Log10 Plaque-forming Units Research Articles

Role of Nestling Mourning Doves and House Finches as Amplifying Hosts of St. Louis Encephalitis Virus

Nestling mourning doves and house finches produced elevated viremias after inoculation with 2-3 log10 plaque-forming units (PFU) of St Louis encephalitis (SLE) virus and infected 67 and 70% of Culex tarsalis Coquillett that engorged upon them, respectively. Mosquito infection rates as well as the quantity of virus produced after extrinsic incubation increased as a function of the quantity of virus ingested and peaked during days 3-5 postinoculation in mourning doves and days 2-4 in house finches. Only female Cx. tarsalis with body titers > or = 4.6 log10 PFU were capable of transmitting virus. Overall, 38% of females infected by feeding on mourning doves and 22% feeding on house finches were capable of transmission. The quantity of virus expectorated was variable, ranging from 0.8 to 3.4 log10 PFU and was greatest during periods when avian viremias were elevated. Our data indicated that nestling mourning doves and house finches were competent hosts for SLE virus and that the quantity of virus ingested from a viremic avian host varies during the course of the infection and determines transmission rates by the mosquito vector.

Western Equine Encephalomyelitis Virus Infection Affects the Life Table Characteristics ofCulex tarsalis(Diptera: Culicidae)

The life table attributes of Culex tarsalis Coquillett females infected experimentally by feeding on 4 and 6 log10 plaque-forming units (PFU) of western equine encephalomyelitis virus (WEEV) per milliliter of heparinized chicken blood were compared with an uninfected control group. Females continually were offered 10% sucrose and an oviposition substrate and daily a blood meal through a biomembrane feeder. Mortality (dead females) and fecundity (female eggs per female) were monitored daily until all females died. Overall, 94% of 198 females in the two virus-infected groups were positive for WEEV at death when tested by plaque assay; the average body virus titer at death did not differ between groups. WEEV infection significantly altered the life table characteristics of Cx. tarsalis. Life expectancy at infection in days (ex), reproductive effort in female eggs per female per generation (Ro), and generation time (T) in days for the infected cohorts were significantly lower than for the uninfected controls, whereas the reproductive rate (rc) in female eggs per female per day was higher for infected than uninfected cohorts. In agreement with the WEEV infection data that showed similar body titers, there were few differences between the life table parameters for the 4 and 6 log10 PFU treatment groups. Greatest differences were observed for survivorship between days 17-40 when virus titers in infected dying females were greatest. Our data extend recent studies that indicate mosquito infection with encephalitis viruses has a cost of reduced life expectancy and fitness.

Experimental infection of California birds with western equine encephalomyelitis and St. Louis encephalitis viruses.

A total of 27 bird species from the San Joaquin and Coachella valleys of California were inoculated subcutaneously with sympatric strains of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses. Overall, 133 of 164 birds inoculated with WEE virus developed a viremia detected by plaque assay; significantly greater than 72 of 163 birds inoculated with SLE virus. Host competence was calculated as the average number of days that each avian species had a viremia > or = 2 log10 plaque-forming units per 0.1 ml, the threshold for infecting susceptible Culex tarsalis Coquillett, the primary vector of these viruses in California. Eleven of 20 species inoculated with WEE virus had a value > or = 1 and were considered to be competent hosts, whereas only six of 22 species inoculated with SLE virus had a value > or = 1. Overall, 133 of 164 birds inoculated with WEE virus and 105 of 163 inoculated with SLE virus produced antibody detectable by enzyme immunoassay and/or plaque reduction neutralization test. Six birds infected with WEE virus (one house finch, three mourning doves, one Brewer's sparrow, and one white-crowned sparrow) and nine birds infected with SLE virus (two house finches, three white-crowned sparrows, one song sparrow, two Western scrub-jays, and one orange crowned warbler) contained viral RNA detected by reverse transcription-polymerase chain reaction at necropsy > 6 wk postinoculation; infectious WEE and SLE viruses were only recovered from three mourning doves and an orange-crowned warbler, respectively, after blind passage in mosquito cells. Our study indicated that birds with elevated field antibody prevalence rates may not be the most competent hosts for encephalitis viruses and that relatively few birds developed chronic infections that could be important in virus persistence and dispersal.

