VTechWorks Repository :: Browsing by Author "Meng, Xiang-Jin"

Browsing by Author "Meng, Xiang-Jin"

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Assembly, characterization and evaluation of a 3rd generation nanoparticle based drug carrier for metastatic breast cancer treatment
Huang, Wei (Virginia Tech, 2013-06-03)
Cancer is one of the leading causes of death in the world. For women in the U.S. and the European countries, breast cancer is the most common type and it continuously threatens the lives of the patients and causes huge economic losses. Chemotherapy and endocrine therapy are the common treatments for recurrence prevention and metastatic cancer symptom palliation. However, the uses of these therapies are meanwhile largely limited because their toxic side effects and non-specificity usually lead to low quality lives of the patients. Low aqueous solubility, multi-drug resistance, degradation of drug, limited intra-tumor diffusion and etc. are other limitations of conventional chemotherapies and endocrine therapies. Nanoparticle based drug carriers were extensively studied for therapeutic drug delivery. Many carriers could be loaded with high dose of hydrophobic and hydrophilic drugs, protect the drug from the surrounding in vivo environment during the transportation, specifically target and enter the tumor cells and slowly release the drug thereafter. Advanced nanoparticle drug carriers are studied driven by the need of a more efficient drug delivery. The 3rd generation of nanoparticle based drug carriers are recently developed. They usually consist of more than one type of nanoparticles. Different part of the particle has more specialized functions. Therefore, by carefully selecting from the conventional nanoparticle carriers, a 3rd generation particle could have the properties such as high loading capacity of multiple drugs, prolonged half-life in circulation, higher tendency of accumulating at the tumor site, improved specificity to the tumor cells, higher cell uptake rate and accurately triggered controlled release, and combination of the above-mentioned properties. In our study, a paclitaxel loaded nanoparticle supported immunoliposome was assembled for metastatic breast cancer drug delivery. Functionalized single walled carbon nanohorn or poly(lactic-co-glycolic acid) was encapsulated in the polyethylene glycol (PEG) coated liposome for high drug loading and controlled release. Anti-Her2 antibody or Herceptin® was grafted onto the surface of the liposome for a higher affinity to the Her2 overexpressing breast cancer cells. Firstly, the conjugation of protein to the surface of liposome and PEGylated liposomes were investigated. Proteins with or without membrane binding domain were conjugated to liposome and PEGylated liposomes through covalent and non-covalent binding for comparison. A modified enzyme-linked immune sorbent assay was developed for surface grafted protein quantification. Secondly, the encapsulation of solid nanoparticle into PEGylated immunoliposome was investigated. Results showed a new structure of solid nanoparticle in PEGylated immunoliposome at a 1:1 ratio was formed during the repeated freeze-thawing process. Supported immunoliposomes with high homogeneity in size and structure were purified by sucrose density gradient centrifugation. Thirdly, the drug loading, triggered release, cell binding, cell uptake and cell toxicities of the supported immunoliposome were studied. Release results showed a minimum drug leakage in serum at body temperature from the particle. The release was initiated with a minor burst trigged by low pH inside the tumor cell and followed with a long term linear pattern. Cell assay results showed the highest binding affinity of the antibody or Herceptin® grafted nanoparticles to Her2 overexpressing cell lines and a lysosomal intracellular distribution of the endocytosised particles. In the final study, a fabrication process for polymeric material nanoparticles was established. The process was capable of providing accurate control of the particle size with significant high output rates, thus largely extends the scope of materials for supporting the immunoliposome.
