Analysis of the 5'UTR of HCV genotype 3 grown in vitro in human B cells, T cells, and macrophages.

BACKGROUND: Previously, we have reported the isolation and molecular characterization of human Hepatitis C virus genotype 1 (HCV-1) from infected patients. We are now reporting an analysis of HCV obtained from patients infected with HCV genotype 3 (HCV-3) as diagnosed by clinical laboratories. RESULTS: HCV was cultured in vitro using our system. HCV RNA was isolated from patients' blood and from HCV cultured in various cell types for up to three months. The 5'UTR of these isolates were used for comparisons. Results revealed a number of sequence changes as compared to the serum RNA. The HCV RNA produced efficiently by infected macrophages, B-cells, and T-cells had sequences similar to HCV-1, which suggests that selection of the variants was performed at the level of macrophages. Virus with sequences similar to HCV-1 replicated better in macrophages than HCV having a 5'UTR similar to HCV-3. CONCLUSIONS: Although HCV-3 replicates in cell types such as B-cells, T-cells, and macrophages, it may require a different primary cell type for the same purpose. Therefore, in our opinion, HCV-3 does not replicate efficiently in macrophages, and patients infected with HCV-3 may contain a population of HCV-1 in their blood.

Coccidioidomycosis and Pulmonary Emboli: A Report of 5 Cases.

BACKGROUND Coccidioidomycosis is endemic to the Sonoran life zone, which extends from Latin America to the western United States. The principle manifestation is pneumonia but disseminated disease also occurs. Venous thromboembolism occurring in association with this disease has not been reported. We encountered 5 cases of coccidioidomycosis, each complicated by pulmonary emboli, during a single year. We report these cases with the intent of making those caring for patients with coccidioidomycosis aware of this association. CASE REPORT A 35-year-old man developed fever and respiratory symptoms. He was initially treated with antibiotics as an outpatient and during a subsequent hospitalization. He was readmitted because of persistent respiratory symptoms and treated presumptively for coccidioidomycosis pneumonia. Hypoxemia persisted and multiple acute pulmonary emboli were evident on imaging. Serological study and organism identification confirmed a diagnosis of coccidioidomycosis infection. Details of this case and 4 additional cases are described. CONCLUSIONS Venous thromboembolism occurred in 5 patients with pulmonary coccidioidomycosis. The etiology of this rare association remains unclear but could be related to regional environmental changes that preceded the appearance of these cases.

The simultaneous presence and expression of human hepatitis C virus (HCV), human herpesvirus-6 (HHV-6), and human immunodeficiency virus-1 (HIV-1) in a single human T-cell.

We have developed a system that isolates and replicates HCV in vitro. These isolates are called CIMM-HCV. This system has made it possible to analyze the biology, nature, and extent of HCV variability, among other things. Individuals that are infected with HIV-1 are often also infected with HCV and HHV-6. In addition to HCV, our lab has systems for replicating HIV-1 and HHV-6. We asked whether all these viruses could infect the same cells. We report here the successful infection of a T-cell (CEM) by CIMM-HCV, HHV-6, and HIV-1. PCR analyses demonstrated that the CEM cells were productively infected by HHV-6A. RT-PCR showed that the same cell culture was positive for HCV and HIV-1. Co-infection of a T-cell by all three viruses was confirmed by transmission electron microscopy (TEM). All these viruses are highly cytolytic; therefore, triply-infected cells were short lived. However, HIV-1 and HCV co-infected cells unexpectedly lasted for several weeks. Viral replication was unhindered and the phenomenon of 'dominance' was not observed in our experiments. In addition, CIMM-HCV was present in the perinuclear space, suggesting their possible synthesis in the nucleus. This report is based entirely on viruses produced in vitro in our laboratories. As part of the determinations of host ranges of these viruses, studies were designed to demonstrate the infection of a single cell by these viruses and to study the consequences of this phenomenon. All measurements were made on cultured cells and cell culture supernatants.