West Nile virus in blood: stability, distribution, and susceptibility to PEN110 inactivation.

The outbreak of West Nile virus (WNV) is the most recent reminder that the blood supply continues to be vulnerable to emerging and reemerging pathogens. A potentially prospective approach to reducing the risk of transfusion-transmitted infections of a known or newly emerging microbe is implementation of a broad-spectrum pathogen reduction technology. The purpose of this study was to evaluate the susceptibility of WNV to PEN110 inactivation in RBCs and to characterize the WNV interaction with blood, including the stability of WNV in RBCs stored at 1 to 6 degrees C, its distribution and infectivity, and its ability to infect WBCs. Inactivation was performed with three WNV isolates spiked into WBC-reduced RBCs. The stability of the virus was evaluated by spiking two viral loads into RBCs followed by storing at 1 to 6 degrees C for up to 42 days. The distribution of the virus in plasma, RBCs, and PBMCs was evaluated with whole blood from infected hamsters. Finally, in vitro propagation of WNV was evaluated with the THP-1 cell line and primary monocytes. The kinetics of PEN110 inactivation of WNV isolates RI-44, NJ-176, and 99-3494031 were fast and complete within 24 hours with reduction factors of 5 to 7 log plaque-forming units per mL. WNV remained infectious for up to 42 days at 1 to 6 degrees C. The WNV titers in whole blood, plasma, RBCs, and PBMC fractions were equally distributed and ranged from 2 to 3 log tissue culture infectious dose 50 percent per mL. Productive infection of stimulated monocytes and THP-1 cells was also demonstrated. These studies demonstrated that PEN110 efficiently inactivated WNV in RBCs and whole blood from infected hamsters to the limit of detection. WNV survived in RBCs stored at 1 to 6 degrees C with a gradual loss of titer but infectivity could still be observed for up to 42 days. In addition, it was observed that WNV was equally distributed in all blood fractions including PBMCs and it was possible to establish productive infection of a human monocytic cell line and stimulated human monocytes.

Viremia and Immunogenicity in Nonhuman Primates of a Tetravalent Yellow Fever–Dengue Chimeric Vaccine: Genetic Reconstructions, Dose Adjustment, and Antibody Responses against Wild-type Dengue Virus Isolates

Chimeric yellow fever (YF)–dengue (DEN) viruses (ChimeriVax–DEN) were reconstructed to correct amino acid substitutions within the envelope genes of original constructs described by Guirakhoo et al. (2001, J. Virol. 75, 7290–7304). Viruses were analyzed and compared to the previous constructs containing mutations in terms of their growth kinetics in Vero cells, neurovirulence in mice, and immunogenicity in monkeys as monovalent or tetravalent formulations. All chimeras grew to high titers [∼7 to 8 log10, plaque-forming units (PFU)/ml] in Vero cells and were less neurovirulent than YF 17D vaccine in mice. For monkey experiments, the dose of DEN2 chimera was lowered to 3 log10 PFU in the tetravalent mixture in an effort to reduce its dominant immunogenicity. The magnitude of viremia in ChimeriVax–DEN immunized monkeys was similar to that of YF-VAX, but significantly lower than those induced by wild-type DEN viruses. All monkeys developed high levels of neutralizing antibodies against homologous (chimeras) or heterologous (wild-type DEN viruses isolated from different geographical regions) viruses after a single dose of monovalent or tetravalent vaccine. Administration of a second dose of tetravalent vaccine 2 months later increased titers to both homologous and heterologous viruses. A dose adjustment for dengue 2 chimera resulted in a more balanced response against dengue 1, 2, and 3 viruses, but a somewhat higher response against chimeric dengue 4 virus. This indicates that further formulations for dose adjustments need to be tested in monkeys to identify an optimal formulation for humans.