Avian hepatitis E virus, vaccines and methods of protecting against avian hepatitis-splenomegaly syndrome and mammalian hepatitis E
(United States Patent and Trademark Office, 2006-02-28)
The present invention relates to a novel isolated avian hepatitis E virus having a nucleotide sequence set forth in SEQ ID NO:1 or its complementary strand. The invention further concerns immunogenic compositions comprising this new virus or recombinant products such as the nucleic acid and vaccines that protect an avian or mammalian species from viral infection or hepatitis-splenomegaly syndrome caused by the hepatitis E virus. Also included in the scope of the invention is a method for propagating, inactivating or attenuating a hepatitis E virus comprising inoculating an embryonated chicken egg with a live, pathogenic hepatitis E virus and recovering the virus or serially passing the pathogenic virus through additional embryonated chicken eggs until the virus is rendered inactivated or attenuated. Further, this invention concerns diagnostic reagents for detecting an avian hepatitis E viral infection or diagnosing hepatitis-splenomegaly syndrome in an avian or mammalian species comprising an antibody raised or produced against the immunogenic compositions and antigens such as ORF2 proteins expressed in a baculovirus vector, E. coli, etc. The invention additionally encompasses methods for detecting avian HEV nucleic acid sequences using nucleic acid hybridization probes or oligonucleotide primers for polymerase chain reaction (PCR).
Avian hepatitis E virus, vaccines and methods of protecting against avian hepatitis-splenomegaly syndrome and mammalian hepatitis E
(United States Patent and Trademark Office, 2010-11-30)
The present invention relates to a novel isolated avian hepatitis E virus having a nucleotide sequence set forth in SEQ ID NO:1 or its complementary strand. The invention further concerns immunogenic compositions comprising this new virus or recombinant products such as the nucleic acid and vaccines that protect an avian or mammalian species from viral infection or hepatitis-splenomegaly syndrome caused by the hepatitis E virus. Also included in the scope of the invention is a method for propagating, inactivating or attenuating a hepatitis E virus comprising inoculating an embryonated chicken egg with a live, pathogenic hepatitis E virus and recovering the virus or serially passing the pathogenic virus through additional embryonated chicken eggs until the virus is rendered inactivated or attenuated. Further, this invention concerns diagnostic reagents for detecting an avian hepatitis E viral infection or diagnosing hepatitis-splenomegaly syndrome in an avian or mammalian species comprising an antibody raised or produced against the immunogenic compositions and antigens such as ORF2 proteins expressed in a baculovirus vector, E. coli, etc. The invention additionally encompasses methods for detecting avian HEV nucleic acid sequences using nucleic acid hybridization probes or oligonucleotide primers for polymerase chain reaction (PCR).
Avian hepatitis E virus, vaccines and methods of protecting against avian hepatitis-splenomegaly syndrome and mammalian hepatitis E
(United States Patent and Trademark Office, 2009-09-01)
The present invention relates to a novel isolated avian hepatitis E virus having a nucleotide sequence set forth in SEQ ID NO:1 or its complementary strand. The invention further concerns immunogenic compositions comprising this new virus or recombinant products such as the nucleic acid and vaccines that protect an avian or mammalian species from viral infection or hepatitis-splenomegaly syndrome caused by the hepatitis E virus. Also included in the scope of the invention is a method for propagating, inactivating or attenuating a hepatitis E virus comprising inoculating an embryonated chicken egg with a live, pathogenic hepatitis E virus and recovering the virus or serially passing the pathogenic virus through additional embryonated chicken eggs until the virus is rendered inactivated or attenuated. Further, this invention concerns diagnostic reagents for detecting an avian hepatitis E viral infection or diagnosing hepatitis-splenomegaly syndrome in an avian or mammalian species comprising an antibody raised or produced against the immunogenic compositions and antigens such as ORF2 proteins expressed in a baculovirus vector, E. coli, etc. The invention additionally encompasses methods for detecting avian HEV nucleic acid sequences using nucleic acid hybridization probes or oligonucleotide primers for polymerase chain reaction (PCR).
