A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2.

An outbreak of coronavirus disease 2019 (COVID-19)1-3, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, has spread globally. Countermeasures are needed to treat and prevent further dissemination of the virus. Here we report the isolation of two specific human monoclonal antibodies (termed CA1 and CB6) from a patient convalescing from COVID-19. CA1 and CB6 demonstrated potent SARS-CoV-2-specific neutralization activity in vitro. In addition, CB6 inhibited infection with SARS-CoV-2 in rhesus monkeys in both prophylactic and treatment settings. We also performed structural studies, which revealed that CB6 recognizes an epitope that overlaps with angiotensin-converting enzyme 2 (ACE2)-binding sites in the SARS-CoV-2 receptor-binding domain, and thereby interferes with virus-receptor interactions by both steric hindrance and direct competition for interface residues. Our results suggest that CB6 deserves further study as a candidate for translation to the clinic.

A Chimeric Classical Insect-Specific Flavivirus Provides Complete Protection Against West Nile Virus Lethal Challenge in Mice.

West Nile virus (WNV), an arthropod-borne flavivirus, can cause severe symptoms, including encephalitis, and death, posing a threat to public health and the economy. However, there is still no approved treatment or vaccine available for humans. Here, we developed a novel vaccine platform based on a classical insect-specific flavivirus (cISF) YN15-283-02, which was derived from Culicoides. The cISF-WNV chimera was constructed by replacing prME structural genes of the infectious YN15-283-02 cDNA clone with those of WNV and successfully rescued in Aedes albopictus cells. cISF-WNV was nonreplicable in vertebrate cells and nonpathogenic in type I interferon receptor (IFNAR)-deficient mice. A single-dose immunization of cISF-WNV elicited considerable Th1-biased antibody responses in C57BL/6 mice, which was sufficient to offer complete protection against lethal WNV challenge with no symptoms. Our studies demonstrated the potential of the insect-specific cISF-WNV as a prophylactic vaccine candidate to prevent infection with WNV.

Protection of the receptor binding domain (RBD) dimer against SARS-CoV-2 and its variants.

IMPORTANCE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants achieved immune escape and became less virulent and easily transmissible through rapid mutation in the spike protein, thus the efficacy of vaccines on the market or in development continues to be challenged. Updating the vaccine, exploring compromise vaccination strategies, and evaluating the efficacy of candidate vaccines for the emerging variants in a timely manner are important to combat complex and volatile SARS-CoV-2. This study reports that vaccines prepared from the dimeric receptor-binding domain (RBD) recombinant protein, which can be quickly produced using a mature and stable process platform, had both good immunogenicity and protection in vivo and could completely protect rodents from lethal challenge by SARS-CoV-2 and its variants, including the emerging Omicron XBB.1.16, highlighting the value of dimeric recombinant vaccines in the post-COVID-19 era.

HostNet: improved sequence representation in deep neural networks for virus-host prediction.

BACKGROUND: The escalation of viruses over the past decade has highlighted the need to determine their respective hosts, particularly for emerging ones that pose a potential menace to the welfare of both human and animal life. Yet, the traditional means of ascertaining the host range of viruses, which involves field surveillance and laboratory experiments, is a laborious and demanding undertaking. A computational tool with the capability to reliably predict host ranges for novel viruses can provide timely responses in the prevention and control of emerging infectious diseases. The intricate nature of viral-host prediction involves issues such as data imbalance and deficiency. Therefore, developing highly accurate computational tools capable of predicting virus-host associations is a challenging and pressing demand. RESULTS: To overcome the challenges of virus-host prediction, we present HostNet, a deep learning framework that utilizes a Transformer-CNN-BiGRU architecture and two enhanced sequence representation modules. The first module, k-mer to vector, pre-trains a background vector representation of k-mers from a broad range of virus sequences to address the issue of data deficiency. The second module, an adaptive sliding window, truncates virus sequences of various lengths to create a uniform number of informative and distinct samples for each sequence to address the issue of data imbalance. We assess HostNet's performance on a benchmark dataset of "Rabies lyssavirus" and an in-house dataset of "Flavivirus". Our results show that HostNet surpasses the state-of-the-art deep learning-based method in host-prediction accuracies and F1 score. The enhanced sequence representation modules, significantly improve HostNet's training generalization, performance in challenging classes, and stability. CONCLUSION: HostNet is a promising framework for predicting virus hosts from genomic sequences, addressing challenges posed by sparse and varying-length virus sequence data. Our results demonstrate its potential as a valuable tool for virus-host prediction in various biological contexts. Virus-host prediction based on genomic sequences using deep neural networks is a promising approach to identifying their potential hosts accurately and efficiently, with significant impacts on public health, disease prevention, and vaccine development.

