Secretory pathways and multiple functions of nonstructural protein 1 in flavivirus infection.

The genus Flavivirus contains a wide variety of viruses that cause severe disease in humans, including dengue virus, yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus. Nonstructural protein 1 (NS1) is a glycoprotein that encodes a 352-amino-acid polypeptide and has a molecular weight of 46-55 kDa depending on its glycosylation status. NS1 is highly conserved among multiple flaviviruses and occurs in distinct forms, including a dimeric form within the endoplasmic reticulum, a cell-associated form on the plasma membrane, or a secreted hexameric form (sNS1) trafficked to the extracellular matrix. Intracellular dimeric NS1 interacts with other NSs to participate in viral replication and virion maturation, while extracellular sNS1 plays a critical role in immune evasion, flavivirus pathogenesis and interactions with natural vectors. In this review, we provide an overview of recent research progress on flavivirus NS1, including research on the structural details, the secretory pathways in mammalian and mosquito cells and the multiple functions in viral replication, immune evasion, pathogenesis and interaction with natural hosts, drawing together the previous data to determine the properties of this protein.

Linear epitope identification of monoclonal antibodies against the duck Tembusu virus NS1.

Since 2010, the duck Tembusu virus (DTMUV) has caused a severe outbreak of egg drop syndrome in laying ducks in China, which has resulted in substantial financial losses in the poultry industry. DTMUV nonstructural protein 1 (NS1), as the only secreted protein, could aid in the development of therapeutic antibodies and diagnostic techniques; however, there are few studies on the preparation and epitope identification of monoclonal antibodies (mAbs) against DTMUV NS1. In this study, by indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, and indirect immunofluorescence assay, we screened 6 mAbs (8A4, 8E6, 10F12, 1H11, 3D5, 5C11) that could specifically recognize DTMUV NS1. For epitope mapping of mAbs, a series of GST-tagged truncated fusion proteins of DTMUV NS1 were constructed by prokaryotic expression. Finally, the 4 shortest linear epitopes were identified by indirect ELISA and Western blotting. The epitope 133FVIDGPK139 was recognized by 8A4, the epitope 243IPKTLGGP250 was recognized by 8E6, the epitope 267PWDEK271 was recognized by 10F12, and 156EDFGFGVL163 was recognized by 1H11, 3D5, and 5C11. By sequence alignment and cross-reaction tests, we found that 8A4 and 8E6 had high specificity for DTMUV NS1 compared with that of other mAbs, but 10F12, 1H11, 3D5, and 5C11 exhibited a clear degree of cross-reaction with dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) NS1. Finally, the predicted crystal structure analysis showed the approximate spatial positions of the 4 epitopes on the NS1 dimer. In summary, our study revealed 2 specific mAbs for DTMUV NS1 recognition and 4 multiflavivirus mAbs for DENV, JEV, WNV, and ZIKV NS1 recognition.

Nonstructural proteins 2B and 4A of Tembusu virus induce complete autophagy to promote viral multiplication in vitro.

Tembusu virus (TMUV) is an emerging flavivirus that has broken out in different regions of China. TMUV infection has been reported to induce autophagy in duck embryo fibroblast cells. However, the molecular mechanisms underlying this autophagy induction remain unclear. Here, we explored the interactions between autophagy and TMUV and the effects of the structural and nonstructural proteins of TMUV on autophagy in vitro. Among our results, TMUV infection enhanced autophagy to facilitate viral replication in HEK293T cells. After pharmacologically inducing autophagy with rapamycin (Rapa), the replication of TMUV increased by a maximum of 14-fold compared with the control group. To determine which TMUV protein primarily induced autophagy, cells were transfected with two structural proteins and seven nonstructural proteins of TMUV. Western blotting showed that nonstructural proteins 2B (NS2B) and 4 A (NS4A) of TMUV significantly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3) from LC3-I to LC3-II in HEK293T cells. In addition, through immunofluorescence assays, we found that NS2B and NS4A significantly increased the punctate fluorescence of GFP-LC3-II. Furthermore, we found that both NS2B and NS4A interacted with polyubiquitin-binding protein sequestosome 1 (SQSTM1/p62) in a coimmunoprecipitation assay. Moreover, the autophagic degradation of p62 and LC3 mediated by NS2B or NS4A was inhibited by treatment with the autophagic flux inhibitor chloroquine (CQ). These results confirmed the vital effects of NS2B and NS4A in TMUV-induced complete autophagy and clarified the importance of complete autophagy for viral replication, providing novel insight into the relationship between TMUV and autophagy.

Stem-Loop I of the Tembusu Virus 3'-Untranslated Region Is Responsible for Viral Host-Specific Adaptation and the Pathogenicity of the Virus in Mice.

Tembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3'-untranslated region (3'UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3'UTRs. Compared with rMM 1775, rMM-CQ3'UTR (recombinant MM 1775 virus carrying the 3'UTR of CQW1) exhibited enhanced proliferation in vitro, with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3'UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads. In contrast, rCQ-MM3'UTR showed similar growth kinetics in vitro and neurovirulence in mice compared with those of rCQW1. Then, the Stem-loop I (SLI) structure, which showed the highest variation within the 3'UTR between CQW1 and MM 1775, was further chosen for making chimeric viruses. The peak titers of rMM-CQ3'UTRSLI displayed a 15- or 4-fold increase in vitro, and the neurovirulence in mice was attenuated, compared with that of rMM 1775; rCQ-MM3'UTRSLI displayed comparable multiplication ability in vitro but was significantly attenuated in mice, in contrast with rCQW1. In conclusion, we demonstrated that the TMUV SLI structure of the 3'UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old mice, providing a new understanding of the functions of TMUV 3'UTR in viral host switching and the pathogenicity changes in mice. IMPORTANCE Mosquito-borne flaviviruses (MBFVs) constitute a large number of mosquito-transmitted viruses. The 3'-untranslated region (3'UTR) of MBFV has been suggested to be relevant to viral host-specific adaptation. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3'UTR are largely unknown. Here, using Tembusu virus (TMUV), an avian MBFV as models, we observed that the duck-derived SLI of the 3'UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3'UTR in viral host switching and pathogenicity changes in mice.

Assembly-defective Tembusu virus ectopically expressing capsid protein is an approach for live-attenuated flavivirus vaccine development.

Live-attenuated vaccines (LAVs) represent a promising approach for flavivirus vaccine development. In the present study, we demonstrated a method for generating flavivirus LAVs based on breaking spatially and temporally regulated C-prM cleavage to disturb the viral assembly process, using an avian flavivirus (Tembusu virus) as the model. Using reverse genetics technology, we successfully generated two recombinant viruses (CQW1-IRES-mC and CQW1-MINI-mC) with bicistronic genomic RNA in which native capsid genes were deleted and instead expressed in the 3'UTR under the control of an internal ribosome entry site (IRES) or minimum IRES. Both viruses showed a significantly attenuated phenotype in vitro due to impaired viral assembly, and the engineered mutations were genetically stable in vitro within ten passages. Importantly, their virulence was also highly attenuated in ducklings and suckling mice and did not cause any overt clinical symptoms or mortality. In addition, a single dose of immunization with any of these mutant viruses could completely protect ducklings from a lethal challenge, and no viremia was detected after immunization and challenge, even though the viruses induced a relatively moderate immune response in terms of the T-lymphocytes proliferative response and the level of neutralization antibodies compared with that obtained with the wild-type virus. Besides, a recombinant virus ectopically expressing the prM-E protein was also generated in the present study, but this virus was too attenuated with severely decreased proliferation. Our results indicated that the use of a recombinant flavivirus that ectopically expresses structural proteins could be an effective and universal method for flavivirus LAVs development.

Replication/Assembly Defective Avian Flavivirus With Internal Deletions in the Capsid Can Be Used as an Approach for Living Attenuated Vaccine.

Avian Tembusu virus (TMUV) is a novel flavivirus causing severe egg drop and fatal encephalitis in avian in Asia. In the present study, we screened the structural and functional requirements of TMUV capsid protein (CP) for viral morphogenesis using reverse genetics methods in combination with replicon packaging assays. TMUV-CP showed dramatic functional and structural flexibility, and even though 44 residues were removed from the N-terminus, it was still capable of packaging replicon RNA; in addition, 33 residues were deleted from the C-terminus (containing nearly the entire α4-helix), and infectious particles were still produced, although α4-α4' is supposedly vital for CP dimerization and nucleocapsid formation. We further analyzed two mutants (ΔC20-43 and ΔC64-96 viruses) with relatively large deletions that still replicated well in BHK-21 cells. Our data indicate that internal deletions within CP impaired viral replication or assembly, resulting in attenuated virus proliferation in cells and attenuated virulence in duck embryos, and these deletion mutations are quite stable in cell culture. An in vivo assay indicated that both ΔC20-43 virus and ΔC64-96 virus were highly attenuated in ducklings but still immunogenic. Single-dose immunization with ΔC20-43 virus or ΔC64-96 virus could protect ducklings from a lethal challenge with good antigen clearance. Together, our data shed light on replication/assembly defective TMUV with internal deletions in CP and provide an effective approach to attenuate viral virulence in live vaccines without changing the antigen composition.

