Protection against developmental retardation in apolipoprotein E-deficient mice by a fatty neuropeptide: Implications for early treatment of Alzheimer's disease

Stearyl-Nle-¹⁷-VIP (SNV) is a novel agonist of vasoactive intestinal peptide (VIP) exhibiting a 100-fold greater potency than the parent molecule and specificity for a receptor associated with neuronal survival. Here, mice deficient in apolipoprotein E (ApoE), a molecule associated with the etiology of Alzheimer's disease, served as a model to investigate the developmental and protective effects of SNV. In comparison to control animals, the deficient mice exhibited (a) reduced amounts of VIP messenger RNA; (b) decreased cholinergic activity (c) significant retardation in the acquisition of developmental milestones: forelimb placing behavior and cliff avoidance behavior; and (d) learning and memory impairments. Daily injections of SNV to ApoE-deficient newborn pups resulted in increased cholinergic activity and marked improvements in the time of acquisition of behavioral milestones, with peptide-treated animals developing as fast as control animals and exhibiting improved cognitive functions after cessation of peptide treatment. Specificity was demonstrated in that treatment with a related peptide (PACAP), pituitary adenylate cyclase-activating peptide, produced only limited amelioration. As certain genotypes of ApoE increase the probability of Alzheimer's disease, early counseling and preventive treatments may now offer an important route for therapeutics design. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 329–342, 1997     

The effect of ontogenetic development on the anticonvulsant activity of midazolam.

The anticonvulsant effects and duration of protective action of midazolam against Metrazol induced seizures were studied in 528 rats aged 7,12,18,25 and 90 days. The doses of 0.025, 0.05, 0.25, 0.5 and 1.0 mg/kg were administered immediately before Metrazol (100 mg/kg in all but 18-day-old animals where 90 mg/kg were given) for detection of antimetrazol activity at all age groups. The doses of 0.05, 0.25, and/or 0.5 mg/kg were used to study the time course of the protective action of midazolam. Each experimental group consisted of eight animals. Dose-dependent antimetrazol effects of midazolam till now described only in adult animals were demonstrated at all developmental stages studied. There were no qualitative differences in these effects among age groups studied. Midazolam action was better expressed against major Metrazol seizures than against minimal Metrazol seizures. Duration of the protective action depended on the dose tested at all developmental stages, as a rule, lasted longer in young animals than in adult rats. Only quantitative changes of action were found.     

Chemical modification of ascorbic acid and evaluation of its lipophilic derivatives as inhibitors of secretory phospholipase A2 with anti-inflammatory activity

This investigation aims to evaluate the antitumor and antioxidant potential of Chrysaora quinquecirrha (sea nettle) nematocyst venom on Ehrlich ascites carcinoma (EAC) tumor model. Tumor was induced in mice by intraperitoneal injection of EAC cells. The antitumor effect of sea nettle nematocyst venom (SNV) peptide was evaluated by assessing in vitro cytotoxicity, survival time, hematological, and antioxidant parameters. Intraperitoneal injection of SNV peptide increased the survival time of the EAC-bearing mice. The SNV peptide brought back the altered levels of the hematological and antioxidant parameters in a dose dependent manner in EAC-bearing mice. The results were comparable to that of the result obtained from the animals treated with the standard drug 5-fluorouracil (20 mg/kg bw). Thus, present study revealed that SNV peptide possessed significant antitumor and antioxidant activity.     