The Potential Role of Nitric Oxide as a Therapeutic Agent against SARS-CoV-2 Infection.

The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become the greatest worldwide public health threat of this century, which may predispose multi-organ failure (especially the lung) and death despite numerous mild and moderate symptoms. Recent studies have unraveled the molecular and clinical characteristics of the infectivity, pathogenicity, and immune evasion of SARS-CoV-2 and thus improved the development of many different therapeutic strategies to combat COVID-19, including treatment and prevention. Previous studies have indicated that nitric oxide (NO) is an antimicrobial and anti-inflammatory molecule with key roles in pulmonary vascular function in the context of viral infections and other pulmonary disease states. This review summarized the recent advances of the pathogenesis of SARS-CoV-2, and accordingly elaborated on the potential application of NO in the management of patients with COVID-19 through antiviral activities and anti-inflammatory properties, which mitigate the propagation of this disease. Although there are some limits of NO in the treatment of COVID-19, it might be a worthy candidate in the multiple stages of COVID-19 prevention or therapy.

Environmental surveillance of SARS-CoV-2 RNA in wastewater systems and related environments in Wuhan: April to May of 2020.

Wastewater-based epidemiology (WBE) has emerged as an effective environmental surveillance tool in monitoring fecal-oral pathogen infections within a community. Congruently, SARS-CoV-2, the etiologic agent of COVID-19, has been demonstrated to infect the gastrointestinal tissues, and be shed in feces. In the present study, SARS-CoV-2 RNA was concentrated from wastewater, sludge, surface water, ground water, sediment, and soil samples of municipal and hospital wastewater systems and related environments in Wuhan during the COVID-19 middle and low risk periods, and the viral RNA copies quantified using reverse transcription quantitative polymerase chain reaction (RT-qPCR). From the findings of this study, during the middle risk period, one influent sample and three secondary effluents collected from waste water treatment plant 2, as well as two samples from Jinyintan Hospital wastewater system influent were SARS-CoV-2 RNA positive. One sludge sample collected from Guanggu Branch of Tongji Hospital, which was obtained during the low risk period, was also positive for SARS-CoV-2 RNA. These study findings demonstrate the significance of WBE in continuous surveillance of SARS-CoV-2 at the community level, even when the COVID-19 prevalence is low. Overall, this study can be used as an important reference for contingency management of wastewater treatment plants and COVID-19 prevention and control departments of Wuhan.

Characterization of a novel reassortment Tibet orbivirus isolated from Culicoides spp. in Yunnan, PR China.

Orbiviruses are arboviruses with 10 double-stranded linear RNA segments, and some have been identified as pathogens of dramatic epizootics in both wild and domestic ruminants. Tibet orbivirus (TIBOV) is a new orbivirus isolated from hematophagous insects in recent decades, and, currently, most of the strains have been isolated from insects in PR China, except for two from Japan. In this study, we isolated a novel reassortment TIBOV strain, YN15-283-01, from Culicoides spp. To identify and understand more characteristics of YN15-283-01, electrophoresis profiles of the viral genome, electron microscopic observations, plaque assays, growth curves in various cell lines, and bioinformatic analysis were conducted. The results indicated that YN15-283-01 replicated efficiently in mosquito cells, rodent cells and several primate cells. Furthermore, the maximum likelihood phylogenetic trees and simplot analysis of the 10 segments indicated that YN15-283-01 is a natural reassortment isolate that had emerged mainly from XZ0906 and SX-2017a.

Horizontal transfer of large plasmid with type IV secretion system and mosquitocidal genomic island with excision and integration capabilities in Lysinibacillus sphaericus.

