Viral infections of the central nervous system (CNS) in infants are rare; however, they are associated with high morbidity and mortality rates. These virus infections often induce strong innate immune responses in the brain including: the production of cytokines and chemokines, the activation of astrocytes and microglia and the recruitment of macrophages. Innate immune responses are often initiated by toll-like receptors (TLR). Several studies have demonstrated that toll-like receptor 7 (TLR7) can be stimulated by single-stranded RNA from multiple viruses. In the current study, we examined the mechanism by which TLR7 contributes to neuroinflammation in the neonatal brain using a mouse model of polytropic retrovirus infection. We found that TLR7 deficiency had no effect on neurologic disease, viral replication, or induction of interferon beta mRNA. However, TLR7 deficiency significantly altered neuroinflammatory responses including proinflammatory cytokine production, astrocyte activation, and microglial/macrophage activation. To our knowledge, this is the first demonstration of the necessity of TLR7 for innate immune responses to retrovirus infection in vivo. Additionally, this indicates that the immune response to retrovirus in the CNS may not be essential for disease pathogenesis in neonates.

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortions in goats and sheep and Malta fever in humans. The zoonotic disease brucellosis causes severe economic losses in the Mediterranean region and parts of Africa, Asia, and Latin America. With the completion of the genomic sequences of B. abortus 2308 and B. melitensis 16M, no classical virulence factors were found; and the chromosomes were virtually identical. However, in B. melitensis, a putative hemagglutinin gene was identified which is absent in B. abortus. The possibility of the hemagglutinin gene being a potential virulence factor was evaluated via gene replacement/deletion in B. melitensis and expression in trans in B. abortus. The hemagglutinin gene was PCR-amplified, cloned into pBBR1MCS-4, and electroporated into B. abortus 2308 yielding B. abortus 2308-QAE. A kanamycin-Region E-kanamycin disrupted gene fragment (KAN-E-KAN) was also generated and electroporated into B. melitensis 16M. The resulting mutants were characterized biochemically to confirm its Brucella origin and screened by antibiotic selective pressure. A colonization study of non-pregnant goats infected with B. abortus 2308, B. melitensis 16M, B. abortus 2308-QAE, or B. melitensis 16MÄE revealed no attenuation of the 16MÄE mutant when compared to 16M at 4, 7, and 21 days post inoculation. The study also showed that both the variant and the mutant were capable of infecting and disseminating throughout the host. All four strains were introduced into the pregnant goat model and evaluated for pathogenicity. Pregnancy/delivery results revealed 27%, 78%, 67%, and 50% abortion rates in goats infected with 2308, 16M, 2308-QAE, and 16MÄE, respectively. Bacterial culture of tissues from 2308, 16M, 2308-QAE, 16MÄE -exposed goats revealed 45 %, 79%, 75%, and 100% colonization of dam/kid pairs, respectively. The expression of the B. melitensis 16M hemagglutinin gene in trans in 2308-QAE revealed a significant (p<0.05) increase in colonization and abortion rates when compared to B. abortus 2308, mimicking the virulence of B. melitensis 16M in pregnant goats. The B. melitensis 16MÄE disruption mutant colonization and abortion rates demonstrated no attenuation in colonization but did show a 28% reduction in abortions when compared to B. melitensis 16M.

Haemonchus contortus is one of the major nematode parasites causing substantial economic losses in small ruminant farming worldwide. Recently, effect of anthelmintic treatment has decreased due to an increasing problem of nematode populations that have developed resistance to anthelmintics. There are certain breeds of sheep that are identified as being relatively resistant to the parasite including Gulf Coast Native (Native) sheep. Understanding the mode of immune response that helps these breeds of sheep control infection could help design vaccines and enhance control programs. This experiment was designed to evaluate the immunological responses of Native, compared with susceptible Suffolk sheep that might be responsible for this resistance. In Experiment 1, groups (n = 5) of 6 month old Native and Suffolk lambs were given infective larvae as one time (bolus) or trickle experimental infections. Fecal, blood, and serum samples were collected. Abomasal mucosa samples were collected at the time of necropsy on day 14 and day 21. There was no significant difference in number of worms recovered at necropsy but the ratio of adult vs larvae was significantly greater in bolus infected Suffolk than Native. Native lambs had significantly greater numbers of mast cells and eosinophils in the abomasal mucosa and serum IgG production was significantly greater compared to Suffolk lambs. Native lambs also showed a trend of increased level of serum IgA and IgE compared to Suffolk lambs. In Experiment 2, immune responses were evaluated in naturally infected Native and Suffolk lambs that grazed pasture contaminated predominantly with H. contortus. Ten lambs of each breed grazed together for 42 days. Fecal, blood and serum samples were collected. Five lambs of each breed were necropsied on day 35 and five on day 42 for nematode recovery and abomasal tissue sample collection. Native lambs had significantly lower FEC, significantly lower PCV reduction percent, and significantly higher serum IgE after day 14 and increased expression of Il-4 on day 10 post exposure compared to Suffolk lambs. At both necropsy time points, Native lambs had significantly greater numbers of mucosal mast cells, eosinophils and globule leukocytes in abomasal mucosa than Suffolk lambs.

