Evaluation of dengue virus strains for human challenge studies.

Discordance between the measured levels of dengue virus neutralizing antibody and clinical outcomes in the first-ever efficacy study of a dengue tetravalent vaccine (Lancet, Nov 2012) suggests a need to re-evaluate the process of pre-screening dengue vaccine candidates to better predict clinical benefit prior to large-scale vaccine trials. In the absence of a reliable animal model and established correlates of protection for dengue, a human dengue virus challenge model may provide an approach to down-select vaccine candidates based on their ability to reduce risk of illness following dengue virus challenge. We report here the challenge of flavivirus-naïve adults with cell culture-passaged dengue viruses (DENV) in a controlled setting that resulted in uncomplicated dengue fever (DF). This sets the stage for proof-of-concept efficacy studies that allow the evaluation of dengue vaccine candidates in healthy adult volunteers using qualified DENV challenge strains well before they reach field efficacy trials involving children. Fifteen flavivirus-naïve adult volunteers received 1 of 7 DENV challenge strains (n=12) or placebo (n=3). Of the twelve volunteers who received challenge strains, five (two DENV-1 45AZ5 and three DENV-3 CH53489 cl24/28 recipients) developed DF, prospectively defined as ≥2 typical symptoms, ≥48h of sustained fever (>100.4°F) and concurrent viremia. Based on our study and historical data, we conclude that the DENV-1 and DENV-3 strains can be advanced as human challenge strains. Both of the DENV-2 strains and one DENV-4 strain failed to meet the protocol case definition of DF. The other two DENV-4 strains require additional testing as the illness approximated but did not satisfy the case definition of DF. Three volunteers exhibited effusions (1 pleural/ascites, 2 pericardial) and 1 volunteer exhibited features of dengue (rash, lymphadenopathy, neutropenia and thrombocytopenia), though in the absence of fever and symptoms. The occurrence of effusions in milder DENV infections counters the long-held belief that plasma leakage syndromes are restricted to dengue hemorrhagic fever/dengue shock syndromes (DHF/DSS). Hence, the human dengue challenge model may be useful not only for predicting the efficacy of vaccine and therapeutic candidates in small adult cohorts, but also for contributing to our further understanding of the mechanisms behind protection and virulence.

White particulate matter found in blood collection bags consist of platelets and leukocytes.

BACKGROUND: In late January 2003, some blood centers and hospitals throughout the US voluntarily sus-pended the use of some RBC and plasma units for trans-fusion due to the presence of unknown white particulate matter (WPM) in these units. To better understand the WPM phenomena, a number of technologies were used to establish the nature of the particulates observed in Terumo Collection sets. STUDY DESIGN AND METHODS: All AS-5 nonleuko-reduced RBCs and plasma units were visually inspected for WPM by placing the bags on a flat counter, undisturbed, for approximately 10 minutes and then perform-ing a visual examination for particles. Particles were isolated and placed on microscope slides or in plastic tubes for further analysis. Electron microscopy, bright field microscopy, differential interference contrast microscopy, infrared spectroscopy, and flow cytometry procedures were performed to establish the nature of the particulate matter. In addition, leukoreduction filters and blood transfusion sets were used on RBCs units with WPM. RESULTS: The particles were mostly composed of PLTs and WBCs, and fragments of these cells. All macroscopic WPM was removed from RBCs with leukoeduction and transfusion filters. CONCLUSIONS: WPM originated from PLTs and WBCs. Foreign matter (e.g., plastic) was not observed in any of the units. Leukoreduction and transfusion filters can be used to remove macroscopic WPM.

Antiserum generated by DNA vaccine binds to hepatitis E virus (HEV) as determined by PCR and immune electron microscopy (IEM): application for HEV detection by affinity-capture RT-PCR.