We identified a ~30-kb genomic island (named GI8) carrying the binary toxin gene operon binA/binB on both the chromosome and large pBsph plasmid in the mosquitocidal Lysinibacillus sphaericus C3-41 strain. We found that GI8 is related to the occurrence of binA/binB within L. sphaericus and displays excision and integration capability by recognizing the attB region, which consists of a 2-nt target site (AT) flanked by an 11-nt imperfect inverted repeat. pBsph and two pBsph-like plasmids (p2362 and p1593) were found to carry a type IV secretion system (T4SS) and displayed transmissibility within a narrow host range specific to L. sphaericus. GI8 can be co-transferred with pBsph as a composite element by integration into its attB site, then excised from pBsph and re-integrated into the chromosomal attB site in the new host. The potential hosts of GI8, regardless of whether they are toxic or non-toxic to mosquito larvae, share good collinearity at the chromosomal level. Data indicated that the appearance of the mosquitocidal L. sphaericus lineage was driven by horizontal transfer of the T4SS-type conjugative plasmid and GI8 with excision and specific integration capability.

Different Degrees of 5'-to-3' DAR Interactions Modulate Zika Virus Genome Cyclization and Host-Specific Replication.

Mosquito-borne flaviviruses, which include many important human pathogens, such as West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV), have caused numerous emerging epidemics in recent years. Details of the viral genome functions necessary for effective viral replication in mosquito and vertebrate hosts remain obscure. Here, using ZIKV as a model, we found that the conserved "downstream of AUG region" (DAR), which is known to be an essential element for genome cyclization, is involved in viral replication in a host-specific manner. Mutational analysis of the DAR element showed that a single-nucleotide mismatch between the 5' DAR and the 3' DAR had little effect on ZIKV replication in mammalian cells but dramatically impaired viral propagation in mosquito cells. The revertant viruses passaged in mosquito cells generated compensatory mutations restoring the base pairing of the DAR, further confirming the importance of the complementarity of the DAR in mosquito cells. We demonstrate that a single-nucleotide mutation in the DAR is sufficient to destroy long-range RNA interaction of the ZIKV genome and affects de novo RNA synthesis at 28°C instead of 37°C, resulting in the different replication efficiencies of the mutant viruses in mosquito and mammalian cells. Our results reveal a novel function of the circular form of the flavivirus genome in host-specific viral replication, providing new ideas to further explore the functions of the viral genome during host adaptation.IMPORTANCE Flaviviruses naturally cycle between the mosquito vector and vertebrate hosts. The disparate hosts provide selective pressures that drive virus genome evolution to maintain efficient replication during host alteration. Host adaptation may occur at different stages of the viral life cycle, since host-specific viral protein processing and virion conformations have been reported in the individual hosts. However, the viral determinants and the underlying mechanisms associated with host-specific functions remain obscure. In this study, using Zika virus, we found that the DAR-mediated genome cyclization regulates viral replication differently and is under different selection pressures in mammalian and mosquito cells. A more constrained complementarity of the DAR is required in mosquito cells than in mammalian cells. Since the DAR element is stably maintained among mosquito-borne flaviviruses, our findings could provide new information for understanding the role of flavivirus genome cyclization in viral adaptation and RNA evolution in the two hosts.

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.

Replication-Defective West Nile Virus with NS1 Deletion as a New Vaccine Platform for Flavivirus.

