Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida.

Infectious pancreatic necrosis (IPN) virus (IPNV) infection in Atlantic salmon Salmo salar L. post-smolts and its influence on the outcome of secondary infections with infectious salmon anaemia (ISA) virus (ISAV) or Vibrio salmonicida were studied. The infections with ISAV or V salmonicida were performed both in a period of acute IPN and in the following IPNV carrier stage, 3 and 6 to 8 wk after experimental IPNV challenge, respectively. An IPNV carrier condition at low virus titre did not influence the mortality rates after secondary infections. Neither the ISAV infection nor the V. salmonicida infection in experimentally induced IPNV carriers resulted in mortalities different from those observed after challenge of IPNV-free fish. At higher IPNV titres in Atlantic salmon with acute IPN, the outcome of secondary infections was quite different from that observed in IPNV-free fish and in IPNV carriers. In 2 different experiments significantly more fish died when fish with acute IPN were infected with V salmonicida than when fish were infected with V salmonicida alone. Mortality also started earlier in the double-infected group than in the group challenged with V. salmonicida alone, 3 to 4 and 8 d after V salmonicida infection, respectively. Similar results were observed independent of whether mortalities due to IPN alone were registered in the experiments. When Atlantic salmon with acute IPN were infected with ISAV, significantly fewer fish died than when fish were infected with ISAV alone. The ongoing IPNV infection seemed to provide some protection against development of ISA.     

Immunofluorescent Cell Assay of Infectious Pancreatic Necrosis Virus

An immunofluorescent cell (IFC) assay technique was developed for the quantification of infectious pancreatic necrosis (IPN) virus of salmonid fishes. Cover slip cultures of rainbow trout gonad (RTG-2) cells were infected with diluted virus preparations. After incubation to permit antigen development, the cells were stained with antiviral fluorescent antibody, and the number of fluorescing (infected) cells was counted. Optimal conditions for the IFC assay procedure are: (i) the use of RTG-2 cells cultured for at least 3 days at 20 C; (ii) 1-h absorption of IPN virus to RTG-2 cells at 20 C or alternatively, 4 h at 4 C; (iii) staining the infected cell cultures at 10 to 12 h postinfection. A linear relationship between the relative concentration of virus in the inoculum and the number of fluorescent cells in the first cycle of infection was observed. The IFC assay method is more sensitive than the plaque method for the assay of IPN virus.     

The effects of oestrogen and progesterone on re-excretion of infectious bronchitis virus strain G (correction of strainG) in SPF chickens.

The effects of oestrogen and progesterone on the re-excretion of IB virus strain G in chickens infected at day-old was evaluated in this study. Following infection of the chickens at day-old, the birds were swabbed regularly from the trachea and cloaca until no more virus was isolated from either site. Between 10 and 14 weeks of age oestrogen and progesterone were administered by intramuscular injection to infected or control chickens. An infected but non-hormone injected group was maintained. All the birds were monitored for virus re-excretion and/or increase in serum neutralising (SN) antibody levels during this period of treatment. Similar parameters, and the egg production of these birds were evaluated when they reached their natural sexual maturity. At the end of the experiment, the birds were sacrificed and their internal organs especially the reproductive organs were examined for any pathological lesions. The acute phase of the disease following infection at day-old was typical of IB virus infection in terms of clinical signs and virus excretions from trachea and cloaca [1]. The injected hormones failed to induce virus re-excretion as no virus was isolated during this period. There was no change in the level of antibody during this period either. However, when the hens attained their natural sexual maturity, IB virus was re-excreted by all the birds. Isolations was also more frequent from the cloaca than from the trachea. The SN antibody levels of individual showed no definite pattern to correspond with periods of re-excretion in the birds up to the end of the experiment. The control birds remained normal throughout the period of the study.     

