Transfer and expression of the lacZ gene in rat brain neurons mediated by herpes simplex virus mutants

Three mutants of herpes simplex virus type 1 (HSV-1) were used to deliver and express the Escherichia coli lacZ gene in cells of the rat central nervous system. Because the lacZ gene was inserted in place of the genes encoding one of the immediate-early viral proteins ICP0 or ICP4 or the early viral protein thymidine kinase, these mutants were compromised or defective in their ability to replicate. All mutant vectors exhibited reduced pathogenesis in animals as compared to the wild type HSV-1 strain KOS. In all cases lacZ was under the control of immediate-early or early viral promoters that are active in the early phase of infection. Expression of beta-galactosidase was observed in cortical neurons following stereotactic inoculation of mutant viruses into adult rat brains; distinct patterns of expression were observed with each mutant vector. Injection of the ICP0 mutant in the frontal cortex and caudate nucleus resulted in beta-galactosidase expression in a substantial number of cells around the inoculation site and at some distance from it for 14 days, with maximum expression after 3 days. The ICP0 vector appeared to have reached the ipsilateral and contralateral cingulate cortex by retrograde transport. Following inoculations of the ICP4 and thymidine kinase vectors into the same brain regions, only a few cells in areas immediately adjacent to the injection track expressed beta-galactosidase and they did so for only a few days. These herpes virus-derived vectors provide a means for the in situ delivery and expression of specific genes in neurons in the central nervous system with little adverse effect on animals.     

Analysis of a naturally-occurring deletion mutant of Spodoptera frugiperda multiple nucleopolyhedrovirus reveals sf58 as a new per os infectivity factor of lepidopteran-infecting baculoviruses

The Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, is structured as a mixture of nine genotypes (A-I). Occlusion bodies (OBs) of SfMNPV-C, -D and -G pure genotypes are incapable of oral transmission; a phenotype which in SfMNPV-C and -D is due to the absence of pif1 and pif2 genes. The complete sequence of the SfMNPV-G genome was determined to identify possible factors involved in this phenotype. Deletions of 4860 bp (22,366-27,225) and 60 bp (119,759-119,818) were observed in SfMNPV-G genome compared with that of the predominant complete genotype SfMNPV-B (132,954 bp). However no genes homologous to previously described per os infectivity factors were located within the deleted sequences. Significant differences were detected in the nucleotide sequence in sf58 gene (unknown function) that produced changes in the amino acid sequence and the predicted secondary structure of the corresponding protein. This gene is conserved only in lepidopteran baculoviruses (alpha- and betabaculoviruses). To determine the role of sf58 in peroral infectivity a deletion mutant was constructed using bacmid technology. OBs of the deletion mutant (Sf58null) were not orally infectious for S. frugiperda larvae, whereas Sf58null rescue virus OBs recovered oral infectivity. Sf58null DNA and occlusion derived virions (ODVs) were as infective as SfMNPV bacmid DNA and ODVs in intrahemocelically infected larvae or cell culture, indicating that defects in ODV or OB morphogenesis were not involved in the loss of peroral infectivity. Addition of optical brightener or the presence of the orally infectious SfMNPV-B OBs in mixtures with SfMNPV-G OBs did not recover Sf58null OB infectivity. According to these results sf58 is a new per os infectivity factor present only in lepidopteran baculoviruses.     

A double deletion prevents replication of the pestivirus bovine viral diarrhea virus in the placenta of pregnant heifers

In contrast to wild type bovine viral diarhea virus (BVDV) specific double deletion mutants are not able to establish persistent infection upon infection of a pregnant heifer. Our data shows that this finding results from a defect in transfer of the virus from the mother animal to the fetus. Pregnant heifers were inoculated with such a double deletion mutant or the parental wild type virus and slaughtered pairwise on days 6, 9, 10 and 13 post infection. Viral RNA was detected via qRT-PCR and RNAscope analyses in maternal tissues for both viruses from day 6 p.i. on. However, the double deletion mutant was not detected in placenta and was only found in samples from animals infected with the wild type virus. Similarly, high levels of wild type viral RNA were present in fetal tissues whereas the genome of the double deletion mutant was not detected supporting the hypothesis of a specific inhibition of mutant virus replication in the placenta. We compared the induction of gene expression upon infection of placenta derived cell lines with wild type and mutant virus via gene array analysis. Genes important for the innate immune response were strongly upregulated by the mutant virus compared to the wild type in caruncle epithelial cells that establish the cell layer on the maternal side at the maternal–fetal interface in the placenta. Also, trophoblasts which can be found on the fetal side of the interface showed significant induction of gene expression upon infection with the mutant virus although with lower complexity. Growth curves recorded in both cell lines revealed a general reduction of virus replication in caruncular epithelial cells compared to the trophoblasts. Compared to the wild type virus this effect was dramtic for the mutant virus that reached only a TCID₅₀ of 1.0 at 72 hours post infection.     