Broadening the Heterologous Cross-Neutralizing Antibody Inducing Ability of Porcine Reproductive and Respiratory Syndrome Virus by Breeding the GP4 or M genes
Zhou, Lei; Ni, Yan-Yan; Piñyero, Pablo E.; Cossaboom, Caitlin M.; Subramaniam, Sakthivel; Sanford, Brenton J.; Dryman, Barbara A.; Huang, Yao-Wei; Meng, Xiang-Jin (PLOS, 2013-06-24)
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens, which causes reproductive failure in sows and respiratory disease in piglets. A major hurdle to control PRRSV is the ineffectiveness of the current vaccines to confer protection against heterologous strains. Since both GP4 and M genes of PRRSV induce neutralizing antibodies, in this study we molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from six genetically different strains of PRRSV in an attempt to identify chimeras with improved heterologous cross-neutralizing capability. The shuffled GP4 and M genes libraries were each cloned into the backbone of PRRSV strain VR2385 infectious clone pIR-VR2385-CA. Three GP4-shuffled chimeras and five M-shuffled chimeras, each representing sequences from all six parental strains, were selected and further characterized in vitro and in pigs. These eight chimeric viruses showed similar levels of replication with their backbone strain VR2385 both in vitro and in vivo, indicating that the DNA shuffling of GP4 and M genes did not significantly impair the replication ability of these chimeras. Cross-neutralization test revealed that the GP4-shuffled chimera GP4TS14 induced significantly higher cross-neutralizing antibodies against heterologous strains FL-12 and NADC20, and similarly that the M-shuffled chimera MTS57 also induced significantly higher levels of cross-neutralizing antibodies against heterologous strains MN184B and NADC20, when compared with their backbone parental strain VR2385 in infected pigs. The results suggest that DNA shuffling of the GP4 or M genes from different parental viruses can broaden the cross-neutralizing antibody-inducing ability of the chimeric viruses against heterologous PRRSV strains. The study has important implications for future development of a broadly protective vaccine against PRRSV.
Characterization of an Amphipathic Alpha-Helix in the Membrane Targeting and Viral Genome Replication of Brome Mosaic Virus
Sathanantham, Preethi (Virginia Tech, 2022-03-01)
Positive-strand RNA viruses associate with specific organelle membranes of host cells to establish viral replication complexes. The replication protein 1a of brome mosaic virus associates strongly with the nuclear endoplasmic reticulum (ER) membranes, invaginates membranes into the lumen, and recruits various host proteins to establish replication complexes termed spherules. 1a has a strong affinity towards the perinuclear ER membrane, however, the structural features in 1a that dictate its membrane associations and thereby membrane remodeling activities are unclear. This study examined the possible role of an amphipathic α-helix, helix B, in BMV 1a's membrane association. Deletion or single substitution of multiple amino acids of helix B abolished BMV 1a's localization to nuclear ER membranes. Additional reporter-based, gain-of-function assays showed that helix B is sufficient in targeting several soluble proteins to the nuclear ER membranes. Furthermore, we found that the helix B-mediated organelle targeting is a functionally conserved feature among positive-strand RNA viruses of the alphavirus-like superfamily that includes notable human viruses such as Hepatitis E virus and Rubella virus as well as plant viruses such as cucumber mosaic virus and cowpea chlorotic mottle virus. Our results demonstrate a critical role for helix B across members of the alphavirus-like superfamily in anchoring viral replication complexes to the organelle membranes. We anticipate our findings to be a starting point for the development of sophisticated models to use helix B as a novel target for the development of antivirals for positive-strand RNA viruses that belong to the alphavirus-like superfamily.
