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Background: Although the SARS-CoV-2 virus is transmitted mainly through the respiratory tract, possible transmission by transfusion from asymptomatic carriers should be explored. As yet there are no reports of transfusion transmission of COVID-19. Haemovigilance findings within a three-month surveillance period during the new coronavirus pandemic are presented., Materials and Methods: Due to great demand and shortage, blood sessions in outpatient facilities were organized during the high prevalence period of COVID-19, alongside a national plan to monitor the evolving public health situation by random molecular screening of high-risk groups of the population. Haemovigilance protocols were implemented as well as surveillance for any COVID-19 case reported post-transfusion. A 14-day quarantine and follow-up molecular and antibody testing of any COVID-19 positive case was obligatory., Results: Post-donation, post-transfusion information and molecular testing of swab samples collected from three asymptomatic donors at risk for COVID-19, revealed the case of an immunosupressed patient who had been transfused with whole blood derived platelets from a donor subsequently diagnosed with COVID-19. The recipient exhibited no symptoms of the disease. Molecular and antibody testing results were negative., Conclusion: Haemovigilance provided information supporting the absence of transfusion transmission of COVID-19, thus strengthening the hypothesis that, even if it cannot yet be definitively ruled out, COVID-19 is not transmitted through blood transfusion. As of early June 2020, a perfect test does not exist, therefore haemovigilance along with the implementation of strict proactive measures is crucial to identify eluding asymptomatic individuals and ensure blood safety during the pandemic., (Copyright © 2020 Société française de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved.)

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration and high mortality in neonatal piglets. Two different genogroups of PEDV, S INDEL [PEDV variant containing multiple deletions and insertions in the S1 subunit of the spike (S) protein, G1b] and non-S INDEL (G2b) strains were detected during the diarrheal disease outbreak in US swine in 2013-2014. Similar viruses are also circulating globally. Continuous improvement and update of biosecurity and vaccine strains and protocols are still needed to control and prevent PEDV infections worldwide. Although the non-S INDEL PEDV was highly virulent and the S INDEL PEDV caused milder disease, the latter has the capacity to cause illness in a high number of piglets on farms with low biosecurity and herd immunity. The main PEDV transmission route is fecal-oral, but airborne transmission via the fecal-nasal route may play a role in pig-to-pig and farm-to-farm spread. PEDV infection of neonatal pigs causes fecal virus shedding (alongside frequent detection of PEDV RNA in the nasal cavity), acute viremia, severe atrophic enteritis (mainly jejunum and ileum), and increased pro-inflammatory and innate immune responses. PEDV-specific IgA effector and memory B cells in orally primed sows play a critical role in sow lactogenic immunity and passive protection of piglets. This review focuses on the etiology, transmission, pathogenesis, and prevention and control of PEDV infection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Several successive arbovirus outbreaks have affected French Polynesia (FP) in the recent past years due to different dengue serotypes (DENV) present for several decades, Zika (ZIKV) (2013-2014) and chikungunya (CHIKV) (2014-2015) viruses with a potential impact on blood safety and blood supply due to the geographical isolation of these islands. This study reports an assessment of the impact of these outbreaks on blood products supply and infectious safety in FP and discuss the effectiveness of implemented preventive measures., Methods: To ensure the infectious safety of blood products during outbreaks, several measures have successively been introduced as the selection of donors suspected of infection, the nucleic acid testing (NAT) and the pathogen reduction of platelets and plasmas., Results: The donor deferral rate increased by 6% between 2012 and 2014 without changes in the number of collected donations. NAT excluded five blood donations reactive for DENV RNA, 42 for ZIKV and 34 for CHIKV. As Zika screening could not been implemented before the third month of the outbreak, 36 blood products from ZIKV-infected donors were transfused to 26 recipients. However, no transfusion-transmitted arbovirus has been reported., Conclusion: The last past arboviruses outbreaks did not have a significant impact on blood supply in FP. The measures introduced to prevent arbovirus transmission by transfusion were able to maintain infectious safety for all blood products without impairing self-sufficiency., (Copyright © 2019 Société française de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved.)

