The Chimeric Chaoyang-Zika Vaccine Candidate Is Safe and Protective in Mice.

Zika virus (ZIKV) is an emerging flavivirus that causes congenital syndromes including microcephaly and fetal demise in pregnant women. No commercial vaccines against ZIKV are currently available. We previously generated a chimeric ZIKV (ChinZIKV) based on the Chaoyang virus (CYV) by replacing the prME protein of CYV with that of a contemporary ZIKV strain GZ01. Herein, we evaluated this vaccine candidate in a mouse model and showed that ChinZIKV was totally safe in both adult and suckling immunodeficient mice. No viral RNA was detected in the serum of mice inoculated with ChinZIKV. All of the mice inoculated with ChinZIKV survived, while mice inoculated with ZIKV succumbed to infection in 8 days. A single dose of ChinZIKV partially protected mice against lethal ZIKV challenge. In contrast, all the control PBS-immunized mice succumbed to infection after ZIKV challenge. Our results warrant further development of ChinZIKV as a vaccine candidate in clinical trials.

Rapid Generation of Recombinant Flaviviruses Using Circular Polymerase Extension Reaction.

The genus Flavivirus is a group of arthropod-borne single-stranded RNA viruses, which includes important human and animal pathogens such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), Dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Tick-borne encephalitis virus (TBEV). Reverse genetics has been a useful tool for understanding biological properties and the pathogenesis of flaviviruses. However, the conventional construction of full-length infectious clones for flavivirus is time-consuming and difficult due to the toxicity of the flavivirus genome to E. coli. Herein, we applied a simple, rapid, and bacterium-free circular polymerase extension reaction (CPER) method to synthesize recombinant flaviviruses in vertebrate cells as well as insect cells. We started with the de novo synthesis of the JEV vaccine strain SA-14-14-2 in Vero cells using CPER, and then modified the CPER method to recover insect-specific flaviviruses (ISFs) in mosquito C6/36 cells. Chimeric Zika virus (ChinZIKV) based on the Chaoyang virus (CYV) backbone and the Culex flavivirus reporter virus expressing green fluorescent protein (CxFV-GFP) were subsequently rescued in C6/36 cells. CPER is a simple method for the rapid generation of flaviviruses and other potential RNA viruses. A CPER-based recovery system for flaviviruses of different host ranges was established, which would facilitate the development of countermeasures against flavivirus outbreaks in the future.

Pathogenicity and Structural Basis of Zika Variants with Glycan Loop Deletions in the Envelope Protein.

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

Evaluation of Zika virus DNA vaccines based on NS1 and domain III of E.

BACKGROUND: A large-scale outbreak of Zika virus (ZIKV) has occurred in Brazil and other South American countries, and has rapidly spread to 60 countries and regions worldwide since 2015, but no approved anti-ZIKV vaccines are available as of 2021. METHODS: We developed four types of anti-ZIKV DNA vaccine candidates: VPC-NS1, VPC-prME, VPC-prME-NS1, and VPC-EIII-NS1. They were developed against the structural proteins prM and E, and non-structural protein 1 (NS1) of ZIKV using the mammalian cell expression vector pcDNA3.1(+) as the backbone. For immunization, we intramuscularly injected mice with each vaccine candidate (n = 12 to 15 per group) on day 0 and day 14, with mice injected with phosphate-buffered saline (PBS) and pcDNA3.1(+) backbone vector as controls. On day 7, 21, and 35 after initial immunization, the effect of DNA vaccines was evaluated by ZIKV-specific humoral immunity determined by enzyme-linked immunosorbent assay (ELISA), ZIKV-specific T cell immunity determined by intracellular cytokine staining by flow cytometry and serum neutralization capacity determined by plaque reduction neutralization test (PRNT50) assay. RESULTS: The sequencing results showed that DNA vaccine vectors were successfully constructed. Western blotting and immunofluorescence results demonstrated the successful expression of immunogens carried by the DNA vaccines. On day 21 and 35 after the initial immunization, the levels of serum total immunoglobulin (Ig)G in all vaccine-given groups were slightly higher (approximately 1.5- to 2-fold) than those in the control groups. By contrast, ZIKV-specific IgG levels of all vaccine-given groups were significantly higher (approximately 10- to 1000- fold) than those of the control groups. The PRNT50 assay showed that the average serum dilution factors for neutralizing half ZIKV virions from vaccine-given groups were at least 32-fold (highest, 93-fold), while the sera from control group showed no protection. For cellular immunity, the proportions of CD11b+ myeloid cells, CD19+ B lymphocytes and CD3+ T lymphocytes in the mouse spleens as well as the percentages of CD4+ and CD8+ subsets of T cell were not changed 35 days after initial immunization. By contrast, the proportions of ZIKV-specific CD4+T cell and CD8+T cell in all vaccine-given groups were 2- to 10-folds and 2- to 30-fold than those in the control groups, respectively. CONCLUSION: All four DNA vaccines designed for the ZIKV induced neutralizing IgGs and cellular immune responses against ZIKV. Particularly, VPC-EIII-NS1 induced high level of humoral response comparable to the vaccine candidate containing prM, E and NS1 polyprotein, suggesting a potent reduced ADE effect and reserved neutralizing activity. Our findings may provide guidance for improving safety of anti-ZIKV vaccines in the future.

