Microbial succession, composition and ecological distribution within the gastro-intestinal tract are critical areas of study since commensal bacteria have been shown to affect animal health and development. A series of experiments were conducted to determine whether altered microbial succession in neonatal animals would modulate the development and health of pigs later in life. An initial experiment in conventional pigs was conducted to establish the early postnatal microbial succession profile and to identify early colonizing bacterial species. Culture-independent analysis of digesta and mucosal microbiota showed distinct variation between the proximal and distal gastro-intestinal tract (GIT) indicating that fecal or distal gut profiles cannot be used to predict succession in the upper GIT. Temporally, Clostridium spp. were found to be most prevalent in the GIT microbiota of the neonatal pig up to 0.5 d of age, accompanied by a high abundance of Escherichia and Shigella spp. These genera were transiently displaced by Streptococcus spp. followed by a preponderance of Lactobacillus spp. between 3 and 20 d of age. Subsequently, a “snatch-farrow” model was employed to modulate early postnatal microbial succession and investigate the effects on postweaning microbial composition. Pigs were collected into sterile towels directly from the vaginal canal and transferred to a sterile isolator environment for the first 4 days. Pigs were either inoculated with sow feces or not at 1 d of age resulting in significant differences in fecal microbial profile at 4 days of age, prior to removal from isolators. Analysis using terminal restriction fragment length polymorphisms (TRFLP) of intestinal microbiota at 28 d of age did not show significant clustering or variation in diversity indices for either group during the 4-d postnatal isolator phase. However, enumeration of selected taxa using quantitative PCR did indicate significant treatment differences in postweaning microbiota. Despite these results, this approach was rejected for further use as the protocol provided only moderate control of early postnatal colonization and variation and unpredictability of the timing of natural farrowing contributed to significant litter effects. Finally, a gnotobiotic monoassociation model was used investigate the effects of modulating early postnatal microbial succession on postweaning physiology, microbial composition and mucosal gene expression. Twenty-four cesarean-section derived piglets were monoassociated for the first 4 days of life with either L. mucosae (L), S. infantarius (S), C. perfringens (C) or E. coli (E). Pigs from treatments E and L animals showed the highest growth rate during the conventional rearing period (7-28 d of age). Monoassociation with different bacterial species during the first 4 d of life resulted in significant changes in postweaning microbial composition in small intestine and colon as assessed by quantitative PCR, although TRFLP did not identify unique clustering by treatment or variation in diversity. L. mucosae was the only inoculant species with significant variation, with a reduction in the colonic mucosa at 28 days of age. Monoassociation with L. mucosae was also associated with increased nutrition related gene expression in small intestine. Pigs monoassociated with E. coli had low expression of microbial sensing (TLR2 and 4), NFkappaB complex genes and mucins at 28 d of age. This study clearly showed that controlled early microbial succession in neonatal pigs altered post-weaning commensal microbiota composition, postweaning physiology and host gene expression in small and large intestine. The findings suggest the importance of peri-natal management and feeding strategies in promoting postweaning health and performance.

All animals exist in lifelong relations with a complement of bacteria. Because of the ubiquity of these symbioses as well as the derived biomedical applications, the study of both beneficial and pathogenic host-microbe associations has long been established. The monospecific light organ association between the Hawaiian sepiolid squid Euprymnascolopes and the marine luminous bacterium Vibrio fischeri has been used as a experimental model for the study of the most common type of animal-bacterial interaction, i.e., the association of coevolved Gram-negative bacteria with the extracellular apical surfaces of polarized epithelia. A fundamental step for understanding the mechanisms of host-symbiont associations lies in defining the genetic components involved; specifically defining changes in host gene expression. The studies presented in this dissertation identify and characterize V. fischeri-induced changes in host gene expression at both the transcript and protein level.