Xiangmin Li

Genome comparison of a novel classical swine fever virus isolated in China in 2004 with other CSFV strains

The genome of a novel classical swine fever virus (CSFV), SWH/CA/2004, isolated from a hog pen in Henan Province, central China, is 12296 nucleotides (nt) in length. It is composed of a 373-nt 5′ terminal non-translated region (NTR), a 11697-nt open reading frame (ORF) encoding a polyprotein of 3898 amino acids (aa), and a 226-nt 3′-NTR. Genome comparison of the SWH/CA/2004 isolate (GenBank Accession: DQ127910) with other known CSFV isolates was performed and analyzed. Corresponding segments from SWH/CA/2004 and other reported strains shared 80.4–99.8% identity at the nucleotide level and 89.5–99.8% identity at the amino acid level. From an evolutionary point of view, isolate SWH/CA/2004 is closely related to the highly virulent isolate cF114/CA/2001, with a pairwise distance of 0.013; and distantly related to the moderately virulent isolate GXWZ02/CA/2003, with pairwise distance 0.170. The phylogenetic trees of the full-length genome and the following region Erns, E1, E2, and NS5B-based neighbor-joining (NJ) method were constructed and approximately divided into different genetic groups according to avirulence, moderate virulence and high virulence, while other region-based NJ trees demonstrated sequence conservation between these groups. The four genomic regions may constitute important criteria for genetic typing of diverse CSFV isolates. Based on these analyses, isolate SWH/CA/2004 was deduced to belong to the highly virulent isolate group. However, SWH/CA/2004 also contains a 14-U deletion in the 3′-NTR that is characteristic of avirulent isolates. These analyses constitute a comprehensive study of the phylogenetics of CSF based on distinct regions of the genome and may provide the basis for future molecular epidemiology research to identify virulent strain outbreaks and trigger implementation of appropriate control measures.

A recombinant pseudorabies virus co-expressing capsid proteins precursor P1-2A of FMDV and VP2 protein of porcine parvovirus: a trivalent vaccine candidate

Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK−/gE−/LacZ+ mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK−/gE−/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.

Avian Influenza (H5N1) Virus in Waterfowl and Chickens, Central China

In 2004, 3 and 4 strains of avian influenza virus (subtype H5N1) were isolated from waterfowl and chickens, respectively, in central People’s Republic of China. Viral replication and pathogenicity were evaluated in chickens, quails, pigeons, and mice. We analyzed the sequences of the hemagglutinin and neuraminidase genes of the isolates and found broad diversity among them.

Apigenin restricts FMDV infection and inhibits viral IRES driven translational activity.

Foot-and-mouth disease (FMD) is a highly contagious disease of domestic and wild ruminants that is caused by FMD virus (FMDV). FMD outbreaks have occurred in livestock-containing regions worldwide. Apigenin, which is a flavonoid naturally existing in plant, possesses various pharmacological effects, including anti-inflammatory, anticancer, antioxidant and antiviral activities. Results show that apigenin can inhibit FMDV-mediated cytopathogenic effect and FMDV replication in vitro. Further studies demonstrate the following: (i) apigenin inhibits FMDV infection at the viral post-entry stage; (ii) apigenin does not exhibit direct extracellular virucidal activity; and (iii) apigenin interferes with the translational activity of FMDV driven by internal ribosome entry site. Studies on applying apigein in vivo are required for drug development and further identification of potential drug targets against FDMV infection.

TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A

The members of tripartite-motif containing (TRIM) protein participate in various cellular processes and play an important role in host antiviral function. TRIM proteins exert their antiviral activity either directly by degrading viral proteins through their E3 ligase activity, or indirectly by promoting host innate immunity. This study demonstrated for the first time that TRIM52 is a novel antiviral TRIM protein against Japanese encephalitis virus (JEV) infection. Overexpression of TRIM52 restricted JEV replication in BHK-21 and 293T cells. In addition, JEV nonstructural protein 2A (NS2A) is a protein that interacts with TRIM52. Their interaction degraded NS2A in a proteasome-dependent manner via the E3 ligase activity of TRIM52. Thus, TRIM52 is a novel antiviral TRIM protein, and it exerted antiviral activity against JEV infection by targeting and degrading viral NS2A.

