No abstract

Targeted recombination within the S (spike) gene of transmissible gastroenteritis coronavirus (TGEV) was promoted by passage of helper respiratory virus isolates in cells transfected with a TGEV-derived defective minigenome carrying the S gene from an enteric isolate. The minigenome was efficiently replicated in trans and packaged by the helper virus, leading to the formation of true recombinant and pseudorecombinant viruses containing the S proteins of both enteric and respiratory TGEV strains in their envelopes. The recombinants acquired an enteric tropism, and their analysis showed that they were generated by homologous recombination that implied a double crossover in the S gene resulting in replacement of most of the respiratory, attenuated strain S gene (nucleotides 96 to 3700) by the S gene of the enteric, virulent isolate. The recombinant virus was virulent and rapidly evolved in swine testis cells by the introduction of point mutations and in-phase codon deletions in a domain of the S gene (nucleotides 217 to 665) previously implicated in the tropism of TGEV. The helper virus, with an original respiratory tropism, was also found in the enteric tract, probably because pseudorecombinant viruses carrying the spike proteins from the respiratory strain and the enteric virus in their envelopes were formed. These results demonstrated that a change in the tropism and virulence of TGEV can be engineered by sequence changes in the S gene.

Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n ‫؍‬ 5), PRRSV inoculated (n ‫؍‬ 5), PCV2 inoculated (n ‫؍‬ 7), and PRRSV and PCV2 inoculated (n ‫؍‬ 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV-and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.

-Porcine circovirus type 2 (PCV2) is considered as the causative agent of postweaning multisystemic wasting syndrome (PMWS) in domestic pigs, where the virus is ubiquitous as evidenced by serological surveys. We present the results of the first nationwide sero-survey on the presence of PCV2 antibodies in European wild boars, and report the first PMWS case in a wild boar from Spain. Sera from 656 hunter harvested wild boars from 45 different geographical sites and 22 additional imported animals were analysed by means of an immunoperoxidase monolayer assay (IPMA). We also examined the tissues from 55 healthy and one diseased wild boars for the presence of PCV2 nucleic acid and PMWS lesions by in situ hybridisation and histopathology, respectively. Additionally, abundance estimates of wild boars and field interviews were carried out on 30 sampling sites. The prevalence of medium to high PCV2 serological titres among the examined wild boars was 47.89 ± 1.9%. Seropositive wild boars appeared in all but one of the geographical regions analysed. Seroprevalence and titre of PCV2 antibodies were closely related to the management of the wild boar populations. Wild boars from intensively managed, farm-like populations had higher prevalence than wild boars living in more natural situations. The effect of wild boar abundance and management on PCV2 antibody prevalence was further evidenced by the high correlation existing between the relative abundance estimates of animals and the percentage of wild boars with medium to high levels of PCV2 antibodies. PCV2 nucleic acid was detected in the tissues of three wild boars. One of these was diagnosed as PMWS. The results, in addition to information on piglet mortalities, suggest a potential role of PMWS in piglet mortality in intensively managed wild boar populations.

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (PCV) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of PCV (which recognises both PCV-1 and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twenty-eight of the 30 Spanish pigs and all three American pigs had PCV in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyer's patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that PCV is present in tissues of almost all pigs affected by PDNS, and PCV has to be considered as a possible agent involved in the pathogenesis of the syndrome.

Transmissible gastroenteritis coronavirus (TGEV) contains eight overlapping genes that are expressed from a 3'-coterminal nested set of leader-containing mRNAs. To facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (TRSs) and introduction of unique restriction endonuclease sites at the 5' end of each gene using an infectious cDNA clone. The recombinant TGEV (rTGEV) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. In contrast, the rTGEV replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rTGEV in which gene 7 expression was abrogated (rTGEV-delta7) were recovered from cDNA constructs, indicating that TGEV gene 7 was a nonessential gene for virus replication. Interestingly, in vivo infections with rTGEV-delta7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that TGEV gene 7 influences virus pathogenesis.

Summary Bacteroides thetaiotaomicron (Bt) is a human colonic symbiont that degrades many different complex carbohydrates (glycans), the identities and amounts of which are likely to change frequently and abruptly from meal-to-meal. To understand how this organism reacts to dynamic growth conditions, we challenged it with a series of different glycan mixtures and measured responses involved in glycan catabolism. Our results demonstrate that individual Bt cells can simultaneously respond to multiple glycans and that responses to new glycans are extremely rapid. The presence of alternative carbohydrates does not alter response kinetics, but reduces expression of some glycan utilization genes as well as the cell’s sensitivity to glycans that are present in lower concentration. Growth in a mixture containing twelve different glycans revealed that Bt preferentially uses some before others. This metabolic hierarchy is not changed by prior exposure to lower priority glycans because re-introducing high priority substrates late in culture re-initiates repression of genes involved in degrading those with lower priority. At least some carbohydrate prioritization effects occur at the level of monosaccharide recognition. Our results provide insight into how a bacterial glycan generalist modifies its responses in dynamic glycan environments and provide essential knowledge to interpret related metabolic behavior in vivo.

The genome of the coronavirus transmissible gastroenteritis virus (TGEV) has been engineered as an expression vector with an infectious cDNA. The vector led to the efficient (>40 g/10 6 cells) and stable (>20 passages) expression of a heterologous gene (green fluorescent protein [GFP]), driven by the transcriptionregulating sequences (TRS) of open reading frame (ORF) 3a inserted in the site previously occupied by the nonessential ORFs 3a and 3b. Expression levels driven by this TRS were higher than those of an expression cassette under the control of regulating sequences engineered with the N gene TRS. The recombinant TGEV including the GFP gene was still enteropathogenic, albeit with a 10-to 10 2 -fold reduction in enteric tissue growth. Interestingly, a specific lactogenic immune response against the heterologous protein has been elicited in sows and their progeny. The engineering of an additional insertion site for the heterologous gene between viral genes N and 7 led to instability and to a new genetic organization of the 3 end of the recombinant viruses. As a consequence, a major species of subgenomic mRNA was generated from a TRS with the noncanonical core sequence 5-CUAAAA-3. Extension of the complementarity between the TRS and sequences at the 3 end of the viral leader was associated with transcriptional activation of noncanonical core sequences. The engineered vector led to expression levels as high as those of well-established vectors and seems very promising for the development of vaccines and, possibly, for gene therapy.