[Show abstract][Hide abstract] ABSTRACT: While H2N2 viruses have been sporadically isolated from wild and domestic birds, H2N2 viruses have not been detected among human populations since 1968. Should H2N2 viruses adapt to domestic poultry they may pose a risk of infection to people, as most anyone born after 1968 would likely be susceptible to their infection. We report the isolation of a novel influenza A virus (H2N2) cultured in 2013 from a healthy domestic duck at a live poultry market in Wuxi City, China. Sequence data revealed that the novel H2N2 virus was similar to Eurasian avian lineage avian influenza viruses, the virus had been circulating for ≥ two years among poultry, had an increase in α2,6 binding affinity, and was not highly pathogenic. Approximately 9% of 100 healthy chickens sampled from the same area had elevated antibodies against the H2 antigen. Fortunately, there was sparse serological evidence that the virus was infecting poultry workers or had adapted to infect other mammals. These findings suggest that a novel H2N2 virus has been circulating among domestic poultry in Wuxi City, China and has some has increased human receptor affinity. It seems wise to conduct better surveillance for novel influenza viruses at Chinese live bird markets.
[Show abstract][Hide abstract] ABSTRACT: Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6 mg of a recombinant peptide (RVG29-9R-6His) from 0.4 g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.
Protein Expression and Purification 12/2014; 104. DOI:10.1016/j.pep.2014.09.001 · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rabies is a viral infection of the CNS that is almost always fatal once symptoms occur. No effective treatment of the disease is available and novel antiviral strategies are urgently required. Street rabies viruses are field isolates known to be highly neurotropic. Aptamers are single-stranded oligonucleotides that bind their targets with high affinity and specificity and thus have potential for use in diagnostic and therapeutic applications. In this study, we demonstrate that the aptamers FO24 and FO21, which target RABV-infected cells, can significantly protect mice from a lethal dose of the street rabies virus FJ strain in vivo. Groups receiving preexposure prophylaxis had higher survival rates than the groups receiving postexposure prophylaxis. When mice were inoculated with aptamers (4nmol) for 24h by intracranial or intramuscular injection prior to intramuscular inoculation with the FJ strain, approximately 60% of the mice survived. These results indicate that the FO21 and FO24 aptamers may be used to develop preventative antiviral therapy against rabies disease.
International immunopharmacology 04/2014; 21(2). DOI:10.1016/j.intimp.2014.03.020 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rabies is a fatal central nervous system (CNS) disease caused by the neurotropic rabies virus (RABV). The therapeutic management of RABV infections is still problematic, and novel antiviral strategies are urgently required. We established the RVG-BHK-21 cell line, which expresses RABV glycoprotein on the cell surface, to select aptamers. Through 28 iterative rounds of selection, single-stranded DNA (ssDNA) aptamers were generated by exponential enrichment (SELEX). A virus titer assay and a real-time quantitative reverse transcription PCR (qRT-PCR) assay revealed that four aptamers could inhibit the replication of RABV in cultured baby hamster kidney (BHK)-21 cells. However, the aptamers did not inhibit the replication of other virus, e.g., canine distemper virus (CDV) and canine parvovirus (CPV). In addition, the GE54 aptamer was found to effectively protect mice against lethal RABV challenge. After inoculation with aptamers for 24h or 48h, followed by inoculation with CVS-11, approximately 25% to 33% of the mice survived. In summary, we selected aptamers that could signiﬁcantly protect from a lethal dose of RABV in vitro and in vivo.
Virus Research 01/2014; 184. DOI:10.1016/j.virusres.2014.01.021 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.
