Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus

To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner protein. Based on a total of 32 horse sera analyzed for the presence of EAV antibodies by immunoblot, using the MBP-N or -M fusion proteins and the Xa factor cleavage EAV products, and in the serum neutralization test, there was 100% concordance between the assays. Sera from horses experimentally infected with EAV were reactive in the immunoblot test with both the MBP-N and the MBP-M fusion proteins by day 14 after EAV exposure. The reactivity continued to the end of the experiment at day 145 after infection. This immune reactivity correlated with the detection of neutralizing antibodies in the serum samples. Based on these findings, the recombinant N and M proteins might be useful for serodetection of EAV-infected animals.     

Enzyme-linked immunosorbent assay for serological survey of equine arteritis virus in racehorses

To examine antibodies against equine arteritis virus (EAV), an enzyme-linked immunosorbent assay (ELISA) using purified virus antigen was developed. The results of ELISA were compared with those of serum neutralization (SN) tests. The ELISA absorbance values and the SN titers in sera collected weekly from EAV-infected horses showed a similar pattern. The ELISA could detect antibody to EAV in horses experimentally infected with not only a homologous virus strain, which was used as the ELISA antigen, but also a heterologous strain. Using the ELISA, serum samples collected in 1996 from racehorses in three prefectures (Hokkaido, Ibaraki, and Shiga) were examined and there was no evidence of recent EAV infection among these racehorse populations in Japan. The ELISA should be a simple and highly specific method for rapid screening of EAV infection in racehorses.     

Deficits in discriminated learning remain despite clearance of long-term persistent viral infection in mice

Mice persistently infected with lymphocytic choriomeningitis virus (LCMV) exhibit impaired learning ability. In this report, we determined whether clearance of the virus was associated with restoration of behavioral function. Neonatal Balb/cByJ mice were persistently infected with LCMV and tested as adults in a nonconditional spatial discrimination task. The presence of viral proteins in neurons was confirmed immunohistochemically and infectious virus was quantified in the blood by plaque assay. LCMV-infected adult mice made more errors in a Y-maze avoidance task compared to sham-inoculated controls. After the initial behavioral analysis, infected and control mice received a dose of cytotoxic T-lymphocytes sufficient to clear virus from these mice. Following complete clearance of the virus, mice were re-tested in the behavioral task, 5 months after the original test. No reversal of the learning deficit was seen following viral clearance; mice that had been cleared of the virus and those that remained persistently infected behaved similarly. These data indicate that persistent LCMV infection of the CNS lasting up to 7 months results in discriminated learning impairments that are not reversed by subsequent anti-viral immunocytotherapy.     

Major types of epilepsy surgery

We evaluated the impact of concomitant infection with Hepatitis B virus (HBV) and Hepatitis C virus (HCV) on the clinical course after renal transplantation (Tx). In 335 patients (pts) transplanted between 1991 and 1993 we found 30 (9%) recipients who were positive for Hepatitis B surface antigen (HBsAg) (ELISA, Organon) and anti-HCV antibodies (immunoblot assay Lia Tek) preTx. Chronic liver disease (CLD) (two-fold or greater increase in serum ALT and AST levels for at least six months) developed in 40.7% coinfected pts as compared to 24.4% and 25.7% pts infected only with HCV or HBV, respectively. Maintenance immunosuppression consisted of P + Aza + CsA, mean follow-up time was 28 +/- 15 months. The mean time of the onset of CLD was 3.0 months (range: 1-18 months) after Tx. Percutaneous liver biopsy performed in 5 CLD pts revealed chronic active hepatitis (CAH) in 4 and chronic persistent hepatitis (CPH) in 1 pt. Four pts who had CAH and were positive for HCV RNA (RT PCR) in serum and for HBcAg in liver tissue, received interferon-alpha therapy for 6 months. Clinical improvement of liver function was observed in all of them, but none cleared HBsAg or HCV RNA. One pt lost his graft due to acute rejection. Concomitant infection with HBV and HCV is associated with the high risk of development of CLD early after Tx. We recommend that pretransplant evaluation of both anti-HCV and HBsAg positive pts should include liver biopsy to exclude potential recipients with CAH.     

