Induction of latent human cytomegalovirus by conventional gamma irradiation and prevention by treatment with INACTINE PEN110.

BACKGROUND AND OBJECTIVES: Two different leucocyte-inactivation technologies--gamma irradiation and INACTINE PEN110--were evaluated for their effects on cell-associated human cytomegalovirus (CMV). MATERIALS AND METHODS: In vitro CMV-infected cells were spiked into leucoreduced red blood cell concentrates (RCC) or medium at a final concentration of 0.5 - 1 x 10(7) cells/ml to mimic non-leucoreduced levels of leucocytes. The spiked RCC/medium was divided into three equal units and treated with gamma irradiation at the US Food and Drug Administration (FDA)-approved dose of 25 Gy, with 0.1% v/v PEN110 at 22 degrees C for 24 h, or stored at 4 degrees C as a control. The treated and control cells were recovered and tested using infectivity, viability and polymerase chain reaction (PCR) assays. RESULTS: Gamma-irradiated CMV-infected cells produced active virus, as shown by both infectivity assays and PCR quantification of viral DNA. PCR analysis demonstrated higher CMV DNA levels in gamma-irradiated, latently infected monocytic THP-1 cells than untreated control cells. The increased virus production in gamma-irradiated cells was paralleled by an increased metabolic rate and the development of enlarged multinuclear cells. In contrast, PEN110 treatment terminated virus replication and completely inactivated the infected cell. CONCLUSIONS: These results demonstrate that gamma irradiation, at levels currently used to treat RCC, has the capacity to induce expression of CMV, whereas PEN110 inhibits CMV replication and efficiently inactivates the infected cells.

Broad-spectrum virus reduction in red cell concentrates using INACTINE PEN110 chemistry.

BACKGROUND AND OBJECTIVES: The risk of transmission of blood-borne pathogens by transfusion is a persistent problem in medicine. To address this safety issue, INACTINE PEN110 chemistry is being utilized to develop a process for preparing pathogen-reduced red blood cell concentrates (RBCC). The purpose of this study was to characterize the virucidal effectiveness of the INACTINE PEN110 chemistry in full units of RBCC by using a panel of viruses with diverse properties in composition, size and shape. MATERIALS AND METHODS: The panel included four enveloped (bovine viral diarrhoea virus, pseudorabies virus, vesicular stomatitis Indiana virus and sindbis virus), six non-enveloped (porcine parvovirus, human adenovirus 2, reovirus 3, vesicular exanthema of swine virus, bluetongue virus, and foot and mouth disease virus) and cell-associated (human immunodeficiency) viruses. All viruses were individually spiked into CPD/AS-1, CP2D/AS-3 and CPD/AS-5 RBCC units and treated with 0.1% PEN110 (vol/vol) at 22 +/- 2 degrees C for up to 22 +/- 2 h. The PEN110 treatment reaction was stopped by chemical quenching, and residual virus was assayed. The cytotoxicity effect of PEN110-treated RBCC on indicator cells and the potential interference with the ability of the virus to infect indicator cells was determined and taken into consideration for calculating the virus-reduction factors, to avoid underestimation or overestimation of the virus reduction. RESULTS: The kinetics of inactivation for viruses spiked into CPD/AS-1, CP2D/AS-3 and CPD/AS-5 RBCC were equivalent. All viruses analysed in this study were reduced to the limit of detection of the assay. The reduction factors for the virus panel ranged from 4.2 to 7.5 log10/ml. CONCLUSIONS: The results from the study demonstrate for the first time that a pathogen-reduction technology for RBCC can achieve a broad-spectrum virucidal effect against both enveloped and non-enveloped viruses. The broad spectrum of virucidal activity of INACTINE PEN110, and equivalent kinetics of virus inactivation in RBCC prepared using different commercially available RBC storage solutions, demonstrate the robustness of this pathogen-reduction process.

Poliomyelitis in intraspinally inoculated poliovirus receptor transgenic mice.

Mice transgenic with the human poliovirus receptor gene develop clinical signs and neuropathology similar to those of human poliomyelitis when neurovirulent polioviruses are inoculated into the central nervous system (CNS). Factors contributing to disease severity and the frequencies of paralysis and mortality include the poliovirus strain, dose, and gender of the mouse inoculated. The more neurovirulent the virus, as defined by monkey challenge results, the higher the rate of paralysis, mortality, and severity of disease. Also, the time to disease onset is shorter for more neurovirulent viruses. Male mice are more susceptible to polioviruses than females. TGM-PRG-3 mice have a 10-fold higher transgene copy number and produce 3-fold more receptor RNA and protein levels in the CNS than TGM-PRG-1 mice. CNS inoculations with type III polioviruses differing in relative neurovirulence show that these mouse lines are similar in disease frequency and severity, demonstrating that differences in receptor gene dosage and concomitant receptor abundance do not affect susceptibility to infection. However, there is a difference in the rate of accumulation of clinical signs. The time to onset of disease is shorter for TGM-PRG-3 than TGM-PRG-1 mice. Thus, receptor dosage affects the rate of appearance of poliomyelitis in these mice.

DNA sequence analysis of conserved and unique regions of swinepox virus: identification of genetic elements supporting phenotypic observations including a novel G protein-coupled receptor homologue.

Swinepox virus (SPV) contains a double-stranded cross-linked linear DNA genome of approximately 175 kilobase pairs with terminal inverted repetitions (TIRs) of 4.3 kb. The nucleotide sequence was determined for fragments from several regions of the genome including a 2.85-kb fragment from the central potentially conserved portion and two fragments within the presumed variable near-terminal regions which tend to be unique to a given poxvirus. The core sequence contains one partial and two complete open reading frames that are highly conserved and colinear with three contiguous ORFs within the HindIII D fragment of vaccinia virus (VV). The two near-terminal fragments, encompassing 14.2 and 3.6 kb, are respectively located 2.1 kb internal to the left and right cross-linked termini of the DNA and span the TIR junctions. The sequences encode 25 open reading frames including numerous proteins predicted to be membrane-bound or secreted in infected cells. Several ORFs unique to SPV were identified that may be involved in cell attachment, immune modulation, and pathogenesis including a novel poxvirus G protein-coupled receptor. In addition, several polypeptides encoded within the near-terminal regions of vaccinia virus DNA that function as host range or virulence factors are lacking within this region of swinepox virus including the VV growth factor, complement-binding protein, and ORFs C7L and K1L, associated with host range. The lack of these functional homologues could explain the characteristic attenuated phenotype and limited host range of SPV.