PLoS ONE 01/2014; 9(1):e79575. DOI:10.1371/journal.pone.0079575 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as antiviral agent against rabies, a recombinant AAV2 vectors expressing siRNA targeting nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was 37.8 ± 3.4 to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. Whatever NA cells was treated with rAAV-N796 before or after challenged with RABV, significant reduction was observed in both administrations. When mice treated intracerebrally with rAAV-N796 were challenged intracerebrally or intramuscally with lethal RABV, there was 50 ± 5.3 and 62.5 ± 4.7% protection, respectively. When mice treated intramuscally with rAAV-N796 were challenged intramuscally with lethal RABV, there was 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscally with rAAV-N796 after exposed to RABV infection 24 hr later, there was 25 ± 4.1% protection The N gene mRNA level in challenged mice brain from three different administrations was reduced with 55, 68, 32 and 25%, respectively. These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication; in vivo inhibited RABV multiplication in mice brain and imparted partial protection against lethal rabies. So it may have a potential to be used as an alternative antiviral approach against rabies.
Journal of Veterinary Medical Science 06/2013; 75(10). DOI:10.1292/jvms.13-0127 · 0.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aptamers, functional nucleic acids, capable of binding a variety of molecular targets with high affinity and specificity, have emerged as promising therapeutic agents. In this study, the cell surface-systematic evolution of ligands by exponential enrichment (Cell-SELEX) strategy was used to generate DNA aptamers which targeted to the intact rabies virus-infected live cells. Through 35 iterative rounds of selection, five high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Virus titer assay and real-time quantitative reverse transcription PCR (qRT-PCR) assay revealed that all five aptamers could inhibit replication of rabies virus (RABV) in cultured baby hamster kidney (BHK)-21 cells; and T14 and F34 aptamers were most effective. The qRT-PCR also showed a dose-dependent inhibitory effect in BHK-21 cells. Collectively, these data show the feasibility of generating functionally effective aptamers against rabies virus-infected cells by the Cell-SELEX iterative procedure. These aptamers may prove clinically useful as therapeutic molecules with specific antiviral potential against RABV infections.
International immunopharmacology 07/2012; 14(3):341-7. DOI:10.1016/j.intimp.2012.06.019 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The H5N1 avian influenza virus (AIV) causes widespread infections in bird and human respiratory tracts, and vaccines and drug therapy are limited in their effectiveness. Recent studies of AIV structures have been published and provide new targets for designing antiviral drugs such as antisense oligonucleotides (AS ODNs), which effectively inhibit gene replication. In this study, we designed and synthesized three AS ODNs (NP267, NP628, NP749) that were specific for the RNA binding region of nucleoprotein (NP) based on AIV structure. Results showed that all three AS ODNs could inhibit viral replication in MDCK cells. The NP628 showed the best antiviral effect of all through viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. In addition, the liposome mediated NP628 could partially protect the mice from a lethal H5N1 influenza virus challenge. Moreover, the NP628 group had a lower viral titer and lung index in the infected mice when compared with the viral control. Our results showed that AS ODN targeting of the AIV NP gene could potently inhibit AIV H5N1 reproduction, thus, formulating a candidate for an emergent therapeutic drug for the pathogenic H5N1 influenza virus infection.
International immunopharmacology 09/2011; 11(12):2057-61. DOI:10.1016/j.intimp.2011.08.019 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: H5N1 avian influenza virus (AIV) causes widespread infections in poultry and wild birds, and has the potential to emerge as a pandemic threat to human. Antisense oligonucleotides (AS ODNs) are highly effective at inhibiting gene replication. Antibody-mediated delivery is a novel approach to target specific cells and tissues. In this study, we designed and synthesized three AS ODNs (PA4, PA492 and PA1203) specific for conserved region of AIV PA protein, and all the three AS ODNs could inhibit viral replication. The PA492 ODN showed the best antiviral effect by viral titers and quantitative RT-PCR in MDCK cells. The fusion protein scFv-tP was constructed as a single chain variable fragment (scFv) against AIV hemaglutinin antigen with a truncated protamine (tP). The results showed that scFv-tP fusion improved the antiviral effectiveness of PA492 in MDCK cells as measured by viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. In addition, scFv-tP-delivered PA492 was also found to partially protect mice from lethal H5N1 influenza virus challenge. Using scFv-tP delivery, fluorescein isothiocyanate labeled-PA492 was found to be significantly localized in the lungs, compared to liposome-delivered PA492. Moreover, the fusion protein mediated PA492 had a lower lung index and viral titers in the infected mice as compared with the liposome method. These results provided a potential method for using anti-HA fusion protein for the targeted delivery of AS ODNs against AIV H5N1.