Increased yield of AP-3 by inactivation of asm25 in Actinosynnema pretiosum ssp. auranticum ATCC 31565.

Asamitocins are maytansinoids produced by Actinosynnema pretiosum ssp. auranticum ATCC 31565 (A. pretiosum ATCC 31565), which have a structure similar to that of maytansine, therefore serving as a precursor of maytansine in the development of antibody-drug conjugates (ADCs). Currently, there are more than 20 known derivatives of ansamitocins, among which ansamitocin P-3 (AP-3) exhibits the highest antitumor activity. Despite its importance, the application of AP-3 is restricted by low yield, likely due to a substrate competition mechanism underlying the synthesis pathways of AP-3 and its byproducts. Given that N-demethylansamitocin P-3, the precursor of AP-3, is regulated by asm25 and asm10 to synthesize AGP-3 and AP-3, respectively, asm25 is predicted to be an inhibitory gene for AP-3 production. In this study, we inactivated asm25 in A. pretiosum ATCC 31565 by CRISPR-Cas9-guided gene editing. asm25 depletion resulted in a more than 2-fold increase in AP-3 yield. Surprisingly, the addition of isobutanol further improved AP-3 yield in the asm25 knockout strain by more than 6 times; in contrast, only a 1.53-fold increase was found in the WT strain under the parallel condition. Thus, we uncovered an unknown function of asm25 in AP-3 yield and identified asm25 as a promising target to enhance the large-scale industrial production of AP-3.

Visualization of yellow fever virus infection in mice using a bioluminescent reporter virus.

Yellow fever virus (YFV) is a re-emerging flavivirus, which can lead to severe clinical manifestations and high mortality, with no specific antiviral therapies available. The live-attenuated yellow fever vaccine 17D (YF17D) has been widely used for over eighty years. However, the emergence of yellow fever vaccine-associated viscerotropic disease (YFL-AVD) and yellow fever vaccine-associated neurotropic disease (YFL-AND) raised non-negligible concerns. Additionally, the attenuation mechanism of YF17D is still unclear. Thus, the development of convenient models is crucial to understand the mechanisms behind YF17D attenuation and its adverse effects. In this work, we generated a reporter YF17D expressing nano-luciferase (NLuc). In vitro and in vivo characterization demonstrated that the NLuc-YF17D shared similar biological properties with its parental strain and the NLuc activity can reflect viral infectivity reliably. Combined with in vivo bioluminescence imaging, a series of mice models of YF17D infection was established, which will be useful for the evaluation of antiviral medicines and novel vaccine candidates. Especially, we demonstrated that intraperitoneally (i.p.) infection of NLuc-YF17D in type I interferon receptor-deficient mice A129 resulted in outcomes resembling YEL-AVD and YEL-AND, evidenced by viral replication in multiple organs and invasion of the central neuronal system. Finally, in vitro and in vivo assays based on this reporter virus were established to evaluate the antiviral activities of validated antiviral agents. In conclusion, the bioluminescent reporter virus described herein provides a powerful platform to study YF17D attenuation and vaccine-associated diseases as well as to develop novel countermeasures against YFV.

Computer-Aid Directed Evolution of GPPS and PS Enzymes.