HearNPV Pseudotyped with PIF1, 2, and 3 from MabrNPV: Infectivity and Complex Stability

Effective oral infection is set off by interaction of a group of conserved per os infectivity factors (PIFs) with larval midgut columnar epithelial cells. We constructed pseudotyped viruses by substituting pif1, pif2 or pif3 genes of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) with their homologs from Mamestra bracissae multiple nucleopolyhedrovirus and tested their infectivity to tissue culture cells and to larvae. Transfection and infection assays revealed that all recombinant viruses generated infectious budded virus in both cell culture and in larvae. Electron microscopy showed synthesized occlusion body and occlusion derived virus (ODV) were morphologically indistinguishable from those of the parental virus. By contrast, feeding assays revealed that pseudotyped viruses could not rescue oral infectivity except for pif3 pseudotyped virus that only partially rescued oral infectivity but at a mortality rate much lower than that of the parental HearNPV. Consistent with the bioassay result, PIF complex was detected in ODVs of pif3 pseudotyped virus only but not in pif1 or pif2 pseudotyped viruses. Our results suggest that PIF complex is essential for oral infectivity, and in the formation of the PIF complex, PIF1, 2 are virus-specific while PIF3 does not appear to be as specific and can function in heterologous environment, albeit to a much more limited extent.     

Activity of the simian virus 40 early promoter-enhancer in herpes simplex virus type 1 vectors is dependent on its position, the infected cell type, and the presence of Vmw175.

We have studied some of the parameters governing the expression of a foreign promoter-reporter gene construct incorporated into herpes simplex virus (HSV) type 1. These include the genetic background of the parental virus, the site of transgene insertion within the HSV genome, and the infected cell type. The genetic background of the vector constructs denoted delta 3 was an HSV type 1 mutant deleted for nearly the entire coding portion of Vmw175 (ICP4), the product of the essential immediate-early gene IE3. For vectors denoted +, the IE3 deletion had been repaired by marker rescue. We used as a reporter gene the bacterial chloramphenicol acetyltransferase (CAT) gene, driven by the simian virus 40 (SV40) early promoter and enhancer region. The SV40-cat hybrid gene was inserted either into the HSV thymidine kinase (TK) locus to create the vectors TKScat delta 3 and TKScat+ or into an intergenic site within the BamHI z fragment of the short unique portion of the viral genome to create the vectors GScat delta 3 and GScat+. In Vero and BHK cells infected with TKScat delta 3, CAT activity was first detected at 10 h postinfection and continued to accumulate until 36 h postinfection. In cells of primate origin infected with the replication-competent vector TKScat+, or in primate cells which complement the IE3 deficiency and which were infected with TKScat delta 3, CAT activity was significantly lower than in cells of rodent origin. However, levels of CAT were increased in the presence of cycloheximide, suggesting that the low production of CAT in primate cells was due to repression of SV40-cat hybrid gene expression. In contrast with results with TKScat delta 3 and TKScat+, CAT activity was not detectable in any of the tested cell types infected with GScat delta 3 or GScat+ except under conditions of cycloheximide reversal. These results show that while HSV gene products expressed in the presence of Vmw175 inhibited SV40-cat expression in the tk locus in a cell-type-specific manner, HSV gene products expressed in the presence or absence of Vmw175 inhibited SV40-cat expression in the BamHI z locus independently of cell type.     

Characterization of novel components of the baculovirus per os infectivity factor complex

Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.     

A deletion in the chromosome of Bacteroides thetaiotaomicron that abolishes production of chondroitinase II does not affect survival of the organism in gastrointestinal tracts of exgermfree mice.