Discovery of significant variants containing large deletions in the 5'UTR of human hepatitis C virus (HCV).

We recently reported the isolation and in vitro replication of hepatitis C virus. These isolates were termed CIMM-HCV and analyzed to establish genotypes and subtypes, which are reported elsewhere. During this analysis, an HCV isolated from a patient was discovered that had large deletions in the 5'UTR. 57% of the HCV RNA found in this patient's sera had 113 or 116 bp deletions. Sequence data showed that domains IIIa to IIIc were missing. Previous studies have suggested that these domains may be important for translation. In vitro replicated HCV from this patient did not contain these deletions, however, it contained a 148 bp deletion in the 5'UTR. Whereas the patient HCV lacked domains IIIa through IIIc, the isolate lacked domains IIIa through IIId. HCV from this patient continues to produce large deletions in vitro, suggesting that the deletion may not be important for the assembly or replication of the virus. This is the first report describing these large deletions.

Analysis of in vitro replicated human hepatitis C virus (HCV) for the determination of genotypes and quasispecies.

Isolation and self-replication of infectious HCV has been a difficult task. However, this is needed for the purposes of developing rational drugs and for the analysis of the natural virus. Our recent report of an in vitro system for the isolation of human HCV from infected patients and their replication in tissue culture addresses this challenge. At California Institute of Molecular Medicine several isolates of HCV, called CIMM-HCV, were grown for over three years in cell culture. This is a report of the analysis of CIMM-HCV isolates for subtypes and quasispecies using a 269 bp segment of the 5'UTR. HCV RNA from three patients and eleven CIMM-HCV were analyzed for this purpose. All isolates were essentially identical. Isolates of HCV from one patient were serially transmitted into fresh cells up to eight times and the progeny viruses from each transmission were compared to each other and also to the primary isolates from the patient's serum. Some isolates were also transmitted to different cell types, while others were cultured continuously without retransmission for over three years. We noted minor sequence changes when HCV was cultured for extended periods of time. HCV in T-cells and non-committed lymphoid cells showed a few differences when compared to isolates obtained from immortalized B-cells. These viruses maintained close similarity despite repeated transmissions and passage of time. There were no subtypes or quasispecies noted in CIMM-HCV.

Mycobacterium szulgai in a patient with advanced acquired immunodeficiency syndrome: an unusual pathogen with unusual multidrug resistance.

A 37-year-old Hispanic man with advanced acquired immunodeficiency syndrome developed extensive pulmonary disease with persistent cough, fever, night sweats, worsening dyspnea, and weight loss. Sputum samples showed scant growth of acid-fast bacilli. He failed to respond to the standard tuberculosis regimen of isoniazid, rifampin, ethambutol, and pyrazinamide. Subsequently, Mycobacterium szulgai was identified, and susceptibility tests showed it to be resistant to all four of those agents. Therapy was changed to clarithromycin, doxycycline, ciprofloxacin, and amikacin. Within 2 weeks, the patient's condition improved significantly, and 6 months after treatment, extensive pulmonary infiltrates had nearly resolved. Fewer than 1% of all human isolates of mycobacteria consist of M. szulgai, which is relatively susceptible to standard antimycobacterial agents. To our knowledge, this is the first reported case of M. szulgai with resistance to all primary antituberculosis drugs.

Transmission of human hepatitis C virus from patients in secondary cells for long term culture.

