Effect of Tachinid Parasitoid Exorista japonica on the Larval Development and Pupation of the Host Silkworm Bombyx mori.

The Tachinidae are natural enemies of many lepidopteran and coleopteran pests of crops, forests, and fruits. However, host-tachinid parasitoid interactions have been largely unexplored. In this study, we investigated the effects of tachinids on host biological traits, using Exorista japonica, a generalist parasitoid, and the silkworm Bombyx mori, its lepidopteran host, as models. We observed that E. japonica parasitoidism did not affect silkworm larval body weight gain and cocooning rate, whereas they caused shortened duration of molting from the final instar to the pupal stage, abnormal molting from larval to pupal stages, and a subsequent decrease in host emergence rate. Moreover, a decrease in juvenile hormone (JH) titer and an increase in 20-hydroxyecdysone (20E) titer in the hemolymph of parasitized silkworms occurred. The transcription of JH and 20E responsive genes was downregulated in mature parasitized hosts, but upregulated in parasitized prepupae while Fushi tarazu factor 1 (Ftz-f1), a nuclear receptor essential in larval ecdysis, showed dramatically reduced expression in parasitized hosts at both the mature and prepupal stages. Moreover, the transcriptional levels of BmFtz-f1 and its downstream target genes encoding cuticle proteins were downregulated in epidermis of parasitized hosts. Meanwhile, the content of trehalose was decreased in the hemolymph, while chitin content in the epidermis was increased in parasitized silkworm prepupae. These data reveal that the host may fine-tune JH and 20E synthesis to shorten developmental duration to combat established E. japonica infestation, while E. japonica silences BmFtz-f1 transcription to inhibit host pupation. This discovery highlights the novel target mechanism of tachinid parasitoids and provides new clues to host/tachinid parasitoid relationships.

H5N1 influenza A virus with K193E and G225E double mutations in haemagglutinin is attenuated and immunogenic in mice.

Live-attenuated influenza vaccines (LAIVs) are now available for the prevention of influenza, with LAIV strains generally derived from serial passage in cultures or by reverse genetics (RG). The receptor-binding domain (RBD) in haemagglutinin (HA) of influenza virus is responsible for viral binding to the avian-type 2,3-α-linked or human-type 2,6-α-linked sialic acid receptor; however, the virulence determinants in the RBD of H5N1 virus remain largely unknown. In the present study, serial passage of H5N1 virus A/Vietnam/1194/2004 in Madin-Darby canine kidney cells resulted in the generation of adapted variants with large-plaque morphology, and genomic sequencing of selected variants revealed two specific amino acid substitutions (K193E and G225E) in the RBD. RG was used to generate H5N1 viruses containing either single or double substitutions in HA. The RG virus containing K193E and G225E mutations (rVN-K193E/G225E) demonstrated large-plaque morphology, enhanced replication and genetic stability after serial passage, without changing the receptor-binding preference. Importantly, in vivo virulence assessment demonstrated that rVN-K193E/G225E was significantly attenuated in mice. Microneutralization and haemagglutination inhibition assays demonstrated that immunization with rVN-K193E/G225E efficiently induced a robust antibody response against WT H5N1 virus in mice. Taken together, our experiments demonstrated that K193E and G225E mutations synergistically attenuated H5N1 virus without enhancing the receptor-binding avidity, and that the RG virus rVN-K193E/G225E represents a potential H5N1 LAIV strategy that deserves further development. These findings identify the RBD as a novel attenuation target for live vaccine development and highlight the complexity of RBD interactions.

Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection of novel avian influenza A (H7N9) virus.