Characterization of Avirulent Turkey Hemorrhagic Enteritis Virus: A Study of the Molecular Basis for Variation in Virulence and the Occurrence of Persistent Infection
Beach, Nathan Matthew (Virginia Tech, 2006-08-04)
Hemorrhagic enteritis is a disease of turkeys caused by virulent strains of Turkey Hemorrhagic Enteritis Virus (THEV) resulting in depression, splenomegaly, intestinal hemorrhage, immunosuppression, and mortality. Avirulent strains that do not produce intestinal lesions and mortality are used in live-virus vaccines that protect turkeys from virulent field challenge. The cause for the difference in phenotype between virulent and avirulent strains is unknown. The full-length genome of the Virginia Avirulent Strain (VAS) of THEV was sequenced and compared to the genome sequence of a virulent field isolate from Israel. Genetic differences were found in seven viral genes. Further sequencing narrowed the focus from seven genes to three: ORF1, E3, and Fiber. Consistent variation in these genes between strains of THEV with different phenotypes strongly indicates these genes as key factors affecting virulence. THEV is an officially recognized member of the viral family Adenoviridae, genus Siadenovirus. The genomes of the members of the genus, THEV and Frog Adenovirus 1, are not well-characterized. The genome sequences of both members were compared for the prediction of genetic and structural elements. Common features were found that distinguish this genus from all other adenoviruses, and differences were found that possibly contribute to host specificity of the members. The VAS is known to stimulate a life-long protective antibody response, though viral replication is only of short duration. Several studies were undertaken to determine changes in virus location and serology over time. Viral DNA was detected in various tissues through 15 weeks post-infection in the presence of high antibody titers. THEV infection was found to be similar to the non-lytic persistent infections seen with human adenoviruses. Regardless of the mechanism involved in the persistent stimulation of antibodies in infected turkeys, the VAS was shown to be an ideal vector for use in a recombinant live-virus vaccine. The next step in THEV research should be the creation of a full-length infectious DNA clone, which could be used in the creation of a recombinant vaccine. The infectious clone would also allow for the systematic testing of genes that are suspected to be involved in virulence.
Characterization of the Bacteriophage Felix O1 Endolysin and Potential Application for Salmonella Bioremediation
Settle, Lori L. (Virginia Tech, 2012-08-09)
There is an increasing incidence of antimicrobial-resistant organisms isolated from food and food products. Coupled with that rising incidence is increased media scrutiny and coverage of outbreaks of foodborne illnesses. Consequently, consumers increasingly demand safer food, and that the antimicrobial measures used be other than antimicrobial drugs. A possible solution is to use bacteriophages, or the purified holin and endolysin proteins that make them lethal and lytic, as antimicrobial food treatments or additives. The bacteriophage Felix O1 is a promising candidate for development as an anti-Salmonella food treatment. This dissertation describes the work done to determine if these proteins could be of value as bioremedial agents. Endolysin treatments of Gram negative bacteria require two agents: the lytic endolysin, and a second agent to permeabilize the outer membrane of the bacterium. The holin protein was proposed as an outer membrane permeabilization agent. Methods used to locate the holin gene included BLAST analysis, analysis of putative Felix O1 proteins for transmembrane domains, and examination of the lysin sequence for an N-terminal signal sequence. Analyses did not reveal a promising candidate. Cloning of rIIA as a potential holin was attempted without success. Results of various analyses are discussed, as are chemical alternatives to the use of purified holin as a permeabilization agent. The endolysin, LysO1, was successfully cloned and characterized. PHYRE analysis predicted that the enzyme structure is composed of Î± helices arranged into two lobes, with the active site in a cleft between them. The enzyme lysed all tested strains of Salmonella and a tested strain of the foodborne pathogen Escherichia coli. Campylobacter jejuni susceptibility remains ambiguous, and the enzyme had no effect on Listeria monocytogenes or Micrococcus luteus. LysO1 was most active at alkaline pH and low ionic strength; optimal activity was observed in 25 mM buffer at pH 10. If removed from frozen storage, the enzyme was most thermostable at 30 °C. Lytic activity was adversely affected by the presence of the divalent cations calcium, magnesium, and zinc, and by high ionic strength. Considerable time was devoted to development of the activity assay used to further characterize the enzyme, and details of those experiments are provided. Logical extensions of the research project, such as further characterization and testing needed to obtain government approval for widespread use of the treatment, and possible pursuit of treatment based on an enzyme derivative such as an antimicrobial peptide, are discussed.
Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof
(United States Patent and Trademark Office, 2007-10-09)
The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products. In addition, the invention encompasses two mutations in the PCV2 immunogenic capsid gene and protein, and the introduction of the ORF2 mutations in the chimeric clones.
Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof
(United States Patent and Trademark Office, 2018-02-13)
The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products. In addition, the invention encompasses two mutations in the PCV2 immunogenic capsid gene and protein, and the introduction of the ORF2 mutations in the chimeric clones.
Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof
(United States Patent and Trademark Office, 2007-10-02)
The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products.
Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof
(United States Patent and Trademark Office, 2015-12-15)
The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products. In addition, the invention encompasses two mutations in the PCV2 immunogenic capsid gene and protein, and the introduction of the ORF2 mutations in the chimeric clones.
Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof
(United States Patent and Trademark Office, 2019-12-17)
The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products. In addition, the invention encompasses two mutations in the PCV2 immunogenic capsid gene and protein, and the introduction of the ORF2 mutations in the chimeric clones.
Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof
(United States Patent and Trademark Office, 2009-08-18)
The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products. In addition, the invention encompasses two mutations in the PCV2 immunogenic capsid gene and protein, and the introduction of the ORF2 mutations in the chimeric clones.
Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof
(United States Patent and Trademark Office, 2011-11-15)
The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products. In addition, the invention encompasses two mutations in the PCV2 immunogenic capsid gene and protein, and the introduction of the ORF2 mutations in the chimeric clones.
A commercial porcine circovirus (PCV) type 2a-based vaccine reduces PCV2d viremia and shedding and prevents PCV2d transmission to naive pigs under experimental conditions
Opriessnig, Tanja; Xiao, Chao-Ting; Halbur, Patrick G.; Gerber, Priscilla F.; Matzinger, Shannon R.; Meng, Xiang-Jin (2017-01-05)
Porcine circovirus type 2 (PCV2) vaccination has been effective in protecting pigs from clinical disease and today is used extensively. Recent studies in vaccinated populations indicate a major PCV2 genotype shift from the predominant PCV2 genotype 2b towards 2d. The aims of this study were to determine the ability of the commercial inactivated PCV2a vaccine Circovac (R) to protect pigs against experimental challenge with a 2013 PCV2d strain and prevent transmission. Thirty-eight pigs were randomly divided into four groups with 9-10 pigs per group: NEG (sham-vaccinated, sham-challenged), VAC (PCV2a-vaccinated, sham-challenged), VAC + CHAL (PCV2a-vaccinated and PCV2d-challenged), and CHAL (sham-vaccinated, PCV2d-challenged). Vaccination was done at 3 weeks of age using Circovac (R) according to label instructions. The CHAL and VAC + CHAL groups were challenged with PCV2d at 7 weeks of age and all pigs were necropsied 21 days post-challenge (dpc). The VAC-CHAL pigs seroconverted to PCV2 by 21 days post vaccination (dpv). At PCV2d challenge on 28 dpv, 3/9 VAC and 1/9 VAC + CHAL pigs were seropositive. NEG pigs remained seronegative for the duration of the study. Vaccination significantly reduced PCV2d viremia (VAC + CHAL) at dpc 14 and 21, PCV2d fecal shedding at dpc 14 and 21 and PCV2d nasal shedding at dpc 7, 14 and 21 compared to CHAL pigs. Vaccination significantly reduced mean PCV2 antigen load in lymph nodes in VAC + CHAL pigs compared to CHAL pigs. When pooled serum or feces collected from VAC + CHAL and CHAL pigs at dpc 21 were used to expose single-housed PCV2 naive pigs, a pooled fecal sample from CHAL pigs contained infectious PCV2 whereas this was not the case for VAC + CHAL pigs suggesting reduction of PCV2d transmission by vaccination. Under the study conditions, the PCV2a-based vaccine was effective in reducing PCV2d viremia, tissue loads, shedding and transmission indicating that PCV2a vaccination should be effective in PCV2d-infected herds. (C) 2016 The Author(s). Published by Elsevier Ltd.
