Department of Veterinary Medicine
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Item Recovery of Recombinant Avian Paramyxovirus Type-3 Strain Wisconsin by Reverse Genetics and Its Evaluation as a Vaccine Vector for Chickens(MDPI, 2021-02-19) Elbehairy, Mohamed A.; Khattar, Sunil K.; Samal, Siba K.A reverse genetic system for avian paramyxovirus type-3 (APMV-3) strain Wisconsin was created and the infectious virus was recovered from a plasmid-based viral antigenomic cDNA. Green fluorescent protein (GFP) gene was cloned into the recombinant APMV-3 genome as a foreign gene. Stable expression of GFP by the recovered virus was confirmed for at least 10 consecutive passages. APMV-3 strain Wisconsin was evaluated against APMV-3 strain Netherlands and APMV-1 strain LaSota as a vaccine vector. The three viral vectors expressing GFP as a foreign protein were compared for level of GFP expression level, growth rate in chicken embryo fibroblast (DF-1) cells, and tissue distribution and immunogenicity in specific pathogen-free (SPF) day-old chickens. APMV-3 strain Netherlands showed highest growth rate and GFP expression level among the three APMV vectors in vitro. APMV-3 strain Wisconsin and APMV-1 strain LaSota vectors were mainly confined to the trachea after vaccination of day-old SPF chickens without any observable pathogenicity, whereas APMV-3 strain Netherlands showed wide tissue distribution in different body organs (brain, lungs, trachea, and spleen) with mild observable pathogenicity. In terms of immunogenicity, both APMV-3 strain-vaccinated groups showed HI titers two to three fold higher than that induced by APMV-1 strain LaSota vaccinated group. This study offers a novel paramyxovirus vector (APMV-3 strain Wisconsin) which can be used safely for vaccination of young chickens as an alternative for APMV-1 strain LaSota vector.Item MOLECULAR PATHOGENESIS OF INFLUENZA IN SWINE AND ENGINEERING OF NOVEL RECOMBINANT INFLUENZA VIRUSES(2011) Pena, Lindomar Jose; Perez, Daniel R; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Influenza A viruses (IAVs) belong to the family Orthomyxoviridae and represent major pathogens of both humans and animals. Swine influenza virus is an important pathogen that affects not only the swine industry, but also represents a constant threat to the turkey industry and is of particular concern to public health. In North America, H3N2 triple reassortant (TR) IAVs first emerged in 1998 and have since become endemic in swine populations. In the first part of this dissertation, we focused on the role of surface glycoproteins and PB1-F2 to unravel their roles in the virulence of TR IAVs in this important natural host. We found that surface glycoproteins are necessary and sufficient for the lung pathology, whereas the internal genes play a major role in the febrile response induced by TR H3N2 IAVs in swine. With respect to PB1-F2, we found that PB1-F2 exerts pleiotropic effects in the swine host, which are expressed in a strain-dependent manner. Pathogenicity studies in swine revealed that the presence of PB1-F2 leads the following effects in context of three TR strains tested: no effect in the context of sw/99 strain; increases the virulence of pH1N1; and decreases the virulence of ty/04. Next, we developed temperature-sensitive live attenuated influenza vaccines for use in swine and shown that these vaccines are safe and efficacious against aggressive intratracheal challenge with pH1N1. Lastly, we rearranged the genome of an avian H9N2 influenza virus to generate replication competent influenza virus vectors that provide a robust system for expression and delivery of foreign genes. As a proof-of-principle, we expressed the hemagglutinin from a prototypical highly pathogenic avian influenza virus (HPAIV) H5N1 and shown that this vectored H5 vaccine retained its safety properties in avian and mammalian species, and induced excellent protection against aggressive HPAIV H5N1 challenges in both mice and ferrets. Taken together, these studies have advanced our understanding of molecular basis of pathogenesis of influenza in the swine host and have contributed to the development of improved vaccines and influenza-based vectors with potential applications in both human and veterinary medicine.Item Genetics of Avian Paramyxovirus serotype 2(2010) Subbiah, Madhuri; Samal, Siba K.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian Paramyxovirus (APMV) serotype 2 is one of the nine serotypes of APMV that infect a variety of bird species around the world. In chickens and turkeys, APMV-2 causes respiratory illness and drop in egg production. To understand the molecular characteristics of APMV-2, the complete genome sequences of prototype strain Yucaipa and strains Bangor, England and Kenya were determined. The genome lengths of APMV-2 strains Yucaipa, Bangor, England and Kenya are 14904, 15024, 14904, 14916 nucleotides (nt), respectively. Each genome consists of six non-overlapping genes in the order 3'N-P/V/W-M-F-HN-L5' similar to most of APMVs. Sequence comparison of APMV-2 strains England and Kenya with prototype strain Yucaipa show 94-98% nt and 90-100% aggregate amino acid (aa) identities. However, strain Bangor shares low level of nt and predicted aa sequence identities with the other three strains. The phylogenetic and serological analyses of all four strains indicated the existence of two subgroups: strains Yucaipa, England and Kenya represented one subgroup and strain Bangor represented the other subgroup. All four strains were found to be avirulent for chickens by mean death time and intracerebral pathogenicity test. To further study the molecular biology and pathogenicity of APMV-2, a reverse genetics system for strain Yucaipa was established in which infectious recombinant APMV-2 was recovered from a cloned APMV-2 antigenomic cDNA. The recovered recombinant virus showed in vitro growth characteristics and in vivo pathogenicity similar to wild type virus. Recombinant APMV-2 expressing enhanced green fluorescent protein was also recovered, suggesting its potential use as a vaccine vector. Furthermore, generation and characterization of mutant viruses by replacing the fusion protein (F) cleavage site of APMV-2 with those of APMV serotypes 1 to 9 demonstrated that the amino acid composition at F protein cleavage site does not affect the pathogenicity of APMV-2. Overall, the study conducted here has several downstream applications. The complete genome sequence of APMV-2 is useful in designing diagnostic reagents and in epidemiological studies. The reverse genetics system for APMV-2 would be of considerable utility for introducing defined mutations into the genome of this virus and develop vaccine vector for animal and human pathogens.Item REVERSE GENETICS OF AVIAN METAPNEUMOVIRUS(2005-12-06) Dhanasekaran, Govindarajan; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses to the US turkey industry. With a long-term goal of developing a better vaccine for controlling AMPV in the US, a reverse genetics system to produce infectious AMPV entirely form cloned cDNA was established. To achieve this, the unpublished sequences of the G gene, the L gene, the leader and trailer region were first determined to complete the entire genome sequence of AMPV subgroup C strain Colorado (AMPV/CO). Our results showed that the full-length AMPV/CO genome was 14,150 nucleotides (nt) in length, denoting that AMPV/CO possessed the longest genome among known metapneumoviruses. Subsequently, a cDNA clone encoding the entire 14,150 nt genome was generated by assembling 5 cDNA fragments, representing the entire genome, between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a low-copy number transcription plasmid pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1 and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. The recovered virus was observed to contain the genetic markers that were artificially introduced during cloning. Characterization of the recombinant AMPV/CO showed that its growth characteristics in tissue culture were similar to those of the parental virus. These results demonstrate that infectious AMPV can be generated entirely from cloned DNA using reverse genetics techniques. The potential of AMPV/CO to serve as a viral-vector was examined using green fluorescent protein (GFP) as a reporter. The recovered rAMPV/CO-GFP virus stably expressed GFP for at least five serial passages and showed characteristics similar to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively for various studies involving AMPV molecular biology, pathogenesis and vaccine development.