A novel subunit vaccine co-expressing GM-CSF and PCV2b Cap protein enhances protective immunity against porcine circovirus type 2 in piglets

Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease. Capsid (Cap) protein of PCV2 is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. GM-CSF is an immune adjuvant that enhances responses to vaccines. In this study, recombinant baculoviruses Ac-Cap and Ac-Cap-GM-CSF expressing the Cap protein alone and co-expressing the Cap protein and porcine GM-CSF, respectively, were constructed successfully. The target proteins were analyzed by western blotting and IFA. Further, these proteins were confirmed by electron microscopy, which showed that Cap proteins could self-assemble into virus-like particles having diameters of 17-25nm. Animal experiments showed that pigs immunized with Cap-GM-CSF subunit vaccine showed significantly higher levels of PCV2-specific antibodies and neutralizing antibodies than pigs immunized with the Cap subunit vaccine and a commercial vaccine (Ingelvac CircoFLEX; P<0.05). After PCV2 wild strain challenged, Pigs receiving the Cap-GM-CSF subunit vaccine showed significantly higher average daily weight gain after wild-type PCV2 challenge than pigs receiving the other three vaccines (P<0.05). None of PCV2 DNA was detected in all immunized animals, except control animals immunized with phosphate-buffered saline. These results indicated that GM-CSF was a powerful immunoadjuvant for PCV2 subunit vaccines because it enhanced humoral immune response and improved immune protection against PCV2 infection in pigs. Thus, the novel Cap-GM-CSF subunit vaccine has the potential to be used as an effective and safe vaccine candidate against PCV2 infection.

Swine interferon-induced transmembrane protein, sIFITM3, inhibits foot-and-mouth disease virus infection in vitro and in vivo.

Abstract The interferon-induced transmembrane protein 3 (IFITM3) is a widely expressed potent antiviral effector of the host innate immune system. It restricts a diverse group of pathogenic, enveloped viruses, by interfering with endosomal fusion. In this report, the swine IFITM3 (sIFITM3) gene was cloned. It shares the functionally conserved CD225 domain and multiple critical amino acid residues (Y19, F74, F77, R86 and Y98) with its human ortholog, which are essential for antiviral activity. Ectopic expression of sIFITM3 significantly inhibited non-enveloped foot-and-mouth disease virus (FMDV) infection in BHK-21 cells. Furthermore, sIFITM3 blocked FMDV infection at early steps in the virus life cycle by disrupting viral attachment to the host cell surface. Importantly, inoculation of 2-day-old suckling mice with a plasmid expressing sIFITM3 conferred protection against lethal challenge with FMDV. These results suggest that sIFITM3 is a promising antiviral agent and that can safeguard the host from infection with FMDV.

High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) in Pichia pastoris

High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115. Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5′-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZαA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23 kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0 g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene.

Glycoprotein E2 of classical swine fever virus expressed by baculovirus induces the protective immune responses in rabbits.

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.

Antiviral activity of luteolin against Japanese encephalitis virus.

Japanese encephalitis virus (JEV), a member of family Flaviviridae, is a neurotropic flavivirus that causes Japanese encephalitis (JE). JEV is one of the most important causative agents of viral encephalitis in humans, and this disease leads to high fatality rates. Although effective vaccines are available, no effective antiviral therapy for JE has been developed. Hence, identifying effective antiviral agents against JEV infection is important. In this study, we found that luteolin was an antiviral bioflavonoid with potent antiviral activity against JEV replication in A549 cells with IC50=4.56μg/mL. Luteolin also showed extracellular virucidal activity on JEV. With a time-of-drug addition assay revealing that JEV replication was inhibited by luteolin after the entry stage. Overall, our results suggested that luteolin can be used to develop an antiviral drug against JEV.