, but our limited understanding of the infection process, pathology development, and long-term nature of this disease has made it impossible to develop new drugs, effective treatment, or vaccines. This Review aims to provide a comprehensive update on our understanding of the current life cycle, new morphological forms, and genetic diversity of T. cruzi, as well as identify intervention points in the life cycle where new drugs and treatments could achieve a parasitic cure., Junta de Andalucia E-BIO-464-UGR20, Junta de Andalucia (Proyectos I+D+I FEDER Andalucia 2014-2020), Alfonso Martin Escudero Foundation

Plasmodium falciparum pathogenesis is complex and intimately connected to vascular physiology. This is exemplified by cerebral malaria (CM), a neurovascular complication that accounts for most of the malaria deaths worldwide. P. falciparum sequestration in the brain microvasculature is a hallmark of CM and is not replicated in animal models. Numerous aspects of the disease are challenging to fully understand from clinical studies, such as parasite binding tropism or causal pathways in blood-brain barrier breakdown. Recent bioengineering approaches allow for the generation of 3D microvessels and organ-specific vasculature that provide precise control of vessel architecture and flow dynamics, and hold great promise for malaria research. Here, we discuss recent and future applications of bioengineered microvessels in malaria pathogenesis research.

Staphylococcus aureus have evolved into diverse lineages, known as clonal complexes (“CC”), which exhibit differences in the coding sequences of core virulence factors. Whether these alterations impact functionality is poorly understood. Here, we studied the highly polymorphic pore-forming toxin LukAB. We discovered that the LukAB toxin variants produced by S. aureus CC30 and CC45 kill human phagocytes regardless of whether CD11b, the previously established LukAB receptor, is present, and instead target the human hydrogen voltage-gated channel 1 (HVCN1). Biochemical studies identified the domain within human HVCN1 that drives LukAB species specificity, enabling the generation of humanized HVCN1 mice with enhanced susceptibility to CC30 LukAB and to bloodstream infection caused by CC30 S. aureus strains. Altogether, this work advances our understanding of an important S. aureus toxin and underscores the importance of considering genetic variation to characterizing virulence factors and understand the tug of war between pathogens and the host.

Ovarian cancer is highly metastatic, with a high frequency of relapse, and is the most fatal gynecologic malignancy in women worldwide. It is important to elevate the drug susceptibility and cytotoxicity of ovarian cancer cells, thereby eliminating resident cancer cells for more effective therapeutic efficacy. Here, we developed a bispecific antibody (BsAb; mPEG × HER2) that can easily provide HER2+ tumor tropism to mPEGylated liposomal doxorubicin (PLD) and further increase the drug accumulation in cancer cells via receptor-mediated endocytosis, and improve the cytotoxicity and therapeutic efficacy of HER2+ ovarian tumors. The mPEG × HER2 can simultaneously bind to mPEG molecules on the surface of PLD and HER2 antigen on the surface of ovarian cancer cells. Simply mixing the mPEG × HER2 with PLD was able to confer HER2 specificity of PLD to HER2+ ovarian cancer cells and efficiently trigger endocytosis and enhance cytotoxicity by 5.4-fold as compared to non-targeted PLD. mPEG × HER2-modified PLD was able to significantly increase the targeting and accumulation of HER2+ ovarian tumor by 220% as compared with non-targeted PLD. It could also significantly improve the anti-tumor activity of PLD (P

Biomimetic nanoparticles aim to effectively emulate the behavior of either cells or exosomes. Leukocyte-based biomimetic nanoparticles, for instance, incorporate cell membrane proteins to transfer the natural tropism of leukocytes to the final delivery platform. However, tuning the protein integration can affect the in vivo behavior of these nanoparticles and alter their efficacy. Here we show that, while increasing the protein:lipid ratio to a maximum of 1:20 (w/w) maintained the nanoparticle’s structural properties, increasing protein content resulted in improved targeting of inflamed endothelium in two different animal models. Our combined use of a microfluidic, bottom-up approach and tuning of a key synthesis parameter enabled the synthesis of reproducible, enhanced biomimetic nanoparticles that have the potential to improve the treatment of inflammatory-based conditions through targeted nanodelivery.