We previously produced a replication-defective West Nile virus (WNV) lacking NS1 (WNV-ΔNS1) that could propagate at low levels (105 infectious units [IU]/ml) in a 293T cell line expressing wild-type (WT) NS1. This finding indicates the potential of developing WNV-ΔNS1 as a noninfectious vaccine. To explore this idea, we developed an NS1-expressing Vero cell line (VeroNS1) that significantly improved the yield of WNV-ΔNS1 (108 IU/ml). We evaluated the safety and efficacy of WNV-ΔNS1 in mice. WNV-ΔNS1 appeared to be safe, as no replicative virus was found in naive Vero cells after continuous culturing of WNV-ΔNS1 in VeroNS1 cells for 15 rounds. WNV-ΔNS1 was noninfectious in mice, even when IFNAR-/- mice were administered a high dose of WNV-ΔNS1. Vaccination with a single dose of WNV-ΔNS1 protected mice from a highly lethal challenge with WT WNV. The antibody response against WNV correlated well with the protection of vaccinated mice. Our study demonstrates the potential of the NS1 trans complementation system as a new platform for flavivirus vaccine development.IMPORTANCE Many flaviviruses are significant human pathogens that frequently cause outbreaks and epidemics around the world. Development of novel vaccine platforms against these pathogens is a public health priority. Using WNV as a model, we developed a new vaccine platform for flaviviruses. WNV containing a NS1 deletion (WNV-ΔNS1) could be efficiently trans complemented in Vero cells that constitutively expressed WT NS1 protein. A single-dose immunization with WNV-ΔNS1 elicited robust immune responses in mice. The immunized animals were fully protected against pathogenic WNV infection. No adverse effects related to the WNV-ΔNS1 vaccination were observed. The results have demonstrated the potential of the NS1 complementation system as an alternative platform for flavivirus vaccine development, especially for highly pathogenic flaviviruses.

Infectious Chikungunya Virus (CHIKV) with a Complete Capsid Deletion: a New Approach for a CHIKV Vaccine.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemics of debilitating disease worldwide. Currently, there are no licensed vaccines or antivirals available against CHIKV infection. In this study, we generated a novel live attenuated vaccine (LAV) candidate for CHIKV with a complete deficiency of capsid (ΔC-CHIKV). It could propagate in BHK-21 cells, and had antigenic properties similar to those of native CHIKV. Vaccination of either immunocompromised IFNAR-/- mice or immunocompetent C57BL/6 mice with a single dose of ΔC-CHIKV conferred complete protection upon challenge with wild-type (WT) CHIKV. Taken together, this vaccine candidate appeared to be safe and efficacious, representing a novel strategy for CHIKV vaccine design.IMPORTANCE Currently, there is no licensed vaccine against CHIKV infection. An ideal CHIKV vaccine should generate an optimal balance between efficacy and safety. Live attenuated vaccines that can elicit strong immune responses often involve a trade-off of reduced safety. Here, a novel live attenuated vaccine candidate for CHIKV lacking the entire capsid gene, ΔC-CHIKV, was developed. It was demonstrated to be genetically stable, highly attenuated, immunogenic, and able to confer complete protection against lethal CHIKV challenge after a single dose of immunization. Such an infectious vaccine candidate devoid of capsid provides a novel strategy for the development of a live attenuated CHIKV vaccine.

Application of Bacillus thuringiensis strains with conjugal and mobilizing capability drives gene transmissibility within Bacillus cereus group populations in confined habitats.

BACKGROUND: Bacillus thuringiensis bacteria share similar genetic, physiological, and biochemical characteristics with other members of the Bacillus cereus group. Their diversity and entomopathogenic origin are related to their mobile genetic elements. However, the effects of wide-spread application of B. thuringiensis-based pesticides on genetically related B. cereus group populations present in the environment remain poorly understood. RESULTS: We first identified pBMB76 from B. thuringiensis tenebrionis as a new conjugative plasmid. Mixed mating experiments suggested that pBMB76 may compete with pHT73, another known conjugative plasmid. Applications of single (tenebrionis 4AA1 and kurstaki HD73 carrying pBMB76 and pHT73, respectively) and mixed (4AA1 + HD73) B. thuringiensis strains were performed in confined plot habitats (soil and leaf) over two planting seasons. In total, 684 B. cereus group isolates were randomly selected from different treatment sets, and the transmissibility and occurrence rate of potential conjugative plasmids were surveyed. Results showed that the percentage of isolates with plasmid mobility was markedly enhanced in the B. thuringiensis-sprayed groups. Furthermore, we performed multi-locus sequence typing (MLST) for a subset of 291 isolates, which indicated that the dominant sequence types in the treated habitats were identical or related to the corresponding sprayed formulations. CONCLUSIONS: The application of B. thuringiensis strains with conjugal and mobilizing capability drove gene transmissibility within the B. cereus group populations in confined habitats and potentially modified the population structure.

The discovery and global distribution of novel mosquito-associated viruses in the last decade (2007-2017).