Mouse Hepatitis Virus (MHV-2)

Various factor sinfluencing the plaque formation of mouse hepatitis virus (MHV-2) in DBT cell monolayers were studied and a practical method for plaque assay was developed. Infected DBT cells yielded high-titered virus and were a satisfactory source of complement-fixing viral antigen. The predominant cytopathic effect of MHV-2 in DBT cells was cell rounding and detachment, but no syncytial formation was observed. Fluorescent antibody staining revealed specific fluorescence only in the cytoplasm of infected DBT cells. In one-step growth experiment, newly formed virus was first recognized within 4-hr postinfection and showed subsequently a rapid exponential increase. Release of newly formed virus from the cell was rapid, and a continuous release lasted for a certain period of time. The average per-cell yield of active virus was estimated to be about 6–7 × 10² plaque-forming units.     

Establishment of a Seronegative Human T-Cell Leukemia Virus Type 1 (HTLV-1) Carrier State in Rats Inoculated with a Syngeneic HTLV-1-Immortalized T-Cell Line Preferentially Expressing Tax

Human T-cell leukemia virus type 1 (HTLV-1) causes T-cell malignancies in a small percentage of the population infected with the virus after a long carrier state. In the present study, we established a seronegative HTLV-1 carrier state in rats inoculated with a newly established HTLV-1-infected rat T cell line, FPM1. FPM1 originated from rat thymocytes cocultured with a human HTLV-1 producer, MT-2 cells, and expressed rat CD4, CD5, CD25, and HTLV-1 Tax. However, FPM1 scarcely expressed other major HTLV-1 structural proteins and failed to induce typical antibody responses against HTLV-1 in inoculated rats. In contrast, control rats inoculated with MT-2 cells generated significant levels of anti-HTLV-1 antibodies. HTLV-1 proviruses were detected in peripheral blood cells of syngeneic rats inoculated with FPM1 for more than 1 year. Analysis of the flanking region of HTLV-1 provirus integrated into host cells suggested that FPM1 cells remained in these animals over a relatively long period of time. However, a similar seronegative HTLV-1 carrier state was induced in the rats inoculated with mitomycin C-treated FPM1 cells and also in FPM1-inoculated allogeneic rats, suggesting that FPM1 could also transmit HTLV-1 into host cells in vivo. Our findings indicated that (i) HTLV-1-immortalized T cells which preferentially express HTLV-1 Tax persisted in vivo but failed to induce any diseases in immunocompetent syngeneic rats and that (ii) suboptimal levels of HTLV-1 for antibody responses allowed the establishment of persistent HTLV-1 infection.     

Infectious Bovine Rhinotracheitis Virus Infection in Bulls, with Special Reference to Preputial Infection

In an experiment with infectious bovine rhinotracheitis (IBR) virus in two bulls, observed over a period of 122 weeks, the pattern of virus release was studied. Recurrent, unprovoked release of virus was demonstrated after one year in a nasal washing from one of the bulls and in preputial washings of both on 13 and 4 occasions, respectively, and finally in weeks 113 and 110, although clinical disease was not observed. During periods of recurrent virus release, concentrations of virus in the prepuce were generally much lower than during the period of primary infection; usually, however, they were not of negligible titer. The frequency of such periods and the virus titers observed strongly suggest that an IBR antibody carrier should always be considered as a potential source of infection to other animals. When virus was demonstrated in semen an almost equal amount was found in the preputial washing (50 ml). In week 120, virus replication in the respiratory tract and prepuce was induced in both bulls by prednisolone injections. It is concluded that antibody carriers will rarely attain a state of absolute immunity.     

Purification of infectious pancreatic necrosis (IPN) virus and comparison of polypeptide composition of different isolates.