[Show abstract][Hide abstract] ABSTRACT: The hemagglutinin antigen (HA) of avian influenza virus (AIV) is an immunogen abundant on the surfaces of infected cells, and can be used as a target for specific antibodies to clear viral infection. Protamine has been demonstrated to deliver DNA into cells effectively. Accordingly, a fusion protein of anti-HA single-chain fragment variable (scFv) and truncated protamine (tP) may be used as a vehicle for delivering the anti-AIV siRNA into the AIV-infected cells for gene therapy. To test this hypothesis, we constructed a novel recombinant plasmid, pET28-scFv-tP, by connecting the genes for anti-H5N1 AIV HA-specific scFv with synthesized oligonucleotides encoding the 22 amino acids of human tP and a linker. Furthermore, the recombinant scFV-tP was expressed and purified, with a yield of 7-8mg of scFv-tP and a purity of >92% from 1L of bacterial culture. Characterization of its bioactivity revealed that scFv-tP recognized HA, similar to its scFv control, in a dose-dependent manner and that the scFv-tP, but not its scFv control, bound to DNA and delivered plasmid and oligonucleotide DNA into the AIV-infected MDCK cells effectively. More importantly, transfection with the mixture of the scFv-tP and plasmid for the NP-specific siRNA significantly inhibited the replication of AIV in MDCK cells, as compared with that transfection with the scFv-plasmid mixture, even with the plasmid in liposome. Our data demonstrated that the recombinant scFv-tP retained the functions of both scFv and tP, and might be potentially used for delivering genetic materials for targeting therapy of AIV infection in vivo.
[Show abstract][Hide abstract] ABSTRACT: Two giant pandas (Ailuropoda melanoleuca) died of unknown causes in a Chinese zoo. The clinical disease profile suggested that the pandas may have suffered a viral infection. Therefore, a series of detection including virus isolation, electron microscopy, cytobiological assay, serum neutralization and RT-PCR were used to identify the virus. It was determined that the isolated virus was a canine coronavirus (CCV), on the basis of coronavirus, neutralization by canine anti-CCV serum, and 84.3% to 100% amino acid sequence similarity with CCV. The results suggest that the affected pandas had been infected with CCV.
[Show abstract][Hide abstract] ABSTRACT: C57BL/6 mice were inoculated intranasally (50 microl) with serial 10-fold dilution of HAB/01 H5N1 virus. Three and five days later, three mice of each group were euthanized. Lung injury was assessed by observation of lung histopathology, virus titers and MCD50 were also measured. Our data showed that H5N1 viral infection in mice resulted in mainly epithelial injury and interstitial pneumonia, featuring significant weight loss, dramatically increased lung wet weight:body weight ratio, inflammatory cellular infiltration, alveolar and interstitial edema, hemorrhage in lungs with high virus titers, and MCD50 was 10(-6.5)/ 0.05 mL. These results suggested that a mouse model of H5N1 viral infection was successfully established which may benefit study of H5N1 avian influenza virus and pathogenic mechanism of host.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 12/2008; 24(6):472-7.
[Show abstract][Hide abstract] ABSTRACT: To evaluate emergency prophylactic effects of the avian influenza virus immunized serum on experimentally infected mice.
