Serologic evidence of hantavirus infection in sigmodontine rodents in Mexico

Antibodies to hantaviruses in two species of sigmodontine rodents (Peromyscus maniculatus and Reithrodontomys sumichrasti) collected in central Mexico are reported. Peromyscus maniculatus, a common species throughout much of Mexico, is the reservoir of Sin Nombre virus (SNV), the etiologic agent of the great majority of cases of hantavirus pulmonary syndrome (HPS) in North America. Although the identity of the virus detected in P. maniculatus in Mexico could not be determined by these serologic results, our findings suggest that SNV may occur throughout the range of P. maniculatus in North America. If true, the failure to identify HPS in Mexico is not due to the absence of pathogenic hantaviruses in Mexico.     

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.     

Temporal and Spatial Analysis of Sin Nombre Virus Quasispecies in Naturally Infected Rodents

Sin Nombre virus (SNV) is thought to establish a persistent infection in its natural reservoir, the deer mouse (Peromyscus maniculatus), despite a strong host immune response. SNV-specific neutralizing antibodies were routinely detected in deer mice which maintained virus RNA in the blood and lungs. To determine whether viral diversity played a role in SNV persistence and immune escape in deer mice, we measured the prevalence of virus quasispecies in infected rodents over time in a natural setting. Mark-recapture studies provided serial blood samples from naturally infected deer mice, which were sequentially analyzed for SNV diversity. Viral RNA was detected over a period of months in these rodents in the presence of circulating antibodies specific for SNV. Nucleotide and amino acid substitutions were observed in viral clones from all time points analyzed, including changes in the immunodominant domain of glycoprotein 1 and the 3' small segment noncoding region of the genome. Viral RNA was also detected in seven different organs of sacrificed deer mice. Analysis of organ-specific viral clones revealed major disparities in the level of viral diversity between organs, specifically between the spleen (high diversity) and the lung and liver (low diversity). These results demonstrate the ability of SNV to mutate and generate quasispecies in vivo, which may have implications for viral persistence and possible escape from the host immune system.     

Changes in sin nombre virus antibody prevalence in deer mice across seasons: the interaction between habitat, sex, and infection in deer mice

We examined the impact of season and habitat on Sin Nombre virus (SNV) seroprevalence in deer mice (Peromyscus maniculatus) in Utah's Great Basin Desert from May 2002 through summer 2003. Low mouse captures in 2002 limited analysis for that year. In two seasons during 2003, mouse density and sagebrush cover were positively linked (spring: r = 0.8, P = 0.01; summer: r = 0.8, P = 0.04). In the spring, seroprevalence was negatively correlated with density (r = -0.9, P< 0.01); male and female antibody prevalence did not differ; and scarring was unrelated to antibody status. In the summer, density and antibody prevalence were unrelated; male seroprevalence was higher (chi(2) = 3.6, P = 0.05); and seropositive mice had more scars (t = 2.5, P = 0.02). We speculate nesting behavior could maintain SNV over the winter, whereas summer territoriality could be responsible for transmission.     

Antibody responses to Four Corners hantavirus infections in the deer mouse (Peromyscus maniculatus): identification of an immunodominant region of the viral nucleocapsid protein.

Antibody responses to Four Corners hantavirus (FCV) infections in the deer mouse (Peromyscus maniculatus) were characterized by using FCV nucleocapsid protein (N), glycoprotein 1 (G1), and glycoprotein 2 (G2) recombinant polypeptides in Western immunoblot assays. Strong immunoglobulin G reactivities to FCV N were observed among FCV-infected wild P. maniculatus mice (n = 34) and in laboratory-infected P. maniculatus mice (n = 11). No immunoglobulin G antibody reactivities to FCV G1 or G2 linear determinants were detected. The strongest N responses were mapped to an amino-proximal segment between amino acids 17 and 59 (QLVTARQKLKDAERAVELDPDDVNKSTLQSRRAAVSALETKLG). FCV N antibodies cross-reacted with recombinant N proteins encoded by Puumala, Seoul, and Hantaan viruses.     

