Spatial multi-omics at subcellular resolution via high-throughput in situ pairwise sequencing.

Technology for spatial multi-omics aids the discovery of new insights into cellular functions and disease mechanisms. Here we report the development and applicability of multi-omics in situ pairwise sequencing (MiP-seq), a method for the simultaneous detection of DNAs, RNAs, proteins and biomolecules at subcellular resolution. Compared with other in situ sequencing methods, MiP-seq enhances decoding capacity and reduces sequencing and imaging costs while maintaining the efficacy of detection of gene mutations, allele-specific expression and RNA modifications. MiP-seq can be integrated with in vivo calcium imaging and Raman imaging, which enabled us to generate a spatial multi-omics atlas of mouse brain tissues and to correlate gene expression with neuronal activity and cellular biochemical fingerprints. We also report a sequential dilution strategy for resolving optically crowded signals during in situ sequencing. High-throughput in situ pairwise sequencing may facilitate the multidimensional analysis of molecular and functional maps of tissues.

A Subunit Vaccine Based on the VP2 Protein of Porcine Parvovirus 1 Induces a Strong Protective Effect in Pregnant Gilts.

Porcine parvovirus 1 (PPV1) is one of the most prevalent pathogens that can cause reproductive disorder in sows. The VP2 protein of PPV1 is the most important immunogenic protein that induces neutralizing antibodies and protective immunity. Thus, VP2 is considered an ideal target antigen for the development of a genetically engineered PPV1 vaccine. In this study, the baculovirus transfer vector carrying the HR5-P10-VP2 expression cassette was successfully constructed with the aim of increasing the expression levels of the VP2 protein. The VP2 protein was confirmed using SDS‒PAGE and Western blot analyses. Electronic microscope analysis showed that the recombinant VP2 proteins were capable of self-assembling into VLPs with a diameter of approximately 25 nm. The immunogenicity of the VP2 subunit vaccine was evaluated in pigs. The results showed that VP2 protein emulsified with ISA 201VG adjuvant induced higher levels of HI antibodies and neutralizing antibodies than VP2 protein emulsified with IMS 1313VG adjuvant. Furthermore, the gilts immunized with the ISA 201VG 20 µg subunit vaccine acquired complete protection against PPV1 HN2019 infection. In contrast, the commercial inactivated vaccine provided incomplete protection in gilts. Therefore, the VP2 subunit vaccine is a promising genetically engineered vaccine for the prevention and control of PPV1.

Highly efficient and robust π-FISH rainbow for multiplexed in situ detection of diverse biomolecules.

In the unprecedented single-cell sequencing and spatial multiomics era of biology, fluorescence in situ hybridization (FISH) technologies with higher sensitivity and robustness, especially for detecting short RNAs and other biomolecules, are greatly desired. Here, we develop the robust multiplex π-FISH rainbow method to detect diverse biomolecules (DNA, RNA, proteins, and neurotransmitters) individually or simultaneously with high efficiency. This versatile method is successfully applied to detect gene expression in different species, from microorganisms to plants and animals. Furthermore, we delineate the landscape of diverse neuron subclusters by decoding the spatial distribution of 21 marker genes via only two rounds of hybridization. Significantly, we combine π-FISH rainbow with hybridization chain reaction to develop π-FISH+ technology for short nucleic acid fragments, such as microRNA and prostate cancer anti-androgen therapy-resistant marker ARV7 splicing variant in circulating tumour cells from patients. Our study provides a robust biomolecule in situ detection technology for spatial multiomics investigation and clinical diagnosis.

The Connectome and Chemo-Connectome Databases for Mice Brain Connection Analysis.

The various brain functions rely on the intricate connection networks and certain molecular characteristics of neurons in the brain. However, the databases for the mouse brain connectome and chemo-connectome are still inadequate, hindering the brain circuital and functional analysis. Here, we created mice brain connectome and chemo-connectome databases based on mouse brain projection data of 295 non-overlapping brain areas and in situ hybridization (ISH) data of 50 representative neurotransmission-related genes from the Allen Brain Institute. Based on this connectome and chemo-connectome databases, functional connection patterns and detailed chemo-connectome for monoaminergic nuclei were analyzed and visualized. These databases will aid in the comprehensive research of the mouse connectome and chemo-connectome in the whole brain and serve as a convenient resource for systematic analysis of the brain connection and function.

Construction of a quadruple gene-deleted vaccine confers complete protective immunity against emerging PRV variant challenge in piglets.