Previously, we have described that injection of an expression vector containing hepatitis E virus (HEV) open reading frame 2 (HEV-ORF-2) generated a strong antibody response in mice. To characterize the reaction of this antiserum with native HEV and to evaluate its potential diagnostic application, we tested the antiserum's ability to bind HEV using immune electron microscope (IEM) and affinity-capture reverse transcription polymerase chain reaction (RT-PCR) amplification. Antiserum to ORF-2 aggregated HEV virions as seen by electron microscopy, providing direct evidence that ORF-2 encodes a structural protein. Antiserum also captured HEV for RT-PCR amplification. This antiserum bound HEV from diverse origins (Asia, Africa, Mexico) at virus concentrations found in patient fecal specimens and bile from inoculated non-human primates. The specificity of the affinity binding was demonstrated when pre-immune sera or sera collected from mice injected with control DNA vector (lacking the HEV ORF-2 gene) failed to bind HEV for RT-PCR amplification and IEM. Specific RT-PCR amplification was confirmed by restriction enzyme digestion of PCR products. The sensitivity of the binding was evaluated by RT-PCR amplification of serially diluted bile containing a genetically divergent HEV, Mexico'86. HEV was detected in a 10(-8) dilution of this bile. This is the first report that antibodies elicited by a DNA vaccine recognize native HEV. Our results indicate that ORF-2 encodes a structural protein and that antiserum to this protein enables simple, sensitive, and specific HEV detection by affinity-capture RT-PCR.

Pathogenesis of hepatitis A in orally inoculated owl monkeys (Aotus trivirgatus).

The pathogenesis of hepatitis A virus (HAV) infection was studied in owl monkeys following oral administration of the wild-type HM-175 strain of HAV. Stools were collected daily and blood and pharyngeal swabs twice weekly for viral isolation, and animals were necropsied at various intervals after inoculation. Organs were examined for the presence of virus by isolation in cell culture and for viral antigens by immunofluorescence. Monkeys excreted HAV in the stools for 1-4 days after inoculation, presumably due to the residual unabsorbed inoculum. No virus was found in stools for the next 2-3 days. HAV re-appeared on days 4-7 and then persisted through day 39. Viremia occurred on the 10th day and continued until day 35. Virus was isolated occasionally from throat swabs 1 or 2 weeks after it was detected in stools and blood, and there was no evidence that HAV replicated in the pharyngeal tissues. Animals acquired anti-HAV antibody by the 4th week, and alanine aminotransferase (ALT) was elevated 5-5.5 weeks after inoculation. HAV was isolated from liver 5 days after inoculation; however, viral antigens were first detected in Kupffer cells of the liver at 14 days and in hepatocytes at 21 days. HAV antigen was detected in epithelial cells of the intestinal crypts and in the cells of the lamina propria of the small intestine 3 days postinoculation and thereafter until the 5th week, suggesting that these cells might represent an additional site of HAV replication.

Immunohistochemical detection of cytokines in tissues of Aotus monkeys infected with hepatitis A virus.

Cytokines IL-1-beta, IL-2, and TNF alpha were detected in occasional cells within portal inflammatory infiltrates beginning 3 weeks after oral inoculation of monkeys with HAV. The number of cells secreting those cytokines did not increase, and they were not of importance in the pathogenesis. Production of cytokines IL-6 and IL-4 by T lymphocytes infiltrating portal areas started 4 weeks after inoculation, stimulating local expansion of B cells, probably secreting antibodies to HAV. IL-6 and IL-4 may also stimulate cytotoxic activity of a few CD8+ lymphocytes.

Attenuation phenotype of a cell culture-adapted variant of hepatitis A virus (HM175/p16) in susceptible New World owl monkeys.