BACKGROUND: The emerged human infection with avian influenza A (H7N9) virus in China since 2013 has aroused global concerns. There is great demand for simple and rapid diagnostic method for early detection of H7N9 to provide timely treatment and disease control. The aim of the current study was to develop a rapid, accurate and feasible reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of H7N9 virus. RESULTS: The detection limits of the H7- and N9-specific RT-LAMP assay were both approximately 0.2 PFU per reaction. No cross-reactivity was observed with other subtype of influenza viruses or common respiratory viral pathogens. The assay worked well with clinical specimens from patients and chickens, and exhibited high specificity and sensitivity. CONCLUSIONS: The H7/N9 specific RT-LAMP assay was sensitive and accurate, which could be a useful alternative in clinical diagnostics of influenza A (H7N9) virus, especially in the hospitals and laboratories without sophisticated diagnostic systems.

The first complete genomic characterization of an Amur virus isolate from China.

Amur virus (AMRV) is a member of the genus Hantavirus in the family Bunyaviridae. In this study, we determined for the first time the complete genome sequence of the AMRV H8205 strain, which was isolated from a patient with hemorrhagic fever with renal syndrome (HFRS) in China. The complete nucleotide sequence of the S segment of AMRV H8205 is 1699 nt long, with a 5' noncoding region (5'NC) of 36 nt, followed by a coding sequence of 1290 nt and a 3'NC of 373 nt. The complete sequence of the M segment is 3615 nt long, with a 5'NC of 40 nt, followed by a coding sequence of 3408 nt and a 3'NC of 167 nt. The complete sequence of the L segment is 6536 nt long, with a 5'NC of 37 nt, followed by a coding sequence of 6453 nt and a 3'NC of 40 nt. The major open reading frame (ORF) of each of the three segments (S, nt 37-1326; M, nt 41-3445; L, nt 38-6490) has a coding capacity of 430 aa, 1135 aa, 2151 aa, respectively. Phylogenetic analysis of the nucleotide sequences using the NJ method indicated that H8205 virus, together with the Amur strains isolated from Far-Eastern Russia and Korea, forms a well-supported lineage. Our results will provide insights into the genetic diversity of hantaviruses (HNTV).

The confirmation of three repeated sequence elements in the 3' untranslated region of Chikungunya virus.

In this study, the complete genomic nucleotide sequence of Chikungunya virus (CHIKV) strain S27 African prototype was determined and three 21 nucleotides repeated sequence elements (RSEs) at positions 11398-11418, 11533-11553, and 11620-11640 in the 3' untranslated region (3'UTR) were confirmed. In addition, the 3'UTRs of all CHIKV strains deposited in GenBank were analyzed. The results displayed that the majority of the CHIKV strains consisted of the three 21 nucleotides RSEs in the 3'UTRs, and the third RSE was the most conservative. The conservation of the three RSEs of 21 nucleotides within the 3'UTR of CHIKV genome may play an important role on the virus replication cycle.

Complete genomic characterization of two tick-borne encephalitis viruses isolated from China.

Tick-borne encephalitis (TBEV) is prevalent over a wide area of the Eurasian continent. TBE viruses cause severe encephalitis in humans, with serious sequelae, and have a significant impact on public health in these endemic regions. To gain insight into genetic evolution of tick-borne encephalitis virus (TBEV) in China, the complete genomic sequences of two TBEV strains Senzhang and MDJ01, which were isolated in 1953 and 2001 respectively, were characterized. The complete genome sequences of two strains were all consist 10,784 nucleotides and there are 364 nucleotides deletion in the 3' nontranslated region. Compared with other TBEV strains, homology range from 85.2% (Zausaev) to 99.6% (MDJ02 and MDJ03) on the level of nucleotide. Phylogenetic trees based on the complete genome, open reading frame and E gene nucleotide sequences all showed that the strains Senzhang and MDJ01 belong to Far-Eastern subtype and cluster with other Chinese TBEV strains. All these implied that TBEVs prevalent in China were highly conservative, other measurement should be taken to improve protective efficacy of present vaccine.

A DNA-based West Nile virus replicon elicits humoral and cellular immune responses in mice.