BACKGROUND: Pseudorabies virus (PRV) causes Aujeszky's disease or pseudorabies (PR) in pigs worldwide, which leads to heavy economic losses to the swine industry. Pigs are the natural host, meanwhile, animals such as dogs, cats, foxes, rabbits, cattle and sheep are susceptible to infection. In 2011, the emerging PRV variant led to the outbreak of PR in Bartha-K61 vaccinated pigs. The PR outbreaks demonstrated that the Bartha-K61 vaccine did not provide full protection against the emerging PRV variant. It is widely believed that PRV live attenuated vaccine could control PRV infection. METHODS: In this study, we developed a novel PRV live attenuated vaccine by deleting its gI, gE, US9, and US2 genes through CRISPR/Cas9, which was named PRV GDFS-delgI/gE/US9/US2. RESULTS: Safety experiments confirmed that PRV GDFS-delgI/gE/US9/US2 was safe for 5- to 7-day-old suckling piglets. Piglets immunized with the PRV GDFS-delgI/gE/US9/US2 vaccine did not produce PRV gE-specific antibodies but could generate PRV gB-specific antibodies and high neutralizing titers against the PRV GDFS strain (variant PRV strain) or PRV Ea strain (older PRV strain). After challenge with the emerging PRV GDFS variant, none of the piglets immunized with the PRV GDFS-delgI/gE/US9/US2 vaccine showed any clinical signs, and their rectal temperatures were normal. Moreover, the autopsy and histopathological analyses revealed that the piglets in the PRV GDFS-delgI/gE/US9/US2 vaccine group did not show apparent gross or pathological lesions. Furthermore, the piglets in the PRV GDFS-delgI/gE/US9/US2 vaccine groups did not present weight loss. According to the criteria of the OIE terrestrial manual, the results of the experiment confirmed that the PRV GDFS-delgI/gE/US9/US2 vaccine could provide full protection against the emerging PRV variant strain in piglets. CONCLUSIONS: The PRV GDFS-delgI/gE/US9/US2 strain is a potential new live attenuated vaccine against emerging PRV variant strain infections in China.

Plant-derived Ren's oligopeptide has antiviral effects on influenza virus and SARS-CoV-2.

Influenza virus and SARS-CoV-2 virus are two important viruses that cause respiratory tract diseases. The high-frequency mutation of the two types of viruses leads to failure of the durable immune protection of vaccines, meanwhile it also poses continuous challenges to the development of antiviral drugs. Traditional Chinese medicine contains large number of biologically active compounds, and some of them contain broad-spectrum antiviral ingredients. In this study, we extracted antiviral active ingredients from medicinal and edible plants by biotransformation and enzymatic hydrolysis as a drug, and we named this drug Ren's oligopeptide. Further, we analyzed the antiviral activity of this drug and found that Ren's oligopeptide could inhibit the replication of influenza virus and SARS-CoV-2 virus with high anti-virus activities. In vitro experiments showed that the antiviral activity of the Ren's oligopeptide mainly targets the replication process after virus enters the cell. Therefore, Ren's oligopeptide is a promising drug against influenza and COVID-19.

Inactivated Pseudomonas PE(ΔIII) exotoxin fused to neutralizing epitopes of PEDV S proteins produces a specific immune response in mice.

Porcine epidemic diarrhea (PED) caused by the porcine epidemic diarrhea virus (PEDV), is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide. An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010. S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV. In this study, the construction, expression and purification of Pseudomonas aeruginosa exotoxin A (PE) without domain III (PEΔIII) as a vector was performed for the delivery of PEDV S-A or S-B. PE(ΔIII) PEDV S-A and PE(ΔIII) PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice. The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses, but also stimulate PEDV-specific mucosal immune responses in mice. PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.

Delineating the organization of projection neuron subsets in primary visual cortex with multiple fluorescent rabies virus tracing.

The impressive functions of the brain rely on an extensive connectivity matrix between specific neurons, the architecture of which is frequently characterized by one brain nucleus/region connecting to multiple targets, either via collaterals of the same projection neuron or several, differentially specified neurons. Delineating the fine architecture of projection neuron subsets in a specific brain region could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multiple fluorescent rabies viruses (RV) to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously retrograde labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic projection neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic projection neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic projection neurons targeting two thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. The multiple fluorescent RV tracing tools can be extensively applied to resolve the architecture of projection neuron subsets in certain brain regions, with a strong potential to delineate the computational and functional organization of these brain regions.

AAV-expressed G protein induces robust humoral and cellular immune response and provides durable protection from rabies virus challenges in mice.