Infectious pancreatic necrosis (IPN) virus was partially purified by freon extraction of infected CHSE-214 cells and concentrated by polyethylene glycol (PEG) precipitation of virus from the medium. Both methods resulted in virus concentrates that could be further purified by two CsCl gradient centrifugations with little loss of infectivity. A Recovery of 80 to 100% of the virus infectivity was obtained and over 100-fold concentration of viral infectivity was achieved by these methods. This purification was used to compare 10 isolates of IPN virus with regard to their physiochemical properties by electron microscopy, buoyant density in CsCl, and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified virions. Electron-microscopic observations showed that the virus isolates were identical in that they were isometric, hexagonal in profile, and had a particle diameter of 71 nm. The buoyant densities of the virus isolates in CsCl were found to be 1.33 g/ml. SDS-gel electrophoresis of the virus isolates revealed the presence of three polypeptides of molecular weight 50, 30, and 27 x 10(3) designated as VP50, VP30, and VP27, respectively.     

Pathogenic simian/human immunodeficiency virus SHIV(KU) inoculated into immunized macaques caused infection, but virus burdens progressively declined with time

Using the simian immunodeficiency virus/human immunodeficiency virus (SHIV)-macaque model of AIDS, we had shown in a previous report that a live, nonpathogenic strain of SHIV, further attenuated by deletion of the vpu gene and inoculated orally into adult macaques, had effectively prevented AIDS following vaginal inoculation with pathogenic SHIV(KU). Examination of lymph nodes from the animals at 18 weeks postchallenge had shown that all six animals were persistently infected with challenge virus. We report here on a 2-year follow-up study on the nature of the persistent infections in these animals. DNA of the vaccine virus was present in the lymph nodes at all time points tested, as far as 135 weeks postchallenge. In contrast, the DNA of SHIV(KU) became undetectable in one animal by week 55 and in three others by week 63. These four macaques have remained negative for SHIV(KU) DNA as far as the last time point examined at week 135. Quantification of the total viral DNA concentration in lymph nodes during the observation period showed a steady decline. All animals developed neutralizing antibody and cytotoxic-T-lymphocyte responses to SHIV(KU) that persisted throughout the observation period. Vaccine-like viruses were isolated from two animals, and a SHIV(KU)-like virus was isolated from one of the two macaques that remained positive for SHIV(KU) DNA. There was no evidence of recombination between the vaccine and the challenge viruses. Thus, immunization with the live vaccine not only prevented disease but also contributed to the steady decline in the virus burdens in the animals.     

Mouse hepatitis virus (MHV-2). Plaque assay and propagation in mouse cell line DBT cells.

Various factor sinfluencing the plaque formation of mouse hepatitis virus (MHV-2) in DBT cell monolayers were studied and a practical method for plaque assay was developed. Infected DBT cells yielded high-titered virus and were a satisfactory source of complement-fixing viral antigen. The predominant cytopathic effect of MHV-2 in DBT cells was cell rounding and detachment, but no syncytial formation was observed. Fluorescent antibody staining revealed specific fluorescence only in the cytoplasm of infected DBT cells. In one-step growth experiment, newly formed virus was first recognized within 4-hr postinfection and showed subsequently a rapid exponential increase. Release of newly formed virus from the cell was rapid, and a continuous release lasted for a certain period of time. The average per-cell yield of active virus was estimated to be about 6-7 X 10(2) plaque-forming units.     

Antigenic comparison of a truncated form of VP2 of infectious pancreatic necrosis (IPN) virus expressed in four different cell types

A truncated form of the structural protein VP2 (truncVP2) of infectious pancreatic necrosis (IPN) virus encompassing amino acids 147-307 was expressed in bacterial, yeast, piscine and mammalian cells. All four recombinant antigens were recognised by a VP2-specific monoclonal antibody by ELISA and immunoblot analysis. However, the minimum amount of r-truncVP2 needed for detection by these methods varies depending on the cell type used for expression. Furthermore, all four recombinant preparations, when used to immunise Atlantic salmon, were capable of inducing antibodies reactive with whole IPNV in ELISA.     

Small-scale trial of live-attenuated influenza vaccine (A/Hong Kong/68).