While self-replicating, non-infectious subgenomic flavivirus replicons have been described, most of them are RNA transcripts under the control of an Sp6 or T7 promoter. In this study, using West Nile virus (WNV) as a model, a series of DNA-based reporter replicons under the control of a minimal cytomegalovirus (CMV) immediate-early promoter were constructed, and functional analysis showed that these reporter replicons replicate efficiently in mammalian cells. When the DNA-based WNV replicon was used to immunize mice, NS1-specific IgG antibodies and anti-WNV neutralizing antibodies were both induced. Additionally, immunization with this DNA-based WNV replicon induced high levels of lymphocyte proliferation and enhanced the secretion of IFN-γ. These results suggest that this type of DNA-based replicon can induce humoral and cellular immune responses in mice, indicating that this type of DNA-based replicon may serve as a useful platform for vaccine development and protein expression.

Complete genome sequence analysis of tick-borne encephalitis viruses isolated in northeastern China.

Tick-borne encephalitis virus (TBEV) causes lethal encephalitis in humans, posing a growing public-health problem in many European and Asian countries. TBEV is currently endemic in northeastern China, but the complete genome sequences of Chinese TBEV strains have not been reported. During a TBE outbreak in 2010 in Mudanjiang City, Heilongjiang Province, China, two TBEV strains were isolated from serum samples of two patients, and the complete sequences were determined and compared with other known TBEV strains. Both Mudanjiang isolates consisted of 10,774 nucleotides and encoded a single open reading frame coding for a polyprotein of 3414 amino acids, and a unique deletion of 364 nucleotides in the 3' untranslated region (UTR) was recorded. Phylogenetic analysis based on the amino acid sequence of the E protein and the nucleotide sequence of the 3'UTR revealed that the Mudanjiang isolates are closely related to the Senzhang and Sofjin-HO strains and belong to the Far Eastern subtype of TBEV. These findings provide insight into the evolutionary relationships among Chinese TBEV isolates and are useful for laboratory diagnosis and vaccine development for TBEV.

Cross protection against lethal West Nile virus challenge in mice immunized with recombinant E protein domain III of Japanese encephalitis virus.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that cause severe encephalitic diseases with global impact. Cross protection among JEV and WNV has been previously described, and most cross reactive epitopes were identified within the domain II of E protein (EDII). In this study, the E protein domain III (EDIII) of JEV was successfully expressed in Escherichia coli, purified by a Ni-NTA column and characterized by Western blotting assay. Competitive inhibition assay showed that this recombinant JEV EDIII blocks the entry of JEV into BHK-21 cells. Mice immunized with the recombinant JEV EDIII developed high IgG and neutralizing antibodies titers against JEV. Most importantly, antibodies induced by JEV EDIII could neutralize WNV in vitro and partially protected mice against lethal WNV challenge. These results demonstrate that immunization with JEV EDIII induces cross-protective immunity against WNV infection, indicating a possible role of EDIII for the cross-protection among flavivirus.

Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus.

Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu"-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu"-ts18(cd) were indistinguishable from the reported ts mutant Wu"-ts18. Then we cultured the ts mutant Wu"-ts18(cd) at non-permissive temperature 39.5 °C, which "forced" the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu"-ts18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein.

Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98)DRXW(101) motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.

Establishment of the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.

In order to establish the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression. The recombinant plasmid of pTRE-Tight-SARS-N was constructed by using the plasmid p8S as the PCR template which contains a cDNA clone covering the nucleocapsid gene of SARS-CoV HKU-39449. Restriction enzymes digestion and sequence analysis indicated the recombinant plasmid of pTRE-Tight-SARS-N contained the nucleocapsid gene with the optimized nucleotide sequence which will improve the translation efficiency. Positive cell clones were selected by cotransfecting pTRE-Tight-SARS-N with the linear marker pPUR to BHK-21 Tet-on cells in the presence of puromycin. A set of double-stable eukaryotic cell lines (BHK-Tet-SARS-N) with inducible control of the SARS-CoV neucleocapsid gene expression was identified by using SDS-PAGE and Western-blot analysis. The expression of SARS-CoV nucleocapsid protein was tightly regulated by the varying concentration of doxcycline in the constructed double-stable cell line. The constructed BHK-Tet-SARS-N cell strains will facilitate the rescue of SARS-CoV in vitro and the further reverse genetic research of SARS-CoV.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

Monoclonal antibody induced with inactived EV71-Hn2 virus protects mice against lethal EV71-Hn2 virus infection.