Bacteroides thetaiotaomicron, an obligate anaerobe normally found in high concentrations in the human colon, is one of the few colon bacteria that can ferment host mucopolysaccharides such as chondroitin sulfate. Previously, we found that a directed insertional mutation in the gene that codes for the chondroitinase II gene of B. thetaiotaomicron did not affect growth on chondroitin sulfate despite the fact that chondroitinase II accounts for 70% of the total cellular chondroitinase activity. Thus, the chondroitinase II gene did not seem to contribute significantly to growth on chondroitin sulfate when the bacteria were grown in laboratory medium. To determine whether this enzyme is important for bacteria growing in the intestinal tract, we tested the ability of a strain that does not produce chondroitinase II to colonize the intestinal tracts of germfree mice and to compete with wild-type B. thetaiotaomicron. The mutant used in these experiments carried a 0.5-kilobase deletion in the chondroitinase II gene and was constructed so that, unlike the original insertion mutant, it contained no exogenous DNA. The deletion mutant colonized the intestinal tracts of germfree mice at the same levels as the wild type. When a mixture of the deletion mutant and wild type was used to colonize germfree mice, the percent wild type, measured by colony hybridization with the deleted 0.5-kilobase fragment as the hybridization probe, did not rise to 100% even after periods as long as 9 weeks. In most experiments, the percent wild type did not rise significantly above the percent in the original mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

A deletion in the chromosome of Bacteroides thetaiotaomicron that abolishes production of chondroitinase II does not affect survival of the organism in gastrointestinal tracts of exgermfree mice

Bacteroides thetaiotaomicron, an obligate anaerobe normally found in high concentrations in the human colon, is one of the few colon bacteria that can ferment host mucopolysaccharides such as chondroitin sulfate. Previously, we found that a directed insertional mutation in the gene that codes for the chondroitinase II gene of B. thetaiotaomicron did not affect growth on chondroitin sulfate despite the fact that chondroitinase II accounts for 70% of the total cellular chondroitinase activity. Thus, the chondroitinase II gene did not seem to contribute significantly to growth on chondroitin sulfate when the bacteria were grown in laboratory medium. To determine whether this enzyme is important for bacteria growing in the intestinal tract, we tested the ability of a strain that does not produce chondroitinase II to colonize the intestinal tracts of germfree mice and to compete with wild-type B. thetaiotaomicron. The mutant used in these experiments carried a 0.5-kilobase deletion in the chondroitinase II gene and was constructed so that, unlike the original insertion mutant, it contained no exogenous DNA. The deletion mutant colonized the intestinal tracts of germfree mice at the same levels as the wild type. When a mixture of the deletion mutant and wild type was used to colonize germfree mice, the percent wild type, measured by colony hybridization with the deleted 0.5-kilobase fragment as the hybridization probe, did not rise to 100% even after periods as long as 9 weeks. In most experiments, the percent wild type did not rise significantly above the percent in the original mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the plant bos1 mutant highlights necrosis as an efficient defence mechanism during D. dadantii/Arabidospis thaliana interaction

Dickeya dadantii is a broad host range phytopathogenic bacterium provoking soft rot disease on many plants including Arabidopsis. We showed that, after D. dadantii infection, the expression of the Arabidopsis BOS1 gene was specifically induced by the production of the bacterial PelB/C pectinases able to degrade pectin. This prompted us to analyze the interaction between the bos1 mutant and D. dadantii. The phenotype of the infected bos1 mutant is complex. Indeed, maceration symptoms occurred more rapidly in the bos1 mutant than in the wild type parent but at a later stage of infection, a necrosis developed around the inoculation site that provoked a halt in the progression of the maceration. This necrosis became systemic and spread throughout the whole plant, a phenotype reminiscent of that observed in some lesion mimic mutants. In accordance with the progression of maceration symptoms, bacterial population began to grow more rapidly in the bos1 mutant than in the wild type plant but, when necrosis appeared in the bos1 mutant, a reduction in bacterial population was observed. From the plant side, this complex interaction between D. dadantii and its host includes an early plant defence response that comprises reactive oxygen species (ROS) production accompanied by the reinforcement of the plant cell wall by protein cross-linking. At later timepoints, another plant defence is raised by the death of the plant cells surrounding the inoculation site. This plant cell death appears to constitute an efficient defence mechanism induced by D. dadantii during Arabidopsis infection.     

Influence of infection route on the infectivity of baculovirus mutants lacking the apoptosis-inhibiting gene p35 and the adjacent gene p94.