Seventy-one men who were given live-attenuated A/Hong Kong/68 (H3N2) influenza vaccine during November 1973, and 34 men given placebo were examined for changes in antibody level. Overall, 12 of the 71 men (17%) given the vaccine showed a fourfold rise in haemagglutination-inhibition (HI) antibody titre after 14 days. No such rises were seen in the 34 men given placebo. However, 10 of the men showing a fourfold rise were from 19 who had no detectable HI antibody to this virus before vaccination, representing a conversion rate of 53%. The other two had a HI titre of 1/10 before vaccination. The absence of antibody response, at 14 days, in those with an HI titre of 1/20 or greater may indicated that this represents a protective level against infection. However, the vaccine virus was probably overattenuated and may have constituted a weaker challenge than might occur with a wild strain. No influenza virus was isolated from either group in the week after vaccination and no evidence of transmission to the placebo group was seen. Mild symptoms, chills, muscle pain, and stiffness were more frequently seen in the 12 persons showing a fourfold rise in antibody than in the rest of the volunteers.     

Production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of interleukin-6 (IL-6) but not IL-4, IL-5, and gamma interferon

The activities of cytokines were determined in cerebrospinal fluid (CSF) and serum of mice persistently or intracerebrally acutely infected with lymphocytic choriomeningitis (LCM) virus (LCMV). In contrast to CBA/J (LCMV carrier) mice that responded with low levels of LCMV-specific antibody, high-responder NMRI (carrier) mice showed antibody production by B cells outside of lymphoid organs. The B cells that had infiltrated the brains of LCMV carrier mice exhibited no preferential immunoglobulin isotype or subtype virus-specific antibody production. Phenotypic analysis of the brain infiltrates in virus carrier mice revealed dominance of CD4+ T cells in contrast to virtual absence of CD4+ and dominance of CD8+ in mice with acute LCM. In NMRI but not in CBA/J carrier mice, significant concentrations of interleukin-6 (IL-6) were detected in CSF and serum; IL-2, IL-4, IL-5, granulocyte-macrophage CSF (GM-CSF), and gamma interferon (IFN-gamma) were not elevated. In contrast, during acute, lethal LCM, IL-6 and IFN-gamma were found at high concentrations, and IL-4, IL-5, and GM-CSF were detectable in CSF and serum, but virus-specific antibody-producing cells were not (yet) detectable in the brain. Thus, distinct cytokine patterns are found in acute versus chronic LCMV infection of the brain: in LCM carrier mice, local random-class immunoglobulin production correlated with the absence of IL-2, IL-4, IL-5, and IFN-gamma but active secretion of IL-6.     

The effect of protein-deprivation on the susceptibility to influenza virus infection: a murine model system.

The effect of a 4% albumin diet initiated at weaning on the susceptibility to influenza virus infection was studied in C57Bl mice. Protein-deprivation was found to enhance markedly the susceptibility to a lethal infection with both mouse-virulent and avirulent strains of virus. Viraemia was observed more frequently in protein-deprived mice, and virus persisted longer in the lungs. The humoral immune response following intranasal infection was depressed, with normal levels of IgG antibody but reduced levels of IgM antibody. No difference was found in the seroconversion frequencies between well-nourished and protein-deprived mice. Pre-immunization did not affect the virus titres in the lungs of protein-deprived mice after challenge with the homologous virus, nor did it prevent the spread of virus to the thymus and brain. The results were discussed in terms of the immunocompetence of the malnourished host and of the potential risk of epidemic influenza in children suffering from severe forms of protein-energy malnutrition.     