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus is the causative agent of Japanese encephalitis (JE). The disease affects mostly children and around 30000– 50000 cases of JE and up to 15000 deaths are reported annually. No anti-viral drugs have been discovered against JE so far, but advances in our knowledge of the molecular biology of flaviviruses is propelling flaviviral drug research at an expeditious pace. Since JEV has a small genome which encodes for only ten proteins, there is dearth of potential drug targets. Researchers are now focusing on cellular interactomes, a complex and dynamic molecular biosystem which identifies host proteins which interact with either viral proteins or viral genomes, leading to the generation of an astronomical number of potential drug targets involving common cellular pathways that are required for the life cycle of different viruses. Such studies can pave way for the development of ‘broad-spectrum’, ‘silver-bullet’ anti-viral drugs for the treatment of multiple viral diseases. The cellular interactomes can be studied by Genomics tools such as microarray. Systematic profiling of genes involved in virus infection by RNAi, transcriptome sequencing, microRNA profiling and yeast two-hybrid system has allowed us to assess global gene expression changes providing an unprecedented view on the host-side of the virus–host interactions. Advent of these tools has led to identification of plethora anti-viral genes. For example, over expression of IFN-stimulated gene15 (ISG15) results in inhibition of JEV leading to significant reduction of viral titers. Chemokine profiling of JEV-infected cells by microarray can provide possible therapeutic modalities that can mitigate the morbidity associated with JEV infection. Functional classification of interferon-stimulated genes (ISG) identified using innovative methods have been the stepping stone for identification of many anti-viral genes, among them are few Broadly acting effectors like IRF1, C6orf150, HPSE, RIG-I, MDA5 and IFITM3 and some more targeted antiviral specific like DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT, OASL, RTP4, TREX1 and UNC84B. In this study, we have identified a B16F10 murine melanoma cell line that is resistant to JEV infection. DNA microarray analysis of JEV-susceptible and resistant B16F10 cell lines gave us interesting insights into JEV-induced host gene expression changes. Real time PCR validation of microarray data indicates that a number of virus and interferon inducible genes are expressed constitutively at high levels in this JEV-resistant cell line. Further, several of the mouse genes induced by JEV in B16F10 cell line were also upregulated in JEV-infected mouse brain. To understand the significance of these host gene expression changes, we attempted to generate stable murine cell lines constitutively expressing select JEV-inducible genes and study the JEV infection pattern in these cell lines. One of the JEV-inducible genes encoding thymidylate kinase (Tyki), a mitochondrial protein involved in the sysnthesis of nucleoside diphosphates, when overexpressed in NIH3T3 cells confers resistance to JEV infection as evident from reduced JEV-induced cytopathic effects and significant reduction in viral titer. Since TYKI has two distinct domains: the N-terminal domain with unknown function and the C-terminal domain with the nucleoside monophosphate kinase function, suggest that TYKI may be a bifunctional protein with other biological functions in addition to its UMP-CMP kinase activity. In order to examine whether N-terminal domain is responsible for antiviral activity of the protein, a stable cell line constitutively expressing N-terminal domain of gene was made, but the overexpression of N-terminal domain didn't confer any antiviral immunity. Thus signifying importance of kinase activity in confering antiviral immunity. Our studies indicate for the first time that Tyki may have a role in host resistance to JEV and understanding the mechanism of action Tyki may pave way for novel anti-JEV therapy. Stable cell lines constitutively expressing other JEV-inducible genes (Atf3, Gimap3, Rtp4, Glipr2, Tmem140 and Garg49) couldn't be generated. Therefore, to study the effect of overexpression of these genes on JEV infection, expression vectors encoding these genes were transfected individually to human 293T cells by nucleofection, then infected with JEV and viral titres were examined by plaque assay. Nucleofection was opted as a method of choice since it is the only non-viral method, which transfects DNA directly enter the nucleus. In contrast, other commonly used non-viral transfection methods rely on cell division for the transfer of DNA into the nucleus. Nucleofection of vectors encoding different JEV-inducible genes followed by JEV infection and assay of viral titer led to identification of one more anti-viral gene and three pro-viral genes. Garg49, an interferon and JEV inducible mitochondrial gene was identified as antiviral gene. Further studies led to the identification of GARG49 as a mitochondrial protein. Three genes, Atf3, encoding a cAMP responsive element binding protein family transcription factor, Glipr2, encoding a Glioma related pathogenesis protein and Gimap3, encoding an outer mitochondrial membrane GTPase were identified as pro viral genes. Overexpression of Tmem140, encoding a transmembrane protein and Rtp4, encoding a golgi chaperone did not significantly affect JEV titer. Conclusions: . A JEV-resistant B16F10 murine melanoma cell line was identified and several JEV-inducible genes were found to be expressed constitutively at high levels in this cell line. .We demonstrate for the first time that Tyki/Ump-Cmpk2 encoding a mitochondrial nucleoside monophosphate kinase has an anti-JEV function and the C-terminal domain is essential for anti-viral activity. .Garg49/Ifit3 encodes an interferon and JEV-inducible mitochondrial protein and it has an anti-JEV function. . Activating transcription factor 3 (ATF3), GTPase, IMAP family member 3 (GIMAP3) and GLI pathogenesis-related 2 (GLIPR2) are pro-viral proteins which facilitate virus multiplication resulting in enhanced JEV titer.