Herpes simplex virus type-1 (HSV-1) morphogenesis occurs in multiple stages within infected cells. Initially, the virion capsid assembles within the nucleus and buds through the nuclear membrane into the cytoplasm. Within the cytoplasm, additional tegument proteins attach to the capsid and the fully tegumented capsids bud into trans-Golgi network (TGN) derived vesicles. Enveloped virions are ultimately secreted to extracellular spaces. The process by which the cytoplasmic capsids bud into TGN-derived vesicles is not well understood. The prevalent model calls for specific interactions among viral tegument proteins and membrane proteins and glycoproteins embedded within TGN membranes. To further investigate the roles of tegument proteins in cytoplasmic virion envelopment, we constructed deletion mutants of UL11, UL20, both UL11 and UL20, and UL16. UL11 is involved in cytoplasmic virion envelopment. The ΔUL11 virus exhibits large amounts of unenveloped capsids in the cytoplasm of infected cells. The phenotype of the double null virus most closely resembled that of the UL20 single null virus (ΔUL20) in all areas: plaque phenotype, growth kinetics, and ultrastructural characteristics. To asses whether UL11 has any affect on UL20/gK localization, confocal experiments to determine the localization of UL11, UL20 and gK were undertaken, revealing that UL11 transport was completely independent of UL20/gK. Taken together these results indicate that UL11 acts at a step in cytoplasmic envelopment downstream of UL20, and UL20 is required for proper UL11 function. However, UL11 is not dependent upon the UL20/gK heterodimer for its transport. To assess the role of UL16 in virion morphogenesis and egress, the YEbac102ΔUL16 virus was constructed using a recently described RED markerless recombination system. ΔUL16 showed a large accumulation of intranuclear capsids not seen in the ΔUL11 virus. This result indicates a two-fold role for UL16 in virion morphogenesis and egress: 1) The nuclear accumulation of capsids seems to suggest that the first and most important role of UL16 is in intranuclear capsid assembly/egress. 2) The cytoplasmic accumulation of capsids suggests that UL16 also plays a role in cytoplasmic envelopment. These results indicate a possible pathway for the juxtaposition of cytoplasmic capsids with TGN-derived vesicles for final cytoplasmic envelopment.

Although the biochemical characterization of E. ictaluri, the subsequent disease progression of enteric septicemia of catfish (ESC), and the associated pathologic lesions are well characterized, the mechanism of invasion of E. ictaluri into a susceptible host is poorly understood. Identification and confirmation of virulence factors and associated genes of E. ictaluri is crucial to elucidating the pathogenesis of this important disease. A signature tagged mutagenesis (STM) study conducted by Thune et al. (2006) identified 50 E. ictaluri clones with transposon insertions in genes potentially involved in pathogenesis. A specific STM mutant, 233PR, carrying a transposon insertion in a gene encoding a hypothetical adhesin protein, was selected for further characterization. In addition, an isogenic mutant was created by inserting a kanamycin resistance cassette 222 bp downstream from the site of the transposon insertion in 233PR in order to examine the effects of differential protein truncation on function. Bioinformatic analysis of the E. ictaluri genome revealed a pathogenicity island encoding genes with similarity to a gene cluster encoding putative adhesin/hemolysin genes in E. coli CL3 (Shen et al. 2004). In vivo results demonstrated the importance of the putative adhesins role in E. ictaluri pathogenesis and that protein length correlated to the level of attenuation. In vitro data did not support a role in adhesion, invasion, or intracellular replication in cell culture. The E. ictaluri PAI genes were designated eacA-H for Edwardsiella attenuation complex Results demonstrate that EacF, the putative adhesin, is a virulence factor, but further investigation is required to determine its specific role in E. ictaluri pathogenesis.