Influence of viral infection on expression of cell surface antigens in human retinal pigment epithelial cells

BACKGROUND: Subacute viral infection is known to change the phenotype of infected cells, thereby causing immune-mediated tissue damage. The aim of this study was to investigate the expression of different cell surface molecules on human retinal pigment epithelial cells (RPEC) following viral infection, with special emphasis on those having immune-regulatory functions. METHODS: Cultured RPEC were infected with cytomegalovirus (CMV), coxsackie-virus B3 (CVB) or herpes simplex virus type I (HSV). Double-staining fluorescence technique was used for visualization of virus infection and cell surface markers in the same cells by laser microscopy. RESULTS: CMV downregulated MHC class I antigens on RPEC, whereas CVB and HSV did not alter MHC class I antigen expression. No induction of class II antigens was observed in RPEC infected with CVB, HSV or CMV. The intercellular adhesion molecule ICAM-1 (CD54) was strongly expressed in uninfected RPEC, and a slight increase was observed after virus infection. Vascular cell adhesion molecule 1 (VCAM-1) was expressed in low amounts in both uninfected and infected RPEC. No expression of intercellular adhesion molecule 2 (ICAM-2), E-selectin ELAM-1 or lymphocyte-function-associated antigen 1 (LFA-1) was observed on RPEC before or after virus infection. CONCLUSION: Downmodulation of immune-regulating cell surface antigens has been suggested to provide a means of long-term survival of viruses in the infected cell, favoring establishment of persistent infection. Our observation in cultured human RPEC indicates that this mechanism might indeed contribute to the development of disease affecting retinal tissue.     

Viral teratogenesis: brain developmental damage associated with maturation state at time of infection

The rat brain continues to mature after birth and is particularly vulnerable to developmental damage following perinatal insult. Borna disease virus (BDV) infection of postnatal day one (PND-1) rat brain causes a non-encephalitic, persistent infection associated with developmental neuroanatomical and behavioral abnormalities. To test the hypothesis that BDV infection during different brain developmental stages yields variable pathological and clinical disease sequelae, rats were examined for BDV-induced neuroanatomical and behavioral abnormalities following inoculation with BDV on PND-15, and the findings were compared to those resulting from inoculation on PND-1. Similar to rats inoculated with BDV on PND-1, PND-15 inoculated rats developed a persistent infection associated with body weight stunting, abnormal salt taste preference and hippocampal neuron degeneration. However, unlike rats infected with BDV on PND-1, PND-15 inoculated rats did not show signs of cerebellar hypoplasia or hyperactivity. Thus, the risk of BDV-induced damage to specific brain regions, and their associated behaviors, appears, in part, dependent upon the brain's developmental stage at time of BDV-infection. These studies provide evidence of the selective vulnerability of specific neuroanatomic regions and behaviors in developing nervous system to virus-induced damage.     

Incidence and influence of GB virus C and hepatitis C virus infection in patients undergoing bone marrow transplantation

Markers of GB virus C (GBV-C) and hepatitis C virus (HCV) were sought in 80 patients before and after they underwent BMT in a metropolitan hospital in Tokyo between 1990 and 1996. RNA of GBV-C was detected in 14 (18%) patients before BMT. Of the 55 patients who had been transfused, 14 (25%) possessed GBV-C RNA at a frequency significantly higher than in the 25 untransfused patients who were all negative (P < 0.01). HCV RNA was detected in three of the 55 (5%) transfused patients, but in none of the 25 untransfused patients. Sera at 3 months after BMT were available for 57 patients. GBV-C RNA persisted in all 10 patients who were infected before BMT, while it was detected in five of the remaining 47 (11%) patients who were not. However, persistent and/or ongoing GBV-C infection had no appreciable influence on patient morbidity or mortality. Two of the 57 patients were positive for HCV RNA before BMT and this persisted after BMT in both. HCV RNA became positive in eight of the remaining 55 (15%) patients who were negative before BMT. Of the 14 patients who received transfusions screened by the first-generation test at BMT, seven (50%) became positive for HCV RNA, a rate significantly higher than the one of 41 (2%) patients who received transfusions screened by the second-generation test (P < 0.001). These results indicate that BMT patients are at increased risk of GBV-C infection transmitted by transfusions received before and at the time of BMT, and that the risk of HCV infection has decreased after the implementation of the second-generation anti-HCV test.     

