In vitro replication of epizootic hemorrhagic disease and bluetongue viruses in white-tailed deer peripheral blood mononuclear cells and virus-cell association during in vivo infections

In vitro and in vivo infections were conducted to determine if the epizootic hemorrhagic disease (EHD) and bluetongue (BT) viruses would replicate in peripheral blood mononuclear (PBM) cells of white-tailed deer (Odocoileus virginianus). All of the North American EHD and BT viruses (EHD virus serotypes 1 and 2, and BT virus serotypes 2, 10, 11, 13, and 17) replicated in vitro in cultures of white-tailed deer PBM cells. However, this replication appeared to be monocyte-dependent and was not enhanced by lymphocyte blastogenesis induced by the addition of concanavalin A. In white-tailed deer infected with either EHD virus serotype 2 or BT virus serotype 10, virus could be isolated consistently from PBM cells only from post-infection day 4 through 8, although they remained viremic through post-infection day 21. In deer, highest viral titers were associated with the erythrocyte fraction, and in no cases did viral titers detected in the platelet, PBM cell or polymorphonuclear cell fractions approach titers observed in whole blood. In the in vitro infections of white-tailed deer erythrocytes, the EHD and BT viruses were associated with pits in the erythrocyte membrane. This association may be important in the long-term viremia observed in deer.     

Histamine and eicosanoid levels in plasma and peripheral blood leucocyte cultures during experimental infection of cattle with bluetongue virus serotype 11

Three calves were sensitized with three doses of inactivated BTV-11 UC8 strain and then experimentally infected with the homologous virus. In addition, four BTV-seronegative heifers were also experimentally infected with BTV-11. Granulocyte rich fractions of peripheral blood leucocyte (PBL-GRF) cultures from BTV-sensitized/infected calves and from control unexposed cattle were exposed in vitro with BTV-11. Histamine, leukotriene (LT) C4 and prostaglandin (PG) D2 were assayed in supernatant fluids. Plasma histamine levels increased in BTV-infected heifers from 10.1 +/- 2 ng/ml at Day 0 to 23.1 +/- 6.6 ng/ml at Day 12 following virus exposure. In addition, in this experimental group the concentration of PGF2 alpha (mean 551.97 +/- 243.54 pg/ml) increased significantly (P < or = 0.05) compared with control cattle (mean 467.3 +/- 73.9 pg/ml). Bluetongue virus induced histamine and LTC4 release after in vitro infection of PBL-GRF. Release of LTC4 was significantly (P < or = 0.05) higher in PBL-GRF cultures from sensitized and control animals than in unexposed PBL-GRF cultures. In contrast to these results, PGD2 was not released after BTV infection of PBL-GRF in vitro. The histamine release caused by BTV was virus-specific and mainly mediated by an immunological reaction, since the release was significantly reduced by removal of cell surface immunoglobulins.     

Long-term treatment with desmopressin in children with primary monosymptomatic nocturnal enuresis: an open multicentre study. Swedish Enuresis Trial (SWEET) Group

Cache Valley virus (CVV) and Potosi virus (POTV) are two closely related mosquito-borne viruses (Bunyaviridae: Bunyamwera group) that appear to circulate in several regions of the United States, especially the Midwest. We determined the prevalence of specific neutralizing antibodies to both viruses in Indiana white-tailed deer and conducted infection experiments to assess whether deer could serve as an vertebrate-amplifying host. Cross-infection experiments also were carried out to investigate the level of antibody cross-reactivity and cross-protection between the two viruses. The seroprevalence rate was high for both CVV (> 66%) and POTV (> 43%) in adult deer statewide. Antibodies neutralizing CVV were more common among deer harvested in the northern part of Indiana whereas the prevalence of POTV antibodies suggested a more southern distribution for this virus. Experimental infections of captive deer showed that they may serve as amplifying hosts for either virus. Deer infected with CVV or POTV developed a 1-3-day viremia with 3.0 and 4.1 log10 plaque-forming units/ml mean peak titers, respectively. However, significant levels of antibody cross-reactivity between the two viruses were observed. Viremia was lower and shorter when animals immune to either CVV or POTV were cross-infected with the alternate virus and antibody responses following cross-infections resembled original antigenic sin with higher titers of antibodies against the primary agent.     

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.     

