Chengqiang He

Vaccination influences the evolution of classical swine fever virus

Classical swine fever is a serious, economically damaging disease caused by classical swine fever virus (CSFV). The CSFV is composed of two clades, according to phylogenetic estimates. Attenuated live vaccine such as HCLV, has been widely used to protect pigs from CSFV, but the influence of vaccination on the evolution of CSFV has not been studied. We conducted a systemic analysis of the impact of vaccination on the evolution of CSFV by comparing vaccine-related and non-vaccine-related CSFV groups. We found that vaccination may affect strain diversity and immune escape through recombination and point mutation. We also found that vaccination may influence the population dynamics, evolutionary rate and adaptive evolution of classical swine fever virus. Our evidence suggests that the vaccination might also change host adaptation through influencing codon usage of the virus in swine. These findings suggest that it is necessary to avoid excessive use of CSFV attenuated vaccines.

Identification of Short Hairpin RNA Targeting Foot-And-Mouth Disease Virus with Transgenic Bovine Fetal Epithelium Cells

Background Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. Principal Finding Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. Conclusion RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

Studying classical swine fever virus: making the best of a bad virus.

Classical swine fever (CSF) is a highly contagious and often fatal disease that affects domestic pigs and wild boars. Outbreak of CSF can cause heavy economic losses to the pig industry. The strategies to prevent, control and eradicate CSF disease are based on containing the disease through a systematic prophylactic vaccination policy and a non-vaccination stamping-out policy. The quest for prevention, control and eradication of CSF has moved research forward in academia and industry, and has produced noticeable advances in understanding fundamental aspects of the virus replication mechanisms, virulence, and led to the development of new vaccines. In this review we summarize recent progress in CSFV epidemiology, molecular features of the genome and proteome, the molecular basis of virulence, and the development of anti-virus technologies.

Identification of three H1N1 influenza virus groups with natural recombinant genes circulating from 1918 to 2009.

In this study, we identify a recombinant pb1 gene, a recombinant MP segment and a recombinant PA segment. The pb1 gene is recombined from two Eurasia swine H1N1 influenza virus lineages. It belongs to a H1N1 swine clade circulating in Europe and Asia from 1999 to 2009. The mosaic MP segment descends from H7 avian and H1N1 human virus lineages and pertains to a large human H1N1 virus family circulating in Asia, Europe and America from 1918 to 2007. The recombinant PA segment originated from two swine H1N1 lineages is found in a swine H1N1 group prevailing in Asia and Europe from 1999 to 2003. These results collectively falsify the hypothesis that influenza virus do not evolve by homologous recombination. Since recombination not only leads to virus genome diversity but also can alter its host adaptation and pathogenecity; the genetic mechanism should not be neglected in influenza virus surveillance.

Identification of a natural human serotype 3 parainfluenza virus.

Parainfluenza virus is an important pathogen threatening the health of animals and human, which brings human many kinds of disease, especially lower respiratory tract infection involving infants and young children. In order to control the virus, it is necessary to fully understand the molecular basis resulting in the genetic diversity of the virus. Homologous recombination is one of mechanisms for the rapid change of genetic diversity. However, as a negative-strand virus, it is unknown whether the recombination can naturally take place in human PIV. In this study, we isolated and identified a mosaic serotype 3 human PIV (HPIV3) from in China, and also provided several putative PIV mosaics from previous reports to reveal that the recombination can naturally occur in the virus. In addition, two swine PIV3 isolates transferred from cattle to pigs were found to have mosaic genomes. These results suggest that homologous recombination can promote the genetic diversity and potentially bring some novel biologic characteristics of HPIV.

New genetic mechanism, origin and population dynamic of bovine ephemeral fever virus

Bovine ephemeral fever virus (BEFV) is a typical species of the genusEphemerovirus in the family Rhabdoviridae. Today, prevailing BEFV can be divided into three phylogeographic lineages, East Asia, Mideast, and Australia. In this study, we provide evidence that the whole East Asia lineage originates from a homologous recombination (HR) between the Mideast and Australia lineages that probably occurred in the 1940s. To our knowledge, HR has not been proposed before as the genetic mechanism of BEFV. According to the HR event and Bayesian estimation, the three BEFV lineages might originate from Africa, and may have spread to Asia and Australia through the Mideast. In addition, the population of the virus may have augmented significantly in the 2000s, suggesting that the risk for outbreaks of BEFV may be high at present.

Isolation and Identification of a Novel Rabies Virus Lineage in China with Natural Recombinant Nucleoprotein Gene

Rabies virus (RABV) causes severe neurological disease and death. As an important mechanism for generating genetic diversity in viruses, homologous recombination can lead to the emergence of novel virus strains with increased virulence and changed host tropism. However, it is still unclear whether recombination plays a role in the evolution of RABV. In this study, we isolated and sequenced four circulating RABV strains in China. Phylogenetic analyses identified a novel lineage of hybrid origin that comprises two different strains, J and CQ92. Analyses revealed that the virus 3′ untranslated region (UTR) and part of the N gene (approximate 500 nt in length) were likely derived from Chinese lineage I while the other part of the genomic sequence was homologous to Chinese lineage II. Our findings reveal that homologous recombination can occur naturally in the field and shape the genetic structure of RABV populations.

Rapid detection of infectious bovine Rhinotracheitis virus using recombinase polymerase amplification assays

BackgroundInfectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV.MethodsDistinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens.ResultsRPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%.ConclusionIBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field.

A permanent host shift of rabies virus from Chiroptera to Carnivora associated with recombination

Bat virus host shifts can result in the spread of diseases with significant effects. The rabies virus (RABV) is able to infect almost all mammals and is therefore a useful model for the study of host shift mechanisms. Carnivore RABVs originated from two historical host shifts from bat viruses. To reveal the genetic pathways by which bat RABVs changed their host tropism from bats to carnivores, we investigated the second permanent bat-to-carnivore shift resulting in two carnivore variants, known as raccoon RABV (RRV) and south-central skunk RABV (SCSKV). We found that their glycoprotein (G) genes are the result of recombination between an American bat virus and a carnivore virus. This recombination allowed the bat RABV to acquire the head of the G-protein ectodomain of the carnivore virus. This region is involved in receptor recognition and binding, response to changes in the pH microenvironment, trimerization of G proteins, and cell-to-cell transmission during the viral infection. Therefore, this recombination event may have significantly improved the variant’s adaptability to carnivores, altering its host tropism and thus leading to large-scale epidemics in striped skunk and raccoon.

Biopanning of polypeptides binding to bovine ephemeral fever virus G 1 protein from phage display peptide library

BackgroundThe bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G1 protein with high-affinity and inhibit BEFV replication.MethodsThe purified BEFV G1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G1 was assayed by ELISA. Then the roles of specific G1-binding peptides in the context of BEFV infection were analyzed.ResultsThe results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner.ConclusionTwo antiviral peptide ligands binding to bovine ephemeral fever virus G1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.