Effects of p,p'-DDE on male reproductive organs in peripubertal Wistar rats following a single intraperitoneal injection

The effects of p,p'-DDE on male reproductive organs were investigated in detail in peripubertal Wistar rats following a single intraperitoneal injection. 220 mg/kg of p,p'-DDE (1/4 of LD50) were injected once into prepubertal and postpubertal Wistar rats and its effects were observed until 20 weeks of age. Weights of the body and reproductive organs in p,p'-DDE-injected rats were similar to those in control rats, who were injected with corn oil only. Sperm profile parameters such as spermatid number within the testis, sperm number within the epididymis, sperm motility and its morphology were not different between the prepubertal or postpubertal p,p'-DDE-exposed group and the control group. Like-wise, the histopathological examination at stage VII of the seminiferous epithelium cycle, when the germ cells are sensitive to testosterone, was similar in all three groups during the observation period. Serum levels of testosterone also showed no significant changes by exposure to p,p'-DDE under the conditions of this study. From these results, the antiandrogenic or estrogenic activity attributed to p,p'-DDE was not confirmed in male reproductive organs and no impairment of sperm profile was observed. This study confirmed that the reproductive functions of matured animals are scarcely affected by p,p'-DDE exposure during the peripubertal period and revealed that they might be relatively resistant to exogenous endocrine-disrupting chemicals. p,p'-DDE may threaten the hormonal equilibrium required for normal gonadal development during the organogenesis period, at an earlier stage of life. Further studies are necessary to fully reveal all the effects of p,p'-DDE on male reproductive organs and sperm profile.     

Human immunodeficiency virus infection and women: a survey of missed opportunities for testing and diagnosis

OBJECTIVE: Our purpose was to describe factors that prompted testing of women infected with the human immunodeficiency virus and health encounters in which missed opportunities for diagnosis occurred. STUDY DESIGN: An observational investigation of 81 human immunodeficiency virus-infected women in the Chicago area was performed by means of an interviewer-administered survey. Patient demographic data, health history, and health care contacts were elicited. RESULTS: Sixty-five women (80%) had at least one documented missed opportunity during the 12 months before their diagnosis. Seventy-eight percent of those women with missed opportunities had them occur at reproductive health encounters. Of 25 pregnant women pregnant in the year before their eventual diagnosis, 12 failed to be diagnosed during that pregnancy. CONCLUSION: Despite visits to reproductive health care providers, the presence of symptoms suspicious for human immunodeficiency virus disease, high-risk behaviors, and even specific requests for testing by many of the women, numerous opportunities for the earlier diagnosis of human immunodeficiency virus infection were missed.     

Congenital infection of pigs with ruminant-type pestiviruses

Congenital infections of pigs were induced with two ruminant-type pestiviruses isolated from pigs. One of the viruses was bovine viral diarrhoea virus-like and the other border disease virus-like. Both produced symptoms similar to those observed with low virulence strains of classical swine fever virus. A striking effect of persistent virus infection in post-natal life was stunting in viraemic animals. It was also shown that a congenitally infected pig shed virus for 2.5 years and in sufficient quantity to infect other pigs, even by indirect contact. Unlike ruminants, congenitally infected pigs sometimes had persistent viraemia but eventually eliminated the virus. Clearance of virus from the blood was related to the appearance of neutralizing antibodies. However, clearance from the tissues sometimes took as much as 5 months longer than from the blood.     

A comparative study of gastric motility and secretion after stimulation by 2-deoxy-D-glucose in dogs (author''s transl)

The pathogenesis of Parker's Rat Coronavirus (PRCV) was studied in axenic CD rats. Three to four 9 to 10 week old rats were euthanized daily for eight days after intranasal inoculation. Rats remained free of clinical disease. Virus was recovered from the nasopharynx and trachea after twenty-four hours and from the lung by day three but was not detected in respiratory tract after seven days. Viral antigen was detected by indirect immunofluorescence in the mucosal epithelium of upper respiratory tract and in pulmonary alveolar septae from day two to six postinoculation. Acute rhinitis developed by day two and was associated with mild focal necrosis of respiratory mucosal epithelium. Mild nonsuppurative tracheitis and multifocal interstitial pneumonia appeared by day five and persisted through day eight. Dacryoadenitis did not occur, sialoadenitis was detected in only three rats and virus was recovered from only one submaxillary salivary gland. This experiment indicates that PRCV can be a primary pathogen for the respiratory system of adult rats. In contrast to sialodacryoadenitis (SDA) virus the tropism of PRCV for salivary and lacrimal glands is low.     