The infectivity of Autographa californica nuclear polyhedrosis virus mutants lacking the apoptosis-inhibiting gene p35 is decreased 1,000-fold or more in larvae of the insect Spodoptera frugiperda if the budded form of the virus is administered by hemocoelic injection; this decrease is correlated with the antiviral effects of apoptosis (R. J. Clem and L. K. Miller, J. Virol. 67:3730-3738, 1993). We have extended this correlation by showing that the infectivity of p35 mutant budded virus is restored to wild-type levels by expression of an unrelated baculovirus apoptosis-inhibiting gene, Cp-iap. We have also examined the oral infectivity of the occluded form of mutants lacking p35, the neighboring p94 gene, or both genes by feeding insects occluded virus. The oral infectivity of the p35 mutant was significantly reduced in S. frugiperda larvae, but this reduction (25-fold) was less than that observed for the hemocoelic route of infection (1,000-fold). The disruption of p94 alone had no apparent effect on infectivity by either route. Unexpectedly, however, the disruption of both p35 and p94 restored oral infectivity to nearly wild-type levels but did not exert this compensatory effect on infectivity by hemocoelic injection. Thus, the infectivity of the double p35/p94 mutant is affected in a route-specific manner in S. frugiperda larvae, suggesting a tissue-specific response to p35 and/or p94. Infectivity in a different host, Trichoplusia ni, was unaffected by all the mutants tested, consistent with previous studies indicating a lack of sensitivity to apoptosis in this species. However, T. ni and S. frugiperda larvae infected with p35 mutants failed to exhibit the symptom of morphological disintegration ("melting") typical of a wild-type infection, suggesting that p35 is required for the infection of some tissues in both species.     

InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals

Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.     

尖镰孢古巴专化型1号生理小种致病相关基因敲除突变体△Focr1-328的生物学特性

由尖镰孢古巴专化型1号生理小种Fusarium oxysporum f. sp. cubense race 1（Focr1）引起的香蕉枯萎病是粉蕉类香蕉品种（AAB）不能规模化种植的最主要因素,其致病机理至今尚不十分清楚。本实验室前期通过T-DNA插入获得Focr1致病性丧失突变体Focr1-328,从Focr1全基因组序列中定位了因T-DNA插入失活的基因,并从野生型菌株Focr1-N2中敲除了该致病相关基因,获得了敲除突变体△Focr1-328。为了明确该致病相关基因的生物学功能,通过活体叶片、活体根     

Deletions in the putative cell receptor-binding domain of Sindbis virus strain MRE16 E2 glycoprotein reduce midgut infectivity in Aedes aegypti

The Sindbis virus (Alphavirus; Togaviridae) strain MRE16 efficiently infects Aedes aegypti mosquitoes that ingest a blood meal containing 8 to 9 log(10) PFU of virus/ml. However, a small-plaque variant of this virus, MRE16sp, poorly infects mosquitoes after oral infection with an equivalent titer. To determine the genetic differences between MRE16 and MRE16sp viruses, we have sequenced the MRE16sp structural genes and found a 90-nucleotide deletion in the E2 glycoprotein that spans the 3' end of the coding region for the putative cell-receptor binding domain (CRBD). We examined the role of this deletion in oral infection of mosquitoes by constructing infectious clones pMRE16icDeltaE200-Y229 and pMRE16ic, representing MRE16 virus genomes with and without the deletion, respectively. A third infectious clone, pMRE16icDeltaE200-C220, was also constructed that contained a smaller deletion extending only to the 3' terminus of the CRBD coding region. Virus derived from pMRE16ic replicated with the same efficiency as parental virus in vertebrate (BHK-21) and mosquito (C6/36) cells and orally infected A. aegypti. Viruses derived from pMRE16icDeltaE200-Y229 and pMRE16icDeltaE200-C220 replicated 10- to 100-fold less efficiently in C6/36 and BHK-21 cells than did MRE16ic virus. Each deletion mutant poorly infected A. aegypti and dramatically reduced midgut infectivity and dissemination. However, all viruses generated nearly equal titers (approximately 6.0 log(10) PFU/ml) in mosquitoes 4 days after infection by intrathoracic inoculation. These results suggest that the deleted portion of the E2 CRBD represents an important determinant of MRE16 virus midgut infectivity in A. aegypti.     

Infection and transmission of Rift Valley fever viruses lacking the NSs and/or NSm genes in mosquitoes: potential role for NSm in mosquito infection

Background

Rift Valley fever virus is an arthropod-borne human and animal pathogen responsible for large outbreaks of acute and febrile illness throughout Africa and the Arabian Peninsula. Reverse genetics technology has been used to develop deletion mutants of the virus that lack the NSs and/or NSm virulence genes and have been shown to be stable, immunogenic and protective against Rift Valley fever virus infection in animals. We assessed the potential for these deletion mutant viruses to infect and be transmitted by Aedes mosquitoes, which are the principal vectors for maintenance of the virus in nature and emergence of virus initiating disease outbreaks, and by Culex mosquitoes which are important amplification vectors.

