Chung Chau Hon

Molecular epidemiology of PRRSV: a phylogenetic perspective.

Since its first discovery two decades ago, porcine reproductive and respiratory syndrome virus (PRRSV) has been the subject of intensive research due to its huge impact on the worldwide swine industry. Thanks to the phylogenetic analyses, much has been learned concerning the genetic diversity and evolution history of the virus. In this review, we focused on the evolutionary and epidemiological aspects of PRRSV from a phylogenetic perspective. We first described the diversity and transmission dynamics of Type 1 and 2 PRRSV, respectively. Then, we focused on the more ancient evolutionary history of PRRSV: the time of onset of all existing PRRSV and an origin hypothesis were discussed. Finally, we summarized the results from previous recombination studies to assess the potential impact of recombination on the virus epidemiology.

Phylogeny-Based Evolutionary, Demographical, and Geographical Dissection of North American Type 2 Porcine Reproductive and Respiratory Syndrome Viruses

ABSTRACT Type 2 (or North American-like) porcine reproductive and respiratory syndrome virus (PRRSV) was first recorded in 1987 in the United States and now occurs in most commercial swine industries throughout the world. In this study, we investigated the epidemiological and evolutionary behaviors of type 2 PRRSV. Based on phylogenetic analyses of 8,624 ORF5 sequences, we described a comprehensive picture of the diversity of type 2 PRRSVs and systematically classified all available sequences into lineages and sublineages, including a number of previously undescribed lineages. With the rapid growth of sequence deposition into the databases, it would be technically difficult for veterinary researchers to genotype their sequences by reanalyzing all sequences in the databases. To this end, a set of reference sequences was established based on our classification system, which represents the principal diversity of all available sequences and can readily be used for further genotyping studies. In addition, we further investigated the demographic histories of these lineages and sublineages by using Bayesian coalescence analyses, providing evolutionary insights into several important epidemiological events of type 2 PRRSV. Moreover, by using a phylogeographic approach, we were able to estimate the transmission frequencies between the pig-producing states in the United States and identified several states as the major sources of viral spread, i.e., “transmission centers.” In summary, this study represents the most extensive phylogenetic analyses of type 2 PRRSV to date, providing a basis for future genotyping studies and dissecting the epidemiology of type 2 PRRSV from phylogenetic perspectives.

Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

n Abstractn n The immunological characteristics of SARS-CoV spike protein were investigated by administering mice with plasmids encoding various S gene fragments. We showed that the secreting forms of S1, S2 subunits and the N-terminus of S1 subunit (residues 18–495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S1 fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S1 and S2 subunits cooperatively neutralized SARS-CoV but neither the S1 nor S2 specific antibodies did, suggesting the possible role of both S1 and S2 subunits in host cell docking and entry. These results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against SARS-CoV.n n

Adenovirus-mediated expression of the C-terminal domain of SARS-CoV spike protein is sufficient to induce apoptosis in Vero E6 cells

The pro‐apoptotic properties of severe acute respiratory syndrome coronavirus (SARS‐CoV) structural proteins were studied in vitro. By monitoring apoptosis indicators including chromatin condensation, cellular DNA fragmentation and cell membrane asymmetry, we demonstrated that the adenovirus‐mediated over‐expression of SARS‐CoV spike (S) protein and its C‐terminal domain (S2) induce apoptosis in Vero E6 cells in a time‐ and dosage‐dependent manner, whereas the expression of its N‐terminal domain (S1) and other structural proteins, including envelope (E), membrane (M) and nucleocapsid (N) protein do not. These findings suggest a possible role of S and S2 protein in SARS‐CoV induced apoptosis and the molecular pathogenesis of SARS.

Phylogenetic perspectives on the epidemiology and origins of SARS and SARS-like coronaviruses.

n Abstractn n Severe Acute Respiratory Syndrome (SARS) is a respiratory disease caused by a zoonotic coronavirus (CoV) named SARS-CoV (SCoV), which rapidly swept the globe after its emergence in rural China during late 2002. The origins of SCoV have been mysterious and controversial, until the recent discovery of SARS-like CoV (SLCoV) in bats and the proposal of bats as the natural reservior of the Coronaviridae family. In this article, we focused on discussing how phylogenetics contributed to our understanding towards the emergence and transmission of SCoV. We first reviewed the epidemiology of SCoV from a phylogenetic perspective and discussed the controversies over its phylogenetic origins. Then, we summarized the phylogenetic findings in relation to its zoonotic origins and the proposed inter-species viral transmission events. Finally, we also discussed how the discoveries of SCoV and SLCoV expanded our knowledge on the evolution of the Coronaviridae family as well as its implications on the possible future re-emergence of SCoV.n n

