Switching species tropism: an effective way to manipulate the feline coronavirus genome

Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.     

Industrial microbiology of solar salt production

Solar salterns can be modeled as giant outdoor chemostats, much like a series of dams on a slow-moving river. Microorganisms and their products play an essential, but sometimes uncharacterized, role in salt production in these ponds, from seawater salinity up through NaCl saturation. They may physically affect the evaporation process and their by-products may chemically modify or bind with dissolved ions. Many solar salt facilities engage microbiologists to establish monitoring programs for analyses of nutrients, standing crop and associated biological variables in the ponds. Other solar salt companies engage microbiologists only when there are “crises” in the ponds that interfere with salt production. Journal of Industrial Microbiology & Biotechnology (2002) 28, 42–47 DOI: 10.1038/sj/jim/7000173 Received 20 May 2001/ Accepted in revised form 13 June 2001     

Rapid and unexpected effects of piscivore introduction on trophic position and diet of perch (Perca flavescens) in lakes recovering from acidification and metal contamination

1. Yellow perch (Perca flavescens) are often the only surviving fish species in acidified lakes. We studied four lakes along a gradient of recovery from acidification and that had different food web complexities. All had abundant yellow perch, two had low piscivore abundance, one had a well‐established piscivore population and one was manipulated by introducing piscivorous smallmouth bass (Micropterus dolomieu). We hypothesised that there would be strong effects on perch abundance, behaviour and diet induced by the presence of piscivores. 2. In the manipulated lake, the bass reduced yellow perch abundance by 75% over a 2‐year period. Concomitantly, perch use of the pelagic habitat fell from 48 to 40%. 3. In contrast to findings from less disturbed systems, yellow perch in the littoral zone of the manipulated lake did not strongly shift from zooplankton to benthic food sources after the arrival of piscivores. Diet analysis using stable carbon isotopes revealed a strong continued reliance on zooplankton in all lakes, independent of the degree of piscivory. The failure to switch to benthos in the refuge area of the littoral zone is most likely related to the depauperate benthos communities in these formerly acidified lakes. 4. Yellow perch in lakes recovering from acidification face a considerable ecological challenge as the necessary switch to benthic diet is hindered by a low abundance of benthos. The arrival of piscivores in these recovering lakes imposes further restrictions on perch access to food items. We infer that future recovery of perch populations (and higher trophic levels) will have to be preceded by the re‐establishment of diverse benthic macroinvertebrate communities in these lakes.     

Predation on Moose Calves by European Brown Bears

Abstract: In North America, brown bears (Ursus arctos) can be a significant predator on moose (Alces alces) calves. Our study in Sweden is the first in which brown bears are the only predator on moose calves. Bears and moose occurred at densities of about 30/1,000 km² and 920/1,000 km², respectively, and bears killed about 26% of the calves. Ninety-two percent of the predation took place when calves were <1 month old. Bear predation was probably additive to other natural mortality, which was about 10% in areas both with and without bears. Females that lost their calves in spring produced more calves the following year (1.54 calves/F) than females that kept their calves (1.11 calves/F), which reduced the net loss of calves due to predation to about 22%.     

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

Acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein

In feline coronavirus (FCoV) pathogenesis, the ability to infect macrophages is an essential virulence factor. Whereas the low-virulence feline enteric coronavirus (FECV) isolates primarily replicate in the epithelial cells of the enteric tract, highly virulent feline infectious peritonitis virus (FIPV) isolates have acquired the ability to replicate efficiently in macrophages, which allows rapid dissemination of the virulent virus throughout the body. FIPV 79-1146 and FECV 79-1683 are two genetically closely related representatives of the two pathotypes. Whereas FECV 79-1683 causes at the most a mild enteritis in young kittens, FIPV 79-1146 almost invariably induces a lethal peritonitis. The virulence phenotypes correlate with the abilities of these viruses to infect and replicate in macrophages, a feature of FIPV 79-1146 but not of FECV 79-1683. To identify the genetic determinants of the FIPV 79-1146 macrophage tropism, we exchanged regions of its genome with the corresponding parts of FECV 79-1683, after which the ability of the FIPV/FECV hybrid viruses to infect macrophages was tested. Thus, we established that the FIPV spike protein is the determinant for efficient macrophage infection. Interestingly, this property mapped to the C-terminal domain of the protein, implying that the difference in infection efficiency between the two viruses is not determined at the level of receptor usage, which we confirmed by showing that infection by both viruses was equally blocked by antibodies directed against the feline aminopeptidase N receptor. The implications of these findings are discussed.     

The effects of selection,drift and genetic variation on life‐history trait divergence among insular populations of natterjack toad,Bufo calamita

Although loss of genetic variation is frequently assumed to be associated with loss of adaptive potential, only few studies have examined adaptation in populations with little genetic variation. On the Swedish west coast, the northern fringe populations of the natterjack toad Bufo calamita inhabit an atypical habitat consisting of offshore rock islands. There are strong among‐population differences in the amount of neutral genetic variation, making this system suitable for studies on mechanisms of trait divergence along a gradient of within‐population genetic variation. In this study, we examined the mechanisms of population divergence using Q_ST–F_ST comparisons and correlations between quantitative and neutral genetic variation. Our results suggest drift or weak stabilizing selection across the six populations included in this study, as indicated by low Q_ST–F_ST values, lack of significant population × temperature interactions and lack of significant differences among the islands in breeding pond size. The six populations included in this study differed in both neutral and quantitative genetic variation. Also, the correlations between neutral and quantitative genetic variation tended to be positive, however, the relatively small number of populations prevents any strong conclusions based on these correlations. Contrary to the majority of Q_ST–F_ST comparisons, our results suggest drift or weak stabilizing selection across the examined populations. Furthermore, the low heritability of fitness‐related traits may limit evolutionary responses in some of the populations.     

Polar bear (Ursus maritimus) cub mortality at a den site in northern Alaska

In March 2009, we documented the death of one member of a triplet polar bear (Ursus maritimus) litter at its den site in the southern Beaufort Sea (SBS) of Alaska. We used a self-contained video camera unit to document activity between den emergence and departure. All three cubs showed low activity levels relative to other cubs observed, and one died within one week of den emergence. Necropsy confirmed that the dead cub had a low body weight and was malnourished. Capture later confirmed that the two surviving cubs were also undersized. Polar bear cub survival is influenced by many factors including litter size and sea ice conditions. Triplet litters are often smaller and suffer higher mortality rates than singletons and twins. This cub was not only a triplet but also born following 2 years of record minimum sea ice extent, both of which may have played a role in this cub’s demise.