Pinene, a natural active monoterpene, is widely used as a flavoring agent, perfume, medicine, and biofuel. Although genetically engineered microorganisms have successfully produced pinene, to date, the biological yield of pinene is much lower than that of semiterpenes (isoprene) and sesquiterpenes (farnesene). In addition to the low heterologous expression of geranyl pyrophosphate synthase (GPPS) and pinene synthase (PS), cytotoxicity due to accumulation of the monoterpene also limits the production of pinene in microorganisms. In this study, we attempted to use two strategies to increase the biological yield of pinene. By deleting the random coils of GPPS and PS alone or in combination, a strain with a 335% yield increase was obtained. Additionally, upon computer-guided molecular modeling and docking of GPPS with isopentenyl pyrophosphate (IPP), its substrate, the key sites located within the catalytic pocket for substrate binding, was predicted. After screening, a strain harboring the T273R mutation of GPPS was selected among a batch of mutations of the key sites with a 154% increase in pinene yield.

Short Direct Repeats in the 3' Untranslated Region Are Involved in Subgenomic Flaviviral RNA Production.

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation.IMPORTANCE Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5'-3' exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3'-UTR. The 3'-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.

Visualization of chikungunya virus infection in vitro and in vivo.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has become an important re-emerging pathogen with its rapid spread to many non-endemic areas. The lack of effective vaccines and antiviral agents is largely attributed to the elusive infection and dissemination dynamics in vivo. In this study, we designed and developed a novel, replication-competent, CHIKV reporter virus (CHIKV-iRFP) encoding a near infrared fluorescent protein (iRFP). In vitro and in vivo characterization demonstrated that CHIKV-iRFP retained similar replication and virulence phenotypes to its parental virus. Neonatal BABL/c mice and IFNAR-/- A129 mice were highly susceptible to CHIKV-iRFP infection. Following intracranial (i.c.) inoculation, CHIKV-iRFP efficiently replicated and disseminated into whole body, resulting in rapid death in an age-dependent manner. Remarkably, upon footpad injection, CHIKV-iRFP readily disseminated from footpad to head and whole skeleton, with a specific tropism for bone marrow. Taken together, this novel reporter virus provides a powerful tool to track real time CHIKV replication and to test the in vivo efficacy of vaccines and antiviral therapeutics.

Zika virus shedding in the stool and infection through the anorectal mucosa in mice.

Zika virus (ZIKV) has elicited global concern due to its unique biological features, unusual transmission routes, and unexpected clinical outcomes. Although ZIKV transmission through anal intercourse has been reported in humans, it remains unclear if ZIKV is detectable in the stool, if it can infect the host through the anal canal mucosa, and what the pathogenesis of such a route of infection might be in the mouse model. Herein, we demonstrate that ZIKV RNA can be recovered from stools in multiple mouse models, as well as from the stool of a ZIKV patient. Remarkably, intra-anal (i.a.) inoculation with ZIKV leads to efficient infection in both Ifnar1-/- and immunocompetent mice, characterized by extensive viral replication in the blood and multiple organs, including the brain, small intestine, testes, and rectum, as well as robust humoral and innate immune responses. Moreover, i.a. inoculation of ZIKV in pregnant mice resulted in transplacental infection and delayed fetal development. Overall, our results identify the anorectal mucosa as a potential site of ZIKV infection in mice, reveal the associated pathogenesis of i.a. infection, and highlight the complexity of ZIKV transmission through anal intercourse.

Development of a chimeric Zika vaccine using a licensed live-attenuated flavivirus vaccine as backbone.

The global spread of Zika virus (ZIKV) and its unexpected association with congenital defects necessitates the rapid development of a safe and effective vaccine. Here we report the development and characterization of a recombinant chimeric ZIKV vaccine candidate (termed ChinZIKV) that expresses the prM-E proteins of ZIKV using the licensed Japanese encephalitis live-attenuated vaccine SA14-14-2 as the genetic backbone. ChinZIKV retains its replication activity and genetic stability in vitro, while exhibiting an attenuation phenotype in multiple animal models. Remarkably, immunization of mice and rhesus macaques with a single dose of ChinZIKV elicits robust and long-lasting immune responses, and confers complete protection against ZIKV challenge. Significantly, female mice immunized with ChinZIKV are protected against placental and fetal damage upon ZIKV challenge during pregnancy. Overall, our study provides an alternative vaccine platform in response to the ZIKV emergency, and the safety, immunogenicity, and protection profiles of ChinZIKV warrant further clinical development.

Intranasal infection and contact transmission of Zika virus in guinea pigs.

