Cho-Hua Wan

Molecular characterization of three newly recognized rat parvoviruses

Rodent parvoviruses have been documented to interfere with both in vivo and in vitro research. In this study, three rat parvoviruses distinct from previously characterized rodent parvoviruses were identified from naturally infected rats obtained from four discrete sources. These three newly recognized parvoviruses were designated rat minute virus (RMV)-1a, -1b and -1c. In this study, the genomic nucleotide sequence and the predicted amino acid sequences of proteins for each of the three RMV-1 variants and Kilham rat virus (KRV) were determined and compared with previously characterized rodent parvoviruses. The three RMV-1 variants were shown to be closely related to each other, to be distinct from but closely related to KRV and H-1 virus, and to be significantly different from the previously identified rat parvovirus isolate, RPV-1a.

Characterization of the biochemical effects of naphthalene on the mouse respiratory system using NMR‐based metabolomics

Naphthalene is a ubiquitous environmental pollutant to which humans are exposed. Previous studies have demonstrated that naphthalene causes bronchiolar epithelial necrosis in the mouse distal airway, after parenteral administration. In this study, metabolic variations in the bronchoalveolar lavage fluid (BALF) and the lung tissues of naphthalene‐treated mice and controls were examined using nuclear magnetic resonance (NMR)‐based metabolomics to identify the toxic mechanism. Male ICR mice were treated with naphthalene [0, 50, 100 and 200 mg kg–1, intraperitoneally (i.p.)]. After 24 h, BALF and lung tissues were collected and prepared for 1H and J‐resolved (JRES) NMR analysis after principal component analysis (PCA). PCA modeling of p‐JRES spectra from the BALF, as well as hydrophilic and hydrophobic lung metabolites, enabled the high‐dose group to be discriminated from the control group; increased levels of isopropanol, ethane, and acetone and lower levels of ethanol, acetate, formate, and glycerophosphocholine were detected in the BALF of mice treated with higher doses of naphthalene. Furthermore, increased isopropanol and phosphorylcholine‐containing lipid levels and decreased succinate and glutamine levels were discovered in the lungs of naphthalene‐exposed mice. These metabolic changes may be related to lipid peroxidation, disruptions of membrane components and imbalanced energy supply, and these results may partially explain the loss of cell membrane integrity in the airway epithelial cells of naphthalene‐treated mice. We conclude that NMR‐based metabolomic studies on BALF and lung tissues are a powerful tool to understand the mechanisms underlying respiratory toxicity. Copyright

The effect of infection order of porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus on dually infected swine alveolar macrophages

BackgroundConcurrent infection with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) is known as one of the major causes for porcine respiratory disease complex (PRDC). Dual infection with PCV2 and PRRSV is consistently to have more severe clinical presentations and pulmonary lesions than infection with PCV2 alone or PRRSV alone. However, it is not known if dual infections with PCV2 and PRRSV in different infection order may lead to different clinical symptoms in the host. To mimic the possible field conditions, swine alveolar macrophages (AMs) were inoculated with PCV2 and PRRSV in vitro simultaneously or with one virus 18 h earlier than the other. The cell viability, cytopathic effects, antigen-containing rates, phagocytotic and microbial killing capabilities, cytokine profiles (IL-8, TNF-α, and IFN-α) and FasL transcripts were determined, analyzed, and compared to prove the hypothesis.ResultsA marked reduction in PRRSV antigen-containing rate, cytopathic effect, and TNF-α expression level was revealed in AMs inoculated with PCV2 and PRRSV simultaneously and in AMs inoculated with PCV2 first then PRRSV 18 h later, but not in AMs inoculated with PRRSV first then PCV2 18 h later. Transient decrease in phagocytosis but constant reduction in microbicidal capability in AMs in the group inoculated with PCV2 alone and constant decrease in phagocytosis and microbicidal capability in AMs in all PRRSV-inoculated groups were noted. The levels of IL-8, TNF-α, IFN-α, and FasL transcripts in AMs in all groups with dual inoculation of PCV2 and PRRSV were significantly increased regardless of the infection orders as compared with infection by PCV2 alone or PRRSV alone.ConclusionsSwine AMs infected with PCV2 first then PRRSV later or infected with PCV2 and PRRSV simultaneously displayed marked reduction in PRRSV antigen-containing rate, cytopathic effect, and TNF-α expression level. The different inoculation orders of PCV2 and PRRSV in AMs leading to different results in viral antigen positivity, cytopathology, and cytokine profile may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions in PRDC exerted by dual infection with PCV2 and PRRSV and the variable clinical manifestations of PRDC-affected pigs in the field.