Background: Confidential unit exclusion (CUE) is a mechanism developed in the1980s to increase blood safety by allowing donors to indicate confidentially if they think their blood is not safe for transfusion. While it has been used widely around the world, the efficacy of this strategy is still unclear. The present study evaluated the efficacy of the CUE system at the Blood Transfusion Center of Kohgiluyeh and Boyer Ahmad Province (KBTC), Iran by comparing the prevalence of transfusion-transmitted infectious (TTIs) in CUE and non-CUE groups., Methods: This descriptive study used data from all volunteer blood donors over a ten-year period. Donors were classified in two groups: CUE and non-CUE. Screening tests were performed for hepatitis B surface antigens (HBs-Ag), hepatitis C virus antibody (HCV-Ab), and Human immunodeficiency virus antibody (HIV-Ab) and any repeatedly reactive results were confirmed by standard methods. Significant differences were determined by Chi 2 test. The sensitivity, specificity and PPV of the CUE system were also calculated., Results: In the present study, the non-CUE and CUE groups consisted of 98.94% and 1.06% of volunteer blood donors, respectively. First-time donors selected the CUE option more often than repeated and regular donors. The prevalence of TTIs was significantly higher in the CUE group and CUE sensitivity, specificity, and PPV were 5.5%, 98.95%, and 0.96%, respectively., Conclusion: We recommend the CUE system be pursued for further enhancement of blood safety. However, further studies are needed to establish the overall usefulness of this procedure throughout the whole country., (Copyright © 2019 Société française de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved.)

Thanks to the significant advent of high throughput sequencing in the last ten years, it is now possible via metagenomics to define the spectrum of the microbial sequences present in human blood samples. Therefore, metagenomics sequencing appears as a promising approach for the identification and global surveillance of new, emerging and/or unexpected viruses that could impair blood transfusion safety. However, despite considerable advantages compared to the traditional methods of pathogen identification, this non-targeted approach presents several drawbacks including a lack of sensitivity and sequence contaminant issues. With further improvements, especially to increase sensitivity, metagenomics sequencing should become in a near future an additional diagnostic tool in infectious disease field and especially in blood transfusion safety., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: CMV viremia is a contributor to poor outcomes in critically ill patients with sepsis., Objectives: To assess the expression levels of genes encoding inflammasome-related proteins in the development of CMV viremia in critically ill patients with sepsis., Study Design: A cohort of CMV-seropositive critically ill patients with sepsis due to bloodstream infection underwent weekly testing for CMV viremia. Blood samples to evaluate mRNA levels of genes encoding CASP1, ASC, NLRP1, NLRP3, and NLRP12 were collected at the time of enrollment. Clinical outcomes were assessed at 30days or until death/discharge from ICU., Results: CMV viremia was documented in 27.5% (8/29) of the patients, a median of 7days after the onset of bacteremia. Patients with sepsis who developed CMV viremia had higher CASP1 although this was not statistically significant (relative mean 3.6 vs 1.8, p=0.13). Development of high grade CMV viremia however, was significantly associated with CASP1; septic patients who developed high grade CMV viremia had significantly higher CASP1than all other patients (relative mean 5.5 vs 1.8, p=0.016)., Conclusions: These data document possible involvement of inflammasome in the pathogenesis of CMV. Regulating the host immune response by agents that target these genes may have implications for improving CMV-related outcomes in these patients., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: CMV infection remains major complication after kidney transplantation, thus diagnostics tools that would improve identification of individuals at risk of development of CMV - related complications are useful. For this reason, searching for proper immunological biomarkers candidates gives hope to individualize antiviral therapy and minimize side effects of antiviral drugs., Objectives: The purpose of this research was to assess immune assays that can be used to predict the likelihood of CMV viremia after kidney allotransplantation., Study Design: In the study, immunological markers of CMV viremia were assessed in 52 kidney transplant recipients during two years lasting follow-up. Immunological markers associated with viral infection, like lymphocytosis, cytotoxic T lymphocytes (CTL) and serum cytokines levels were compared with less common immunological assays, like activated T lymphocytes, CMV-specific CTL stratified according to