Rabies is a highly lethal infectious zoonosis caused by rabies virus (RABV), and the mortality rate is almost 100 % once clinical symptoms appear, which poses a huge threat to public health security across the many parts of the word. Vaccination is reported to be the most effective approach to prevent the disease. G protein is the only protein present on the surface of RABV, it also could induce humoral immunity to produce virus neutralizing antibodies (VNA) and stimulate T cells to produce cellular immunity. Adeno-associated viruses (AAVs) have been used as vectors for gene therapy of different human diseases for its low immunogenicity, high safety and long-term stable expression. To develop a safe and effective vaccine, recombinant AAVs containing different kind of G gene were constructed. After intramuscular (i.m.) immunization in mice, all of these rAAV-G vaccines could induce the production of high levels of VNA and effective cellular immune response. Consistently, all of the rAAV-G vaccines could provide protection against lethal RABV challenge. Our results shown that the rAAV-G vaccines could be potential candidates used in the control of RABV infection. In addition, rAAV-G as a vaccine has many advantages of low preparation cost, simple storage and transportation conditions (4 °C storage and transportation), simple immunization program (only one immunization) and so on. Thence, the rAAV-G vaccines could be potential candidates used in the control of RABV infection.

In Vivo Two-photon Calcium Imaging in Dendrites of Rabies Virus-labeled V1 Corticothalamic Neurons.

Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain. Two-photon Ca2+ imaging in vivo using a cranial window and specific neuronal labeling enables real-time, in situ, and long-term imaging of the living brain. Here, we constructed a recombinant rabies virus containing the Ca2+ indicator GCaMP6s along with the fluorescent protein DsRed2 as a baseline reference to ensure GCaMP6s signal reliability. This functional tracer was applied to retrogradely label specific V1-thalamus circuits and detect spontaneous Ca2+ activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca2+ imaging. Notably, we were able to record single-spine spontaneous Ca2+ activity in specific circuits. Distinct spontaneous Ca2+ dynamics in dendrites of V1 corticothalamic neurons were found for different V1-thalamus circuits. Our method can be applied to monitor Ca2+ dynamics in specific input circuits in vivo, and contribute to functional studies of defined neural circuits and the dissection of functional circuit connections.

Differences in neurotropism and neurotoxicity among retrograde viral tracers.

BACKGROUND: Neurotropic virus-based tracers have been extensively applied in mapping and manipulation of neural circuits. However, their neurotropic and neurotoxic properties remain to be fully characterized. METHODS: Through neural circuit tracing, we systematically compared the neurotropism discrepancy among different multi-trans-synaptic and mono-synaptic retrograde viral tracers including pseudorabies virus (PRV), rabies virus (RV), and the newly engineered retro adeno-associated virus (rAAV2-retro) tracers. The (single-cell) RNA sequencing analysis was utilized for seeking possible attribution to neurotropism discrepancy and comparing cell toxicity caused by viral infection between glycoprotein-deleted RV (RV-∆G) and rAAV2-retro. Viral toxicity induced microglia activation and neuronal protein change were evaluated by immunohistochemistry. RESULTS: Multi-trans-synaptic retrograde viral tracers, PRV and RV, exhibit differential neurotropism when they were used for central neural circuit tracing from popliteal lymph nodes. Mono-synaptic retrograde tracers, including RV-∆G and rAAV2-retro, displayed discrepant neurotropic property, when they were applied to trace the inputs of lateral hypothalamic area and medial preoptic nucleus. rAAV2-retro demonstrated preference in cerebral cortex, whereas RV-∆G prefers to label basal ganglia and hypothalamus. Remarkably, we detected a distinct preference for specific cortical layer of rAAV2-retro in layer 5 and RV-∆G in layer 6 when they were injected into dorsal lateral geniculate nucleus to label corticothalamic neurons in primary visual cortex. Complementation of TVA receptor gene in RV-resistant neurons enabled EnvA-pseudotyped RV infection, supporting receptors attribution to viral neurotropism. Furthermore, both RV-∆G and rAAV2-retro exerted neurotoxic influence at the injection sites and retrogradely labeled sites, while the changes were more profound for RV-∆G infection. Finally, we demonstrated a proof-of-concept strategy for more comprehensive high-order circuit tracing of a specific target nucleus by combining rAAV2-retro, RV, and rAAV tracers. CONCLUSIONS: Different multi-trans-synaptic and mono-synaptic retrograde viral tracers exhibited discrepant neurotropism within certain brain regions, even cortical layer preference. More neurotoxicity was observed under RV-∆G infection as compared with rAAV2-retro. By combining rAAV2-retro, RV, and rAAV tracers, high-order circuit tracing can be achieved. Our findings provide important reference for appropriate application of viral tracers to delineate the landscape and dissect the function of neural network.

Memantine can relieve the neuronal impairment caused by neurotropic virus infection.