Mumps and Enteroviral Meningitis in Toronto, 1966

Of 52 children admitted to hospital for apparently typical mumps meningitis in 1966, 50 had their cerebrospinal fluid (CSF) examined. In only 17 was the mumps virus isolated from the CSF. Mumps antihemagglutinin conversions or increments were detected in 32 subjects including 10 whose CSF yielded virus. Antibody conversions were found in 16 patients and fourfold increments in another nine whose serum pairs were collected only one to four days apart. Initial sera from 20 patients were obtained three days or less after the onset of meningitis. Antibody increments were frequently noted about one day after defervescence and clinical improvement. Interferon was detected in CSF from two of eight patients, both of whom yielded virus. Enteroviruses were isolated from CSF and/or feces in seven of 15 cases of aseptic meningitis which occurred between July and October. Six patients including three virus excretors showed enteroviral neutralizing antibody increments during convalescence. The dominant enteroviral serotype was coxsackievirus A9.     

Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus

Cellular immune responses mediated by CD8+ lymphocytes exert efficient control of virus replication during primary simian immunodeficiency virus (SIV) infection. However, the role that antibodies may play in the early control of virus replication remains unclear. To evaluate how antibody responses may affect virus replication during primary SIVmac infection, we depleted rhesus monkeys of B cells with anti-CD20 antibody. In normal rhesus monkeys immunized with tetanus toxoid, anti-CD20 treatment and resulting depletion of B cells inhibited the generation of antitetanus antibodies, while tetanus-specific T-cell responses were preserved. During the first 4 weeks after inoculation with SIVmac251, development of SIV-specific neutralizing antibody was delayed, and titers were significantly lower in B-cell-depleted monkeys than control-antibody-treated monkeys. Despite the lower neutralizing antibody titers, the levels of plasma SIV RNA and the linear slope of the decline seen in B-cell-depleted monkeys did not differ from that observed in monkeys treated with control antibody. However, beginning at day 28 after SIV infection, the B-cell-depleted monkeys showed a significant inverse correlation between neutralizing antibody titers and plasma virus level. These results suggest that the rapid decline of peak viremia that typically occurs during the first 3 weeks of infection was not significantly affected by SIV-specific antibodies. However, the inverse correlation between neutralizing antibodies and plasma virus level during the postacute phases of infection suggests that humoral immune responses may contribute to the control of SIV replication.     

A prospective study on correlation between the decrease in anti-P17 antibody level and progression to AIDS in asymptomatic carriers of HIV.

As the majority of human immunodeficiency virus (HIV) carriers are in asymptomatic stage for a long period of time, it is important to investigate the factors or surrogate markers for conversion from asymptomatic to symptomatic stage. Our study is designed to evaluate the relationship among virus isolation rate, anti-p17 antibody status and progression to AIDS. We studied anti-p17 antibody status along with virus isolation in 56 asymptomatic carriers and 46 AIDS cases. Progression to AIDS was markedly associated with high rate of virus isolation and loss of anti-p17 antibody. In order to know the meaning of loss of anti-p17 antibody during the clinical course, 15 anti-p17 antibody positive and 16 anti-p17 antibody negative cases were followed up prospectively for the development of AIDS. None of the anti-p17 antibody positive cases developed AIDS while 6 out of 16 anti-p17 negative cases developed AIDS during observation period (P < 0.05). Progression to AIDS was associated with loss of anti-p17 antibody. Identification of cases losing anti-p17 antibody in peripheral blood during asymptomatic period may help high-risk group who are in need of chemoprophylaxis. Moreover, study of anti-p17 antibody may be helpful in designing vaccine in future if it works as a neutralizing antibody to HIV in vivo.     