Cell culture-adapted IBDV uses endocytosis for entry in DF-1 chicken embryonic fibroblasts

Although membrane perforation was suggested as the means of penetration mediated by IBDV, the cellular mechanism being hijacked to facilitate its entry is largely unknown. In this study, the entry pathway of cell culture adapted IBDV (caIBDV) was characterized in DF-1 chicken embryonic fibroblasts. We observed that the entry of caIBDV was inhibited by bafilomycin A1 and CaEGTA which interfere with the function of vacuolar H(+)-ATPase (V-ATPase) and retain endosomal Ca(2+). This result suggests that the intact caIBDV particle was transported to the V-ATPase positive vesicles for uncoating and implicates an essential role of endocytosis during the viral entry. The IBDV-mediated endocytosis was demonstrated to be clathrin-independent. Instead, the entry of caIBDV in DF-1 was reduced under the inhibitions or depletions of lipid raft, c-Src tyrosine kinase, dynamin and actin polymerization. In summary, this study confirmed the role of endocytosis in caIBDV entry and characterized the route of its endocytosis.

Quantitative comparison of the efficiency of antibodies against S1 and S2 subunit of SARS coronavirus spike protein in virus neutralization and blocking of receptor binding: Implications for the functional roles of S2 subunit

Neutralizing effects of antibodies targeting the C‐terminal stalk (S2) subunit of the spike protein of severe acute respiratory syndrome coronavirus have previously been reported, although its mechanism remained elusive. In this study, high titered mouse antisera against the N‐terminal globular (S1) and S2 subunits of the S protein were generated and total immunoglobulin G (IgG) was purified from these antisera. The efficiency of these purified IgGs in virus neutralization and blocking of receptor binding were compared quantitatively using virus neutralization assay and a previously developed cell‐based receptor binding assay, respectively. We demonstrated that anti‐S1 IgG neutralizes the virus and binds to the membrane associated S protein more efficiently than anti‐S2 IgG does. Moreover, both anti‐S1 and anti‐S2 IgGs were able to abolish the binding between S protein and its cellular receptor(s), although anti‐S1 IgG showed a significantly higher blocking efficiency. The unexpected blocking ability of anti‐S2 IgG towards the receptor binding implied a possible role of the S2 subunit in virus docking process and argues against the current hypothesis of viral entry. On the other hand, the functional roles of the previously reported neutralizing epitopes within S2 subunit were investigated using an antigen specific antibody depletion assay. Depletion of antibodies against these regions significantly diminished, though not completely abolished, the neutralizing effects of anti‐S2 IgG. It suggests the absence of a major neutralizing domain on S2 protein. The possible ways of anti‐S2 IgGs to abolish the receptor binding and the factors restricting anti‐S2 IgGs to neutralize the virus are discussed.

Naturally occurring anti-Escherichia coli protein antibodies in the sera of healthy humans cause analytical interference in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for serodiagnosis of severe acute respiratory syndrome.

ABSTRACT We reported the analytical interference of anti-Escherichia coli protein (EP) antibodies in human sera and residual EP in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay as a possible source of false positives in severe acute respiratory syndrome serodiagnosis. The rate of false positives was significantly reduced by adding mouse anti-EP antiserum in the blocking step.

Molecular advances in severe acute respiratory syndrome-associated coronavirus (SARS-CoV).

n n The sudden outbreak of severe acute respiratory syndrome (SARS) in 2002 prompted the establishment of a global scientific network subsuming most of the traditional rivalries in the competitive field of virology. Within months of the SARS outbreak, collaborative work revealed the identity of the disastrous pathogen as SARS-associated coronavirus (SARS-CoV). However, although the rapid identification of the agent represented an important breakthrough, our understanding of the deadly virus remains limited. Detailed biological knowledge is crucial for the development of effective countermeasures, diagnostic tests, vaccines and antiviral drugs against the SARS-CoV. This article reviews the present state of molecular knowledge about SARS-CoV, from the aspects of comparative genomics, molecular biology of viral genes, evolution, and epidemiology, and describes the diagnostic tests and the anti-viral drugs derived so far based on the available molecular information.n n