表达禽流感病毒M2基因的重组马立克氏病病毒的构建

将禽流感病毒M2基因克隆于真核表达质粒pIRES-EGFP中,使其位于pCMV启动子的调控下,并与绿色荧光蛋白基因（EGFP）串联后,将上述串联基因插入到含MDV CVI988的非必需区US基因的重组质粒pUS2中,构建带标记的重组质粒,然后将此重组质粒转染感染了MDV CVI988的鸡胚成纤维细胞,利用同源重组的方法,筛选了表达禽流感病毒M2基因的重组病毒MDV1。经PCR、Dot-blotting,Western-blotting等实验的结果表明,禽流感病毒M2基因的确插入到MDV1（CVI988）基因组中并获得表达。重组MDV1免疫1日龄SPF鸡21天后,用ELISA可检测到M2蛋白的特异性抗体。接种了重组病毒rMDV的鸡体内针对H9N2疫苗血凝素的抗体滴度(p<0.05)明显提高,以禽流感病毒AIV A/Chicken/Guangdong/00（H9N2）攻毒后进行病毒重分离试验的结果发现,重组病毒能有效地降低病毒的排出量(p<0.01),说明该重组病毒可以用于防制禽流感的免疫。     

siRNA表达载体的构建及其表达

本研究用限制性内切酶消化质粒pCMV-tag-2B,除去巨细胞病毒(Cytomegalovirus,CMV)启动子核苷酸序列,剩下的核苷酸序列作为构建表达siRNA(Small interfering RNA,siRNA)载体的前体。依据文献提供的扩增H1RNA启动子核苷酸序列的引物序列合成一对引物,以带有H1RNA启动子序列的质粒DNA为模板扩增HIRNA启动子序列,插入前体,构建SiRNA的表达载体pCH1。另外将H1RNA启动子插入pGEM．1lfz相应位点,构建瞬时表达载体pGHl。依据EGFP的有效SiRNA抑制位点,合成两条分别为64bp的核苷酸链,通过体外退火,形成双链,然后插入已构建的两个表达载体。将这两个载体分别与表达EGFP蛋白的质粒pEGFP．N3共转染Bel．7402细胞,观察siRNA对EGFP的抑制效应。研究结果表明构建的载体有效表达了siRNA,这些载体可以用于与siRNA相关抗病毒治疗性试验研究。     

Effects of an epitope-specific CD8+ T-cell response on murine coronavirus central nervous system disease: protection from virus replication and antigen spread and selection of epitope escape mutants

Both CD4(+) and CD8(+) T cells are required for clearance of the murine coronavirus mouse hepatitis virus (MHV) during acute infection. We investigated the effects of an epitope-specific CD8(+) T-cell response on acute infection of MHV, strain A59, in the murine CNS. Mice with CD8(+) T cells specific for gp33-41 (an H-2D(b)-restricted CD8(+) T-cell epitope derived from lymphocytic choriomeningitis glycoprotein) were infected with a recombinant MHV-A59, also expressing gp33-41, as a fusion protein with enhanced green fluorescent protein (EGFP). By 5 days postinfection, these mice showed significantly (approximately 20-fold) lower titers of infectious virus in the brain compared to control mice. Furthermore mice with gp33-41-specific CD8(+) cells exhibited much reduced levels of viral antigen in the brain as measured by immunohistochemistry using an antibody directed against viral nucleocapsid. More than 90% of the viruses recovered from brain lysates of such protected mice, at 5 days postinfection, had lost the ability to express EGFP and had deletions in their genomes encompassing EGFP and gp33-41. In addition, genomes of viruses from about half the plaques that retained the EGFP gene had mutations within the gp33-41 epitope. On the other hand, gp33-41-specific cells failed to protect perforin-deficient mice from infection by the recombinant MHV expressing gp33, indicating that perforin-mediated mechanisms were needed. Virus recovered from perforin-deficient mice did not exhibit loss of EGFP expression and the gp33-41 epitope. These observations suggest that the cytotoxic T-cell response to gp33-41 exerts a strong immune pressure that quickly selects epitope escape mutants to gp33-41.     

Long-term dynamics of Sin Nombre viral RNA and antibody in deer mice in Montana

Infections with hantaviruses in the natural host rodent may result in persistent, asymptomatic infections involving shedding of virus into the environment. Laboratory studies have partially characterized the acute and persistent infection by Sin Nombre virus (SNV) in its natural host, the deer mouse (Peromyscus maniculatus). However, these studies have posed questions that may best be addressed using longitudinal studies involving sequential sampling of individual wild-caught, naturally infected mice. Using enzyme immunoassay and polymerase chain reaction (PCR) analysis of monthly blood samples, we followed the infection status of deer mice in a mark-recapture study in Montana for 2 yr. Only six of 907 samples without IgG antibody to SNV contained detectable SNV RNA, suggesting that there is a very brief period of viremia before the host develops detectable antibody. The simultaneous presence of both antibody and viral RNA in blood was detected in consecutive monthly samples for as long as 3 mo. However, chronic infection was typified by alternating characteristics of PCR positivity and PCR negativity. Two possible interpretations of these results are that 1) viral RNA may be consistently present in the blood of chronically infected deer mouse, but that viral RNA is near the limits of PCR detectability or 2) SNV RNA sporadically appears in blood as a consequence of unknown physiological events. The occurrence of seasonal patterns in the proportion of samples that contains antibody and that also contained SNV RNA demonstrated a temporal association between recent infection (antibody acquisition) and presence of viral RNA in blood.     

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.     

用大肠杆菌同源重组获得克隆化重组腺病毒基因组

利用大肠杆菌细胞内质粒间同源重组获得克隆化重组腺病毒基因组 DNA,高效构建携带有外源基因的均一重组腺病毒 .将带有狂犬病毒糖蛋白 (GP)基因和加强型 GFP(enhanced GFP,EGFP)表达盒的重组穿梭质粒 p Ad- Track- CMV/ GP与腺病毒骨架载体质粒 p Ad Easy- 1一起同时电击共转化大肠杆菌 BJ51 83.在 BJ51 83细胞内 ,带有同源序列的重组穿梭质粒与骨架载体可进行同源重组 ,得到以质粒形式存在的克隆化重组腺病毒基因组 p Ad- GP’.以 p Ad- GP’为模板 ,经DNA测序确认 GP基因成功整合入此质粒中的腺病毒基因组 E1区外源基因表达盒中 .线形化的p Ad- GP’转染 2 93细胞后可得到基因组结构均一、在 E1区插入有 GP和 EGFP表达盒的重组腺病毒 ,病毒滴度可达 1× 1 0 8pfu/ ml.电镜下此重组病毒颗粒直径约为 70 nm,略呈球形 ,用荧光显微镜观察感染细胞有很强的 EGFP表达 .实验表明 :利用大肠杆菌同源重组获得克隆化的重组腺病毒基因组 DNA,可高效制备高滴度的均一重组腺病毒     

Activation of cytomegalovirus in pig-to-primate organ xenotransplantation

Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were investigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.     

Establishment of a deer mouse (Peromyscus maniculatus rufinus) breeding colony from wild-caught founders: comparison of reproductive performance of wild-caught and laboratory-reared pairs

The deer mouse (Peromyscus maniculatus) is a natural reservoir for several human pathogens, but little is known about the mechanisms by which such pathogens are maintained in nature. As a first step toward developing a colony of deer mice that were permissive for infection with Sin Nombre (SN) hantavirus, we collected 68 wild P. maniculatus rufinus from central New Mexico. Mice from this cohort were used to establish 26 breeding pairs, of which 85% were fertile. In subsequent generations, fertility decreased slightly to 73% (N = 59) in laboratory-reared F1 and F2 pairs. Wild-caught females delivered 7.2 litters on average (range, 1 to 18), whereas laboratory-reared pairs delivered 5.5 (range, 1 to 13). The average time between pairing and first litter was 106 days for wild-caught animals, whereas that for laboratory-reared pairs was 71 days. None of the pairs displayed a seasonal breeding preference. Cannibalistic behavior increased from 5% in founders to 26% in laboratory-reared pairs. Mean litter size for wild-caught females was 4.3, whereas that for laboratory-reared dams was 4. Founding animals have been maintained in captivity for longer than 2 years, with only 2 deaths (4.8%). Our colony is competent for infection with SN virus. Thus, it should be useful for testing of models for maintenance of SN virus in wild rodents, and other aspects of the virus-host relationship.     

Medroxyprogesterone, immunosuppression, and conceptus size in Peromyscus

Effects of exogenous medroxyprogesterone acetate (MPA) on skin transplant retention and conceptus size were studied in the deer mouse, Peromyscus maniculatus, and the oldfield mouse Peromyscus polionotus. Daily injections of 2 mg MPA significantly prolonged survival of allografts in P. maniculatus and transspecific grafts between P. maniculatus and P. polionotus. Allografts were retained significantly longer than transspecific grafts (17.2 vs 12.4 days) on MPA-treated P. maniculatus. Near-term fetal and placental sizes and weights were not detectably influenced by daily 1-mg MPA injections given to the mother from the 5th through the 19th day of pregnancy. The data are discussed relative to the possible immunosuppressive role of progestins in protecting the allogeneic conceptus from maternal immune rejection.     

Sin Nombre virus infection of deer mice in Montana: characteristics of newly infected mice, incidence, and temporal pattern of infection

Sin Nombre virus (SNV), hosted by the deer mouse (Peromyscus maniculatus), is the principal cause of hantavirus pulmonary syndrome (HPS) in North America. To improve our understanding of factors that contribute to the occurrence of HPS, we conducted an extensive field study of the characteristics of newly infected (as determined by recent acquisition of antibody) deer mice and the temporal pattern of antibody acquisition (seroconversion) from 1994 through 2004 in Montana, USA. We sampled 6,584 individual deer mice, of which 2,747 were captured over multiple trapping periods. Among these 2,747 deer mice, we detected 171 instances of seroconversion. There was no relationship between seroconversion and the acquisition of scars. However, recently infected Montana deer mice were more likely to be older, more likely to be males, and more likely to be in breeding condition. In addition, recently infected male deer mice gained less weight over the 1-mo period following seroconversion than did those that did not acquire antibody, suggesting that SNV infection may have negatively impacted the health of infected rodents. Incidence was highly variable among years, and timing of infections was primarily associated with the breeding season (generally early spring through late fall).     

Cross-transmission studies with Eimeria arizonensis, E. arizonensis-like oocysts and Eimeria langebarteli: host specificity at the genus and species level within the Muridae

Cross-transmission experiments were done using sporulated oocysts of Eimeria arizonensis from Peromyscus truei and Peromyscus maniculatus, and oocysts of 2 putative species that resemble E. arizonensis, i.e., Eimeria albigulae from Neotoma albigula, and Eimeria onychomysis from Onychomys leucogaster. Oocysts of each species were inoculated into representatives of P. maniculatus and the latter 2 rodent species. Other experiments were conducted wherein oocysts of Eimeria langebarteli from Peromyscus leucopus were given to P. truei and P. maniculatus. Oocysts of E. arizonensis from P. truei and P. maniculatus could be transmitted only to P. maniculatus; likewise, oocysts of E. albigulae and E. onychomysis produced patent infections only in N. albigula and O. leucogaster, respectively. Oocysts of E. langebarteli from P. leucopus could be transmitted to P. truei, but not P. maniculatus. These results indicate that E. arizonensis, and the morphologically similar E. albigulae and E. onychomysis, are distinct species that are not transmissible between the genera of their respective hosts (Peromyscus, Neotoma, Onychomys), and that some isolates of E. langebarteli, reported from 6 species of Peromyscus and Reithrodontomys megalotis, may not always be infective to P. maniculatus.     

