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Lymphatic defence against neurotropic viruses Microorganisms that breach the body's external defences and enter the lymphatic system are liable to be captured by the lymph nodes, and recent work showed that a subset of macrophages found in the subcapsular sinus (SCS) of lymph nodes is critical for clearance of viruses from the lymph and for initiating antiviral humoral immune responses. Now a third function for SCS macrophages has been identified: the prevention of lymph-borne neurotropic viruses from infecting the CNS. Using vesicular stomatitis virus (VSV) as a model, Iannacone et al . showed that local depletion of SCS macrophages made mice injected subcutaneously with VSV more vulnerable to the virus via a mechanism dependent on type I interferon. VSV is a relative of rabies virus typically transmitted by insect bites, causing a fatal paralytic disease in some mammals. Combined with further experiments in mice lacking the IFN-I receptor, these findings suggest that SCS macrophages are crucial gatekeepers to the CNS that prevent fatal viral neuroinvasion upon peripheral infection. Macrophages that populate the lymph nodes are known to clear viruses from the lymph and to initiate antiviral humoral immune responses. It is now shown that these macrophages also have another function: they prevent lymph-borne neurotropic viruses from entering the central nervous system. The mechanism is dependent on the production of type I interferon. Lymph nodes (LNs) capture microorganisms that breach the body’s external barriers and enter draining lymphatics, limiting the systemic spread of pathogens 1 . Recent work has shown that CD11b + CD169 + macrophages, which populate the subcapsular sinus (SCS) of LNs, are critical for the clearance of viruses from the lymph and for initiating antiviral humoral immune responses 2 , 3 , 4 . Here we show, using vesicular stomatitis virus (VSV), a relative of rabies virus transmitted by insect bites, that SCS macrophages perform a third vital function: they prevent lymph-borne neurotropic viruses from infecting the central nervous system (CNS). On local depletion of LN macrophages, about 60% of mice developed ascending paralysis and died 7–10 days after subcutaneous infection with a small dose of VSV, whereas macrophage-sufficient animals remained asymptomatic and cleared the virus. VSV gained access to the nervous system through peripheral nerves in macrophage-depleted LNs. In contrast, within macrophage-sufficient LNs VSV replicated preferentially in SCS macrophages but not in adjacent nerves. Removal of SCS macrophages did not compromise adaptive immune responses against VSV, but decreased type I interferon (IFN-I) production within infected LNs. VSV-infected macrophages recruited IFN-I-producing plasmacytoid dendritic cells to the SCS and in addition were a major source of IFN-I themselves. Experiments in bone marrow chimaeric mice revealed that IFN-I must act on both haematopoietic and stromal compartments, including the intranodal nerves, to prevent lethal infection with VSV. These results identify SCS macrophages as crucial gatekeepers to the CNS that prevent fatal viral invasion of the nervous system on peripheral infection.

Vesicular stomatitis virus (VSV), comprising vesicular stomatitis New Jersey virus (VSNJV) and vesicular stomatitis Indiana virus (VSIV), emerges from its focus of endemic transmission in Southern Mexico to cause sporadic livestock epizootics in the Western United States. A dearth of information on the role of potential arthropod vectors in the endemic region hampers efforts to identify factors that enable endemicity and predict outbreaks. In a two-year, longitudinal study at five cattle ranches in Chiapas, Mexico, insect taxa implicated as VSV vectors (blackflies, sandflies, biting midges, and mosquitoes) were collected and screened for VSV RNA, livestock vesicular stomatitis (VS) cases were monitored, and serum samples were screened for neutralizing antibodies. VS cases were reported during the rainy (n = 20) and post-rainy (n = 2) seasons. Seroprevalence against VSNJV in adult cattle was very high (75–100% per ranch) compared with VSIV (0.6%, all ranches). All four potential vector taxa were sampled, and VSNJV RNA was detected in each of them (11% VSNJV-positive of 874 total pools), while VSIV RNA was only detected in four pools of mosquitoes. Our findings indicate that VSNJV is the dominant serotype across our sampling sites with a variety of potential insect vectors involved in its transmission throughout the year. Although no livestock cases were reported in Chiapas during the dry season, VSNJV was detected in insects during this period, suggesting that mechanisms other than transmission from livestock support VSV endemicity.