Heat-stabilized albumin microspheres as a sustained drug delivery system for the antimetabolite, 5-fluorouracil

A nested polymerase chain reaction (PCR) test was developed to examine infection with the bovine lentivirus, bovine immunodeficiency-like virus (BIV), in cattle. Primers were designed to amplify 2 separate regions of the pol and env segments of the BIV genome. Two calves were experimentally infected with an isolate derived from the original strain of BIV, R29, or with a recent field isolate, FL491. Serial blood samples were collected and examined by virus isolation, protein immunoblot, and nested PCR. The nested PCR test detected BIV infection by 3 days after inoculation, earlier than the other 2 methods, and continued to identify infected cattle 9 to 15.5 months after inoculation, even when results from virus isolation and serology became negative. Nested PCR also detected multiple-size env products in samples obtained later in the infection from the calf that received FL491, giving evidence that viral quasispecies were selected during in vivo replication of the virus. Results indicated that the nested PCR test is more sensitive than virus isolation or serology for the detection of BIV infection in cattle.     

The role of in vitro-induced lymphocyte apoptosis in feline immunodeficiency virus infection: correlation with different markers of disease progression

Human immunodeficiency virus infection is characterized by a progressive decline in the number of peripheral blood CD4(+) T lymphocytes, which finally leads to AIDS. This T-cell decline correlates with the degree of in vitro-induced lymphocyte apoptosis. However, such a correlation has not yet been described in feline AIDS, caused by feline immunodeficiency virus (FIV) infection. We therefore investigated the intensity of in vitro-induced apoptosis in peripheral blood lymphocytes from cats experimentally infected with a Swiss isolate of FIV for 1 year and for 6 years and from a number of long-term FIV-infected cats which were coinfected with feline leukemia virus. Purified peripheral blood lymphocytes were either cultured overnight under nonstimulating conditions or stimulated with phytohemagglutinin and interleukin-2 for 60 h. Under stimulating conditions, the isolates from the infected cats showed significantly higher relative counts of apoptotic cells than did those from noninfected controls (1-year-infected cats, P = 0.01; 6-year-infected cats, P = 0.006). The frequency of in vitro-induced apoptosis was inversely correlated with the CD4(+) cell count (P = 0. 002), bright CD8(+) cell count (P = 0.009), and CD4/CD8 ratio (P = 0. 01) and directly correlated with the percentage of bright major histocompatibility complex class II-positive peripheral blood lymphocytes (P = 0.004). However, we found no correlation between in vitro-induced apoptosis and the viral load in serum samples. Coinfection with feline leukemia virus enhanced the degree of in vitro-induced apoptosis compared with that in FIV monoinfected cats. We concluded that the degree of in vitro-induced apoptosis was closely related to FIV-mediated T-cell depletion and lymphocyte activation and could be used as an additional marker for disease progression in FIV infection.     

Polymerase chain reaction to detect infectious laryngotracheitis virus in conjunctival swabs from experimentally infected chickens

The polymerase chain reaction (PCR) was standardized and assessed as a potential diagnostic test for infectious laryngotracheitis using conjunctival swabs collected from experimentally infected chickens. Polymerase chain reaction primers based on the sequence of a 1.1-kb BamHI restriction enzyme fragment of the Ontario 1598 (Ont 1598) strain of infectious laryngotracheitis virus were selected and 300 fg of purified viral DNA were detected by ethidium bromide staining of agarose gels or 30 fg were detected by DNA hybridization. At least five different strains (Ont 1598, ATCC N-71851, LT-IVAX, Laryngo-Vac, and a local strain 322) were amplified whereas other avian pathogens and uninfected cell cultures tested negative. Swabs collected from experimentally infected chickens were analyzed by both PCR and virus isolation on various days postinfection. A comparison of virus isolation to PCR indicated that, in the mid-postinfection phase, PCR and virus isolation appeared to be comparable with a kappa value of greater than 0.8. The polymerase chain reaction was shown to be the better test later in infection, when clinical recovery had occurred.     