naïve/memory phenotype. The test to assess expression of CD28 antigen on CTL, as a possible additional marker of CMV-specificity, was developed., Results: CD28-positive CMV-specific CTL have been found the most useful marker for CMV viremia prediction. Tested value of 3 cells/μl was found to be most suitable for CMV activation assessment with acceptable sensitivity and specificity., Discussion: This preliminary report suggests that CD28-positive CMV-specific CTL could be put at the first line, as possible novel marker associated with CMV viremia development., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Viral safety remains a major concern in transfusion of blood products. Over years, the control measures applied to blood products were made more and more sophisticated; however, the number of infectious agents, and notably of viruses, that can be transmitted by transfusion is increasing continuously. The aim of this review paper is to actualize that published in the same journal by the same authors in 2011 with more details on some of actual vs virtual viral threats that were identified recently in the field of blood transfusion. The main subjects that are covered successively concern the transmission via transfusion of hepatitis E virus, the frequency of transfusion transmitted arboviruses, transfusion at the time of the Ebola epidemics in West Africa, the debated role of Marseillevirus (giant viruses infecting amoebae and suspected to infect human blood latently), and, finally, the recent report of the identification in blood donors of a new member of the Flaviviridae family. The addition of these new viral risks to those already identified-partially controlled or not-pleads for the urgent need to move forward to considering inactivation of infectious agents in blood products., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Background: Replacement donors are considered as having a major risk of transmission of infections to recipients mainly by the World Health Organisation., Study Design and Methods: Seroprevalency of HBV, HCV, HIV and syphilis were determined in 19,783 whole blood donations collected in the Tunisian National Blood Transfusion Centre during the year 2010 (12,968 [65.55%] replacement donations and 6815 [34.44%] voluntary blood donations). For HBV, HCV and syphilis, we performed a univariate analysis to determine whether age, sex and type of donation were risk factors, then multivariate logistic regression, to see if these factors were independent., Results: Mean age of donors was 30.1 years (replacement donors 34.5 years, first time non-remunerated donors 34.5 years, P<0.001). The predominant age group was 30-39 years (35.51%) in replacement donors and 20-29 years (54.15%) in first time non-remunerated donors. Male gender was significantly predominant (73.00% men vs 27.00% women, P<10(-6)). There were significantly more men among replacement donors (82.27% vs 55.38%, P<10(-3)). There were more women in the age groups 18-19 and 20-29 years. Only one HIV seropositive donation was noted in a male first time non-remunerated donor aged 18. Replacement type of donation, male sex and age were three independent risk factors for the HBs Ag carriage. For anti-HCV antibodies and TPHA, only replacement type of donation and age were found out to be risk factors and only age was independent., Conclusion: In Tunisia, replacement blood donors were at higher risk of infection transmission, but only for hepatitis B., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background: In recent years chronic hepatitis E virus (HEV) infections have been reported in immunosuppressed patients, including HIV-positive patients with low CD4 cell counts. Because of delayed anti-HEV seroconversion in patients with CD4 cell count<200 cells/ml it is difficult to draw firm conclusions on HEV-seroprevalence in a population of HIV positive patients., Objectives: To determine the HEV seroprevalence in a population of HIV infected patients., Study Design: We retrospectively analysed the HEV prevalence in a population of 256 HIV infected patients with liver enzyme elevations (LEEs), using HEV specific antibody testing and HEV-RNA detection., Results: Within this cohort we observed a HEV-seroprevalence of 11.7%, without any anti-HEV IgM positive or HEV-RNA positive cases. HEV seropositivity was equally prevalent among different CD4(+) cell count groups., Conclusion: Although HIV infected patients in the Netherlands are at risk of acquiring HEV, the number of acute infections is low and no chronic cases were found., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Viral suppression is a key indicator of antiretroviral therapy (ART) response among HIV-infected patients. Dried blood spots (DBS) are an appealing alternative to conventional plasma-based virologic testing, improving access to monitoring in resource-limited settings. However, validity of DBS obtained from fingerstick in field settings remains unknown., Objectives: Investigate