BACKGROUND: Enterovirus 71 (EV71) is a viral pathogen that belongs to the Picornaviridae family, EV71-infected children can develop severe neurological complications leading to rapid clinical deterioration and death. RESULTS: In this study, several monoclonal antibodies (MAbs) were produced by immunizing mice with the inactived EV71 Henan (Hn2) virus strain. The isolated MAbs were characterised by in vitro neutralizing analysis and peptide ELISA. ELISA assay showed that the neutralizing monoclonal antibody 4E8 specifically reacted with synthetic peptides which contain amino acid 240-250 and 250-260 of EV71 VP1. The in vivo protection assay showed that 4E8 can protect two-day-old BALB/c mice against the lethal challenge of EV71 virus. CONCLUSION: The MAb 4E8 could be a promising candidate to be humanized and used for treatment of EV71 infection.

Sequence analysis of six enterovirus 71 strains with different virulences in humans.

Enterovirus 71 (EV71) infection is the main cause of hand, foot and mouth disease (HFMD) and has been associated with severe neurological diseases resulting in high mortalities. In this study, six EV71 strains isolated from patients with different clinical symptoms were sequenced and analyzed in a mouse model of EV71 infection. In a phylogenetic tree, based on the complete VP1 gene sequence, all six strains grouped into the C4 genotype. The sequence analysis revealed that there are nucleotide changes clustered in the internal ribosome entry site (IRES) element of the 5'-nontranslated region (5'-NTR), as well as amino acid differences clustered in the non-structural proteins. Importantly, we identified a unique amino acid difference (Val(1994)-Ile(1994)) that distinguished the more virulent strains, Anhui1 (Ah1), Henan1 (Hn1) and Henan2 (Hn2) from the less virulent strains, Chongqing1 (Cq1), Chongqing2 (Cq2) and Chongqing3 (Cq3). This amino acid difference is located in the finger domain of the viral RNA-dependent RNA polymerase 3D (3D(pol)). Furthermore, two-day-old Balb/c mice were inoculated with the Ah1, Hn1, Hn2, Cq1, Cq2 and Cq3 isolates by the intracerebral or intraperitoneal routes. All of the mice inoculated with Ah1, Hn1 and Hn2 isolates developed hind-leg paralysis and subsequently died. Mice inoculated with the Cq1, Cq2 or Cq3 isolates survived throughout the 21-day observation period. These results show that clinical isolates of EV71 associated with disease of different severity in humans have characteristic sequence differences and cause different mortality rates when inoculated into mice. These data also provide a rational basis to investigate the molecular determinants of EV71 pathogenesis using a reverse genetic approach.

RNA elements within the 5' untranslated region of the West Nile virus genome are critical for RNA synthesis and virus replication.

RNA elements within the flavivirus genome may play essential regulatory roles during virus replication. Here, recombinant West Nile virus (WNV) NS5 protein was used in combination with WNV subgenomic RNA templates to establish in vitro RNA-dependent RNA polymerase and RNA-binding assays. These assays identified mutations in the stem-loop A (SLA) region of the 5' untranslated region (5'UTR) altering NS5 RNA synthesis and RNA-binding capability. These mutations were then introduced into the full-length WNV genome by reverse genetics. Further analysis of the mutant viruses showed that deletion of nt 46-60, which disrupted the stem and side loop of SLA, greatly compromised virus replication, whereas mutations that destroyed the top loop of SLA required for RNA synthesis in vitro did not significantly alter virus replication. These results suggest that SLA present in the 5'UTR of WNV is essential for RNA synthesis in vitro and for virus replication.