Method of Infection Does Not Alter Response of Chicks and House Finches to Western Equine Encephalomyelitis and St. Louis Encephalitis Viruses

The effects of method of infection and virus dose on the viremia and antibody responses of 1-wk-old chicks and after-hatching-year house finches to infection with western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses were studied under laboratory conditions. Using a capillary tube technique, females from 2 strains of Culex tarsalis Coquillett mosquitoes were estimated to expectorate from 1.0 to 1.7 log10 plaque forming units (PFU) of WEE and from 1.9 to 2.2 log10 PFU of SLE. Based on the proportion of parenterally infected females that transmitted and the number that blood fed during each experiment, virus doses per bird were estimated to be 1.0-1.9 log10 PFU for WEE and 1.4-2.3 log10 PFU for SLE. When infected with comparable doses of WEE by subcutaneous inoculation, there was no significant difference in the duration or magnitude of the viremia response between birds infected by mosquito bite or syringe; few birds developed a viremia response after infection with SLE, precluding analysis. In chickens, increasing the syringe dose of WEE from 0.3 to 1.7 log10 PFU/0.1 ml shortened the time when viremia first appeared from 3 to 1 d postinfection and increased the duration of the viremia period from 1 to 3 d, but did not alter the maximum viremia titer. In house finches, increasing the syringe dose of WEE from 2.6 to 3.3 log10 PFU/0.1 ml did not alter markedly the viremia response. Most birds developed antibody detected by enzyme immunoassay (EIA) or plaque reduction neutralization test (PRNT). In chickens, WEE EIA levels and PRNT titers were higher for birds infected by syringe than by mosquito bite, whereas in house finches the pattern was reversed. For birds infected with SLE, there was overlap among groups infected by mosquito bite or syringe. These results indicate that subcutaneous syringe inoculation provides a biologically sound mode of infection that did not alter viremia and antibody responses when compared with infection by mosquito bite.

Nasal quantity of respiratory syncytical virus correlates with disease severity in hospitalized infants.

To evaluate the relationship between nasal quantity of respiratory syncytial virus (RSV) and disease severity in hospitalized infants without underlying cardiopulmonary disease or immunodeficiency. Nasal aspirates were obtained from hospitalized infants <24 months of age with recently identified RSV infection and evaluated for RSV quantity by a standard plaque assay on HEp-2 cell monolayers. Subjects were classified as having "severe" disease if they required mechanical ventilation at the time of sample collection and as having "nonsevere" disease if they did not. Linear modeling was used to determine the relationship between nasal RSV quantity and various independent variables, including disease severity. Nasal aspirates from 39 patients were evaluated. Age, gender and mean duration of time from symptom onset to sample acquisition (5 days) were similar between the severe (n = 15) and nonsevere (n = 24) groups. Significantly more infants were born at <35 weeks gestation in the severe disease group (7 of 15 vs. 3 of 24, P = 0.017), and infants born at <35 weeks gestation were significantly more likely to be of non-Caucasian ethnicity than were infants born at > or =35 weeks gestation (8 of 10 vs. 12 of 29, P = 0.035). The linear model found that higher nasal RSV quantities were associated with severe disease [mean +/- SEM, 5.06 +/- 0.34 log plaque-forming units (pfu)/ml vs. 3.91 +/- 0.35 log pfu/ml, P = 0.022], gestational age > or =35 weeks (5.44 +/- 0.27 log pfu/ml vs. 3.52 +/- 0.45 log pfu/ml, P = 0.002) and non-Caucasian ethnicity (5.16 +/- 0.30 log pfu/ml vs. 3.80 +/- 0.37 log pfu/ml, P = 0.006). Nasal RSV quantity correlates with disease severity in hospitalized infants with recently identified RSV infection.

Experimental infection of the cane mouse Zygodontomys brevicauda (family Muridae) with guanarito virus (Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever.

Chronic infections in specific rodents appear to be crucial to the long-term persistence of arenaviruses in nature. The cane mouse, Zygodontomys brevicauda, is a natural host of Guanarito virus (family Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever. The purpose of this study was to elucidate the natural history of Guanarito virus infection in Z. brevicauda. Thirty-nine laboratory-reared cane mice each were inoculated subcutaneously with 3.0 log10 plaque-forming units of the Guanarito virus prototype strain INH-95551. No lethality was associated with infection in any animal, regardless of age at inoculation. The 13 newborn, 14 weanling, and 8 of the 12 adult animals developed chronic viremic infections characterized by persistent shedding of infectious virus in oropharyngeal secretions and urine. These findings indicate that Guanarito virus infection in Z. brevicauda can be chronic and thus support the concept that this rodent species is the natural reservoir of Guanarito virus.