Relationships of deer mouse movement, vegetative structure, and prevalence of infection with Sin Nombre virus

The effects of vegetative structure on movement of deer mice (Peromyscus maniculatus) were examined in two distinct vegetation associations, one near Hesperus and the other near Molina in western Colorado (USA) from June-October 1994 to October 1998. We monitored movement by live-trapping small mammals within Gambel's oak/mixed-grass (Hesperus) and sage brush/juniper (Molina) vegetation types. Vegetative structure differed between the sites with Molina having more cover provided by shrubs and Hesperus having more cover provided by forbs. Adult male deer mice moved greater distances at Hesperus than at Molina. Sub-adult males tended to move greater distances than did adult females. Relative abundances of deer mice tended to differ by season, but the average relative abundance of deer mice was greater at Molina. Long-term prevalence of infection with SNV was greater at Hesperus and was greatest in adult males at Hesperus (36.1%). Adult males at Molina exhibited a prevalence of infection with SNV of 25.0%. Infection with SNV was highly associated with scars or wounds for adult male, adult female, and juvenile male deer mice at Hesperus, but only for adult female deer mice at Molina. The presence of scars or wounds tended to be associated with greater age, but male deer mice at Hesperus were more likely to have wounds than female deer mice of the same age class. A similar pattern, excluding juveniles, was observed at Molina. Intraspecific interactions and environmentally elicited long-distance movements of deer mice may play a role in prevalence of infection with SNV in these animals.     

SNV, a lipophilic superactive VIP analog, acts through cGMP to promote neuronal survival.

The current study explored whether the neuroprotective effects of vasoactive intestinal peptide (VIP) and its analog Stearyl-Nle17-VIP (SNV) were mediated through cGMP. SNV, was previously found to be 100-fold more potent than VIP in providing neuroprotection. Neuronal survival was assessed in rat cerebral cortical cultures. A cGMP antagonist (RP-8-pCPT-cGMPS, 10(-12)-10(-9) M) reduced the number of surviving neurons (40-60%), this decline was spared in the presence of SNV (10(-13)M). A cGMP agonist (Sp-8-pCPT-cGMPS, 10(-14)-10(-8)M) and SNV (10(-16)-10(-8)M) both provided significant neuroprotection against 10(-12) M of the cGMP antagonist. Immunoassays indicated that SNV induced increases in cGMP (two-threefold) in these cultures, whereas VIP was 1000-fold less potent. These results implicate cGMP as a second messenger for VIP/SNV-mediated effects on neuronal survival.     

Generation of a recombinant cytomegalovirus for expression of a hantavirus glycoprotein

A cytomegalovirus (CMV) was isolated from its natural host, Peromyscus maniculatus, and was designated Peromyscus CMV (PCMV). A recombinant PCMV was constructed that contained Sin Nombre virus glycoprotein G1 (SNV-G1) fused in frame to the enhanced green fluorescent protein (EGFP) gene inserted into a site homologous to the human CMV UL33 (P33) gene. The recombinant CMV was used for expression and immunization of deer mice against SNV-G1. The results of the study indicate that P. maniculatus could be infected with as few as 10 virus particles of recombinant virus. Challenge of P. maniculatus with either recombinant or wild-type PCMV produced no overt pathology in infected animals. P. maniculatus immunized with recombinant virus developed an antibody response to SNV and EGFP. When rechallenged with recombinant virus, animals exhibited an anamnestic response against SNV. Interestingly, a preexisting immune response against PCMV did not prevent reinfection with recombinant PCMV.     

The Nucleocapsid Domain Is Responsible for the Ability of Spleen Necrosis Virus (SNV) Gag Polyprotein To Package both SNV and Murine Leukemia Virus RNA

Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Psi) and another viral vector RNA containing the SNV packaging signal (E). The chimeric gag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Psi. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLV pol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNV cis-acting elements are not ideal substrates for MLV pol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and other cis-acting elements.     

Purification and properties of spleen necrosis virus DNA polymerase.

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.     

A Lethal Disease Model for Hantavirus Pulmonary Syndrome in Immunosuppressed Syrian Hamsters Infected with Sin Nombre Virus

Sin Nombre virus (SNV) is a rodent-borne hantavirus that causes hantavirus pulmonary syndrome (HPS) predominantly in North America. SNV infection of immunocompetent hamsters results in an asymptomatic infection; the only lethal disease model for a pathogenic hantavirus is Andes virus (ANDV) infection of Syrian hamsters. Efforts to create a lethal SNV disease model in hamsters by repeatedly passaging virus through the hamster have demonstrated increased dissemination of the virus but no signs of disease. In this study, we demonstrate that immunosuppression of hamsters through the administration of a combination of dexamethasone and cyclophosphamide, followed by infection with SNV, results in a vascular leak syndrome that accurately mimics both HPS disease in humans and ANDV infection of hamsters. Immunosuppressed hamsters infected with SNV have a mean number of days to death of 13 and display clinical signs associated with HPS, including pulmonary edema. Viral antigen was widely detectable throughout the pulmonary endothelium. Histologic analysis of lung sections showed marked inflammation and edema within the alveolar septa of SNV-infected hamsters, results which are similar to what is exhibited by hamsters infected with ANDV. Importantly, SNV-specific neutralizing polyclonal antibody administered 5 days after SNV infection conferred significant protection against disease. This experiment not only demonstrated that the disease was caused by SNV, it also demonstrated the utility of this animal model for testing candidate medical countermeasures. This is the first report of lethal disease caused by SNV in an adult small-animal model.     

A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA.

We conducted a mutational analysis within the previously defined encapsidation sequence (E) for spleen necrosis virus (SNV), an avian retrovirus. We found that two regions are necessary for efficient SNV replication. The first region is a double hairpin structure as proposed by Konings et al. (1992, J. Virol., 66, 632-640); the second region is located downstream of the hairpins. We showed further that the double hairpin structure is required for efficient SNV RNA encapsidation. Our work is the first to demonstrate, via linker-scanning and site-directed mutagenesis, that a specific RNA secondary structure is required for the encapsidation of retroviral RNA. Analysis of a series of mutations within the E region indicates (i) that preserving the secondary structure of the two hairpins is important for efficient encapsidation and (ii) that the stem regions of the hairpins contain specific sequences critical for encapsidation. Within the hairpins, the presence of at least one of the two conserved GACG four-residue loops, but not the moderately conserved bulge sequence of the first hairpin, is crucial for function. The function of the hairpins is independent of the relative order of the two hairpins. However, the two hairpins are not redundant and are not functionally identical. Replacement of SNV double hairpin sequences with those of Moloney murine leukemia virus (M-MLV) has no detectable effect on the replication of SNV-based retrovirus vectors with reticuloendotheliosis virus strain A (REV-A) helper virus. Furthermore, replacement of the entire E sequence of SNV with that of Moloney murine sarcoma virus (M-MSV) and M-MLV results in retroviral vectors that replicate as well as SNV vectors with wild type SNV E. This result indicates that the encapsidation sequences of M-MSV/M-MLV and SNV are not virus specific and that, during packaging of SNV and MLV RNA with viral proteins from REV-A, the encapsidation sequences are recognized largely by their secondary or tertiary structures.     

Nonreciprocal Pseudotyping: Murine Leukemia Virus Proteins Cannot Efficiently Package Spleen Necrosis Virus-Based Vector RNA

It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Ψ) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Ψ, but MLV can recognize only MLV Ψ. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.     

Temporal analysis of Andes virus and Sin Nombre virus infections of Syrian hamsters