Neurotropic viruses, such as the rabies virus (RABV) and Japanese encephalitis virus (JEV), induce neuronal dysfunction and complication, causing neuronal damage. Currently, there are still no effective clinical treatments for neuronal injury caused by neurotropic viruses. Memantine, a drug capable of passing through the blood-brain barrier, noncompetitively and reversibly binds to n-methyl- d-aspartic acid (NMDA) receptors. Memantine is used to treat Alzheimer's disease by blocking the activation of extra axonal ion channels, thus preventing neuronal degeneration by inhibiting the abnormal cytosolic Ca 2+ increase. To explore whether memantine can alleviate neurological disturbances caused by RABV and JEV, the following experiments were carried out: (1) for primary neurons cultured in vitro infected with RABV, the addition of memantine showed neuroprotection. (2) In the RABV challenge experiments, memantine had limited therapeutic effect, mildly extending the survival time of mice. In contrast, memantine significantly prolonged the survival time of mice infected with JEV, by reducing the intravascular cuff and inflammatory cell infiltration in mice. Furthermore, memantine decreases the amount of JEV virus in mice brain.

A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus.

Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate's protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development.

Etanercept reduces neuroinflammation and lethality in mouse model of Japanese encephalitis.

BACKGROUND: Japanese encephalitis virus (JEV) is a neurotropic flavivirus that causes Japanese encephalitis (JE), which leads to high fatality rates in human. Tumor necrosis factor alpha (TNF-α) is a key factor that mediates immunopathology in the central nervous system (CNS) during JE. Etanercept is a safe anti-TNF-α drug that has been commonly used in the treatment of various human autoimmune diseases. METHODS: The effect of etanercept on JE was investigated with a JEV-infected mouse model. Four groups of mice were assigned to receive injections of phosphate-buffered saline, etanercept, JEV, or JEV plus etanercept. Inflammatory responses in mouse brains and mortality of mice were evaluated within 23 days post infection. RESULTS: The in vitro assay with mouse neuron/glia cultures showed that etanercept treatment reduced the inflammatory response induced by JEV infection. In vivo experiments further demonstrated that administration of etanercept protected mice from JEV-induced lethality. Neuronal damage, glial activation, and secretion of proinflammatory cytokines were found to be markedly decreased in JEV-infected mice that received etanercept treatment. Additionally, etanercept treatment restored the integrity of the blood-brain barrier and reduced viral load in mouse brains. CONCLUSIONS: Etanercept effectively reduces the inflammation and provides protection against acute encephalitis in a JEV-infected mouse model.

Identification of three antiviral inhibitors against Japanese encephalitis virus from library of pharmacologically active compounds 1280.

Japanese encephalitis virus (JEV) can cause severe central nervous disease with a high mortality rate. There is no antiviral drug available for JEV-specific treatment. In this study, a cytopathic-effect-based, high-throughput screening assay was developed and applied to screen JEV inhibitors from Library of Pharmacologically Active Compounds 1280. The antiviral effects of three hit compounds including FGIN-1-27, cilnidipine, and niclosamide were evaluated in cells by western blotting, indirect immunofluorescence assay, and plaque reduction assay. A time-of-addition assay proved that all three compounds inhibited JEV at the stage of replication. The EC50s of FGIN-1-27, cilnidipine, and niclosamide were 3.21, 6.52, and 5.80 µM, respectively, while the selectivity indexes were 38.79, 30.67, and 7.49. FGIN-1-27 and cilnidipine have high efficiency and selectivity against JEV. This study provided two JEV antiviral inhibitors as candidates for treatment of JEV infection.

[Roles of N-glycosylation in immunity of prME and NS1 gene of JEV].

PrME and NS1 gene were the two main immuneprotect proteins of Japanese encephalitis virus (JEV), and they were also N-linked glycosylation proteins. To clear the effect of N-glycosylation on JEV immunity, the N-glycosylation site of prME and NS1 gene were eliminated by site-directed mutant PCR, subtituting the N to Q. And the the mutant genes were subcloned into eukaryotic expression plasmid. Four-weeks female mice were immuned with the wildtype and mutant gene by twice. The antibodies against prME were detected by ELISA and the neutralization antibodies were tested by viral neutralizing assay. The immunoprotection were determined by attack with JEV virulent strain. Compare with the wild-type gene immuned-groups, one N-glycan eliminated prME gene could induce a little higher ELISA antibody, neutralization antibody and immunoprotection, but the immunity of gene with both N-glycan absence was decreased. The similar status were observed in the wildtype and mutant NS1 groups. Thus these results show that the N-linked glycosylation in the prME and NS1 gene were correlated with the immunity, one glycan absent would enhance the immunity but both two loss would impair it.