Measles virus replication in lungs of hispid cotton rats after intranasal inoculation.

Hispid cotton rats were inoculated intranasally with either measles virus (MV) Edmonston, a multipassaged, tissue culture-adapted strain of MV, or with one of three clinical MV isolates that had limited passages (three to five times) in tissue culture cells. MV Edmonston was recovered from the lungs of every (n = 37) hispid cotton rat inoculated with this virus for at least 7 days after virus inoculation. Peak pulmonary titers occurred on Day +4 (3.3-4.4 log10/g lung). Scattered areas of inflammation were observed interstitially in lung sections from infected animals stained with hematoxylin and eosin, and a similar pattern of diffuse fluorescence was seen in cryostat sections stained with an indirect fluorescent antibody procedure specific for virus antigens. Fluorescent antibody and virus isolation studies on lung lavage cells both suggested that lung leukocytes were a primary target of the virus. In contrast to these findings, virus was isolated only sporadically from hispid cotton rats inoculated with any of the clinical measles virus isolates. Despite the restricted growth of MV in these animals, cotton rats may be useful for studying certain aspects of measles virus pathogenesis and for screening potential antiviral compounds in vivo.     

Replication and persistence of measles virus in defined subpopulations of human leukocytes.

Replication of Edmonston strain measles virus was studied in several human lymphoblast lines, as well as in defined subpopulations of circulating human leukocytes. It was found that measles virus can productively infect T cells, B cells, and monocytes from human blood. These conclusions were derived from infectious center studies on segregated cell populations, as well as from ultrastructural analyses on cells labeled with specific markers. In contrast, mature polymorphonuclear cells failed to synthesize measles virus nucleocapsids even after infection at a relatively high multiplicity of infection. Measles virus replicated more efficiently in lymphocytes stimulated with mitogens than in unstimulated cells. However, both phytohemagglutinin and pokeweed mitogen had a negligible stimulatory effect on viral synthesis in purified populations of monocytes. In all instances the efficiency of measles virus replication by monocytes was appreciably less than that of mitogenically stimulated lymphocytes or of continuously culture lymphoblasts. Under standard conditions of infection, all of the surveyed lymphoblast lines produced equivalent amounts of measles virus regardless of the major histocompatibility (HL-A) haplotype. Hence, no evidence was found that the HL-A3,7 haplotype conferred either an advantage or disadvantage with respect to measles virus synthesis in an immunologically neutral environment. A persistent infection with measles virus could be established in both T and B lymphoblasts. The release of infectious virus from such persistently infected cells was stable over a period of several weeks and was approximately 100-fold less than peak viral titers obtained in each respective line after acute infection.     

Fluorosis risks to resident hispid cotton rats on land-treatment facilities for petrochemical wastes

Land-treatment of petroleum wastes is a widely used industrial practice, yet there has been no comprehensive evaluation of the long-term risks to human or terrestrial ecosystems from such practices. We evaluated cotton rat (Sigmodon hispidus) populations on three sites in Oklahoma (USA) that historically used land-treatment for disposal of various petroleum wastes (July 1995-March 1997). Average concentrations of fluoride in soil from these sites ranged from 878 to 4317 mg/kg. A census of resident cotton rats on land-treatment sites revealed a high incidence (40% overall) of dental lesions compared to reference populations (<1% dental lesions). During winter there was a 34% to 65% increase compared to summer in frequency of dental lesions in cotton rats on two of the three land-treatment sites. Incidence of dental lesions on two land-treatment sites was greater (9-16%) in female cotton rats compared to males. Cotton rats from land-treatment sites had higher concentrations of fluoride in bone and greater severity of dental lesions compared to reference animals. Dental lesions were considered to be most consistent with dental fluorosis because of elevated fluoride in bone. Neither concentration of fluoride in soil nor level of fluoride in bone was a good predictor of severity of dental lesions in cotton rats on land-treatment sites.     

Replication of measles virus in Vero cells at elevated temperatures

In one-step growth experiment of measles virus (MV) in Vero cells at 39 C, the appearance of MV infectivity was delayed for 24 hr and the maximum titer was reduced by approximately 1,000-fold, when compared with those at 35 C. MV infectivity was thermolabile at the high temperature. Penetration was rather enhanced at 39 C. By Northern blot hybridization, viral RNAs including 50S genome-sized RNA and mRNAs were first detectable 24 hr post-infection (PI) at 35 C and 36 hr PI at 39 C, respectively. Rapid degradation of viral mRNAs was not observed in the infected cells at 39 C. The synthesis of N, F, and M proteins was relatively reduced at the high temperature and appearance of the other viral protein was delayed, in agreement with the time course of viral RNA synthesis. All these data suggest that less efficient synthesis of viral RNA, restriction of synthesis of N, F, and M proteins at translational level and thermolability of infectivity are all involved in the suppressed MV production in Vero cells at 39 C.     

Differential downregulation of CD46 by measles virus strains.