Infection by human hepatitis C virus (HCV) is the principal cause of post-transfusion hepatitis and chronic liver diseases worldwide. A reliable in vitro culture system for the isolation and analysis of this virus is not currently available, and, as a consequence, HCV pathogenesis is poorly understood. We report here the first robust in vitro system for the isolation and propagation of HCV from infected donor blood. This system involves infecting freshly prepared macrophages with HCV and then transmission of macrophage-adapted virus into freshly immortalized B-cells from human fetal cord blood. Using this system, newly isolated HCV have been replicated in vitro in continuous cultures for over 130 weeks. These isolates were also transmitted by cell-free methods into different cell types, including B-cells, T-cells and neuronal precursor cells. These secondarily infected cells also produced in vitro transmissible infectious virus. Replication of HCV-RNA was validated by RT-PCR analysis and by in situ hybridization. Although nucleic acid sequencing of the HCV isolate reported here indicates that the isolate is probably of type 1a, other HCV types have also been isolated using this system. Western blot analysis shows the synthesis of major HCV structural proteins. We present here, for the first time, a method for productively growing HCV in vitro for prolonged periods of time. This method allows studies related to understanding the replication process, viral pathogenesis, and the development of anti-HCV drugs and vaccines.

Oral doxycycline for treatment of neurosyphilis in two patients infected with human immunodeficiency virus.

The frequency of syphilis has been increasing during the past 5 years primarily among men who have sex with men, many of whom are infected with the human immunodeficiency virus (HIV). Data on treatment options other than intravenous or intramuscular penicillin for syphilis are very limited. We describe two HIV-infected patients with asymptomatic neurosyphilis who were successfully treated with oral doxycycline. The first patient was a 45-year-old Hispanic man with well-suppressed HIV RNA who had a positive Venereal Disease Research Laboratory (VDRL) titer of 1:128. His cerebral spinal fluid (CSF) revealed a positive VDRL titer of 1:16, and an elevated white blood cell count of 96 cells/mm(3) and protein level of 89 mg/dl. He received high-dose doxycycline 200 mg twice/day for 28 days. Two months later, his CSF VDRL titer, white blood cell count, and protein level decreased to 1:4, 5 cells/mm(3), and 60 mg/dl, respectively. The second patient was a 37-year-old Caucasian man with complications from acquired immunodeficiency disease. A routine VDRL titer was found to be 1:64. Although the CSF VDRL was nonreactive, both his white blood cell count and protein level were elevated at 29 cells/mm(3) and 46 mg/dl, respectively. High-dose doxycycline 200 mg twice/day was prescribed for 28 days. Three months later, the patient's VDRL titer decreased to 1:2; his CSF white blood cell count decreased significantly to 1 cell/mm(3), and his protein level was within normal limits. Clinicians should be aware that an extended course of high-dose, oral doxycycline may be an effective and safe alternative regimen to intravenous or intramuscular penicillin, without requiring hospitalization or home health care, for the treatment of neurosyphilis in HIV-infected patients. Prospective trials are needed to assess the long-term efficacy oral doxycycline for neurosyphilis.

Serum tumor markers.

Monoclonal antibodies are used to detect serum antigens associated with specific malignancies. These tumor markers are most useful for monitoring response to therapy and detecting early relapse. With the exception of prostate-specific antigen (PSA), tumor markers do not have sufficient sensitivity or specificity for use in screening. Cancer antigen (CA) 27.29 most frequently is used to follow response to therapy in patients with metastatic breast cancer. Carcinoembryonic antigen is used to detect relapse of colorectal cancer, and CA 19-9 may be helpful in establishing the nature of pancreatic masses. CA 125 is useful for evaluating pelvic masses in postmenopausal women, monitoring response to therapy in women with ovarian cancer, and detecting recurrence of this malignancy. Alpha-fetoprotein (AFP), a marker for hepatocellular carcinoma, sometimes is used to screen highly selected populations and to assess hepatic masses in patients at particular risk for developing hepatic malignancy. Testing for the beta subunit of human chorionic gonadotropin (beta-hCG) is an integral part of the diagnosis and management of gestational trophoblastic disease. Combined AFP and beta-hCG testing is an essential adjunct in the evaluation and treatment of nonseminomatous germ cell tumors, and in monitoring the response to therapy. AFP and beta-hCG also may be useful in evaluating potential origins of poorly differentiated metastatic cancer. PSA is used to screen for prostate cancer, detect recurrence of the malignancy, and evaluate specific syndromes of adenocarcinoma of unknown primary.