Autophagy is an essential process for physiological homeostasis, but its role in viral infection is only beginning to be elucidated. We show here that the Atg5-Atg12 conjugate, a key regulator of the autophagic process, plays an important role in innate antiviral immune responses. Atg5-deficient mouse embryonic fibroblasts (MEFs) were resistant to vesicular stomatitis virus replication, which was largely due to hyperproduction of type I interferons in response to immunostimulatory RNA (isRNA), such as virus-derived, double-stranded, or 5'-phosphorylated RNA. Similar hyperresponse to isRNA was also observed in Atg7-deficient MEFs, in which Atg5-Atg12 conjugation is impaired. Overexpression of Atg5 or Atg12 resulted in Atg5-Atg12 conjugate formation and suppression of isRNA-mediated signaling. Molecular interaction studies indicated that the Atg5-Atg12 conjugate negatively regulates the type I IFN production pathway by direct association with the retinoic acid-inducible gene I (RIG-I) and IFN-β promoter stimulator 1 (IPS-1) through the caspase recruitment domains (CARDs). Thus, in contrast to its role in promoting the bactericidal process, a component of the autophagic machinery appears to block innate antiviral immune responses, thereby contributing to RNA virus replication in host cells.

Differentiation of CD4 + T cells into either follicular helper T (T FH ) or type 1 helper T (T H 1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4 + T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove T FH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into T H 1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4 + T cell differentiation and might instruct vaccine design strategies. Iannacone and colleagues show that the spatiotemporal regulation of type I interferon expression shapes the differentiation of antiviral CD4 + T cells into T FH or T H 1 cells.

For the first time, we describe phylogenomic signatures of an epidemic lineage of vesicular stomatitis Indiana virus (VSIV). We applied multiple evolutionary analyses to a dataset of 87 full-length genome sequences representing the circulation of an epidemic VSIV lineage in the US between 2019 and 2020. Based on phylogenetic analyses, we predicted the ancestral relationship of this lineage with a specific group of isolates circulating in the endemic zone of Chiapas, Mexico. Subsequently, our findings indicate that the lineage diversified into at least four different subpopulations during its circulation in the US. We identified single nucleotide polymorphisms (SNPs) that differentiate viral subpopulations and assessed their potential relevance using comparative phylogenetic methods, highlighting the preponderance of synonymous mutations during the differentiation of these populations. Purifying selection was the main evolutionary force favoring the conservation of this epidemic phenotype, with P and G genes as the main drivers of the evolution of this lineage. Our analyses identified multiple codon sites under positive selection and the association of these sites with specific functional domains at P, M, G, and L proteins. Based on ancestral reconstruction analyses, we showed the potential relevance of some of the sites identified under positive selection to the adaptation of the epidemic lineage at the population level. Finally, using a representative group of viruses from Colorado, we established a positive correlation between genetic and geographical distances, suggesting that positive selection on specific codon positions might have favored the adaptation of different subpopulations to circulation in specific geographical settings. Collectively, our study reveals the complex dynamics that accompany the evolution of an epidemic lineage of VSIV in nature. Our analytical framework provides a model for conducting future evolutionary analyses. The ultimate goal is to support the implementation of an early warning system for vesicular stomatitis virus in the US, enabling early detection of epidemic precursors from Mexico.

We evaluated the in vitro effects of lyophilization for 2 vesicular stomatitis virus-based vaccines by using 3 stabilizing formulations and demonstrated protective immunity of lyophilized/reconstituted vaccine in guinea pigs. Lyophilization increased stability of the vaccines, but specific vesicular stomatitis virus-based vaccines will each require extensive analysis to optimize stabilizing formulations.