Andes virus (ANDV) and Sin Nombre virus (SNV) are rodent-borne hantaviruses that cause a highly lethal hemorrhagic fever in humans known as hantavirus pulmonary syndrome (HPS). There are no vaccines or specific drugs to prevent or treat HPS, and the pathogenesis is not understood. Syrian hamsters infected with ANDV, but not SNV, develop a highly lethal disease that closely resembles HPS in humans. Here, we performed a temporal pathogenesis study comparing ANDV and SNV infections in hamsters. SNV was nonpathogenic and viremia was not detected despite the fact that all animals were infected. ANDV was uniformly lethal with a mean time to death of 11 days. The first pathology detected was lymphocyte apoptosis starting on day 4. Animals were viremic and viral antigen was first observed in multiple organs by days 6 and 8, respectively. Levels of infectious virus in the blood increased 4 to 5 logs between days 6 and 8. Pulmonary edema was first detected ultrastructurally on day 6. Ultrastructural analysis of lung tissues revealed the presence of large inclusion bodies and substantial numbers of vacuoles within infected endothelial cells. Paraendothelial gaps were not observed, suggesting that fluid leakage was transcellular and directly attributable to infecting virus. Taken together, these data imply that HPS treatment strategies aimed at preventing virus replication and dissemination will have the greatest probability of success if administered before the viremic phase; however, because vascular leakage is associated with infected endothelial cells, a therapeutic strategy targeting viral replication might be effective even at later times (e.g., after disease onset).     

Epizootiology of Sin Nombre and El Moro Canyon hantaviruses, southeastern Colorado, 1995-2000

Sin Nombre virus (SNV) is an etiologic agent of hantavirus pulmonary syndrome. To better understand the natural history of this virus we studied population dynamics and temporal pattern of infection of its rodent hosts in southeastern Colorado (USA) from 1995 to 2000. We present evidence for the presence of two hantaviruses, SNV in deer mice (Peromyscus maniculatus) and El Moro Canyon virus in western harvest mice (Reithrodontomys megalotis), at our study sites. Sin Nombre virus appeared only sporadically in deer mouse populations; overall prevalence of antibody to SNV was 2.6%. El Moro Canyon virus was enzootic: seroconversions occurred throughout the year; antibody prevalence (11.9% overall) showed a delayed-density-dependent pattern, peaking as relative abundance of mice was declining. Males of both host species were more frequently infected than were females. An apparently lower mean survivorship (persistence at the trapping site) for SNV antibody-positive deer mice could indicate a detrimental effect of SNV on its host, but might also be explained by the fact that antibody-positive mice were older when first captured.     

Experimental parasite community perturbation reveals associations between Sin Nombre virus and gastrointestinal nematodes in a rodent reservoir host

Individuals are often co-infected with several parasite species, yet measuring within-host interactions remains difficult in the wild. Consequently, the impacts of such interactions on host fitness and epidemiology are often unknown. We used anthelmintic drugs to experimentally reduce nematode infection and measured the effects on both nematodes and the important zoonosis Sin Nombre virus (SNV) in its primary reservoir (Peromyscus spp.). Treatment significantly reduced nematode infection, but increased SNV seroprevalence. Furthermore, mice that were co-infected with both nematodes and SNV were in better condition and survived up to four times longer than uninfected or singly infected mice. These results highlight the importance of investigating multiple parasites for understanding interindividual variation and epidemiological dynamics in reservoir populations with zoonotic transmission potential.     

Deer mouse movements in peridomestic and sylvan settings in relation to Sin Nombre virus antibody prevalence

Prevalence of antibody to Sin Nombre virus (SNV) has been found to be nearly twice as high in deer mice (Peromyscus maniculatus) in peridomestic settings as in sylvan settings in two studies in Montana and one in New Mexico. We investigated whether this difference may be related to a difference in deer mouse movements in the two settings. We used radiotelemetry to determine home range size and length of movement for 22 sylvan (1991-1992) and 40 peridomestic deer mice (1995-1999). We also determined the percentage of locations inside versus outside of buildings for peridomestic mice. Though variable, average home range size for female deer mice was significantly smaller for peridomestic deer mice than for sylvan deer mice. The smaller home range in peridomestic settings may concentrate shed SNV, and protection from solar ultraviolet radiation inside buildings may increase environmental persistence of SNV. Both these factors could lead to increased SNV exposure of deer mice within peridomestic populations and result in higher antibody prevalence. Peridomestic deer mice moved between buildings and outside areas, which is evidence that SNV can be transmitted between peridomestic and sylvan populations.