Lethargy, ulcers, bronchopneumonia and death in two aged female bonnet macaques presumed to be caused by Cercopithicine herpes virus I

Over the course of 4 weeks, two female aged bonnet macaque (Macaca radiata) group‐housed females died after the dominant male was removed from the group and the newly dominant male persistently chased, caught and bred all females in the pen. The two aged affected females were observed exhibiting lethargy, dyspnea, with widespread necroulcerative lesions in and around the mouth, muzzle and bridge of their noses. Extensive ulcerative glossitis, necrotic bronchopneumonia with intra‐nuclear inclusions and the absence of other evidence is highly suggestive that death was caused by an alphaherpes virus commonly known as herpes B virus. Herpes B virus is a potentially zoonotic disease periodically shed by macaques, which is structurally related to herpes simplex viruses I and II of humans. The emergence of fatal B virus to primates in this pen may have been associated with the combination of age and stress in the affected individuals.

Detection of rat parvovirus type 1 and rat minute virus type 1 by polymerase chain reaction.

Two newly recognized parvovirus species, rat parvovirus 1 (RPV-1) and rat minute virus 1 (RMV-1), were recently identified in naturally infected rats. In this study, two polymerase chain reaction (PCR) assays were developed to specifically detect RPV-1 and RMV-1. The RPV-1 PCR assay amplified the expected 487-bp deoxyribonucleic acid (DNA) fragment only in the presence of RPV-1 DNA; the RMV-1 PCR assay amplified the expected 843-bp product only from RMV-1 DNA, not from other rodent parvoviruses. The RPV-1 and the RMV-1 PCR assays detected approximately 18 and 70 copies of DNA template, respectively. These two PCR assays were shown to be sensitive, specific and rapid methods for detecting RPV-1 and RMV-1 infections in rats. These assays may also be valuable for evaluation of biological specimens for parvovirus contamination.

An eight-year epidemiologic study based on baculovirus-expressed type-specific spike proteins for the differentiation of type I and II feline coronavirus infections

BackgroundFeline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV). FCoVs are divided into two serotypes with markedly different infection rates among cat populations around the world. A baculovirus-expressed type-specific domain of the spike proteins of FCoV was used to survey the infection of the two viruses over the past eight years in Taiwan.ResultsAn immunofluorescence assay based on cells infected with the recombinant viruses that was capable of distinguishing between the two types of viral infection was established. A total of 833 cases from a teaching hospital was surveyed for prevalence of different FCoV infections. Infection of the type I FCoV was dominant, with a seropositive rate of 70.4%, whereas 3.5% of cats were infected with the type II FCoV. In most cases, results derived from serotyping and genotyping were highly agreeable. However, 16.7% (4/24) FIP cats and 9.8% (6/61) clinically healthy cats were found to possess antibodies against both viruses. Moreover, most of the cats (84.6%, 22/26) infected with a genotypic untypable virus bearing a type I FCoV antibody.ConclusionA relatively simple serotyping method to distinguish between two types of FCoV infection was developed. Based on this method, two types of FCoV infection in Taiwan was first carried out. Type I FCoV was found to be predominant compared with type II virus. Results derived from serotyping and genotyping support our current understanding of evolution of disease-related FCoV and transmission of FIP.

Immune gene expression profiles in swine inguinal lymph nodes with different viral loads of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) infection has been suggested as an acquired immunodeficiency disorder. However, the immunopathogenesis of PCV2 infection is still not fully clarified. In the present study, 35 inguinal lymph nodes (LNs) with different levels of PCV2 load obtained from postwaening multisystemic wasting syndrome (PMWS)-affected pigs and 7 from healthy subclinically PCV2-infected pigs were selected. The LNs were subsequently ranked by their PCV2 loads to mimic the progression of PCV2 infection-associated lesion development. The expressions of 96 selected immune genes in these LNs were assessed by the integration of several reverse transcription quantitative real-time polymerase chain reaction experiments. Hierarchical cluster analysis of the gene expression profiles resulted in 5 major clusters (A, B, C, D, and E). Different clusters of immune gene expression profiles were compatible with the divergent functions of various immune cell subpopulations. 61 out of 96 selected genes belonged to cluster C and were mainly involved in the activation of dendritic cells and B and T lymphocytes. The expression levels of these genes were generally up-regulated in the LNs obtained from PMWS-affected pigs with relatively lower PCV2 loads. However, the up-regulated level tended to reduce or turned into down-regulation as the PCV2 load increased. Genes belonging to cluster B, involved in T cell receptor signaling, became silenced as the PCV2 load increased. The expression profiles of macrophage-associated genes were either independent from or positively correlated with the PCV2 load, such as those in clusters A and E and in cluster D, respectively. In addition, the principle component analysis of the expression of the 96 selected genes in the 42 inguinal LNs revealed that 53.10% and 72.29% of the total data variants could be explained by the top-3 and top-7 principle components, respectively, suggesting that the disease development of PCV2 infection may be associated with a few major and some minor factors. In conclusion, assessment of immune gene expression profiles in LNs supports a close interaction between immune activation and suppression during the progression of PMWS development.