Pathogenesis of chronic hepatitis C and associated clinical manifestations

In 20% of patients exposed to hepatitis C virus, infection is transient but, after a few months, the patient remains susceptible to infection with the same strain. Protective immunity is short-lived. This suggests that recovery is related to the cellular immune response, which presumably lyses infected cells, and that the need during recovery for a virus-neutralizing anti-envelope response, is transient. In 80% of patients the infection is persistent, and it seems that antigenic variation of the envelope proteins allows the virus to escape neutralization by anti-envelope responses. The fact that this antigenic variation occurs at a much lower rate in agammaglobulinaemic subjects suggests that the major immune pressure producing this variation is humoral. How the virus-infected cells avoid lysis by cytotoxic T cells, which can be demonstrated in small numbers in the infected liver, remains unclear. The recent observation, that HCV infects CD8 lymphocytes, raises the possibility that virus infection of CD8 cells may impair their function and contribute to persistent infection. The mechanisms of production of cryoglobulin and of autoantibody formation are both unclear.     

Radiolabelled monoclonal antibodies (McAb): an alternate approach to the conventional methods for the assessment of cardiomyocyte damage in an experimental brain-death pig model

BACKGROUND: According to the Ministry of Health and Welfare AIDS Surveillance Committee's report on vertically transmitted human immunodeficiency virus (HIV) infection, there have been eight children with acquired immune deficiency syndrome (AIDS) and 18 children with HIV infection in Japan, totalling 26 in all as of February 1997. A search of the literature fails to reveal any report that deals with many cases of vertically transmitted HIV infection in Japan. METHODS: A primary questionnaire survey was taken of the main medical institutions across the country, followed by a secondary questionnaire survey of physicians and pediatricians who treated the disease. A clinical review was made of 19 children with vertically transmitted HIV infection (including eight AIDS children) according to the 1994 Revised Classification System for HIV Infection in Children. RESULTS: The mean age at diagnosis was 14.5 months and the diagnosis was made at less than 18 months of life in approximately 70% of infected children. In the mean observation period of 16 months, six of eight AIDS children (75%), and one child of group B died. The mean period of observation for the seven dead children was 7 months, and six of seven children died by 36 months of life. The survival period after the diagnosis of AIDS was 15 months. The diagnosis of HIV infection was made based on the clinical symptoms of all children with AIDS. Of 11 children, six (45%) presented with symptoms of HIV infection by 6 months of life, and 10 of 11 children (91%) presented with symptoms by 26 months of life. The noteworthy clinical findings included hepatomegaly, splenomegaly, recurrent respiratory tract infection, lymph node swelling, oral candidiasis, hepatitis, wasting syndrome, HIV encephalopathy and severe pneumonia. The favored age for the start of complications and the magnitude of decrease in the HIV helper cell count varied with each case of complications of HIV infection (wasting syndrome, HIV encephalopathy) or opportunistic infections (cytomegalovirus infection, Mycobacterium avium complex infection). Anti-HIV drugs (mainly zidovudine) had been used in five of eight children with AIDS and were effective in two long survivors alone. CONCLUSIONS: Children who are diagnosed with HIV infection, based on their clinical symptoms, carry a poor prognosis. In this respect, early diagnosis and progress in anti-HIV therapy are necessary.     

Influence of cold or hyperbaric helium-oxygen environments on mouse response to a respiratory viral infection

In investigating the stress effects of chilling (2-3 degrees C) and hypothermia (2-3 degrees C drop in body core temperature mediated by exposure to hyperbaric helium-oxygen atmosphere) on mouse resistance to "influenza," it was noted that these stresses adversely affected the course of pulmonary infection produced by aerosols of the NWS strain of influenza virus. Comparatively, respiratory LD50 values for control animals were about 25 virus plaque-forming units (PFU) with median mortality occurring on day 13. The LD50 values for mice chilled at 2-3 degrees C were about 15 PFU with median mortality on day 7, and for mice exposed to hyperbaric helium, about 12 PFU with median mortality on day 6. Cold or hyperbaric stress impaired interferon production. Impairment was observed at 24 h but not at 12 h post-challenge and persisted for several days until mice became moribund.     

Oral immunization of macaques with attenuated vaccine virus induces protection against vaginally transmitted AIDS