Methodology and Principal Findings

Aedes aegypti and Culex quinquefasciatus mosquitoes were fed bloodmeals containing the deletion mutant viruses. Two weeks post-exposure mosquitoes were assayed for infection, dissemination, and transmission. In Ae. aegypti, infection and transmission rates of the NSs deletion virus were similar to wild type virus while dissemination rates were significantly reduced. Infection and dissemination rates for the NSm deletion virus were lower compared to wild type. Virus lacking both NSs and NSm failed to infect Ae. aegypti. In Cx. quinquefasciatus, infection rates for viruses lacking NSm or both NSs and NSm were lower than for wild type virus.

Conclusions/Significance

In both species, deletion of NSm or both NSs and NSm reduced the infection and transmission potential of the virus. Deletion of both NSs and NSm resulted in the highest level of attenuation of virus replication. Deletion of NSm alone was sufficient to nearly abolish infection in Aedes aegypti mosquitoes, indicating an important role for this protein. The double deleted viruses represent an ideal vaccine profile in terms of environmental containment due to lack of ability to efficiently infect and be transmitted by mosquitoes.     

Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides.

Synthetic oligonucleotide linkers containing translational termination codons in all possible reading frames were inserted at various positions in the cloned gene encoding the herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein, ICP4. It was determined that the amino-terminal 60 percent of the ICP4 gene was sufficient for trans-induction of a thymidine kinase promoter-CAT chimera (pTKCAT) and negative regulation of an ICP4 promoter-CAT chimera (pIE3CAT); however, it was relatively inefficient in complementing an ICP4 deletion mutant. The amino-terminal ninety amino acids do not appear to be required for infectivity as reflected by the replication competence of a mutant virus containing a linker insertion at amino acid 12. The size of the ICP4 molecule expressed from the mutant virus was consistent with translational restart at the next methionine codon corresponding to amino acid 90 of the deduced ICP4 amino acid sequence.     

Identification of feline immunodeficiency virus rev gene activity.

We constructed 16 deletion mutants from an infectious molecular clone of feline immunodeficiency virus (FIV) and a reporter plasmid carrying the bacterial chloramphenicol acetyltransferase (CAT) gene to identify the rev transactivator activity of the virus. Cotransfections of various mutants and the rev reporter clone bearing a portion of FIV env in addition to the CAT gene revealed that the sequence important for the augmentation of CAT production was located in three separate parts of the virus genome. This enhancement was FIV specific in that the human retrovirus rev and rex gene products did not activate the reporter. The phenotypic properties of an FIV proviral mutant containing a small deletion in the genome were similar to those of rev mutants derived from primate immunodeficiency viruses. These results indicate that FIV, like the other lentiviruses, contains the rev gene in its genome.     

Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus.

The infection of epithelia] swine testicle and intestinal porcine epithelial (IPEC-1) cell lines by adenovirus type 5 (Ad5) has been studied in vitro by using an Ad5-luciferase recombinant containing the firefly luciferase gene as a reporter. Porcine cell lines supported Ad5 replication, showing virus titers, kinetics of virus production, and luciferase expression levels similar to those obtained in human 293 cells, which constitutively express the 5'-end 11% of the Ad5 genome. The tropism of Ad5-based vectors in swine and its ability to induce an efficient immune response against heterologous antigens expressed by foreign genes inserted in these vectors has been determined. Ad5 vectors replicate and express heterologous antigens in porcine lungs and mediastinal and mesenteric lymph nodes. Significant levels of heterologous antigen expression were also demonstrated in the small intestine (jejunum and ileum), but Ad5 replication in this organ was very poor, suggesting that Ad vectors undergo an abortive replication in the porcine small intestine. The tissues infected by Ad5 were dependent on the inoculation route. The oronasal route appeared to be best for inoculation of bronchus-associated lymphoid tissue infection, while the intraperitoneal route was best for gut-associated lymphoid tissue infection. Epithelial cells of bronchioles, macrophages, type II pneumocytes, and follicular dendritic cells were identified as targets for Ad5, while epithelial cells of the intestine were not infected by Ad5. Viruses with a deletion from 79.5 to 84.8 map units in the E3 region, with or without heterologous inserted genes, replicated to lower levels in porcine tissues than did wild-type Ad5. It was also shown that an Ad5 recombinant expressing the four antigenic sites (A, B, C, and D) of transmissible gastroenteritis coronavirus (TGEV) spike protein induced in swine immune responses which neutralized TGEV infectivity. In addition, porcine serum from Ad-TGEV-immune animals provide passive protection when mixed with fully virulent TGEV and orally administered to highly susceptible newborn piglets. These results taken together indicate that swine may be a good animal model for human Ad5 lung infection to aid in the evaluation of candidate adenovirus vaccines and that Ad5 may be suitable as a recombinant viral vaccine or for other applications in swine.