Reduced abundance of Ixodes scapularis (Acari: Ixodidae) with exclusion of deer by electric fencing

To assess the effect of deer exclusion on populations of Ixodes scapularis Say (formerly I. dammini Spielman, Clifford, Piesman & Corwin) in the northeastern United States, host-seeking ticks and ticks on white-footed mice, Peromyscus leucopus (Rafinesque), were monitored inside and outside a wooded, residential deer exclosure (approximately 3.5 ha) in Lyme, CT, in 1991 and 1992. Another deer exclosure was added in Lyme (approximately 7.4 ha) during 1992. Additional sample sites at other residences served as secondary controls. A seven-wire, slanted, high-tensile electric deer fence was used at both areas. Larvae of I. scapularis were 81.5% (1991) and 97.8% (1992) less abundant within the exclosure than immediately outside the deer exclosures. Nymphs of I. scapularis were 47.4% (1991) and 55.8% (1992) less abundant within the deer fence. The effect on adult ticks was mixed. No difference in tick abundance was seen at the 3.5-ha site. However, larvae, nymphs, and adults were 100, 83.8, and 74.1% less abundant, respectively, in plots at the 7.4-ha exclosure > or = 70 m from the deer fence and isolated from woodlands outside the fence by lawns, driveways, and buildings. The recovery of larvae and nymphs of I. scapularis from mice captured within the deer exclosures indicates that infestations of nymphs and adults are probably, at least in part, a result of the movement of these rodents. Based upon the number of nymphs per 100 m2 infected with Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner, the causal agent of Lyme disease, there were 73 and 82% fewer infected nymphs within the deer exclosures in 1992 in comparison with the number of infected ticks outside the fence and at the secondary control sites, respectively. The exclusion of deer in conjunction with other tick control strategies in large areas could substantially reduce populations of I. scapularis and the risk of acquiring Lyme disease.     

Cytokine production by cats infected with feline immunodeficiency virus: a longitudinal study

The immune responsiveness of cats naturally or experimentally infected with feline immunodeficiency virus (FIV) was studied. Peripheral blood mononuclear cells (PBMC) from naturally infected, symptomatic animals displayed depressed proliferation and interleukin-2 (IL-2) production in response to mitogens, which was accompanied by a significant increase in IL-1, IL-6 and tumour necrosis factor (TNF) production. Longitudinal studies were performed over a period of 4 years in experimentally infected animals. The responses of cells from these cats to concanavalin A (Con A) were consistently less than those from uninfected cats throughout the period but, owing to variation between cats, were significantly lower on only a few occasions. By contrast, the responses of cells to pokeweed mitogen (PWM) were severely affected and declined progressively throughout the 4-year period. In general, responses to Con A but not PWM could be restored by the addition of exogenous IL-2. The decline in immune responsiveness was concurrent with a decline in feline (f)CD4+ cells and an inversion in the CD4:CD8 ratio. Peak production of IL-1, IL-6 and TNF coincided with periods of depressed immune responses. Additionally, immunodeficient responses and elevated levels of proinflammatory cytokines were concurrent with the presence of clinical signs. We conclude that, like human immunodeficiency virus (HIV), FIV infection results in significant perturbation of the immune response. Responses to PWM appear to correlate with disease progression which suggests that the CD3 pathway is affected in the earlier stages of the disease and that additional activation pathways such as CD2 may not be affected until the animal enters the acquired immune deficient syndrome (AIDS) stage of the disease.     

A model for respiratory syncytial virus (RSV) infection based on experimental aerosol exposure with bovine RSV in calves

Five conventionally kept calves aged between 17 and 24 days were experimentally infected with bovine respiratory syncytial virus (BRSV) by aerosol in order to mimic the natural infection route. The calves were killed and autopsies performed 7 days after the first virus challenge. The BRSV isolate used induced tracheitis, bronchitis and atelectasis in infected calves. The only virus which could be isolated from the lungs of the calves was BRSV. In addition, Mycoplasma bovirhinis was isolated from the lungs or/and trachea of two calves. The clinical and histopathological findings, as well as the detection of BRSV antigens by immunofluorescence in the epithelial cells of lung and trachea, and the reisolation of the virus from bronchoalveolar lavage fluids of all inoculated calves, provided confirmation of successful infection with BRSV.     