Comparative and Functional Genomic Studies of Histophilus somni (Haemophilus somnus)
Siddaramappa, Shivakumara Swamy (Virginia Tech, 2007-04-09)
Histophilus somni is a commensal of the mucosal surfaces of respiratory and reproductive tracts of cattle and sheep. However, as an opportunistic pathogen, H. somni can cause diseases such as pneumonia, myocarditis, abortion, arthritis, and meningo-encephalitis. Previously, several virulence factors/mechanisms had been identified in H. somni of which the phase-variable lipooligosaccharide, induction of host cell apoptosis, intraphagocytic survival, and immunoglobulin Fc binding proteins were well characterized. To further understand the biological properties of H. somni, the genomes of pneumonia strain 2336 and preputial strain 129Pt have been sequenced. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, putative genes that encode proteases, restriction-modification enzymes, hemagglutinins, glycosyltransferases, kinases, helicases, and adhesins have been identified in H. somni. Most of the H. somni strain-specific genes were found to be associated with prophage-like sequences, plasmids, and/or transposons. Therefore, it is likely that these mobile genetic elements played a significant role in creating genomic diversity and phenotypic variability among strains of H. somni. Functional characterization of H. somni luxS in the genomic context revealed that the gene encodes S-ribosylhomocysteinase that can complement biosynthesis of AI-2 quorum sensing signal molecules in Escherichia coli DH5alpha. It was also found that several pathogenic isolates of H. somni form a prominent biofilm and that luxS as well as phosphorylcholine expression can influence biofilm formation by H. somni. In conclusion, comparative analyses of the genomes and functional characterization of putative genes have shed new light on the versatility and evolution of H. somni.
Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types
Gerber, Priscilla F.; O'Neill, Kevin; Owolodun, Olajide; Wang, Chong; Harmon, Karen; Zhang, Jianqiang; Halbur, Patrick G.; Zhou, Lei; Meng, Xiang-Jin; Opriessnig, Tanja (American Society for Microbiology, 2012-12-05)
The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) real-time reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days -2, 1, 3, 5, 7, 14, and 21 postinoculation (p.i.) and tested by one of three commercially available real-time RT-PCR assays(VetMax from Applied Biosystems, Foster City, CA [ abbreviated AB]; VetAlert from Tetracore, Rockville, MD[TC]; and AcuPig from AnDiaTec GmbH, Kornwestheim, Germany [ AD]). At day 1 p.i., all assays detected at least one positive sample in each group. The highest detection rates were on days 3 and 5 p.i. Between days 1 and 7 p.i., serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (kappa value of 0.97) and semen (kappa value of 0.80). Oral fluids had the lowest detection rates (AB, 55%; TC, 41%; AD, 46%) and the highest disagreement between kits (kappa value of 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a large amount (cycle threshold,< 30) of viral RNA in the pool. Serum and blood swab samples had shorter turnaround times for RNA extraction. The AB assay had a 1.6-times-shorter PCR time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and the shortest turnaround times.