In the last decade, virus hunting and discovery has gained pace. This achievement has been driven by three major factors: (a) advancements in sequencing technologies, (b) scaled-up routine arbovirus surveillance strategies, and (c) the "hunt" for emerging pathogens and novel viruses. Many novel viruses have been discovered from a myriad of hosts, vectors, and environmental samples. To help promote understanding of the global diversity and distribution of mosquito-associated viruses and facilitate future studies, we review mosquito-associated viruses discovered between years 2007 and 2017, across the world. In the analyzed period, novel mosquito-associated viruses belonging to 25 families and a general group of unclassified viruses were categorized. The top three discovered novel mosquito-associated viruses belonged to families Flaviviridae (n=32), Rhabdoviridae (n=16), and Peribunyaviridae (n=14). Also, 67 unclassified viruses were reported. Majority of these novel viruses were identified from Culex spp, Anopheles spp, Aedes spp, and Mansonia spp mosquitoes, respectively. Notably, the number of these discovered novels is not representative of intercontinental virus diversity but rather is influenced by the number of studies done in the study period. Some of these newly discovered mosquito-associated viruses have medical significance, either directly or indirectly. For instance, in the study period, 14 novel mosquito-borne viruses that infect mammalian cells in vitro were reported. These viruses pose a danger to the global health security on emerging viral diseases. On the other hand, some of the newly discovered insect specific viruses described herein have potential application as future biocontrol and vaccine agents against known pathogenic arboviruses. Overall, this review outlines the crucial role played by mosquitoes as viral vectors in the global virosphere.

Effect of an Inactivated Vaccine Against SARS-CoV-2 on Safety and Immunogenicity Outcomes: Interim Analysis of 2 Randomized Clinical Trials.

Importance: A vaccine against coronavirus disease 2019 (COVID-19) is urgently needed. Objective: To evaluate the safety and immunogenicity of an investigational inactivated whole-virus COVID-19 vaccine in China. Interventions: In the phase 1 trial, 96 participants were assigned to 1 of the 3 dose groups (2.5, 5, and 10 µg/dose) and an aluminum hydroxide (alum) adjuvant-only group (n = 24 in each group), and received 3 intramuscular injections at days 0, 28, and 56. In the phase 2 trial, 224 adults were randomized to 5 µg/dose in 2 schedule groups (injections on days 0 and 14 [n = 84] vs alum only [n = 28], and days 0 and 21 [n = 84] vs alum only [n = 28]). Design, Setting, and Participants: Interim analysis of ongoing randomized, double-blind, placebo-controlled, phase 1 and 2 clinical trials to assess an inactivated COVID-19 vaccine. The trials were conducted in Henan Province, China, among 96 (phase 1) and 224 (phase 2) healthy adults aged between 18 and 59 years. Study enrollment began on April 12, 2020. The interim analysis was conducted on June 16, 2020, and updated on July 27, 2020. Main Outcomes and Measures: The primary safety outcome was the combined adverse reactions 7 days after each injection, and the primary immunogenicity outcome was neutralizing antibody response 14 days after the whole-course vaccination, which was measured by a 50% plaque reduction neutralization test against live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results: Among 320 patients who were randomized (mean age, 42.8 years; 200 women [62.5%]), all completed the trial up to 28 days after the whole-course vaccination. The 7-day adverse reactions occurred in 3 (12.5%), 5 (20.8%), 4 (16.7%), and 6 (25.0%) patients in the alum only, low-dose, medium-dose, and high-dose groups, respectively, in the phase 1 trial; and in 5 (6.0%) and 4 (14.3%) patients who received injections on days 0 and 14 for vaccine and alum only, and 16 (19.0%) and 5 (17.9%) patients who received injections on days 0 and 21 for vaccine and alum only, respectively, in the phase 2 trial. The most common adverse reaction was injection site pain, followed by fever, which were mild and self-limiting; no serious adverse reactions were noted. The geometric mean titers of neutralizing antibodies in the low-, medium-, and high-dose groups at day 14 after 3 injections were 316 (95% CI, 218-457), 206 (95% CI, 123-343), and 297 (95% CI, 208-424), respectively, in the phase 1 trial, and were 121 (95% CI, 95-154) and 247 (95% CI, 176-345) at day 14 after 2 injections in participants receiving vaccine on days 0 and 14 and on days 0 and 21, respectively, in the phase 2 trial. There were no detectable antibody responses in all alum-only groups. Conclusions and Relevance: In this interim report of the phase 1 and phase 2 trials of an inactivated COVID-19 vaccine, patients had a low rate of adverse reactions and demonstrated immunogenicity; the study is ongoing. Efficacy and longer-term adverse event assessment will require phase 3 trials. Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR2000031809.