The Lyme disease spirochete Borrelia burgdorferi has a very unusual genome composed of one linear chromosome and up to 21 linear and circular plasmids. Several plasmids are known to be important either for mammalian infection or tick colonization. A single spirochete harbors up to 7 different cp32 plasmids; however, nothing is known about their role in mammalian infection. The plasmids in this family are well maintained during in vitro cultivation, making it difficult to study their functions. To effectively deplete the plasmids, an 8kbp fragment containing essential elements for replication and partitioning in B. burgdorferi was amplified from one of the cp32 plasmids, cp32-3, and cloned into the vector pGE22 that carries a gentamycin resistance cassette and essential elements for replication in Escherichia coli. The resulting construct, pG22cp32-3plus, was electroporated into borrelial cells. By increasing gentamycin selection pressure, the spirochetes were forced to lose the corresponding cp32 plasmid. This strategy can be used to knock out other members of the cp32 family.

A conditional silencing system using anti-gB siRNAs was devised to investigate the structure and function of the KSHV gB. Transient co-transfection of plasmids constitutively expressing gB and anti-gB siRNAs in 293 cells substantially inhibited gB mRNA levels and protein production. Similarly, transient expression of siRNAs into the basal cavity-based lymphoma cells (BCBL-1) caused substantial reduction of gB transcription and protein synthesis. TaqMan real-time PCR and infectivity assays showed that gB was essential for virion egress and infectivity. Transfection of a codon-optimized gB not recognized by the anti-gB siRNAs, efficiently rescued virion egress and infectivity in BCBL-1 cells in the presence of siRNAs inhibiting wild-type gB expression. Virion egress experiments with truncated gBs revealed that removal of the entire predicted cytoplasmic domain of gB increased virion egress suggesting the presence of a egress-regulation domain located proximal to the intramembrane sequence within the cytoplasmic domain of gB. All supernatant virions were infectious on 293 cells indicating that the carboxyl terminus of gB is not essential for either virion egress or virus infectivity. To investigate the potential role of gB in tumorigenesis, BCBL-1 cells transiently transfected with anti-gB siRNAs and codon optimized gB were mixed with Matrigel and injected subcutaneously in nude mice. Direct measurement of tumors revealed that BCBL-1 cells transfected with anti-gB siRNAs produced tumors significantly smaller than mock-transfected BCBL-1 cells. Co-transfection of codon optimized gB appeared to abrogate the inhibition of tumor formation by siRNAs. These results show that gB is important for infectivity, virion egress and pleural effusion lymphoma (PEL) formation in mice. Current work focuses on the use of a lentiviral expression system to generate BCBL-1 cells that constitutively express anti-gB siRNAs to improve inhibition of endogenous gB expression.

HSV-1 acquires its final envelope by budding into cytoplasmic vesicles thought to be derived from Trans-Golgi Network (TGN) membranes. This process is facilitated by interactions among the carboxyl termini of viral glycoproteins and tegument proteins. To investigate the relative importance of different viral glycoproteins in cytoplasmic virion morphogenesis, a set of recombinant viruses were constructed silencing expression of the glycoprotein E (ΔgE), the carboxyl terminus of glycoprotein D (gDΔcp), and the membrane protein UL20 (ΔUL20). In addition, recombinant viruses were constructed having the ΔgE+gDΔcp, and the ΔgE+ΔgM (glycoprotein M) deletions. These recombinant viruses were constructed using the double-red, site-directed mutagenesis system implemented on the HSV-1 genome cloned into a bacterial artificial chromosome (bac). The ΔgE, ΔgE+ΔgM, and gDΔcp viruses produced viral plaques that were approximately 50% smaller to those produced by the wild-type virus HSV-1(F strain). The gDΔcp+ΔgE recombinant virus produced viral plaques that were on the average 50% smaller to those produced by either the ΔgE, ΔgE+ΔgM, or the gDΔcp viruses. However, these viral plaques were substantially larger than those produced by previously constructed UL20-null or gK-null viruses. Kinetics of viral replication revealed that all recombinant viruses appeared to produce similar viral titers at late times post infection. However, both the gDΔcp and the ΔgE+gDΔcp viruses appeared to replicate slower than the wild-type virus or the ΔgE and ΔgE+ΔgM viruses. Electron microscopy revealed that all viruses regardless of their different gene mutations produced enveloped virions that were secreted outside, with no apparent accumulation of unenveloped capsids in the cytoplasm of infected cells. These results suggest that the gD and gE carboxyl termini, either alone or in a redundant manner, are not essential in cytoplasmic virion envelopment and egress from infected cells. Furthermore, the results show that gK/UL20 complex serves preeminent roles among all viral glycoproteins in cytoplasmic virion morphogenesis and egress.