The virulence of a clonally isolated, cell culture-adapted hepatitis A virus (HM175/p16) was assessed in 4 seronegative owl monkeys inoculated intravenously with 2.8 x 10(4) radioimmunofocus-forming units of virus. The virus was highly attenuated, even though its complete nucleotide sequence contains only 19 mutations from the wild-type genome. Only 3 monkeys developed antibodies to hepatitis A virus (only 2 within 96 days of virus inoculation). One monkey had viremia and significantly elevated serum aminotransferase levels. In this animal, maximum viremia and fecal shedding of virus occurred 30-33 days after inoculation. In contrast, in earlier studies of a related cell culture-adapted but still hepatovirulent virus (HM175/S18), viremia was documented in 6 of 6 animals and peak viremia and fecal shedding of virus occurred 18 or 19 days after intravenous inoculation of about one-tenth as much virus.

Experimental hepatitis E: pathogenesis in cynomolgus macaques (Macaca fascicularis).

The pathogenesis of experimental hepatitis E has not been thoroughly investigated. The purpose of this study was to more accurately document the events in this disease. Cynomolgus macaques were inoculated intravenously with bile or feces containing hepatitis E virus (HEV). Serum, bile, and liver specimens were evaluated with light microscopy, immune electron microscopy, immunofluorescence microscopy, EIA, and polymerase chain reaction. In the third week, there were histopathologic changes and HEV antigen (HEVAg) in liver, HEV in bile, and alanine aminotransferase (ALT) elevations. Widespread pathologic changes were detected during the fourth week and antibody to HEV (anti-HEV) and peak ALT values in the fifth or sixth week. By the sixth week, HEVAg had disappeared but pathologic changes persisted. This study supports the concept that experimental hepatitis E has an initial phase in which hepatic HEV replication is accompanied by the onset of hepatitis and a later phase in which the appearance of anti-HEV is accompanied by progression of the hepatitis.

Infection of owl monkeys (Aotus trivirgatus) and cynomolgus monkeys (Macaca fascicularis) with hepatitis E virus from Mexico.

Owl and cynomolgus monkeys were inoculated with hepatitis E virus (HEV) to compare disease models and produce antibody and virus. By immune electron microscopy (IEM), all six owl monkeys were shown to have serologic responses manifested by unusually high levels of anti-HEV at 6 months, but only three developed hepatitis. Virus-related antigen in liver (HEV Ag) was detected by immunofluorescence microscopy of biopsies from two of four owl monkeys; one with HEV Ag also had HEV in acute-phase bile (detected by IEM) and feces (detected by infecting another owl monkey). In contrast, cynomolgus monkeys propagated HEV to higher levels and all five had hepatitis. Moderate-to-high levels of HEV Ag correlated with detectable HEV in bile for both species. Thus, the value of using HEV-infected cynomolgus was confirmed. Owl monkeys were shown to be HEV-susceptible and sources of high-level anti-HEV; Sustained anti-HEV in these monkeys may also be useful for understanding immune responses.

Virus-like particles in the liver of a patient with fulminant hepatitis and antibody to hepatitis E virus.

In earlier studies, hepatitis E virus (HEV) particles were detected in the stools of patients with enterically transmitted non-A, non-B (ENANB) hepatitis, and HEV was etiologically associated with this disease. Such particles have not been observed in the liver, however. We describe the pathological findings in the liver of a young pregnant woman from Nepal who died as a result of fulminant NANB hepatitis. IgM antibody to HEV was detected in the patient's serum by immune electron microscopy, suggesting that she was acutely infected with that virus. On light microscopic examination of the liver we observed cholestatic hepatitis with proliferation of bile ductules and pseudoglandular arrangement of hepatocytes around distended bile canaliculi. Three types of virus-like particles were detected by electron microscopy. The most frequently observed particles were in cells lining small bile ductules; they measured 32-37 nm and were enclosed by a membrane. Particles of a second type were seen in clusters in the sinusoidal cells; they were uniform in size, without a membrane, and measured about 32 nm in diameter. Particles of a third type (65 nm) were found in epithelial cells of the small bile ductules. Among the particles we detected, the 32 nm particles most closely resembled those of HEV.

In vivo replication and reversion to wild type of a neutralization-resistant antigenic variant of hepatitis A virus.