[Laboratory confirmation of the first influenza A (H1N1) imported case in Mainland China].

The clinical throat swab specimen of an imported suspected case of influenza A (H1N1) was detec ted with real-time PCR, RT-PCR and subsequently confirmed by gene sequencing. The presence of influ enza A (H1N1) virus confirmed the first case with A (H1N1) infection in Mainland China.

[Histologic and ultrastructural studies of the patient died of highly pathogenic H5N1 avian influenza virus infection in China].

OBJECTIVE: To explore histopathologic and ultrastructural characteristics of human avian influenza (AI) infection and related etiological pathogenesis. METHODS: Postmortem lung and heart samples were collected from the patient who died of avian influenza virus infection on November 29, 2003 in China. Light and electron microscopy, immunohistochemistry and histochemistry were used to investigate the pathological changes. RESULTS: The main pathological findings included extensive pulmonary consolidation, hemorrhage, pulmonary edema and local hemorrhagic infarct. The lamina of alveoli and bronchioles were abundantly filled with protein-rich fluid, erythrocytes, fibrin and cell debris admixed with many neutrophilis, macrophages, lymphocytes and a few of monokaryon and multinuclear giant cells. Hyaline membranes were formed. Local pulmonary tissues were heavily damaged by hemorrhage and necrosis. Alveolar septum was disintegrated. Mesenchymal edema with a few of macrophages infiltration of heart was found. Electron microscopy showed the avian influenza A virus-like particles (type C and type A) of 80 - 120 nm diameter and envelopes in the cytoplasm of pneumocytes and endothelial cells. CONCLUSIONS: Fatal pneumonia associated with highly pathogenic avian influenza A virus (H5N1) infection leads to extensive pulmonary consolidation, edema and marked hemorrhagic necrosis and inflammation. Electron microscopy can identify avian influenza A virus-like particles. The findings may offer an important theoretical basis for clinical diagnosis and treatment.

[Transfection with a novel cationic gene carrier: PEI-PBLG].

This study mainly deals with cell transfection and cytotoxicity for PEI(10kD)-PBLG, a novel cationic copolymer, to observe its potential as a gene carrier. Size measurement and SEM were used to show the modality of the PEI-PBLG/pDNA complexes. Cytotoxicity of PEI (10kD)-PBLG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and compared with PEI(25kD)-PBLG, PEI(10kD), and PEI(25kD). Furthermore, pEGFP that can express the enhanced green fluorescent protein was chosen as a reporter to observe the transfection efficiency directly. Then, PEI (10kD)-PBLG/pEGFP complexes were transfected into several cell lines, such as Hela, COS-7, Vero-E6, and ECV-304, and effects of the transfection conditions were evaluated. The efficiencies were measured by FACS. Size measurement of complex particles indicated that PEI-PBLG/pDNA tended to form smaller nanoparticles compared with PEI/pDNA. The representative size of the PEI(10kD)-PBLG/pDNA complex was approximately 100 - 200 nm. SEM images showed that the particles were condense and compact. This can be suitable for their entry into cells. Cytotoxicity studies suggested that PEI (10kD)-PBLG had considerably lower toxicity than the other three materials. In the transfection tests, PEI (10kD)-PBLG/pDNA complexes could be transfected into all the cell lines that were tested. These provided the highest level of EGFP expression (45.02%) in Hela cells, which was considerably higher than that of PEI(10kD)/pEGFP (29.16%). Being less affected by the serum during transfection, PEI-PBLG/pDNA complexes offered greater biocompatibility than PEI. PEI-PBLG copolymer reduces the cytotoxicity of PEI, improves the transfection efficiency, and offers greater biocompatibility than PEI. It shows considerable potential as an efficient nonviral carrier for gene delivery.