African swine fever virus specific porcine cytotoxic T cell activity

African swine fever virus (ASFV) specific, cytotoxic T lymphocyte (CTL) activity has been studied in a protection model in which SLA inbred miniature swine are experimentally inoculated with a naturally occurring, non-fatal ASFV isolate (NHV). Peripheral blood mononuclear cells (PBMC) from such infected swine show significant activity in CTL assays, using cultured ASFV-infected porcine blood derived macrophages as target cells. This CTL activity is elicited from PBMC by in vitro restimulation of effector cells with low doses (multiplicity of infection = 0.1) of the homologous virus isolate for 48 to 72 h. For SLAc/c effectors, this CTL activity appears to be SLA class I restricted because (1) blocking target cell antigens with monoclonal antibodies (mAb) against SLA class I antigens causes a major reduction in CTL activity; (2) there is preferential lysis of SLA class I matched, ASFV infected targets; and (3) depletion of effector cells with CD8 specific mAb and complement causes a reduction in CTL activity. The CTL activity is ASFV specific for all pigs tested in that infected macrophages are preferentially lysed as compared to normal (non-infected) cultured macrophages or macrophages infected with hog cholera virus (HCV). Lysis of macrophages infected with different ASFV isolates revealed that there is marked lysis of macrophages infected with the virulent L60 isolate but less lysis of macrophages infected with the DR-II and Tengani isolates. In summary, our data show that ASFV specific CTL activity is triggered in swine infected with the NHV ASFV isolate.     

PCR detection of North American and Central African isolates of epizootic hemorrhagic disease virus (EHDV) based on genome segment 10 of EHDV serotype 1

PCR amplification technology for the detection of epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell culture and clinical specimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for two nonstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV-2 prototype, and a number of EHDV field isolates, including the Central African isolates of EHDV-5 and EHDV-318 propagated in cell cultures, were detected by this PCR-based assay. The specific 535-bp PCR products were visualized onto agarose gels, and the identity of the PCR products was confirmed by chemiluminescent hybridization with a 352-bp internal probe. The sensitivity of the EHDV PCR assay was increased by chemiluminescent hybridization; by this EHDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification product was not detected when the PCR-based assay was applied to RNAs from North American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfractionated blood from calves and deer that were EHDV seronegative and virus isolation negative. The described EHDV PCR-based assay with primers derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural and experimental EHDV infections and provides a valuable tool to study the epidemiology of EHDV infection in susceptible ruminants.     

Immunity to porcine rubulavirus infection in adult swine

The immune response against the porcine rubulavirus was analyzed in experimentally infected adult pigs. High titers of virus neutralizing and hemagglutinating inhibitory antibodies were identified in infected animals. The antibody specificity was directed towards HN, M, and NP rubula virion proteins; immunodominance of HN proteins was demonstrated. Peripheral blood mononuclear cells from infected, but not from non-infected pigs proliferated in vitro in response to virus antigenic stimuli, showing a bell-shaped plot with the highest peak at 5 weeks post-infection. Virus-induced lymphoblasts expressed CD4+ CD8+ phenotype, whereas lectin-induced lymphoblasts were mainly identified as CD4+ CD8- cells. Phenotype analysis of freshly prepared PBMC revealed increased number of both monocytes (PoM1+) and total T lymphocytes (CD2+) early during infection, with reduced values of B lymphocytes at 4 weeks post-infection. Decrease in CD4+ CD8- blood cells was observed at 3 weeks post-infection, whereas both CD4- CD8+ and CD4+ CD8+ cells increased 1 and 4 weeks post-infection, respectively. This work discusses the relevance of CD4+ CD8+ T cells in the control of porcine rubulavirus infection.     

Incubation time for feline immunodeficiency virus cultures

In a recent report, Fiscus et al. (S. A. Fiscus, S. L. Welles, S. A. Spector, and J. L. Lathey, J. Clin. Microbiol. 33:246-247, 1995) have shown that qualitative human immunodeficiency virus cultures can be terminated at day 21 with minimal false-negative results. We have evaluated a large number of qualitative and quantitative feline immunodeficiency virus (FIV) isolations to determine how long FIV cultures should be incubated to obtain reasonably certain results. The rate at which FIV cultures became positive was influenced by whether the cats under study were naturally or experimentally infected, the duration of in vivo infection, and the number of infected peripheral blood mononuclear cells seeded. The results show that cultures for FIV isolation should be kept for 5 to 6 weeks.     