•Viral infectivity methods are often slow, tedious, labor intensive, and difficult to standardize.•Laser Force Cytology uses optical and fluidic forces to quantify cellular changes upon infection.•Non-subjective measurements were made in 16 h that correlated with TCID50 results requiring 72 h.•Real-time Laser Force Cytology based measurements were correlated with TCID50 of the supernatant.•Rapid infectivity measurements can improve vaccine research, development and manufacturing. The ability to rapidly and accurately determine viral infectivity can help improve the speed of vaccine product development and manufacturing. Current methods to determine infectious viral titers, such as the end-point dilution (50% tissue culture infective dose, TCID50) and plaque assays are slow, labor intensive, and often subjective. In order to accelerate virus quantification, Laser Force Cytology (LFC) was used to monitor vesicular stomatitis virus (VSV) infection in Vero (African green monkey kidney) cells. LFC uses a combination of optical and fluidic forces to interrogate single cells without the use of labels or antibodies. Using a combination of variables measured by the Radiance™ LFC instrument (LumaCyte), an infection metric was developed that correlates well with the viral titer as measured by TCID50 and shortens the timeframe from infection to titer determination from 3 days to 16 h (a 4.5 fold reduction). A correlation was also developed between in-process cellular measurements and the viral titer of collected supernatant, demonstrating the potential for real-time infectivity measurements. Overall, these results demonstrate the utility of LFC as a tool for rapid infectivity measurements throughout the vaccine development process.

Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. Here we report two crystal structures of VSV G in complex with two distinct cysteine-rich domains (CR2 and CR3) of LDL-R, showing that their binding sites on G are identical. We identify two basic residues on G, which are essential for its interaction with CR2 and CR3. Mutating these residues abolishes VSV infectivity even though VSV can use alternative receptors, indicating that all VSV receptors are members of the LDL-R family. Collectively, our data suggest that VSV G has specifically evolved to interact with receptor CR domains. These structural insights into the interaction between VSV G and host cell receptors provide a basis for the design of recombinant viruses with an altered tropism. Glycoprotein G of vesicular stomatitis virus (VSV) enables viral entry by binding to the major VSV receptor LDL-R. Here the authors present crystal structures of G in complex with two distinct CR domains of LDL-R, identifying structural determinants for VSV infectivity in mammalian and insect cells.

Oncolytic viruses can be neutralized in the bloodstream by antiviral antibodies whose titers increase progressively with each exposure, resulting in faster virus inactivation and further reductions in efficacy with each successive dose. A single dose of cyclophosphamide (CPA) at 370 mg m –2 was not sufficient to control the primary antiviral immune responses in mice, squirrel monkeys and humans. We therefore tested clinically approved multidose CPA regimens, which are known to kill proliferating lymphocytes, to determine if more intensive CPA therapy can more effectively suppress antiviral antibody responses during virotherapy. In virus-susceptible mice, primary antibody responses to intravenously (i.v.) administered oncolytic measles virus (MV) or vesicular stomatitis virus (VSV) were partially or completely suppressed, respectively, by oral (1 mg × 8 days) or systemic (3 mg × 4 days) CPA regimens initiated 1 day before virus. When MV- or VSV-immune mice were re-challenged with the respective viruses and concurrently treated with four daily systemic doses of CPA, their anamnestic antibody responses were completely suppressed and antiviral antibody titers fell significantly below pre-booster levels. We conclude that the CPA regimen of four daily doses at 370 mg m –2 should be evaluated clinically with i.v. virotherapy to control the antiviral antibody response and facilitate effective repeat dosing.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease causing severe hemorrhagic symptoms with a nearly 30 % case-fatality rate in humans. The experimental use of CCHF virus (CCHFV), which causes CCHF, requires high-biosafety-level (BSL) containment. In contrast, pseudotyping of various viral glycoproteins (GPs) onto vesicular stomatitis virus (VSV) can be used in facilities with lower BSL containment, and this has facilitated studies on the viral entry mechanism and the measurement of neutralizing activity, especially for highly pathogenic viruses. In the present study, we generated high titers of pseudotyped VSV bearing the CCHFV envelope GP and analyzed the mechanisms involved in CCHFV infection. A partial deletion of the CCHFV GP cytoplasmic domain increased the titer of the pseudotyped VSV, the entry mechanism of which was dependent on the CCHFV envelope GP. Using the pseudotype virus, DC-SIGN (a calcium-dependent [C-type] lectin cell-surface molecule) was revealed to enhance viral infection and act as an entry factor for CCHFV.