Characterizing the current population structure of potentially invasive species provides a critical context for identifying source populations and for understanding why invasions are successful. Non-native populations inevitably lose genetic diversity during initial colonization events, but subsequent admixture among independently introduced lineages may increase both genetic variation and adaptive potential. Here we characterize the population structure of the gypsy moth (Lymantria dispar Linnaeus), one of the world's most destructive forest pests. Native to Eurasia and recently introduced to North America, the current distribution of gypsy moth includes forests throughout the temperate region of the northern hemisphere. Analyses of microsatellite loci and mitochondrial DNA sequences for 1738 individuals identified four genetic clusters within L. dispar. Three of these clusters correspond to the three named subspecies; North American populations represent a distinct fourth cluster, presumably a consequence of the population bottleneck and allele frequency change that accompanied introduction. We find no evidence that admixture has been an important catalyst of the successful invasion and range expansion in North America. However, we do find evidence of ongoing hybridization between subspecies and increased genetic variation in gypsy moth populations from Eastern Asia, populations that now pose a threat of further human-mediated introductions. Finally, we show that current patterns of variation can be explained in terms of climate and habitat changes during the Pleistocene, a time when temperate forests expanded and contracted. Deeply diverged matrilines in Europe imply that gypsy moths have been there for a long time and are not recent arrivals from Asia.
Two enzyme immunoassays for detection of antibody to rodent coronaviruses were compared. Mouse hepatitis virus (MHV), strain JHM, antigen was in the form of formalin-fixed, infected 17 C1 1 cells. This antigen detected antibody to the homologous strain of MHV as well as to two heterologous MHV strains and a serologically related rat coronavirus, sialodacryodenititis virus. Antibody titers in assays using horseradish peroxidase (HRP)-conjugated or ureiase-conjugated anti-mouse IgG were substantially higher than in an indirect immunofluorescence assay. The ureiase assay was somewhat more sensitive than the HRP assay. MHV-JHM antigen was stable under a variety of storage conditions for at least two months.
Because many strains of mouse hepatitis virus (MHV) infect laboratory mice, no effective vaccine has yet been developed. An alternative approach to control MHV disease is the use of a host cell receptor-targeted ligand. To address the potential usefulness of this approach, a monoclonal antibody directed against the host cell receptor for the coronavirus MHV-A59 was administered to infant mice that were then challenged oronasally with 10(4) intracerebral infant mouse median lethal doses of MHV-A59. Antibody treatment of virus-challenged mice resulted in lower proportions of mice with MHV-A59 in target organs and markedly reduced viral titers in these organs compared with mock-treated infected mice. Some antibody-treated infected mice survived for 7 days after viral challenge, whereas no mock-treated, infected mice survived beyond day 3 after viral inoculation. These results support a receptor-targeted approach to intervention in coronavirus disease.
SummaryThe growth, stability and seroprevalence in laboratory rodents of the two known strains of mouse adenovirus were compared. The FL strain of mouse adenovirus grew in both L 929 murine fibroblasts and in CMT-93 murine rectal carcinoma cells, whereas the K 87 strain grew only in CMT-93 cells. The bulk of the FL progeny virus was released from the host cells. K 87 virus was largely cell-associated. Both virus strains were stable at 37 ° C in liquid medium. The K 87 strain was completely inactivated after 5-15 minutes at 56 ° C, whereas FL infectivity was still detected after two hours at this temperature. Both virus strains were stable in the dessicated state for 14 days, although FL viability was more dependent on the presence of protein in the virus diluent. Seroepidemiologic data suggest that viruses antigenically related to mouse adenovirus are more prevalent among laboratory rats than among laboratory mice and that the virus(es) infecting rats differ from those infecting mice. Results of retrospective serologic testing suggest an association between mouse adenovirus and an outbreak of disease in a mouse breeding colony.
Plaque assays under Sephadex or agarose overlays are described for rat coronaviruses (RCVs) grown in L2 mouse fibroblasts. A plaque assay using Sephadex was simple; however, viable plaques could not be collected for propagation, and fixation was necessary before evaluation. Plaque formation under agarose was optimized using diethylaminoethyl-dextran (DEAE-D) in the pre-treatment and absorption media and trypsin added to the absorption media and agarose overlay. The use of DEAE-D alone, trypsin alone or trypsin combined with DEAE-D significantly increased plaque numbers and visibility. Plaque numbers were highest when pre-treatment media contained DEAE-D, absorption media contained DEAE-D and trypsin, and the agarose overlay contained trypsin. The assay was useful for plaque isolation and quantification of sialodacryoadenitis virus (SDA), Parker's rat coronavirus (PRCV) and other coronavirus isolates from rats and its specificity was demonstrated by plaquereduction neutralization testing. These methods will facilitate production of cloned virus stocks for study of RCV biology and virus quantification for in vitro and in vivo studies of RCVs.
Enterotropic mouse hepatitis virus (MHV) strains have been difficult to grow in cell culture. In an attempt to develop an efficient in vitro cultivation system for enterotropic MHV strains (MHV-RI and MHV-Y), 8 murine cell lines were inoculated with MHV-RI- or MHV-Y-infected infant mouse intestinal homogenates and screened for the production of cytopathic effects. MHV-RI and MHV-Y consistently produced cytopathic effects only in J774A.1 cells. Both strains produced titers of > 10(6) TCID50/ml in subsequent passages in J774.1 cells. MHV strains-1, -3, -A59, -JHM, -S and -DVIM also produced high-titer viral stocks in J774A.1 cells. Therefore J774A.1 cells are the first cells found that support the replication of these 8 enterotropic and respiratory MHV strains. After passage in J774A.1 cells, MHV-RI and MHV-Y could infect previously non-susceptible cell lines (17Cl-1, CMT-93, N18 and NCTC 1469), though cytopathic effects were often negligible with MHV-RI. MHV-Y, but not MHV-RI, grew in L2(Percy) cells. Using L2(Percy) cells, an agarose overlay and Giemsa staining, MHV-Y could be quantified by plaque assay. Infant mouse bioassay, plaque assays and cell culture infections were compared for their sensitivity in detecting MHV-Y in infected intestinal homogenates and cell supernatants.
Rat coronaviruses (RCVs) infect laboratory rats and confound biomedical research results. In vitro systems developed so far have limited the growth in knowledge about RCVs by not permitting generation of plaque-cloned virus stocks, reliable isolation of RCVs from rat tissues, or growth of high titered stocks of all isolates. Due to the fact that less than 20% of L2(Percy) cells were becoming infected, sublines were produced and selected for maximal growth of RCVs. Screening of 238 cell sublines yielded L2p.176 cells which were highly susceptible to all RCVs tested; however, susceptibility declined after 30 passages in vitro. Low-passaged L2p.176 cells were used to isolate virus from natural outbreaks and to propagate individual RCV plaques into high titered stocks. Proteins from six RCV isolates were immunoblotted using polyclonal rat and mouse antibodies to sialodacryoadenitis virus and polyclonal monospecific rabbit and goat antibodies against the peplomer (S) and nucleocapsid (N) proteins of mouse hepatitis virus (MHV). Proteins of two prototype, one Japanese and three wild type RCVs were examined and found to be similar to those of MHV, although the exact sizes and ratios of protein forms were unique for most RCV isolates. This study reports the development of a continuous cell line which reliably supports RCVs opening an opportunity for further in vivo studies of the biology of these agents. As a first step in the characterization of RCVs, we have shown that RCV proteins are very similar to those of MHV.
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