Safety of a mutagen-attenuated Rift Valley fever virus vaccine in fetal and neonatal bovids

Abstract Objective To examine effects of in utero inoculation with a mutagen-attenuated Rift Valley fever virus (RVFV) vaccine (RVF MP-12) on fetal bovids and to assess the safety and efficacy of calfhood vaccination with RVF MP-12. Animals 18 pregnant Hereford and Hereford-type cows in the third or fifth month of gestation, their progeny, and 25 calves from cows immunized with RVF MP-12 during pregnancy. Procedure Bovine fetuses were inoculated, via laparotomy, with 1 ml of RVF MP-12 containing 5 log10 plaque-forming units (PFU) of virus. Blood was obtained from newborn calves prior to their ingestion of colostrum. Immune-naive calves and calves born to RVF MP-12-vaccinated dams, ranging in age from 2 to 45 days, were vaccinated with RVF MP-12, and some were later challenge exposed with 1 ml of 5.7 log10 PFU of virulent RVFV strain ZH-501. Cows were monitored for viremia and antibody responses and for hematologic and serum biochemical alterations through parturition or abortion. Results Surviving in utero-vaccinated calves were healthy, with no noticeable defects. Except for 1 vaccine-inoculated fetus that died on postinoculation day 21, all in utero-vaccinated fetuses had serum neutralizing antibody titer ≥ 1:20 at the time of delivery. All dams of in utero-vaccinated fetuses also developed neutralizing antibody titer. Calves born to cows vaccinated during gestation did not have antibody at birth, and all but 1 quickly acquired colostral antibody. Postparturient inoculation of immune-naive calves and calves with colostral antibodies resulted in no untoward effects, and all calves with detectable neutralizing antibodies were protected against virulent virus challenge exposure. Conclusions Fetal death and abortion would be rare even if fetuses were exposed to RVF MP-12. The trauma and complications associated with in utero inoculation do not make this a practical method of immunization. RVF MP-12 was safe, immunogenic, and protective in calves as young as 2 days of age. (Am J Vet Res 1997;58:1110–1114)

Experimental Infection of Young Turkeys with Eastern Equine Encephalitis Virus and Highlands J Virus

Depression, somnolence, and increased mortality were observed in 2-week-old turkeys inoculated intramuscularly with either eastern equine encephalitis (EEE) virus or Highlands J (HJ) virus. Mortality rates in EEE virus- and HJ virus-inoculated turkeys were 7/30 (23%) and 9/30 (27%), respectively; no sham-inoculated controls died. Both EEE virus- and HJ virus-inoculated turkeys developed viremia that lasted 2 days; peak mean titers were 5.5 and 3.2 log10 plaque-forming units per ml of blood, respectively. Pathologic changes in both EEE virus- and HJ virus-inoculated turkeys consisted primarily of multifocal necrosis in the heart, kidney, and pancreas, and lymphoid necrosis and depletion in the thymus, spleen, and bursa of Fabricius. The findings indicate that EEE virus and HJ virus are pathogenic for young turkeys.

Enhancement of tick-borne encephalitis virus transmission by tick salivary gland extracts.

To investigate the role of ticks in TBE virus transmission, salivary gland extract (SGE) was derived from partially fed female Ixodes ricinus, Dermacentor reticulatus and Rhipicephalus appendiculatus ticks. Guinea-pigs were infested with uninfected R. appendiculatus nymphs and inoculated with a mixture of TBE virus and SGE or with virus alone. The number of ticks which on average acquired virus from feeding on animals inoculated with TBE virus and SGE from partially fed ticks was 4-fold greater than the number that became infected by feeding on animals inoculated with virus alone or virus plus SGE from unfed I. ricinus. Viraemia was detected in 67% of guinea-pigs inoculated with virus plus SGE compared to 30% of guinea-pigs inoculated with virus alone. Virus titres in the blood were similar for both groups of animals [range 2.0-2.8 log10 plaque-forming units (PFU)/ml of blood]; however, the number of ticks that became infected was significantly higher on animals inoculated with virus plus SGE from partially fed ticks. No significant difference was observed with respect to the tick species used to derive SGE. The results indicate that TBE virus transmission is enhanced by factor(s) associated with the salivary glands of feeding ticks, and that these factor(s) may facilitate efficient transmission of TBE virus between infected and uninfected ticks even when they feed on hosts that have no detectable viraemia.