Biosafety Level 4 Laboratory User Training Program, China.

Experienced, qualified personnel certified to work in high-level biocontainment laboratories contribute to the safe operation of these facilities. China began a training program for laboratory users after establishing its first Biosafety Level 4 laboratory, the Wuhan National Biosafety Laboratory (Level 4) of the Chinese Academy of Sciences. We provide an overview of the content of this training program, which can serve as a reference for developing national norms for high-containment laboratory training.

Development and evaluation of one-step multiplex real-time RT-PCR assay for simultaneous detection of Zika virus and Chikungunya virus.

Zika virus (ZIKV) and chikungunya virus (CHIKV) are important human pathogens and mosquito-borne arboviruses, which have resembling history, common vectors, circulating regions, and indistinguishable clinical symptoms. Wide geographical range that is suitable for ZIKV and CHIKV transmission underlines the concern about the impact of epidemic and endemic infections on burden of public health. In the present study, a highly sensitive and specific one-step multiplex real-time RT-PCR assay was developed and evaluated for simultaneous detection and quantification of ZIKV and CHIKV. The single reaction assay employs two pairs of primers and two TaqMan probes that differentiate ZIKV and CHIKV infections. The entire viral genomic RNA in vitro transcribed from full-length infectious clones were used to generate the standard curves for absolute quantification in subsequent tests. The detection limit of the one-step multiplex assay was 1 and 0.5 PFU for infectious ZIKV and CHIKV, respectively. The assessment of specificity indicated this assay is highly specific to targeted viruses showing no amplification of a variety of other flaviviruses. Our assay was able to detect geographically separated and phylogenetically diverse strains of ZIKV and CHIKV. On the applicability of monitoring viral multiplication in cells and testing clinical samples, the one-step multiplex assay provided efficient and accurate determination. The one-step multiplex real-time RT-PCR assay offers a valuable tool for detection of ZIKV and CHIKV and potentially contributes to general surveillance and clinical treatment.

vB_LspM-01: a novel myovirus displaying pseudolysogeny in Lysinibacillus sphaericus C3-41.

Lysinibacillus sphaericus has great application potential not only in the biocontrol of mosquitoes but also in the bioremediation of toxic metals. Phages contribute to the genetic diversity and niche adaptation of bacteria, playing essential roles in their life cycle, but may also cause economic damage for industrially important bacteria through phage contamination during fermentation. In this study, the L. sphaericus phage vB_LspM-01, which belongs to the Myoviridae family, was isolated and characterized. Results showed that vB_LspM-01 could specifically infect most tested L. sphaericus isolates but was not active against isolates belonging to other species. Furthermore, phage-born endolysin exhibited a broader antimicrobial spectrum than the host range of the phage. The vB_LspM-01 genome had no obvious similarity with that of its host, and ca. 22.6% of putative ORFs could not get a match with the public databases. Phylogenic analysis based on the putative terminase large subunit showed high similarity with the phages identified with pac-type headful packaging. The vB_LspM-01 encoding genes were only detected in a tiny percentage of L. sphaericus C3-41 individual cells in the wild population, whereas they showed much higher frequency in the resistant population grown within the plaques; however, the phage genes could not be stably inherited during host cell division. Additionally, the vB_LspM-01 encoding genes were only detected in the host population during the logarithmic growth phase. The mitomycin C induction helped the propagation and lysogeny-lysis switch of vB_LspM-01. The study demonstrated that vB_LspM-01 can be present in a pseudolysogenic state in L. sphaericus C3-41 populations.

Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus.

UNLABELLED: Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. IMPORTANCE: Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein.