中国和日本分离的几株流行性出血热病毒的抗原性比较

采用间接免疫荧光法(IFA)和ELISA法比较了几株中国和日本流行性出血热病毒(EHFV)的抗原性,IFA法不能区分大鼠属和姬鼠属来源的病毒,ELISA竞争试验表明,大鼠型病毒(R22、SR-11和TR-352株)与姬鼠型病毒(A 9株)存在弱单向交叉反应,交叉ELISA证实,A 9株与R22株、SR-11株和TR-352株均有较显著的抗原性差异,但R22,SR-11和TR-352株彼此间抗原性相近,本文讨论了有关EHFV抗原性比较中的一些问题。     

Comparison of virulence between two strains of Rattus serotype hemorrhagic fever with renal syndrome (HFRS) virus in newborn rats

Two strains of hemorrhagic fever with renal syndrome (HFRS) virus from Rattus, SR-11 and KI-262, showed virtually identical antigenicity but differed from prototype strain Hantaan 76-118 (Apodemus origin) in a neutralization test. Wistar newborn rats inoculated intraperitoneally (i.p.) with SR-11, which was isolated from a laboratory rat associated with an outbreak of HFRS, developed clinical signs such as ataxia and limb paralysis and died at about 18 days after inoculation. The LD50 of SR-11 in 1-day-old rats was 10(1.2) focus-forming units (FFU). In contrast, the animals inoculated i.p. or intracerebrally with 10(4) FFU of KI-262, which was from a wild rat in a dumping-ground area--an enzootic focus where no human cases have been recorded--did not show any significant clinical signs. The susceptibility of rats to SR-11 fatal infection was age-dependent. Virus titers in brains, lungs, kidneys, and livers of the rats inoculated with SR-11 were significantly higher than those in the same organs of the animals infected with KI-262. Necrosis of neurons in the brain tissue occurred in the rats infected with SR-11, while it was mild in the animals infected with KI-262.     

Microbiological contamination in genetically modified animals and proposals for a microbiological test standard for national universities in Japan.

The Biosafety Committee of the Japanese Association of Laboratory Animal Facilities of National Universities (JALAN) investigated recent episodes of microbiological contamination in genetically modified mice (GMM), and the countermeasures taken when the contaminated GMM were introduced into animal facilities, by questionnaires addressed to 53 animal facilities belonging to JALAN and serological tests. Although almost all of the contaminated GMM were accepted with conditions such as rederivation after or before reception and housing in designated rooms, contamination with a spectrum of microorganisms was demonstrated in GMM transferred domestically and from abroad. In serological tests, Mycoplasma pulmonis, mouse parvovirus, and mouse encephalomylitis virus were detected in GMM transferred from domestic facilities and from abroad. The present results of the questionnaires and serological tests suggest that GMM are highly and widely contaminated with microorganisms compared with mice from commercial breeders. Thus, we propose a microbiological requirement, including microbiological status--excellent, common, and minimum--as a guide for the transfer and procurement of mice and rats in Japan.     

Transmission of avian influenza A viruses among species in an artificial barnyard

Waterfowl and shorebirds harbor and shed all hemagglutinin and neuraminidase subtypes of influenza A viruses and interact in nature with a broad range of other avian and mammalian species to which they might transmit such viruses. Estimating the efficiency and importance of such cross-species transmission using epidemiological approaches is difficult. We therefore addressed this question by studying transmission of low pathogenic H5 and H7 viruses from infected ducks to other common animals in a quasi-natural laboratory environment designed to mimic a common barnyard. Mallards (Anas platyrhynchos) recently infected with H5N2 or H7N3 viruses were introduced into a room housing other mallards plus chickens, blackbirds, rats and pigeons, and transmission was assessed by monitoring virus shedding (ducks) or seroconversion (other species) over the following 4 weeks. Additional animals of each species were directly inoculated with virus to characterize the effect of a known exposure. In both barnyard experiments, virus accumulated to high titers in the shared water pool. The H5N2 virus was transmitted from infected ducks to other ducks and chickens in the room either directly or through environmental contamination, but not to rats or blackbirds. Ducks infected with the H7N2 virus transmitted directly or indirectly to all other species present. Chickens and blackbirds directly inoculated with these viruses shed significant amounts of virus and seroconverted; rats and pigeons developed antiviral antibodies, but, except for one pigeon, failed to shed virus.     

Persistent infection of chimpanzees with human immunodeficiency virus: serological responses and properties of reisolated viruses.

Persistent infection by human immunodeficiency virus (HIV-1) in the chimpanzee may be valuable for immunopathologic and potential vaccine evaluation. Two HIV strains, the tissue culture-derived human T-cell lymphotropic virus type IIIB (HTLV-IIIB) and in vivo serially passaged lymphadenopathy-associated virus type 1 (LAV-1), were injected intravenously into chimpanzees. Two animals received HTLV-IIIB as either virus-infected H9 cells or cell-free virus. A third animal received chimpanzee-passaged LAV-1. Evaluation of their sera for virus-specific serologic changes, including neutralizations, was done during a 2-year period. During this period all animals had persistently high titers of antibodies to viral core and envelope antigens. All three animals developed a progressively increasing type-specific neutralizing LAV-1 versus HTLV-IIIB antibody titer during the 2-year observation period which broadened in specificity to include HTLV-HIRF, HTLV-IIIMN, and HTLV-IIICC after 6 to 12 months. The antibody titers against both viruses were still increasing by 2 years after experimental virus inoculation. Sera from all animals were capable of neutralizing both homologously and heterologously reisolated virus from chimpanzees. A slightly more rapid type-specific neutralizing response was noted for the animal receiving HTLV-IIIB-infected cells compared with that for cell-free HTLV-IIIB. Sera from all persistently infected chimpanzees were capable of mediating group-specific antibody-mediated complement-dependent cytolysis of HIV-infected cells derived from all isolates tested. Viruses reisolated from all three animals at 20 months after inoculation revealed very similar peptide maps of their respective envelope gp120s, as determined by two-dimensional chymotrypsin oligopeptide analysis. One peptide, however, from the original HTLV-IIIB-inoculated virus was deleted in viruses from all three animals, and in addition, we noted the appearance of a new or modified peptide which was common to LAV-1 as well as to HTLV-IIIB reisolated from infected chimpanzees. It thus appears that a group-specific neutralizing antibody response as well as a group-specific cytotoxic response can develop in chimpanzees after an inoculation of a single HIV variant. This finding suggests that a common, less immunodominant determinant(s) is present on a single HIV strain which could induce group-specific antibodies during viral infection and replication. The identification of this group-specific epitope and the induction of analogous immunity may be relevant to vaccine development against human acquired immunodeficiency syndrome.     

登革病毒对人树突状细胞感染性的研究

探讨登革病毒对人树突状细胞(DC)的感染性。人外周新鲜血常规分离单核细胞,经细胞因子GMCSF、IL4诱导培养成DC,通过形态学特征、细胞表型和淋巴细胞刺激能力鉴定。用登革病毒2型(DV2)感染DC,于作用后6h、24h、48h、72h、96h分别收集上清液和细胞,甲基纤维素微量空斑试验测定病毒滴度,间接免疫荧光法检测细胞上病毒抗原表达,透射电镜观察病毒在细胞内的定位。病毒感染后6h即可在培养上清中测出病毒,病毒滴度在48h达到高峰,以后逐渐下降。间接免疫荧光法证明感染的DC胞浆及胞膜上携带病毒抗原。透射电镜下在病毒感染48h后DC胞浆内可见大量病毒颗粒。树突状细胞是登革病毒感染的靶细胞,病毒可感染DC并产生大量病毒颗粒,可能在其发病机制中起重要作用。     

Persistent infection of macaques with simian-human immunodeficiency viruses.

Chimeric simian-human immunodeficiency viruses (SHIV) containing the human immunodeficiency virus type 1 (HIV-1) tat, rev, env, and, in some cases, vpu genes were inoculated into eight cynomolgus monkeys. Viruses could be consistently recovered from the CD8-depleted peripheral blood lymphocytes of all eight animals for at least 2 months. After this time, virus isolation varied among the animals, with viruses continuing to be isolated from some animals beyond 600 days after inoculation. The level of viral RNA in plasma during acute infection and the frequency of virus isolation after the initial 2-month period were higher for the Vpu-positive viruses. All of the animals remained clinically healthy, and the absolute numbers of CD4-positive lymphocytes were stable. Antibodies capable of neutralizing HIV-1 were generated at high titers in animals exhibiting the greatest consistency of virus isolation. Strain-specific HIV-1-neutralizing antibodies were initially elicited, and then more broadly neutralizing antibodies were elicited. env sequences from two viruses isolated more than a year after infection were analyzed. In the Vpu-negative SHIV, for which virus loads were lower, a small amount of env variation, which did not correspond to that found in natural HIV-1 variants, was observed. By contrast, in the Vpu-positive virus, which was consistently isolated from the host animal, extensive variation of the envelope glycoproteins in the defined variable gp120 regions was observed. Escape from neutralization by CD4 binding site monoclonal antibodies was observed for the viruses with the latter envelope glycoproteins, and the mechanism of escape appears to involve decreased binding of the antibody to the monomeric gp120 glycoproteins. The consistency with which SHIV infection of cynomolgus monkeys is initiated and the similarities in the neutralizing antibody response to SHIV and HIV-1 support the utility of this model system for the study of HIV-1 prophylaxis.     

Dobrava, but not Saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice

Hantaviruses are the causative agents of HFRS and HCPS (hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome), two severe, and often fatal human diseases. Mortality from HFRS varies between hantaviruses; Hantaan and Dobrava show the highest, Seoul intermediate, and Puumala low mortality. Saaremaa, genetically closely related to Dobrava, is also known to induce HFRS, with low or no mortality. In this study, mice were inoculated with Dobrava and Saaremaa viruses to test for infectibility, lethality, viremia, nitric oxide production and antibody responses. Out of suckling mice intracerebrally inoculated with 50, 500 and 5,000 focus-forming units of Dobrava virus, respectively, 1/8, 2/8 and 7/8 died within 18-26 days. In all but one of the lethally infected mice high levels of replicating virus were detected, and most were positive for neutralizing antibodies and showed elevated levels of nitric oxide production. All suckling mice intracerebrally inoculated with 50, 500, or 5,000 focus-forming units of Saaremaa virus survived and all seroconverted. Clearly lower viral titers were observed for the Saaremaa virus-inoculated mice, also when sacrificed at day 18 after infection, compared to those in mice that died following Dobrava virus infection. Dobrava, Saaremaa, Puumala and Hantaan virus infections of adult mice were asymptomatic, and the anti-nucleocapsid protein IgG2a/IgG1-titer ratio was higher in mice inoculated with Dobrava virus than in those inoculated with Saaremaa virus. Elevated nitric oxide production was not detected in asymptomatically infected mice, and iNOS-/- mice, like normal mice, cleared viremia. In conclusion, we show that Dobrava virus and Saaremaa virus induce distinct differences in terms of survival, viremia, nitric oxide production and antibody responses in mice.     

广东省屏障设施小鼠群中小鼠肝炎病毒感染情况

目的了解我省屏障设施小鼠群中小鼠肝炎病毒（MHV）感染情况。方法收集2003-2007年实验动物小鼠血清样品的监测数据,并对MHV感染情况有关数据进行分析。结果在6个屏障设施内抽检小鼠,5个屏障设施内抽检的样品检出MHV毒抗体阳性,检出率分别为1.4%,2.4%,2.8%,2.6%,13.2%。品系方面主要分布在BALB/c,BALB/c-nu/nu,NIH三个品系,检出率分别为3.6%,14%,7.1%。结论屏障设施小鼠群中小鼠肝炎病毒感染较为普遍。     

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.     

Hantaviruses: molecular biology, evolution and pathogenesis

Hantaviruses are tri-segmented negative sense single stranded RNA viruses that belong to the family Bunyaviridae. In nature, hantaviruses are exclusively maintained in the populations of their specific rodent hosts. In their natural host species, hantaviruses usually develop a persistent infection with prolonged virus shedding in excreta. Humans become infected by inhaling virus contaminated aerosol. Unlike asymptomatic infection in rodents, hantaviruses cause two acute febrile diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The mortality rate varies from 0.1% to 40% depending on the virus involved. Hantaviruses are distributed world wide, with over 150,000 HFRS and HPS cases being registered annually. In this review we summarize current knowledge on hantavirus molecular biology, epidemiology, genetic diversity and co-evolution with rodent hosts. In addition, special attention was given in this review to describing clinical manifestation of HFRS and HPS, and advances in our current understanding of the host immune response, treatment, and prevention.     

Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV(162P4). Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.     

Balamuthia mandrillaris, agent of amebic encephalitis: detection of serum antibodies and antigenic similarity of isolates by enzyme immunoassay

ABSTRACT. We report the development of an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Balamuthia mandrillaris , a free-living ameba that is an etiologic agent of granulomatous amebic encephalitis (GAE). As part of the California Encephalitis Project (CEP), we have tested serum and cerebrospinal fluid (CSF) samples from a subgroup of 130 hospitalized encephalitis patients (out of ∼430 samples) over a 16-month period. Case criteria were based on clinical, laboratory, and occupational/recreational histories. All serum samples initially underwent screening by immunofluorescent antibody (IFA) staining with results ranging from no detectable ameba antibodies to titers of 1:256. In addition to the 130 samples tested prospectively, sera and/or CSF from 11 previously confirmed cases of balamuthiasis, six healthy individuals, and earlier CEP submissions with high IFA antibody titers were also tested retrospectively. Among the 130 samples, two cases of balamuthiasis were identified by ELISA and confirmed by the polymerase chain reaction (PCR). The availability of sera from human and animal cases and from varied geographic areas allowed comparisons of serologic similarities of the different Balamuthia strains and human sera. All sera, whether from human or other mammals, reacted with all strains of Balamuthia , as they did with Balamuthia amebae from different geographic areas. Enzyme-linked immunosorbent assay results were consistent with the IFA results. Differences between readings were likely due to cross-reactivity between Balamuthia antigens and unidentified antibodies in serum.     

Adverse effects of mouse hepatitis virus on ascites myeloma passage in the BALB/eJ mouse.

During experimental serial passage of ascites myelomas through BALB/cJ mice, unexpected illness and premature deaths occurred. Postmortem examination of affected mice revealed focal or diffuse discolored depressed areas in the liver and, in some cases, splenomegaly. Histopathologic findings consisted of focal to diffuse areas of necrosis with minimal leukocytic infiltration. Aerobic and anaerobic bacterial cultures of livers and spleens from affected mice were negative. Mouse hepatitis virus (MHV) was isolated from livers of clinically ill mice and from the ascites myeloma lines. An MHV contaminated ascites myeloma line, when passed into nude (nu/nu) mice, killed the animals in 6 days; the virus was isolated from livers of inoculated mice. Attempts to determine the source of the infection were unsuccessful. Serologic survey of newly acquired mice indicated no evidence of antibodies to MHV while mice in holding rooms had titers that ranged from 1:10 to 1:40. Two solid myeloma lines (being maintained by subcutaneous passage) were negative for MHV when tested by virus isolation techniques, and nine lines were negative to 11 murine viruses when tested by mouse antibody production assay. Attempts to demonstrate Eperythrozoon coccoides in control BALB/cJ mice were unsuccessful. Because of the outbreak, changes were made in animal handling procedures. A colony of BALB/cAn mice negative to MHV antibodies was established to provide animals for experimental passage of tumors, and animals in both the breeding and transfer room were placed under filter tops. The results were encouraging. In the four newly established tumor lines, one having been passed 46 times, no illness or unexplained deaths were observed.     

Health surveillance of specific pathogen-free and conventionally-housed mice and rats in Korea.

The present study contains information about proper microbiological monitoring of laboratory animals' health and the standardization of microbiological monitoring methods in Korea. Microbiological quality control for laboratory animals, composed of biosecurity and health surveillance, is essential to guard against research complications and public health dangers that have been associated with adventitious infections. In this study, one hundred and twenty-two mice and ninety rats from laboratory animal breeding companies and one animal facility of the national universities in Korea were monitored in 2000-2003. Histopathologically, thickening of the alveolar walls and lymphocytic infiltration around the bronchioles were observed in mice and rats from microbiologically contaminated facilities. Cryptosporidial oocysts were observed in the gastric pits of only conventionally-housed mice and rats. Helicobacter spp. infection was also detected in 1 of 24 feces DNA samples in mice and 9 of 40 feces DNA samples in rats by PCR in 2003, but they were not Helicobacter hepaticus. This paper describes bacteriological, parasitological, and virological examinations of the animals.     

Hematologic and immunologic responses in common marmosets (Callithrix jacchus) infected with Plasmodium knowlesi and Epstein-Barr virus

The hematologic and immunologic responses to infection with either the Epstein-Barr virus alone or infection with Epstein-Barr virus and Plasmodium knowlesi were studied using common marmosets (Callithrix jacchus). The assays performed included complete blood cell counts, determinations of natural killer cell activity, and determinations of antibody titers to Epstein-Barr virus early antigen, virus capsid antigen and the nuclear antigen. While no animal showed signs of lymphoproliferative disease, it was found that animals infected with Epstein-Barr virus became positive for early antigen, virus capsid antigen and nuclear antigen at low levels. No difference in antibody titers between Epstein-Barr virus infected animals and co-infected animals was observed. An increase also was found in the number of leukocytes in all groups, and an increase in natural killer cells following infection with Epstein-Barr virus. Some depression in natural killer cells was observed in the co-infected animals when compared to Epstein-Barr virus infected animals.     

Detection of lymphocytic choriomeningitis virus antibody in colonies of laboratory animals in Japan

Indirect fluorescent antibody method was applied for a detection of lymphocytic choriomeningitis virus (LCMV) antibody in colonies of laboratory animals in Japan. The results showed that the antibody exist in SPF mice (3/152, 2.2%) and conventional mice (30/539, 5.6%) with the titers ranging from 1:10 to 1:160. The antibody was also detected in 2.2% (2/89) of Syrian hamsters, and 2.9% (2/68) of Apodemus agrarius, 21.4% (3/14) of Japanese harvest mice which have been maintained as laboratory colony for several years. However, the antibody was not demonstrated in Mongolian gerbils, Suncus murinus, guinea pigs and rats, thus far. These results indicate that LCMV infection is present in laboratory animals in Japan, and pointed out the importance of microbiological monitoring for LCMV.     

Pathogenesis and transmission of triple-reassortant swine H1N1 influenza viruses isolated before the 2009 H1N1 pandemic

The 2009 H1N1 pandemic influenza virus represents the greatest incidence of human infection with an influenza virus of swine origin to date. Moreover, triple-reassortant swine (TRS) H1N1 viruses, which share similar host and lineage origins with 2009 H1N1 viruses, have been responsible for sporadic human cases since 2005. Similar to 2009 H1N1 viruses, TRS viruses are capable of causing severe disease in previously healthy individuals and frequently manifest with gastrointestinal symptoms; however, their ability to cause severe disease has not been extensively studied. Here, we evaluated the pathogenicity and transmissibility of two TRS viruses associated with disease in humans in the ferret model. TRS and 2009 H1N1 viruses exhibited comparable viral titers and histopathologies following virus infection and were similarly unable to transmit efficiently via respiratory droplets in the ferret model. Utilizing TRS and 2009 H1N1 viruses, we conducted extensive hematologic and blood serum analyses on infected ferrets to identify lymphohematopoietic parameters associated with mild to severe influenza virus infection. Following H1N1 or H5N1 influenza virus infection, ferrets were found to recapitulate several laboratory abnormalities previously documented with human disease, furthering the utility of the ferret model for the assessment of influenza virus pathogenicity.