Nitrogen dioxide exposure in vivo and human alveolar macrophage inactivation of influenza virus in vitro

Epidemiologic studies have reported an increased incidence of respiratory infections and illness in association with elevated indoor levels of nitrogen dioxide (NO2). Animal exposure studies have found that brief exposures to peak levels of NO2 produce greater morbidity than continuous lower level exposure. In order to examine the effect of NO2 inhalation on human alveolar macrophages, normal volunteers were exposed sequentially to air or NO2, by double-blind randomization, in an environmental chamber. Two exposure protocols with comparable concentration x time products were used: (a) continuous 0.60 ppm NO2 (n = 9), and (b) background 0.05 ppm NO2 with three 15-min peaks of 2.0 ppm (n = 15). Inhalation of NO2 caused no significant changes in pulmonary function or airway reactivity in either exposure protocol. Alveolar macrophages obtained by bronchoalveolar lavage 3 1/2 hr after exposure to continuous 0.60 ppm NO2 tended to inactivate influenza virus in vitro less effectively than cells collected after air exposure (1.96 vs 1.25 log10 plaque-forming units on Day 2 of incubation, P less than 0.07). Four of nine subjects accounted for the observed impairment in virus inactivation; cells from these four subjects demonstrated an increase in interleukin-1 (IL-1) production after NO2 vs air, whereas the five remaining subjects decreased IL-1 production after NO2. In contrast, intermittent peak exposure did not alter the rate of viral inactivation or IL-1 production. This methodology has the potential to identify pollutant effects on mechanisms of respiratory defense in humans.

Testing of Male Sockeye Salmon (Oncorhynchus nerka) and Steel head Trout (Salmo gairdneri) for Infectious Hematopoietic Necrosis Virus

Infectious hematopoietic necrosis (IHN) virus has been isolated only rarely from whole milt samples of male sockeye salmon (Oncorhynchus nerka). In 3 yr of testing, virus incidences in males ranged from 0 to 13% when milt was sampled but were 60–100% with spleen or kidney. When IHN virus was isolated from sockeye salmon milt at titers less than 3.00 log10 plaque-forming units (pfu)/mL, the level of virus in the kidney or spleen exceeded 7.00 log10 pfu/g. Higher rates of IHN virus isolation from kidney or spleen than from milt were also generally found in steelhead trout (Salmo gairdneri), although the differences were less pronounced than in sockeye salmon. Furthermore, virus was sometimes isolated from steelhead trout milt when the level of virus in kidney or spleen samples was very low, and was recovered from some milt samples when none was isolated from the corresponding spleen sample. When male salmonids are tested for IHN virus, kidney or spleen samples are superior to whole milt, but milt should be included for critical examinations.

Experimental murine cytomegalovirus infection of ocular structures.

To study experimental cytomegalovirus (CMV) infection of ocular structures, 3-week-old Swiss-Webster mice were inoculated intraperitoneally with a 0% to 20% lethal inoculum of murine CMV (MCMV). Murine cytomegalovirus was recovered from homogenates of eye tissues on days 3, 5, and 7 after inoculation. Peak virus titers, mean of 2.93 +/- 0.67 log plaque-forming units of MCMV per gram of ocular tissue occurred on day 5. Urine cytomegalovirus was recovered from explant cultures of eye and optic nerve 14, 21, 60, 90, and 120 days after MCMV inoculation. Murine cytomegalovirus also persisted in intraocular fluids for as long as 90 days. Murine cytomegalovirus infection was confirmed by immunofluorescence antibody staining and transmission electron microscopy. These experiments indicate that MCMV infects the ocular tissues of mice during systemic MCMV infection, and persists in ocular tissues for as long as 120 days after infection. These studies may be directly relevant to ocular disorders that occur during acquired CMV infections of humans, and suggest that ocular tissues may be a site of CMV persistence or latency.