Hepatitis C virus infection of salivary gland epithelial cells. Lack of evidence

BACKGROUND: Hepatitis C virus genome (HCV-RNA) has been detected in whole salivary gland tissue of chronically infected patients. However, contamination of the tissue by plasma or blood cells was not excluded by the previous reports. AIMS: To assess whether HCV infects the salivary gland epithelial cells in patients with chronic HCV liver disease. METHODS: Twenty unselected patients with chronic active hepatitis (11 cases) or active cirrhosis (nine cases) were examined. Serum and saliva samples were obtained from all patients, 12 of whom (seven, chronic active hepatitis; five, active cirrhosis) underwent salivary gland biopsy. PCR for HCV-RNA was performed on RNA extracted from serum, saliva and salivary gland epithelial cells collected by isokinetic gradient separation after trypsin digestion of whole salivary gland tissue. Saliva samples were also examined for the presence of secretory IgA anti-HCV by gel chromatography and ELISA testing. RESULTS: HCV-RNA was detected in all sera with titers ranging from 5.42 x 10(5) genome equivalents/ml to 123.2 x 10(5) genome equivalents/ml. Thirteen patients were infected with genotype 1b, four patients had genotype 1a, two patients had genotype 2a and one patient was unclassifiable. Low titer HCV-RNA (<2 x 10(5) genome equivalents/ml) was detected in 3/20 saliva samples (15%) from highly viremic patients infected with 1b genotype. RNA extracted from salivary gland epithelial cells consistently tested negative for HCV-RNA. In addition, all saliva specimens tested negative for secretory-IgA (S-IgA) anti-HCV, even after a 10-fold concentration of the samples. CONCLUSIONS: There was no evidence that HCV infects the salivary gland epithelial cells in our viremic patients with HCV chronic liver disease. Low level HCV-RNA in saliva is most probably due to virus spillover from blood.     

Serological diagnosis of influenza B virus infection: comparison of an enzyme-linked immunosorbent assay and the hemagglutination inhibition test

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to influenza B virus was compared with that of the hemagglutination inhibition test on acute- and convalescence-phase sera obtained from adults and children infected with influenza B virus. Two whole virus, tissue culture-grown antigen preparations were used in the ELISA, influenza B/West Virginia/81 and influenza B/Hong Kong/72. Four antigens were used in the hemagglutination inhibition test. These included the tissue culture-grown whole virus antigens that were used in the ELISA. In addition the standard egg-grown antigens, influenza B/Singapore/79 and influenza B/Hong Kong/72, were included for comparison. The ELISA antibody titer was significantly correlated to the hemagglutination inhibition antibody titer, and 10 of 10 adults and 17 of 21 children infected with influenza B had fourfold antibody increases as detected by ELISA with either antigenic type of tissue culture-grown whole virus. Increases in geometric mean antibody titers of 16- to 71-fold were detected by ELISA. Increases in geometric mean antibody titers of 3- to 10-fold were detected by hemagglutination inhibition depending on the type of antigen utilized. We found that ELISA with whole virus antigens was a sensitive and specific test for the detection of antibody to influenza B virus.     

Aural symptoms and hearing loss in patients with lupus

White-tailed deer participate in the maintenance of the Ixodes tick life cycle and are reservoirs for some tick-borne infectious agents. Deer may be useful sentinels for tick-transmitted agents, such as ehrlichiae. In order to determine whether white-tailed deer are markers of natural transmission or are reservoirs for the human granulocytic ehrlichiosis (HGE) agent, we performed indirect immunofluorescent-antibody (IFA) tests and immunoblotting with the HGE agent and Ehrlichia chaffeensis on sera from 43 and 294 deer captured in northwest Wisconsin during 1994 and 1995, respectively, and 12 deer from southern Maryland. According to IFA testing, 47% of 1994 Wisconsin sera, 60% of 1995 Wisconsin sera, and 25% of Maryland sera contained HGE agent antibodies. All IFA-positive deer sera tested reacted with the 44-kDa band which is unique to the Ehrlichia phagocytophila group. Serologic reactions to E. chaffeensis were detected by IFA testing in 15 of 337 (4%) Wisconsin deer and in 10 of 12 (83%) Maryland deer, while 60 and 80% of E. chaffeensis IFA-positive Wisconsin and Maryland deer sera, respectively, reacted with the E. chaffeensis 28- to 29-kDa antigens by immunoblotting. A total of 4% of deer from Wisconsin and 25% of deer from Maryland were found by IFA testing to have antibodies to both the HGE agent and E. chaffeensis; 75% of these were confirmed to contain E. chaffeensis antibodies by immunoblotting. These results suggest that white-tailed deer in diverse geographical regions of the United States are naturally infected with the HGE agent, E. chaffeensis, or both and that these animals, and potentially humans, are exposed to infected ticks at a high frequency in nature.