Fowlpox virus recombinants expressing the envelope glycoprotein of an avian reticuloendotheliosis retrovirus induce neutralizing antibodies and reduce viremia in chickens.

Eight stable fowlpox virus (FPV) recombinants which express the envelope glycoprotein of the spleen necrosis virus (SNV) strain of reticuloendotheliosis virus (REV), an avian retrovirus, were constructed. These recombinants differ in the genomic location of the inserted genes, in the orientation of the insert relative to flanking viral sequences, and in the promoter used to drive expression of the env gene. Of these variables, promoter strength seems to be the most crucial. The P7.5 promoter of vaccinia virus, which is commonly used in the construction of both vaccinia virus and FPV recombinants, resulted in lower levels of expression of the envelope antigen in infected chicken cells compared with a strong synthetic promoter, as determined by immunofluorescence and enzyme-linked immunosorbent assay. Two peptides encoded by the env gene, the 21-kDa transmembrane peptide and a 62-kDa precursor, were detected by immunoprecipitation of labeled proteins from cells infected with recombinant FPVs, using monoclonal antibodies against REV. These peptides comigrated with those precipitated from REV-infected cells. One of the recombinants (f29R-SNenv) was used for vaccination of 1-day-old chickens. Vaccinated chicks developed neutralizing antibodies to SNV more rapidly than did unvaccinated controls following SNV challenge and were protected against both viremia and the SNV-induced runting syndrome.     

Role of small mammals in the ecology of Borrelia burgdorferi in a peri-urban park in north coastal California

The role of small mammals other than woodrats in the enzootiology of the Lyme disease spirochete, Borrelia burgorferi, was assessed in a peri-urban park. Mammals were collected monthly from September through to April. Following tick removal, the animals were tested for B. burgdorferi by culture of ear-punch biopsies. Larvae and nymphs that were intermediate in morphology between Ixodes spinipalpis and Ixodes neotomae occurred on several species of rodents (Peromyscus truei, Peromyscus californicus, Microtus californicus, Rattus rattus and Reithrodontomys megalotis) and the brush rabbit (Sylvilagus bachmani). Morphometric analyses of these I. spinipalpis-like ticks and the offspring from two I. neotomae females from this site suggest that I. neotomae may be conspecific with I. spinipalpis. Borrelia burgdorferi was isolated from eight out of 109 (7.3%), three out of 16 (18.8%), two out of 38 (5.3%) and two out of six (33.3%) P. truei, P. maniculatus, M. californicus and R. rattus, respectively. One brush rabbit yielded the first isolate of B. burgdorferi from a lagomorph in western North America. This isolate and three others derived from unfed I. spinipalpis-like nymphs failed to produce infection when inoculated intradermally into 11–12 P. maniculatus each. Likewise, no spirochetes were detected in 420 Ixodes pacificus nymphs derived from larvae fed on animals inoculated with these isolates. An additional isolate, derived from an I. spinipalpis-like nymph, was recovered by ear-punch biopsies from five out of 12 (42%) needle-inoculated P. maniculatus. However, spirochetes were not detected in 20 I. pacificus nymphs fed as larvae on each of five mice (two infected and three uninfected) inoculated with this isolate. We conclude that brush rabbits and several species of rodents besides woodrats may contribute to the maintenance of B. burgdorferi because they harbour the spirochete and are fed upon by competent enzootic and bridge vectors.