feasibility and accuracy of DBS vs plasma collected by healthcare workers in real-world settings of remote hospitals in Malawi. Compare venous DBS to fingerstick DBS for identifying treatment failure., Study Design: We recruited patients from ART clinics at two district hospitals in Malawi, collecting plasma, venous DBS (vDBS), and fingerstick DBS (fsDBS) cards for the first 149 patients, and vDBS and fsDBS only for the subsequent 398 patients. Specimens were tested using Abbott RealTime HIV-1 Assay (lower detection limit 40 copies/ml (plasma) and 550 copies/ml (DBS))., Results: 21/149 (14.1%) had detectable viremia (>1.6 log copies/ml), 13 of which were detectable for plasma, vDBS, and fsDBS. Linear regression demonstrated high correlation for plasma vs. DBS (vDBS: β=1.19, R(2)=0.93 (p<0.0001); fsDBS β=1.20, R(2)=0.90 (p<0.0001)) and vDBS vs. fsDBS (β=0.88, R(2)=0.73, (p<0.0001)). Mean difference between plasma and vDBS was 1.1 log copies/ml [SD: 0.27] and plasma and fsDBS 1.1 log copies/ml [SD: 0.31]. At 5000 copies/ml, sensitivity was 100%, and specificity was 98.6% and 97.8% for vDBS and fsDBS, respectively, compared to plasma., Conclusions: DBS from venipuncture and fingerstick perform well at the failure threshold of 5000 copies/ml. Fingerstick specimen source may improve access to virologic treatment monitoring in resource-limited settings given task-shifting in high-volume, low-resource facilities., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Blood transfusion is a life-saving therapeutic act without alternative. Each blood transfusion carries the risk of blood-borne pathogens transmission. The present study, is the first establishing the usefulness of blood donors screening questionnaire in the setting of the Gabonese National Blood Transfusion Center., Study Design: Nine hundred and thirty-four blood donors aged between 18 and 48 years old were initially enrolled and submitted to physical examination (body-mass index and blood pressure). After physical examination 854 donors were judged fit for blood donation and were randomly distributed in two groups. The first group of donors did not take the screening questionnaire; whereas the second group went through the screening questionnaire. Both groups were then tested for human immunodeficiency virus, hepatitis B, hepatitis C and syphilis., Results: Data revealed a seroprevalence among the donors of 2.5%, 2.5%, 1.1%, and 3.3% for the human immunodeficiency virus, hepatitis B, hepatitis C and syphilis markers respectively. In the Gabonese setting, blood donors' screening questionnaire reduced respectively by 0.6%, 0.35% and 1.3% the proportion of hepatitis B, hepatitis C and treponema pallidum seroreactive donors being selected for donation. The questionnaire had no positive effect on discriminating human immunodeficiency virus positive donors., Conclusion: Blood donors' seroprevalence of blood-borne pathogens is relatively important in our setting. Blood donors' screening questionnaire reduced the proportion of hepatitis B, hepatitis C and syphilis seropositive blood donors. The questionnaire did not effectively discriminate human immunodeficiency virus-infected donor candidates., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background: Coherent drug/microbicide/vaccine development research would benefit through a precise knowledge of HIV dissemination and its persistence in the female genital tract. Understanding relationship between plasma viremia and cervicovaginal HIV shedding may help to unveil mechanisms underlying transmission, compartmentalization and pathogenesis., Objectives: To study the association between HIV-1 RNA levels in the plasma and CVL specimens., Study Design: Whole blood, plasma and CVL specimens were collected from 36 ART naïve HIV-1 seropositive women qualifying the study inclusion criteria. Absolute CD4 counts, plasma and CVL HIV-1 RNA levels were estimated using commercially available kits (BD MultiSET™ Kit, Becton Dickinson, US and Abbott RT, Abbott Molecular, Germany). Correlation between plasma and CVL viral load was estimated by the Spearman's rank correlation coefficient. Additionally, the correlation between CVL viral load and absolute CD4 counts was studied., Results: HIV-1 viral load in the CVL specimens was successfully quantified using the Abbott RT. Twenty-seven of 36 women (75%) had detectable HIV-1 RNA levels in plasma and CVL specimens. The CVL viral load did not show any correlation with plasma viral load (ρ=0.281, p=0.096) and showed a 'moderate correlation' (ρ=-0.563, p=0.0004) with absolute CD4 counts., Conclusions: Albeit, the Abbott RT is designed for estimating plasma HIV-1 RNA levels, the study reports its use for estimating HIV-1 RNA levels in the CVL specimens as well. In accordance with the previous studies, our results suggest that plasma and CVL viral load are not correlated and plasma viremia might not solely predict cervico vaginal HIV shedding., (Copyright © 2013 Elsevier B.V. All rights reserved.)