Equine Herpesvirus-1 (EHV-1) is an important ubiquitous enzootic equine pathogen, causing significant economic losses to the horse industry. Despite extensive vaccination protocols, EHV-1 continues to be a major cause of epidemic abortion, perinatal mortality, respiratory disease and neurologic disease. EHV-1 infections are usually dealt with by using management practices that limit spread of the disease and secondary complications, providing symptomatic relief to infected horses, but no specific treatment is available. New therapeutic or virucidal agents could have great utility in slowing both the progression and spread of the disease in an epidemic situation. A number of porphyrins and their derivatives have been tested to have activity against HIV, vaccinia, and coronavirus. Porphyrin based compounds were suggested to inhibit virus infection by reducing the fusogenic potential of the virus (Vzorov et al., 2002). However, the mechanism of action of porphyrin-based compounds is not well understood. While current antiherpetic agents target viral DNA replication, interference with the upstream replicative events such as fusion would not adversely affect the host cell metabolism, and makes them important targets for chemotherapeutic intervention of virus dissemination. We screened a number of porphyrin and platinum compounds for EHV-1 antiviral activity by testing their ability to interfere with EHV-1 infection of rabbit kidney and equine cell cultures during the entry and post entry events of the viral life cycle in order to determine if compounds act at the level of binding, penetration, replication, or egress. We identified Cu (III) tetrasulfonated phenylporphyrin and Fe (II) tetrasulfonated phenylporphyrin as lead candidate antiviral compounds on the basis of their in vitro efficacy, cytotoxicity and therapeutic index. These compounds exhibited high antiviral potency during virus-to-cell fusion events, as well as no apparent cytotoxicity in cell culture assays at EHV-1 inhibitory concentrations. Specifically, selected porphyrin compounds inhibited free virus, gB-mediated virus entry, reduced the extent of virus spread, and cell-to-cell fusion in the virus-free cell fusion system. The EHV-1 antiviral properties and other pharmacological characteristics make porphyrins auspicious candidates for the treatment of EHV-1 infections and may promote understanding of membrane fusion events of EHV-1 life cycle.

Three predictive models were developed within a geographic information system using earth observing satellite remote sensing (RS), the Genetic Algorithm for Rule-Set Prediction (GARP) and the growing degree day-water budget (GDD-WB) concept to predict the distribution and potential risk of visceral leishmaniasis (VL) in the State of Bahia, Brazil. The objective was to define the environmental suitability of the disease as well as to obtain a deeper understanding of the eco-epidemiology of VL by associating environmental and climatic variables with disease prevalence. The RS, the GARP model and the GDD-WB model, using different analysis approaches and with the same human prevalence database, predicted similar distribution and abundance patterns for the Lutzomyia longipalpis-Leishmania chagasi system in Bahia. When applied to the ecological zones of Bahia, all three approaches indicate that the highest VL risk is in the interior region of the state, characterized by a semi-arid and hot climate known as Caatinga, while the risk in the Bahia interior forest and the Cerrado ecological regions is lower. The Bahia coastal forest was predicted to be a low-risk area due to unsuitable conditions for the vector and VL transmission. In a second study in Louisiana, dogs considered to be at high risk of infection with Trypanosoma cruzi, were tested serologically using the indirect fluorescent antibody test (IFAT). Serum samples obtained from a total of 122 dogs from three kennels, and from client dogs from local veterinary practices tested by IFAT revealed a prevalence rate of 22.1%. Fifty randomly selected samples from this group were also tested using two rapid experimental immunochromatographic assays designed as alternative or complementary diagnostic tests for T. cruzi infection. Of the fifty samples tested thirteen animals tested positive using rapid assay A and eleven animals tested positive using rapid assay B. In the same group, 11 animals tested positive by IFAT. The sensitivity of rapid assay A and B were 100%; the specificity of rapid assay A was 95%, and rapid assay B was 100% as compared to the IFAT, the test standard. Clinico-pathological reports revealed that cardiac signs are the main indicators of Chagas disease.