Respiratory infectivity of a recently isolated Egyptian strain of Rift Valley fever virus

The respiratory infectivity of a strain of Rift Valley fever virus isolated in Egypt (strain ZH-501) was compared with that of one isolate from Uganda (Entebbe strain) and two isolates from South Africa (strains SA-51 and SA-75). Studies were performed with ICR mice which were infected by exposure to infectious aerosols composed of particles with a mass median diameter of 0.96 micrometer. The respiratory median lethal doses for ZH-501, Entebbe, SA-51, and SA-75 were 2.2, 1.9, 2.6, and 1.9 log10 plaque-forming units, respectively. Although these values are statistically different, the biological implications of such differences seem unimportant. In an additional study of pathogenesis, a single group of mice was infected with 3.1 log10 plaque-forming units of ZH-501, and tissues were assayed sequentially through 96 h postinfection. Between 6 and 30 h, demonstration of an increasing virus concentration only in the lungs indicated that initial replication occurred there; however, determination of histopathological changes did not reveal evidence of pneumonia. Virus was isolated from the liver by 48 h, and the ultimate outcome of infection was a fulminating and fatal hepatic necrosis.

Enhancement of activity against influenza viruses by combinations of antiviral agents.

In an investigation of alternative therapeutic approaches for the treatment of influenza virus infections, the antiviral activities of rimantadine hydrochloride, amantadine hydrochloride, ribavirin, and combinations of these drugs were assessed in vitro. Madin-Darby canine kidney cell monolayers were inoculated with recent isolates of influenza viruses at low multiplicities of infection, and virus titers were determined after 24 h. The combination of rimantadine and ribavirin resulted in an enhanced antiviral effect (a decrease in virus titer of > 1.0 log10 plaque-forming unit per ml at 24 h relative to the maximal effect of a single drug) against A/USSR/90/77/H1N1, A/Texas/1/77/H3N2, A/New Jersey/76/HSW1N1, and A/PR/834/H0N1 viruses. The degree of inhibition depended on the virus strain used, the drug concentrations, and the virus inoculum. Amantadine and ribavirin showed enhanced activity. Ribavirin in combination with high (50 micrograms/ml), but not low (1.56 to 25 micrograms/ml), concentrations of rimantadine showed an enhanced antiviral effect against B/Hong Kong/72 virus. An assay of Madin-Darby canine kidney cell proliferation in the presence of drugs showed that the enhanced inhibitory effect of drug combinations was not due to increased cytotoxicity.

Pathogenesis of a Venezuelan encephalitis virus strain lethal for adult white rats.

AbstractThe pathogenesis for adult rats of a Venezuelan encephalitis virus (VEE) strain, V‐198, was studied. This virus strain was chosen following virulence‐testing of 12 prototype alphavirus strains, selected from the Semliki Forest, Venezuelan, Eastern, and Western encephalitis virus complexes; only VEE strain V‐198 killed all adult rats when inoculated subcutaneously (sc) with 6.0–6.5 log10 plaque‐forming units (PFU). Lower doses of strain V‐198 by the same route were also lethal: 5.0 and 3.0 log10 PFU killed 100%, and 1.0 log10 PFU killed 70% of rats. Viral infectivity titrations of tissues obtained from V‐198 straininfected rats demonstrated that virus replicated early in thymus and spleen, but did not replicate in bone marrow or liver. Following a brief viremia, virus infectivity titers peaked in brain, but did not persist there. A biphasic neutrophilia also occurred. Lower doses of inoculum virus affected the timing but not the degree of neutrophil fluctuations, virus replication, and clinical disease. Rats, moribund seven days after inoculation of 4.3 log10 PFU, exhibited central nervous system signs and histologic evidence of encephalomyelitis. Significant proportions of rats, otherwise lethally infected with V‐198 virus, were protected by a single intraperitoneal inoculation of hyperimmune V‐198 antiserum, administered 3 or 4, but not 5 days after virus.Since the rat has been well characterized with respect to metabolic alterations following other infectious diseases, it is anticipated that this model for lethal virus‐induced disease will be useful for defining more precisely the functional relationships between viral growth, tissue destruction, metabolic alterations, and clinical course of disease, and that it may suggest specific approaches for effective treatment of viral diseases in general.