Six seronegative owl monkeys were intravenously inoculated with an antigenic variant (S18) of hepatitis A virus that is highly adapted to growth in cell culture and resists neutralization by monoclonal antibodies due to replacement of aspartic acid 70 of capsid protein VP3 with histidine. Each developed hepatitis 22-33 days after inoculation. Virus in feces, serum, and liver was quantified by radioimmunofocus assay. Viremia developed 7-11 days after inoculation, in parallel with fecal shedding of virus, and persisted for a mean of 20.5 days. Although the antigenic variant was recovered from feces or liver of three animals, virus in liver at the time of enzyme elevations was predominantly wild-type antigenic phenotype. Virus was not recovered from liver 96 days after challenge. These studies further define virologic events in hepatitis A and show that in vivo replication of an antigenic variant was restricted compared with that of wild-type virus.

Demonstration of hepatitis A virus in cell culture by electron microscopy with immunoperoxidase staining.

Virus-like particles were demonstrated by electron microscopy in BS-C-1 cells infected with hepatitis A virus (HAV). Particles were usually enclosed within vesicles and accompanied by myelin-like membranous structures. Less often they were seen free in the cytoplasm. They were never observed in the nucleus. By immunoperoxidase staining particles were found to contain HAV antigens. These antigens were also found in the membrane of the vesicles surrounding the masses of particles and adjacent parts of the mitochondrial membranes. Our results demonstrate the usefulness of an electron microscopic immunocytochemical technique to study replication of HAV.

Acute and latent infection of sensory ganglia with herpes simplex virus: immune control and virus reactivation.

The role of antiviral antibody in controlling the acute and latent phases of herpes simplex virus (HSV) infection in sensory ganglia of mice was studied in vitro and in vivo. Organ cultures of ganglia inoculated in vitro with HSV produced infectious virus for at least 3 weeks. In the presence of antiviral antibody, the titre of virus was markedly reduced, but the infection was not eliminated. Similarly, passive administration of antibody to HSV-infected immunodeficient (nude) mice reduced the virus titre but did not eliminate the acute phase of the ganglionic infection. Suppression of the cell-mediated immune response in latently infected immunocompetent mice by treatment with cyclophosphamide and/or X-irradiation resulted in reactivation of HSV in up to 70% of the animals. Reactivation was demonstrated by recovering infectious virus in cell-free homogenates of ganglia and eye globes and by finding virus antigens in ganglia by immunofluorescent staining. Reactivation occurred both in vitro and in vivo in the presence of high concentrations of neutralizing antibody. It is concluded that antibody alone is not sufficient to eliminate the acute phase of the ganglionic infection and that cytotoxic agents known to suppress the host's cellular immune response can reactivate virus in the presence of neutralizing antibody.

Effect of immunization on the development of latent ganglionic infection in mice challenged intravaginally with herpes simplex virus types 1 and 2.

Immunization of BALB/c mice with virulent and avirulent strains of HSV-1 resulted in high levels of neutralizing antibody and protected against both the lethal effect of the virus and the development of a latent ganglionic infection when animals were challenged by the intravaginal route. In animals immunized with avirulent strain of HSV-2 and challenged with a high virulent strain of HSV-2, substantial protection against death was observed despite low levels of neutralizing antibody. Nineteen per cent of the survivors, however, developed a latent ganglionic infection. Relatively little protection was observed in mice immunized with HSV-1 and challenged with HSV-2.

Persistent shedding of adenovirus in urine of chimpanzees.

A new adenovirus, designated Pan 11, was isolated repeatedly from the urine of several chimpanzees for more than 1 year. One chimpanzee had chronic interstitial nephritis; the others were healthy. Most chimpanzees tested had neutralizing antibodies to Pan 11 virus in the serum; three people who worked with chimpanzees also acquired antibodies. Transplantable rhabdomyosarcomas developed in hamsters inoculated as newborns with Pan 11 virus.