N130, N175 and N207 are N-linked glycosylation sites of duck Tembusu virus NS1 that are important for viral multiplication, viremia and virulence in ducklings.

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that has caused acute egg-drop syndrome in egg-laying ducks. DTMUV nonstructural protein 1 (NS1) contains three potential predicted N-linked glycosylation sites at residues 130, 175 and 207. In this study, we found that mutations at these sites affect the molecular weight of recombinant NS1, as assessed by western blot assays; however, the mutations do not affect their subcellular localization in the cytoplasm, as assessed by colocalization assays. Four recombinant viruses substituting the asparagine (N) residues at N130, N175, N207 or N130/N175/N207 of NS1 with alanine (A) residues were generated using rDTMUV-i, an infectious cDNA clone of the DTMUV CQW1 strain. Deglycosylation assays of the mutant virus NS1 were performed using endoglycosidases Endo H or PNGase F treatment in both mammalian and avian cells. The NS1-WT, NS1-N130A, NS1-N175A and NS1-N207A showed a shift in migration to 37 kDa after digestion with both endoglycosidases, which further confirmed that N130, N175 and N207 were the glycosylation sites of DTMUV NS1. Compared to the parental rDTMUV, the single mutants impaired viral multiplication in vitro, while the nonglycosylated virus rDTMUV-NS1-N130A/N175A/N207A showed a 5-fold to 178-fold decrease in viral titers and smaller plaque sizes. Notably, all mutant viruses were still highly virulent to duck embryos, but the embryos inoculated with rDTMUV-NS1-N130A/N175A/N207A started to die on the fourth day, which exhibited a prolonged time to death compared to that of rDTMUV. Moreover, rDTMUV-NS1-N130A/N175A/N207A was attenuated in vivo, showing no mortality and producing significantly lower viral titers in heart, spleen, kidney, brain and thymus as well as 2-fold to 3-fold lower viremia at 3 and 5 days post infection. Overall, our results indicated that N130, N175 and N207 are N-linked glycosylation sites of DTMUV NS1, which play crucial roles in viral multiplication, viremia and virulence in vitro and in vivo.

Efficacy of a chlorocresol-based disinfectant product on Toxocara canis eggs.

Toxocara canis is a common parasite of dogs and can cause zoonotic toxocariasis in humans. As a part of control programs for this agent, optimized hygiene including chemical disinfection is considered essential in the prevention and control of zoonotic toxocariasis in humans. However, commonly used disinfectants at present mostly fail to inhibit the embryogenesis and viability of T. canis eggs. To this effect, the present study was designed to evaluate the effect of a chlorocresol-based disinfectant product Neopredisan®135-1 (NP) on embryonic development of T. canis eggs in vitro and to investigate the infectivity of exposed eggs by assessing larval establishment in a mouse model. Under in vitro conditions, NP at a final concentration of 0.25, 0.50, 1, 2, or 4% all exhibited significant killing effect on T. canis embryogenesis compared with the control eggs (P < 0.05), regardless of contact times (30, 60, 90, or 120 min). Such killing activity increased in a concentration- and time-dependent manner, with a maximum killing efficacy of 95.81% at 4% concentration and 120 min exposure time. Comparisons between low and high concentrations and between short and long contact times concluded that a protocol using the 1% concentration of NP with a 90-min contact could be the most suitable for practical application. Additionally, the lower larval recovery in mice inoculated with eggs treated by either 0.25 or 0.5% NP than that from their corresponding controls (P < 0.05) verified once again that NP had an adverse impact on the larval development of T. canis eggs even at a low concentration. To the best of our knowledge, this is the first study to report the effect of the chlorocresol-based disinfectant NP on the embryonation and larval development of T. canis eggs, and the results presented here would contribute to environmental clearance and control of toxocariasis by providing an alternative disinfectant resource. However, it is highlighted that the clearance of the novel and existing sources of infection including larvated eggs in places treated with NP is not guaranteed and therefore continuous monitoring and additional disinfection are still required.

Characterization of the complete mitochondrial genome of Spirometra decipiens (Cestoda: Diphyllobothriidae) from China.

The plerocercoid larvae (spargana) of Spirometra decipiens (Cestoda: Diphyllobothriidae) can parasitize humans, causing the zoonotic sparganosis. In this study, the complete mitochondrial genome of this tapeworm was determined using an Illumina sequencing platform. The entire genome was 13,642 bp in length and contained 12 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and two non-coding regions. The phylogeny indicated that S. decipiens was closely related to Spirometra erinaceieuropaei and supported the monophyletic relationships between Spirometra, Diphyllobothrium, and Diplogonoporus within the Dipyllobothriidae. These results should contribute to a better understanding of the phylogenetic position of this species.