家养犬、猫及野生动物中狂犬病病毒流行病学调查及我国不同类型狂犬病疫苗免疫效果观测研究

选择我国狂犬病高发地区(湖南、贵州)、低发地区(江苏、武汉)和无狂犬病报告地区(沈阳)等三种不同类型的地区,开展针对家养犬、猫和啮齿类动物,以及蝙蝠等野生动物的狂犬病病毒带毒率的流行病学调查,对分离到的病毒株在抗原型别、基因变异以及与现行疫苗是否匹配等方面进行分析、比较研究。结果显示:我国狂犬病的主要宿主动物为犬,捕获犬总带毒率为2.56%;犬中狂犬病病毒的监测点最高阳性检出率达到20.0%;啮齿类动物及蝙蝠等野生动物在本次研究未检测到狂犬病病毒。我国自主研制成功的以aG株和CTN株为毒种的现行人用狂犬病疫苗,经应用研究发现,CTN株的糖蛋白基因与本研究分离到的街毒核酸序列更接近。为进一步评价现行疫苗的有效性,在5个观察点分别以aG株和CTN株毒种生产的疫苗接种两组人群(每组各约50人),结果显示均未出现中、强度反应,疫苗免疫后第7d和14d产生的中和抗体分别达到0.49IU/mL~0.52IU/mL和6.7IU/mL~7.53IU/mL;抗体阳转率分别为45.1%~47.9%和100%,表明这些疫苗具有良好的安全性和保护效果。     

Inhibition of Cellular DNA Synthesis in Cells Infected with Infectious Pancreatic Necrosis Virus

In asynchronous RTG-2 cell cultures infected with infectious pancreatic necrosis (IPN) virus, inhibition of cellular DNA synthesis, but not protein synthesis, was detected 5 to 6 h postinfection and was 80 to 90% complete by 7 to 8 h. Inhibition of DNA synthesis was largely abolished by UV irradiation of the virus. Sedimentation analyses of phenol-extracted DNA indicated that native cellular DNA was not degraded during infection. Sedimentation on alkaline sucrose gradients of DNA from cells pulsed with radioactive thymidine for varying periods indicated that elongation of nascent DNA chains proceeded normally in infected cells. These and previous results suggest that IPN virus infection results in a reduction of the number of chromosomal sites active in DNA synthesis but does not affect the rate of polymerization at active sites. Cells synchronized with excess thymidine and hydroxyurea and infected with virus at the time of release from the block demonstrated an inhibition of DNA synthesis 3 h postinfection. Cells infected 4 h prior to release continued to synthesize normal amounts of DNA for 1 to 2 h after release. These results indicated that DNA synthesis in early synthetic phase is relatively insensitive to inhibition by IPN virus.     

Serologic survey for pathogens potentially affecting pronghorn (Antilocapra americana) fawn recruitment in Arizona, USA

During the 1990s, pronghorn (Antilocapra americana) populations declined in Arizona, USA. To investigate potential causes of decline, we collected blood samples from hunter-harvested male pronghorn from 2001 to 2003 on four Arizona sites. Sera were tested for antibody to parainfluenza virus type 3 (PI3), bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, epizootic hemorrhagic disease virus (EHDV), bluetongue virus (BTV), and Chlamydia psittaci. Antibody against PI3 was found in 33% of the samples, whereas antibody against BTV/EHDV was found in 77%. Antibodies to other pathogens were found at low prevalence rates. Although pronghorn decline in Arizona is probably not directly related to disease, potential reproductive effects of BTV/EHDV and PI3 infection on pronghorn in Arizona merit further study.     

Immunization of arctic foxes (Alopex lagopus) with oral rabies vaccine

Arctic foxes (Alopex lagopus) were successfully immunized against rabies using an orally-administered, liquid SAD-BHK21 live virus vaccine in a sausage bait. Immunization was determined by serologic response and by resistance to challenge with an arctic rabies virus strain. Virus was not shed in saliva following oral vaccination, indicating that arctic foxes would not infect other foxes after ingesting this vaccine. High antibody levels were present in all experimental foxes 2 wk following initial vaccination. A booster vaccination at 56 wk induced a significant serologic response within 1 wk, suggesting an anamnestic response but titers began to decline within 8 wk in most foxes. Foxes were observed for 16 mo following the challenge and exhibited no symptoms of rabies. The SAD-BHK21 rabies vaccine in a sausage bait system has a strong potential for vaccinating wild populations of arctic fox.