Although they globally cause viral gastroenteritis in children, astroviruses are understudied due to the lack of well-defined animal models. While murine astroviruses (muAstVs) chronically infect immunodeficient mice, a culture system and understanding of their pathogenesis is lacking. Here, we describe a platform to cultivate muAstV using air–liquid interface (ALI) cultures derived from mouse enteroids, which support apical infection and release. Chronic muAstV infection occurs predominantly in the small intestine and correlates with higher interferon-lambda (IFN-λ) expression. MuAstV stimulates IFN-λ production in ALI, recapitulating our in vivo findings. We demonstrate that goblet cells and enterocytes are targets for chronic muAstV infection in vivo, and that infection is enhanced by parasite co-infection or type 2 cytokine signaling. Depletion of goblet cells from ALI limits muAstV infection in vitro. During chronic infection, muAstV stimulates IFN-λ production in infected cells and induces ISGs throughout the intestinal epithelium in an IFN-λ-receptor-dependent manner. Collectively, our study provides insights into the cellular tropism and innate immune responses to muAstV and establishes an enteroid-based culture system to propagate muAstV in vitro.

Purpose of review The current review outlines recent discoveries on the infection of erythroid cells by Plasmodium parasites, focusing on the molecular interactions governing the tropism of parasites for their host cell and the implications of this tropism for parasite biology and erythroid cell maturation. Recent findings Although most studies about the interactions of Plasmodium parasites and their host cell focused on the deadliest human malaria parasite, Plasmodium falciparum, and the erythrocyte, there is increasing evidence that several Plasmodium species, including P. falciparum, also develop within erythroid precursors. These interactions likely modify the remodeling of the host cell by the parasite and affect the maturation of erythroblast and reticulocytes. Summary A better understanding of the remodeling of immature erythroid cells by Plasmodium parasites will have important implications for the development of antimalarial drugs or vaccines. In addition, deciphering how Plasmodium parasites interfere with erythropoiesis will provide new insights on how these parasites contribute to anemia in malaria patients.

Adeno-associated virus (AAV) transduction efficiency and tropism are conventionally determined by high expression of a fluorescent reporter gene. Emerging data has suggested that such conventional methods may underestimate AAV transduction for cells in which reporter expression from AAV vectors is undetectable. To explore an alternative method that captures AAV transduction in cells in which low expression of a cargo is sufficient for the intended activity, we sought after CRISPR/Cas9-mediated gene disruption. In this study, we use AAV to deliver CRISPR/guide RNA designed to abolish the genes NeuN, GFAP, or MOG expressed specifically in neurons, astrocytes, or oligodendrocytes respectively in the central nervous system (CNS) of mice. Abrogated expression of these cell-type-specific genes can be measured biochemically in CNS subregions and provides quantitative assessment of AAV transduction in these CNS cell types. By using this method, we compared CNS transduction of AAV9, AAV-PHP.B, and AAV-PHP.eB delivered via intracerebroventricular injection (ICV) in neonatal mice. We found both AAV-PHP.B and AAV-PHP.eB resulted in marked disruption of the NeuN gene by CRISPR/Cas9, significantly greater than AAV9 in several brain regions and spinal cord. In contrast, only modest disruption of the GFAP gene and the MOG gene was observed by all three AAV variants. Since the procedure of ICV circumvents the blood–brain barrier, our data suggests that, independent of their ability to cross the blood–brain barrier, AAV-PHP.B variants also exhibit remarkably improved neuronal transduction in the CNS. We anticipate this approach will facilitate profiling of AAV cellular tropism in murine CNS.

The tropism of mesenchymal stem cells (MSCs) for tumors forms the basis for their use as delivery vehicles for the tumor-specific transport of therapeutic genes, such as the theranostic sodium iodide symporter (NIS). Hyperthermia is used as an adjuvant for various tumor therapies and has been proposed to enhance leukocyte recruitment. Here, we describe the enhanced recruitment of adoptively applied NIS-expressing MSCs to tumors in response to regional hyperthermia. Hyperthermia (41°C, 1 h) of human hepatocellular carcinoma cells (HuH7) led to transiently increased production of immunomodulatory factors. MSCs showed enhanced chemotaxis to supernatants derived from heat-treated cells in a 3D live-cell tracking assay and was validated in vivo in subcutaneous HuH7 mouse xenografts. Cytomegalovirus (CMV)-NIS-MSCs were applied 6–48 h after or 24–48 h before hyperthermia treatment. Using (123)I-scintigraphy, thermo-stimulation (41°C, 1 h) 24 h after CMV-NIS-MSC injection resulted in a significantly increased uptake of (123)I in heat-treated tumors compared with controls. Immunohistochemical staining and real-time PCR confirmed tumor-selective, temperature-dependent MSC migration. Therapeutic efficacy was significantly enhanced by combining CMV-NIS-MSC-mediated (131)I therapy with regional hyperthermia. We demonstrate here for the first time that hyperthermia can significantly boost tumoral MSC recruitment, thereby significantly enhancing therapeutic efficacy of MSC-mediated NIS gene therapy.