Recently, we found that several lymphotropic wild-type isolates of measles virus (MV) did not lead to the downregulation of CD46 following infection. We hypothesized that either the site of virus isolation, e.g., throat swab versus peripheral blood mononuclear cells, or the cell type used for the isolation may exert selective pressure on a mixed population of viruses, resulting in isolates with the differential properties observed. This hypothesis has been tested by simultaneously isolating MV from a throat swab and peripheral blood mononuclear cells from a single patient by cultivation on B95 and Vero cells. We report that neither the source of MV nor the cell type used for isolation directly influenced the capacity for CD46 modulation of these MV isolates.     

Diversifying animal models: the use of hispid cotton rats (Sigmodon hispidus) in infectious diseases

The hispid cotton rat (Sigmodon hispidus) has been a longstanding laboratory animal model of infectious diseases. In this review, the most common usage of hispid cotton rats as models of infectious diseases is discussed in detail and all organisms, which have been shown to infect cotton rats, are listed. A state of the art overview is given on handling and maintenance of hispid cotton rats as well as experimental techniques such as narcosis and blood withdrawal. Most importantly, through the development of new reagents, the hispid cotton rat can be used to study immune responses against the respective pathogen. Hispid cotton rat cytokine and chemokine genes have been sequenced and cotton rat specific antibodies and cell lines have been produced which in connection with the establishment of immunological methods should facilitate the use of hispid cotton rats as animal models in the pathogenesis of infectious diseases.     

Development of neutralizing antibodies correlates with resolution of interstitial pneumonia after measles virus infection in cotton rats

Cotton rats (Sigmodon hispidus, inbred strain COTTON/NIco) have been shown to be a good animal model to investigate measles virus (MV) immune suppression and to assess alternative vaccine candidates against MV infection. Here we demonstrate that cotton rats develop a bronchusassociated and interstitial pneumonia with necrotic lesions after intranasal infection resembling findings from fatal cases of human MV pneumonia. In the absence of superinfection restitutio ad integrum is observed and overcoming of lung infection correlates with the development of MV specific neutralizing antibodies.     

Evidence of recombination among early-vaccination era measles virus strains

Background  

The advent of live-attenuated vaccines against measles virus during the 1960'ies changed the circulation dynamics of the virus. Earlier the virus was indigenous to countries worldwide, but now it is mediated by a limited number of evolutionary lineages causing sporadic outbreaks/epidemics of measles or circulating in geographically restricted endemic areas of Africa, Asia and Europe. We expect that the evolutionary dynamics of measles virus has changed from a situation where a variety of genomic variants co-circulates in an epidemic with relatively high probabilities of co-infection of the individual to a situation where a co-infection with strains from evolutionary different lineages is unlikely.     

Experimental Helicobacter pylori infection induces antral-predominant, chronic active gastritis in hispid cotton rats (Sigmodon hispidus)

BACKGROUND: The hispid cotton rat has proven to be an excellent animal model for a variety of human infectious disease agents. This study was performed to evaluate the use of the cotton rat as a model of Helicobacter pylori infection. MATERIALS AND METHODS: Thirty-eight inbred cotton rats were orogastrically inoculated with a human strain of H. pylori. Twenty-eight control cotton rats were dosed with vehicle only. Animals were sacrificed at 2, 4, 12, 26, or 38 weeks after inoculation for bacterial and histologic and immunologic examinations. RESULTS: Helicobacter pylori was cultured from the glandular stomach of 89% of the infected cotton rats. The level of colonization was consistently high (approximately 10(4-6) colony-forming units/g tissue). Histologically, the spiral bacteria were demonstrated on the epithelial surface and in the foveolae of the gastric mucosa with highest numbers in the antrum. H. pylori infection was associated with antral-predominant, chronic active gastritis which progressively increased in severity over time. By week 26 of infection, moderate antral gastritis had developed with frequent involvement of the submucosa and formation of lymphocytic aggregates. Splenic T cells from infected cotton rats expressed mRNAs for interferon-gamma, interleukin-4, interleukin-6, and interleukin-10 following in vitro stimulation with H. pylori. Serum levels of H. pylori-specific immunoglobulin G were significantly elevated after 12 weeks of infection. CONCLUSIONS: The H. pylori-infected cotton rat represents a novel animal model that should prove useful for studies of H. pylori-induced chronic active gastritis and factors affecting gastric colonization by this pathogen.     

Infectious clones of cotton leafroll dwarf virus strains used for genotype evaluation in cotton

Cotton blue disease (CBD) and atypical-CBD are the most important viral diseases of cotton plants in the southern region of South America. Common and atypical strains of cotton leafroll dwarf virus (CLRDV and CLRDV-at, respectively) are thought to be causative agents of CBD and atypical-CBD, respectively. Inoculation of test plants via aphid vectors is difficult, as is determining strains via molecular diagnosis; accordingly, it is difficult for breeders to evaluate the effects of blue disease-associated virus infections in cotton lineages. In the present study, we attempted to circumvent these difficulties by producing six full-length cDNA infectious clones from CLRDV and CLRDV-at strains using the Gibson Assembly protocol. For inoculation of the infectious clones, a vacuum chamber-mediated agroinfiltration protocol was adapted and applied. Using this protocol, 90%–100% of cotton plants became infected with the clones, which was not possible via syringe-based agroinfiltration. A genotyping protocol based on RT-qPCR targeting a specific region of the virus P0 protein was also developed, allowing rapid differentiation of CLRDV and CLRDV-at. Applying this protocol to 68 field samples revealed that CLRDV-at was dominant (50%) over CLRDV (5.8%) in single virus infections. These preliminary results imply that CLRDV-at might occupy the ecological niche of CLRDV in the cotton fields of Brazil.     

Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin,inducing specific immune responses and protection in cotton rats

Measles remains a leading cause of child mortality in developing countries. Residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current vaccine in young infants. Because cotton rat respiratory tract is susceptible to measles virus (MV) replication after intranasal (i.n.) challenge, this model can be used to assess the efficacy of MV vaccines. Pursuing a new measles vaccine strategy that might be effective in young infants, we used attenuated Salmonella enterica serovar Typhi CVD 908-htrA and Shigella flexneri 2a CVD 1208 vaccines to deliver mucosally to cotton rats eukaryotic expression plasmid pGA3-mH and Sindbis virus-based DNA replicon pMSIN-H encoding MV hemagglutinin (H). The initial i.n. dose-response with bacterial vectors alone identified a well-tolerated dosage (1 x 10(9) to 7 x 10(9) CFU) and a volume (20 micro l) that elicited strong antivector immune responses. Animals immunized i.n. on days 0, 28, and 76 with bacterial vectors carrying DNA plasmids encoding MV H or immunized parenterally with these naked DNA vaccine plasmids developed MV plaque reduction neutralizing antibodies and proliferative responses against MV antigens. In a subsequent experiment of identical design, cotton rats were challenged with wild-type MV 1 month after the third dose of vaccine or placebo. MV titers were significantly reduced in lung tissue of animals immunized with MV DNA vaccines delivered either via bacterial live vectors or parenterally. Since attenuated serovar Typhi and S. flexneri can deliver measles DNA vaccines mucosally in cotton rats, inducing measles immune responses (including neutralizing antibodies) and protection, boosting strategies can now be evaluated in animals primed with MV DNA vaccines.     

Assessment of space-use patterns in the hispid cotton rat (Sigmodon hispidus)

Summary Ecological interpretation of space use patterns often suffers from two methodological problems: inadequate number of captures per individual and pooling of data over time intervals. Insufficient sample size biases the computation of spatial areas, while pooling data over time intervals may mask shifts in space use due to changes in resource abundance. Radiotelemetry was used to alleviate these problems in an analysis of space use by the hispid cotton rat (Sigmodon hispidus). Home range area was greater for males than females, was largest during summer and winter months, was positively correlated with body hass, and was negatively correlated with population dencity. Exclusivity of home range revealed a high degree of ntolerance (41% exclusivity) and was positively correlated with body mass for males. In addition, like-sex categories (male-male, female-female) were more exclusive than unlike sex categories (male-female).Habitat composition of home ranges of females was significantly different from that of males and from that available. This result suggested home ranges of females were responsive to habitat composition (and quality), while males may respond more to female occurrence than resource availability.Space-use patterns of the hispid cotton rat indicated a solitary existence with greater tolerance of individuals of the opposite sex. Home range size decreased as population size increased, whereas home range overlaps were not affected by population density. These results reinforced the view of a dominance hierarchy in this species and suggested the existence of a polygynous mating system.     

Extent of measles virus spread and immune suppression differentiates between wild-type and vaccine strains in the cotton rat model (Sigmodon hispidus)

Infection of humans with wild-type measles virus leads to strong immune suppression and secondary infections, whereas immunization with an attenuated vaccine strain does not. Using the cotton rat model (Sigmodon hispidus), we investigated whether vaccine and wild-type viruses differ in viral spread and whether this is correlated with inhibition of of proliferation of spleen cells ex vivo after mitogen stimulation. After intranasal infection of cotton rats with wild-type and vaccine strains, it was found that wild-type virus replicates better in lung tissue, spreads to the mediastinal lymph nodes, and induces a more pronounced and longer-lasting inhibition of proliferation of spleen cells ex vivo after mitogen stimulation than does vaccine virus. To induce the same degree of proliferation inhibition, 1,000-fold less wild-type virus was required than vaccine virus. With this system, the virulence of various measles virus isolates and recombinant viruses was tested. Four (in humans and/or monkeys) highly pathogenic virus strains were immunosuppressive, whereas viruses of vaccine virus genotype A were not. Using virus pairs which, due to passage on fibroblasts versus lymphoid cells or due to a point mutation in the hemagglutinin (N481 --> Y), differed in their usage of the two receptor molecules CD46 and CD150 on human cells, it was found that viruses using exclusively CD150 in vitro spread to mediastinal lymph nodes and induced strong immune suppression. These data demonstrate that important parameters of virulence seen in humans, such as viral spread and immune suppression, are reflected in the cotton rat model.     

Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.