The chimeric simian-human immunodeficiency virus SHIVKU-1, bearing the envelope of human immunodeficiency virus type 1 (HIV-1), causes fulminant infection with subtotal loss of CD4(+) T cells followed by development of AIDS in intravaginally inoculated macaques and thus provides a highly relevant model of sexually transmitted disease caused by HIV-1 in human beings. Previous studies using this SHIV model had shown that the vpu and nef genes were important in pathogenesis of the infection, and so we deleted portions of these genes to create two vaccines, DeltavpuDeltanefSHIV-4 (vaccine 1) and DeltavpuSHIVPPc (vaccine 2). Six adult macaques were immunized subcutaneously with vaccine 1, and six were immunized orally with vaccine 2. Both viruses caused infection in all inoculated animals, but whereas vaccine 1 virus caused only a nonproductive type of infection, vaccine 2 virus replicated productively but transiently for a 6- to 10-week period. Both groups were challenged 6 to 7 months later with pathogenic SHIVKU-1 by the intravaginal route. All four unvaccinated controls developed low CD4(+) T-cell counts (<200/microliter) and AIDS. The 12 vaccinated animals all became infected with SHIVKU-1, and two in group 1 developed a persistent productive infection followed by development of AIDS in one. The other 10 have maintained almost complete control over virus replication even though spliced viral RNA was detected in lymph nodes. This suppression of virus replication correlated with robust antiviral cell-mediated immune responses. This is the first demonstration of protection against virulent SHIV administered by the intravaginal route. This study supports the concept that sexually transmitted HIV disease can be prevented by parenteral or oral immunization.     

Oral inoculation of SCID mice with an attenuated herpes simplex virus-1 strain causes persistent enteric nervous system infection and gastric ulcers without direct mucosal infection

BACKGROUND: After placement of herpes simplex virus type 1 (HSV-1) into the esophageal lumen of BALB/c mice, the virus replicated in enteric neurons within the esophagus and stomach and was transported to the sensory ganglia of the vagus nerve (nodose ganglia), where viral replication also occurs and where ultimately a long term latent infection is established. This described infection of immunocompetent mice primarily involved neuronal cells and associated satellite cells. EXPERIMENTAL DESIGN: Severe combined immunodeficient (SCID) mice were orally infected with an attenuated strain of HSV-1 to better identify sites of viral involvement in the gastrointestinal tract, particularly the mucosa. RESULTS: Three to five weeks after oral inoculation of SCID mice with HSV-1 strain in1814, a persistent viral infection of the gastrointestinal tract was established in most of the mice. Extensive viral replication was detected by immunohistochemistry throughout pathways of the vagus nerve and within the intrinsic enteric nervous system. Despite this ultimately fatal infection, viral replication in the gut occurred almost exclusively in enteric neurons and their processes; viral proteins were occasionally seen in smooth muscle cells immediately adjacent to heavily infected enteric ganglia. More than 50% of these persistently infected mice, when killed 18 to 31 days postinoculation, had gastric ulcers that were identified grossly and histologically. Only one of the 40 gastric ulcers was found to contain viral Ag. The remaining ulcers, although devoid of viral proteins, were found adjacent to virus-infected ganglia. CONCLUSIONS: HSV-1 can enter enteric neurons with minimal initial mucosal involvement, and once inside the nervous system, the virus is contained there despite the absence of a specific host immune response. Furthermore, chronically infected enteric neurons may provide an indirect mechanism for the pathogenesis of gastric ulcers in these immune-deficient mice.     

Results of a control programme for the porcine reproductive and respiratory syndrome in the French 'Pays de la Loire' region

The porcine reproductive and respiratory syndrome (PRRS) virus first entered the Pays de la Loire region in November 1992, with variable effects ranging from sub-clinical seroconversion to severe reproductive failure and piglet mortality, and significant reduction of daily weight gains in finishing pigs. An epidemiological survey was carried out in February 1993. Since the infection prevalence was low (11 infected out of 2310 herds), the pig population was of medium density and the eradication programme of Aujeszky's disease had been successful in the Pays de la Loire region, it was decided (in March 1993) to undertake a control programme for PRRS. In 1993, introduction of infected pigs was known to be the most frequent source by which PRRS virus entered a herd. In the absence of vaccination, this source of virus introduction was reduced by a control programme applied to all members of the regional pig industry, through the impetus of the leaders of the Regional Sanitary Defence Confederation (FRGDS). The control programme was applied on purchased animals (sows, boars, piglets), artificial insemination centres and other environmental factors (people, vehicles, materials, slurry,...). Moreover, pigs from many infected herds were slaughtered. Results showed that in a context of low prevalence and limited spreading to nearby herds, efficient control of animal movements limited the infection spread. At the end of 1993, the PRRS prevalence was 2.7% in the region. Two years after the first outbreak, the PRRS infection could be considered as controlled since 98% of the herds remained free. In order to maintain this low infected status, the control programme was renewed. From this study epidemiological investigations have raised two major initial sources of infection, the use of contaminated semen and the introduction of infected pigs. Around an infected herd, serological screening is still running to detect infection in nearby herds.