Multiplex polymerase chain reaction assay for the detection of minute virus of mice and mouse parvovirus infections in laboratory mice

Mouse parvoviruses are among the most prevalent infectious pathogens in contemporary mouse colonies. To improve the efficiency of routine screening for mouse parvovirus infections, a multiplex polymerase chain reaction (PCR) assay targeting the VP gene was developed. The assay detected minute virus of mice (MVM), mouse parvovirus (MPV) and a mouse housekeeping gene (α-actin) and was able to specifically detect MVM and MPV at levels as low as 50 copies. Co-infection with the two viruses with up to 200-fold differences in viral concentrations can easily be detected. The multiplex PCR assay developed here could be a useful tool for monitoring mouse health and the viral contamination of biological materials.

Reduction of Classical Swine Fever Virus-Specific Cell Proliferative Response of Porcine Peripheral Blood Mononuclear Cells by Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) infection reducing the efficacy of Lapinized Philippines Coronel (LPC) vaccine of classical swine fever virus (CSFV) has been demonstrated in the previous study. In order to investigate the possible mechanisms of PCV2-derived interference on the CSFV-specific cell-mediated immune response, an ex vivo model of CSFV-specific cell proliferative response of peripheral blood mononuclear cells (PBMCs) from PCV2-carrier pigs was established. The ALD, a virulent CSFV strain, induced a significantly higher CSFV-specific cell proliferative response than the attenuated LPC strain did. The ALD strain at a dose of 0.1-1 multiplicity of infection (MOI) induced a significantly higher CSFV-specific cell proliferative response than that at 3 MOI did. Pre-inoculated PCV2 significantly reduced the levels of CSFV-specific cell proliferative response and CD25 expression in PBMCs than that of ALD alone. The PCV2-derived interference was not associated with the CpG-ODNs of PCV2 genome neither the cytokine levels of IL-2, IL-4, IL-10, and TNF-α in the supernatant of PBMC cultures. These results indicated that PCV2 could reduce CSFV-specific cell proliferative response in PBMCs partially through the reduction of CD25 expression in stimulated PBMCs.

History of Major Epidemics of Avian Influenza in Humans and Public Health Perspectives

Avian influenza (AI) has had great impacts on global health and socioeconomics in the past two decades. Among many newly emerged influenza viruses, avian influenza viruses (AIVs) have played either indirect or direct role in human infections. The continuous evolvement of AIVs through antigenic drift and genetic reassortment has led to emerging diversities of local virus strains or variants. These dynamic changes of AIVs, even low pathogenic avian influenza (LPAI) viruses, have not only affected poultry health but also resulted in severe and fatal human cases. Moreover, the persistence of these viruses at a population level may lead to outbreaks of AI occurring year after year in a vicious cycle, if they are not completely eradicated. In light of the emergence of novel H7N9 and other subtypes of AIVs in China in recent years, residents of Taiwan who live closely to this neighboring country must be well prepared in case these viruses are imported. Furthermore, the potential for interspecies transmission to humans from either of the two subtypes of avian H5N2 and H6N1 viruses increases the risk assessment of potential public health threats coming from continuous viral mutations. These two AIV subtypes are relevant in chickens and have been endemic in Taiwan for many years, and the newly emerged novel three subtypes (H5N2, H5N3, and H5N8 clade 2.3.4.4) of AIVs that have spread island-wide since January of 2015 must be expeditiously assessed for public health risks. In summary, this article discusses the history, virology, and epidemiology of avian influenza in humans. We believe that fully understanding virological and epidemiological characteristics of avian influenza viruses and the history of past pandemics or major epidemics will help strengthen the effectiveness of prevention and control measures.