Serum HI antibody titers of 30 mice were detected at day 1 to 19 after being inoculated with 0.2 ml immune serum to estimate half life of immune serum. Ten mice clinical symptom was recorded to estimate the serum security after mice injected 1.5 ml immune serum. Seventy mice were randomly divided into 7 groups according to random number table and inoculated with 0.2 ml, 0.1 ml and 0.05 ml immune serum respectively via intraperitoneal injection on day 8, 4 and 1 prior to challenged with 10 LD(50) influenza virus intranasal. Mice were observed continually for 14 days to calculate the morbidity, mortality, average survival days and compare the lung index and viral titers in lung.
Serum HI antibody titers of mice which inoculated with 0.2 ml immune serum maintained 2(6) in 15 days after injection, but drawdown after day 17, the mice injected 1.5 ml immune serum were all alive and none onset. The survival rate of mice which injected 0.2 ml serum on the day 8, 4, 1 before challenge was 80%, 100% and 100%, and the average survival period was 13.1 days, 14.0 days and 14.0 days respectively. The survival rate of mice which injected 0.1 ml and 0.05 ml serum on day 1 before challenge was 100% and 50%, and the average survival days were 14.0 days and 11.7 days respectively. The mice lung index of experimental groups (0.0096 +/- 0.0033 - 0.0145 +/- 0.0060) was smaller than that of viral control group (0.0199 +/- 0.0025), with a statistical significance (P value 0.0022 - 0.0470, < 0.05). The viral titers in lung were significantly decreased by 2 titer as compared to the viral controls.
The avian influenza virus immunized serum might contain the emergency prophylactic effects and could be developed as an agent for possible human-avian influenza pandemic.
Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine] 12/2008; 42(11):814-7.
[Show abstract][Hide abstract] ABSTRACT: In this study, the HPAIV A/Tiger/Harbin/01/2002 (H5N1) used was originated from tigers and propagated in SPF embryonated hen eggs. TCID5, of the virus was 10(-7.36)/0. 05mL on MDCK cell. The cats were inoculated through bronchus route and then, the cats of dead and control were collected for histopathological and immunohistochemistry examination. Meanwhile, the emulsion supernatant fluid of organs and the pharyngeal swab samples of the dead cats were collected for RT-PCR, survived cats and the control cats were tested for the presence of HI antibody by standard method. The results indicated that the damage of lungs from the dead cats were most obvious, the wide range of red consolidation focus emerged on the lobus pulmonis, the fused focus of infection caused injury of lungs. Histology under the microscope revealed diffuse alveolar damage, confluence phlegmasia pathology, infiltration of lymphomonocytes, sackful of infiltration of macrophages and manipulus protein-like effusion in the alveolar. By immunohistochemistry, the positively stained virus particles were found on the epithelial cells of bronchus and alveolus, and also in the endochylema of lymphomonocytes. The specific electophoretic band of 464bp amplified by RT-PCR from samples of pharyngeal swabs, lungs, kidneys, hearts and brains was as same as the theory value. HI antibody titers of the survived cat were 1:32.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 12/2007; 23(6):477-80.
[Show abstract][Hide abstract] ABSTRACT: H5N1 highly pathogenic avian influenza virus was highly pathogenic and sometimes even fatal for tigers and cats. To develop a new type of vaccine for Felidae influenza prevention, recombinant replication-competent canine adenovirus Type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus was constructed. A/tiger/Harbin/01/2003 (HSN1) HA gene was cloned into PVAX1. The HA expression cassette which included CMV and HA and PolyA was ligated into the E3 deletion region of pVAXdeltaE. The recombinant plasmid was named pdeltaEHA. The pdelta EHA and the pPoly2-CAV2 were digested with Nru I /Sal I, respectively. The purified Nru I/Sal I DNA fragment containing the HA expression cassette was cloned into pPoly2-CAV2 to generate the recombinant plasmid pCAV-2/HA. The recombinant genome was released from pCAV-2/HA, and was transfected into MDCK cells by Lipofectamine. The recombinant virus named CAV2/HA was gained. Anti-H5N1 influenza virus HI antibody (1:8 - 1:16) was detected in the cat immunized with CAV-2/HA.