Zika virus (ZIKV) is primarily transmitted to humans through mosquito bites or sexual contact. The excretion and persistence of contagious ZIKV in various body fluids have been well documented in ZIKV patients; however, the risk of direct contact exposure remains unclear. Here, we show that guinea pigs are susceptible to ZIKV infection via subcutaneous inoculation route; infected guinea pigs exhibit seroconversion and significant viral secretion in sera, saliva, and tears. Notably, ZIKV is efficiently transmitted from infected guinea pigs to naïve co-caged animals. In particular, intranasal inoculation of ZIKV is fully capable of establishing infection in guinea pigs, and viral antigens are detected in multiple tissues including brain and parotid glands. Cynomolgus macaques also efficiently acquire ZIKV infection via intranasal and intragastric inoculation routes. These collective results from animal models highlight the risk of exposure to ZIKV contaminants and raise the possibility of close contact transmission of ZIKV in humans.

Visualization of a neurotropic flavivirus infection in mouse reveals unique viscerotropism controlled by host type I interferon signaling.

Flavivirus includes a large group of human pathogens with medical importance. Especially, neurotropic flaviviruses capable of invading central and peripheral nervous system, e.g. Japanese encephalitis virus (JEV) and Zika virus (ZIKV), are highly pathogenic to human and constitute major global health problems. However, the dynamic dissemination and pathogenesis of neurotropic flavivirus infections remain largely unknown. Here, using JEV as a model, we rationally designed and constructed a recombinant reporter virus that stably expressed Renilla luciferase (Rluc). The resulting JEV reporter virus (named Rluc-JEV) and parental JEV exhibited similar replication and infection characteristics, and the magnitude of Rluc activity correlated well with progeny viral production in vitro and in vivo. By using in vivo bioluminescence imaging (BLI) technology, we dissected the replication and dissemination dynamics of JEV infection in mice upon different inoculation routes. Interestingly, besides replicating in mouse brain, Rluc-JEV predominantly invaded the abdominal organs in mice with typical viscerotropism. Further tests in mice deficient in type I interferon (IFN) receptors demonstrated robust and prolonged viral replication in the intestine, spleen, liver, kidney and other abdominal organs. Combined with histopathological and immunohistochemical results, the host type I IFN signaling was evidenced as the major barrier to the viscerotropism and pathogenicity of this neurotropic flavivirus. Additionally, the Rluc-JEV platform was readily adapted for efficacy assay of known antiviral compounds and a live JE vaccine. Collectively, our study revealed abdominal organs as important targets of JEV infection in mice and profiled the unique viscerotropism trait controlled by the host type I IFN signaling. This in vivo visualization technology described here provides a powerful tool for testing antiviral agents and vaccine candidates for flaviviral infection.

25-Hydroxycholesterol Protects Host against Zika Virus Infection and Its Associated Microcephaly in a Mouse Model.

Zika virus (ZIKV) has become a public health threat due to its global transmission and link to severe congenital disorders. The host immune responses to ZIKV infection have not been fully elucidated, and effective therapeutics are not currently available. Herein, we demonstrated that cholesterol-25-hydroxylase (CH25H) was induced in response to ZIKV infection and that its enzymatic product, 25-hydroxycholesterol (25HC), was a critical mediator of host protection against ZIKV. Synthetic 25HC addition inhibited ZIKV infection in vitro by blocking viral entry, and treatment with 25HC reduced viremia and conferred protection against ZIKV in mice and rhesus macaques. 25HC suppressed ZIKV infection and reduced tissue damage in human cortical organoids and the embryonic brain of the ZIKV-induced mouse microcephaly model. Our findings highlight the protective role of CH25H during ZIKV infection and the potential use of 25HC as a natural antiviral agent to combat ZIKV infection and prevent ZIKV-associated outcomes, such as microcephaly.

Characterization of a 2016 Clinical Isolate of Zika Virus in Non-human Primates.

Animal models are critical to understand disease and to develop countermeasures for the ongoing epidemics of Zika virus (ZIKV). Here we report a non-human primate model using a 2016 contemporary clinical isolate of ZIKV. Upon subcutaneous inoculation, rhesus macaques developed fever and viremia, with robust excretion of ZIKV RNA in urine, saliva, and lacrimal fluid. Necropsy of two infected animals revealed that systematic infections involving central nervous system and visceral organs were established at the acute phrase. ZIKV initially targeted the intestinal tracts, spleen, and parotid glands, and retained in spleen and lymph nodes till 10days post infection. ZIKV-specific immune responses were readily induced in all inoculated animals. The non-human primate model described here provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccine and therapeutics.