Development of models and detection methods for different forms of cytomegalovirus for the evaluation of viral inactivation agents.

BACKGROUND: Cytomegalovirus (CMV) is transmitted by transfusion of infected blood products and can cause serious diseases in specific risk groups. CMV can be present in infected blood as cell-free virus (CFV), cell-associated actively replicating virus (CAV), and cell-associated latent virus (LV). STUDY DESIGN AND METHODS: In vitro models for all three infectious forms of CMV and virus detection assays based on both tissue culture and polymerase chain reaction (PCR) were developed. The utility of the CMV model systems and assays were tested by validation studies of a novel pathogen inactivation agent, PEN110, for red blood cells. RESULTS: Reproducible high titers of CFV and CAV were obtained by optimized tissue culture techniques for CMV-infected MRC-5 cells. An LV model was obtained with CMV-infected THP-1 cells and reactivation of virus replication by phorbol ester treatment. The model systems showed that PEN110 treatment is effective against all three forms of CMV as measured by tissue culture-based infectivity assays and a long-range PCR method specific for detection of damage to CMV viral DNA. CONCLUSION: This study describes model systems to the relevant forms of CMV in blood and detection assays that can be used to evaluate the efficacy of viral inactivation agents.

Development of a sensitive PCR inhibition method to demonstrate HBV nucleic acid inactivation.

BACKGROUND: The evaluation of pathogen reduction technologies with relevant viruses currently contaminating the blood supply is limited by the availability of high-titer virus inocula and sensitive in vitro or in vivo infectivity assays. Because HBV infectivity can only be assessed by in vivo studies with chimpanzees, a sensitive PCR inhibition assay was developed to measure PEN110 inactivation of HBV. STUDY DESIGN AND METHODS: PCR amplification of 1.1 kb of HBV genome was optimized to determine DNA damage introduced by treatment with PEN110 in RBCs. Inactivation of duck HBV (DHBV) in RBCs, with measurement of the in vitro infectivity, was performed to validate the PCR assay. RESULTS: The PCR was highly specific and sensitive for amplification of the HBV genome and used to demonstrate a reduction of at least 7.2 and 8.1 log geq per mL within the first 18 hours of PEN110 treatment. PEN110 inactivation of DHBV was also achieved within the first 18 hours with a reduction factor of at least 5.0 log tissue culture infectious dose 50 percent per mL, suggesting that PCR inhibition is an alternative to infectivity assays. CONCLUSION: This study establishes PCR inhibition as a reasonable approach to assess the efficiency of PEN110 inactivation of human pathogens with human plasma donations that have been found to contain high titers of relevant agents during different stages of infection.

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

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Inactivation of HIV in blood.

BACKGROUND: The residual risk of HIV infection after HIV screening tests in combination with the risk of new emerging pathogens entering the blood supply has sparked research on the development of a technology for reduction of pathogens in RBCs. STUDY DESIGN AND METHODS: HIV-1 was treated with PEN110 (INACTINE) and analyzed for the kinetics of virus reduction in RBC, the effect of PEN110 on nucleic acids, the integrity of the virus morphology and viral proteins, and the ability of the virus to bind HIV cell receptors and enter susceptible cells. RESULTS: PEN110 effectively reduced HIV-1 to the limit of detection for a reduction factor of at least 5.57 log 50 percent tissue culture infectious dose per bulk test. The PEN110-treated virions maintained their morphology, protein integrity, and functionality. However, the PEN110-treated HIV-1 RNA genome was neither functional to serve as a template for RT-PCR amplification of about 1 kb nor able to support viral DNA synthesis in cell culture. CONCLUSION: These results suggest that PEN110 inactivates HIV-1 by targeting the viral nucleic acid.

Process for the preparation of pathogen-inactivated RBC concentrates by using PEN110 chemistry: preclinical studies.

BACKGROUND: A pathogen-inactivation process for RBC concentrates is being developed by using PEN110 chemistry (INACTINE, V.I. Technologies). The objective of this study was to characterize the quality of RBCs prepared by using the PEN110 process and to measure the virucidal effect achieved against two viruses. STUDY DESIGN AND METHODS: Virology and RBC studies were conducted with standard RBC units treated with 0.1-percent (vol/vol) PEN110 at 22 degrees C for 6 hours. The quality of PEN110-treated human RBCs was assessed with biochemical and phenotypic variables. The in vivo viability of PEN110-treated RBCs in baboons was studied with the double-label (51)Cr/(125)I method. RESULTS: Decreases in infectious titer by inactivation of greater than a 5 log 50-percent tissue culture infectious doses per mL of bovine viral diarrhea virus (an enveloped RNA virus) and porcine parvovirus (a nonenveloped DNA virus) was observed. RBC hemolysis was less than 1 percent after 42 days of storage, and no changes in RBC antigens were observed. The in vivo viability of PEN110-treated baboon RBCs was unchanged from control. CONCLUSION: The preparation of RBCs by using the PEN110 process achieved a significant viral reduction of two diverse viruses without causing adverse effects to the RBCs. The process appears to be a promising approach, thus justifying further study.