Comparison of Real-Time Reverse Transcriptase PCR Assays for Detection of Swine Hepatitis E Virus in Fecal Samples
Gerber, Priscilla F.; Xiao, Chao-Ting; Cao, Dianjun; Meng, Xiang-Jin; Opriessnig, Tanja (American Society for Microbiology, 2014-01-15)
Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in people in many developing countries and is also endemic in many industrialized countries. Mammalian HEV (mHEV) isolates can be divided into at least four recognized major genotypes. Several nucleic acid amplification techniques have been developed for mHEV detection, with great differences in sensitivity. The aim of this study was to compare the performances of two singleplex real-time reverse transcriptase (RT) PCR assays for broad detection of all four mHEV genotypes (assays A and B) and two duplex real-time RT-PCR assays for detection and differentiation of mHEV genotypes 3 and 4 (assays C and D). RNAs extracted from 28 fecal samples from pigs experimentally inoculated with HEV genotype 3 and 186 fecal samples from commercial pigs with unknown HEV exposure were tested by all four assays. In experimental samples, HEV RNA was detected in 96.4% (assay A), 39.2% (assay B), 14.2% (assay C), and 0% (assay D) of the samples. In field samples with unknown HEV exposure, HEV RNA was detected in 67.2% (assay A), 36.4% (assay B), 1.1% (assay C), and 0.5% (assay D) of the samples. The assays showed overall poor agreement (kappa = 0.19 to 0.03), with differences in detection rates between assays (P < 0.01). Assays A and B, which broadly detect HEV genotypes 1 to 4, had significantly higher detection rates for HEV RNA than the duplex assays C and D, which were both designed to detect and differentiate between HEV genotypes 3 and 4.
Complete Genome Sequence and Pathogenicity of Two Swine Parainfluenzaviruses Isolated from Pigs in the United States
Qiao, Dan (Virginia Tech, 2009-05-21)
Members of the family Paramyxoviridae are non-segmented, negative-strand RNA viruses. A large and diverse host species are infected by paramyxoviruses, including avian, porcine, canine, bovine, equine, ovine, reptiles, aquatic species and humans. In the last few decades, many novel paramyxoviruses have emerged causing catastrophic illnesses in different aquatic and terrestrial species of animals and some of them also made the species jump to humans. Two novel paramyxoviruses 81-19252 (Texas81) and 92-7783 (ISU92) were isolated in the 1980s and 1990s from the brain of pigs that experienced respiratory and central nervous system disease from South and North Central United States. To understand their importance as swine pathogens, molecular characterization and pathogenicity studies were undertaken. The complete genome of Texas81 virus was 15456 nucleotides (nt) and ISU92 was 15480 nt in length consisting of six non-overlapping genes coding for the nucloeo- (N), phospho- (P), matrix (M, fusion (F), hemagglutinin-neuraminidase (HN) and large polymerase (L) proteins in the order 3'-N-P/C/V-M-F-HN-L-5'. The features related to virus replication and found to be conserved in most members of Paramyxoviridae were also found in swine viruses. These include: conserved and complementary 3â leader and 5â trailer regions, trinucleotide intergenic sequences, highly conserved gene start and gene stop signal sequences. The length of each gene of these two viruses was similar except for the F gene, in which ISU92 had an additional 24 nt "U" rich 3â untranslated region (UTR). The P gene of these viruses were predicted to express the P protein from the primary transcript and edit a portion of its mRNA to encode V and D proteins and the C protein was expected to be expressed from alternate translation initiation from the P gene as in Respiroviruses. Sequence specific features related to virus replication and host specific amino acid signatures in P, F, HN and L proteins indicated that these viruses probably originated from bovine parainfluenzavirus 3. Pairwise comparisons of deduced amino acid sequences of swine viral proteins with members of Paramyxoviridae and phylogenetic analysis based on individual genes as well as predicted amino acid sequences suggested that these viruses were novel members of the genus Respirovirus of the Paramyxovirinae subfamily and genotype A of bovine parainfluenzavirus type 3. The mild clinical signs and undetectable gross and microscopic lesions observed in swine parainfluenzavirus (sPIV3)-infected pigs indicate the inapparent nature of these viruses in pigs. Limited seroprevalence studies in serum samples collected from pig farms in Minnesota and Iowa in 2007-2008 by indirect ELISA revealed that sPIV3 are not circulating in these farms. The mild pathogenicity of sPIV3 can facilitate its development as a vaccine vector. The screening ELISA developed by us could be used to detect seroprevalence of sPIV3 in animal and human populations.