There are currently no vaccines or therapeutics to prevent dengue disease which ranges in severity from asymptomatic infections to life-threatening illness. The National Institute of Allergy and Infectious Diseases (NIAID) Division of Intramural Research has developed live, attenuated vaccines to each of the four dengue serotypes (DENV-1-DENV-4). Two doses (10PFU and 1000PFU) of three monovalent vaccines were tested in human clinical trials to compare safety and immunogenicity profiles. DEN4Δ30 had been tested previously at multiple doses. The three dengue vaccine candidates tested (DEN1Δ30, DEN2/4Δ30, and DEN3Δ30/31) were very infectious, each with a human infectious dose 50%≤ 10PFU. Further, infectivity rates ranged from 90 to 100% regardless of dose, excepting DEN2/4Δ30 which dropped from 100% at the 1000PFU dose to 60% at the 10PFU dose. Mean geometric peak antibody titers did not differ significantly between doses for DEN1Δ30 (92 ± 19 vs. 214 ± 97, p=0.08); however, significant differences were observed between the 10PFU and 1000PFU doses for DEN2/4Δ30, 19 ± 9 vs. 102 ± 25 (p=0.001), and DEN3Δ30/31, 119 ± 135 vs. 50 ± 50 (p=0.046). No differences in the incidences of rash, neutropenia, or viremia were observed between doses for any vaccines, though the mean peak titer of viremia for DEN1Δ30 was higher at the 1000PFU dose (0.5 ± 0 vs. 1.1 ± 0.1, p=0.007). These data demonstrate that a target dose of 1000PFU for inclusion of each dengue serotype into a tetravalent vaccine is likely to be safe and generate a balanced immune response for all serotypes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human Immunodeficiency Virus Type-1 (HIV-1) drug-resistance testing is challenging for viral loads below 1,000 copies/mL, but, according to HIV-1 guidelines, it should be considered for improving patient management and treatment options. High-recovery and high-purity extraction methods can enhance standard performances of HIV-1 genotyping assays based on direct full-population sequencing. Aim of the present study was to evaluate performances of the NucliSENS easyMAG (NeM) (BioMerieux, Marcy l'Etoile, F) semi-automated nucleic acid extraction system combined with the direct full-population sequencing ViroSeq HIV-1 genotyping (Abbott, IL, US), for detecting drug resistance in samples with HIV-1 RNA<1,000 copies/mL (n=62). Data were compared with those from the ViroSeq manual extraction in 86 samples with HIV-1 RNA<1,000 copies/mL and studied on HIV-1 reference standards. HIV-1 genotyping was successful in 98% of samples extracted with NeM (61/62) and in 84% of those extracted with ViroSeq (72/86) (X(2)=8.508, p=0.004). HIV-1 RNA levels in samples successfully processed with NeM were significantly lower than those in manually processed ones (mean+/-SD, respectively, 285+/-222 copies/mL vs. 403+/-269 copies/mL) (p=0.004). For HIV-1 RNA levels <300 and 500 copies/mL, performances of HIV-1 genotyping with NeM were significantly high (97% and 98%, vs. 68% and 78% for manual extraction). As assessed on HIV-1 RNA reference standards, the detection rate at 200 copies/mL for HIV-1 genotyping with NeM extraction was 100%. In conclusion, these data support that HIV-1 direct full-population sequencing combined with NeM is associated with a significantly high success rate, thus improving the management of HIV-1 drug resistance in low viremic patients., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Adenovirus serotypes 4 (ADV-4) and 7 (ADV-7) are important causes of febrile acute respiratory disease (ARD) in US military recruits. Previously licensed vaccines, which effectively controlled adenovirus-associated ARD, are no longer available. In the Fall of 2004 we conducted this Phase 1 randomized, double-blind, placebo-controlled trial of the live, oral ADV-4 and ADV-7 vaccines made by a new manufacturer to assess their safety and immunogenicity. The adenovirus vaccines were administered orally together in a single dose to thirty subjects. Twenty eight additional subjects received placebo. Subjects were then observed for 8 weeks. The most commonly reported adverse events were nasal congestion (33%), cough (33%), sore throat (27%), headache (20%), abdominal pain (17%), arthralgia (13%), nausea (13%) and diarrhea (13%). None of these rates differed significantly from placebo. The duration of vaccine virus fecal shedding was 7-21 days. Seventy three percent of vaccine recipients seroconverted to ADV-4 (GMT 23.3) while 63% seroconverted to ADV-7 (GMT 51.1) by Day 28. The new ADV-4 and ADV-7 vaccines were safe and induced a good immune response in the study population. Expanded trials for safety and efficacy are in progress.

As an alternative strategy to classical inactivated viral vaccine against FMDV, naked DNA vaccine is attractive because of safety, flexibility and low cost. However DNA vaccination is usually poorly efficient in target species. Indeed we found that naked DNA plasmids encoding for P1-2A3C3D and GM-CSF proteins did not induce any detectable immunity against FMDV in sheep. Interestingly, we demonstrate herein that formulations of DNA on poly(D,L-lactide-co-glycolide) (PLG) or in lipofectin triggered divergent types of immune responses: PLG stimulated a T cell response and could elicit significant neutralising antibody titers, whereas lipofectin generated even higher antibody titers but no significant T cell response. The DNA/PLG regimen used in five sheep protected against clinical symptoms and viraemia and prevented the carrier state in four of them. Thus formulated DNA can be remarkably efficient against FMDV in a ruminant species that is usually refractory to DNA vaccination.

Hepatitis B virus (HBV) infection is endemic in many Asian countries. Among many transmission routes, transfusion is the one that should be prevented. The first major success in enhancing transfusion safety came with the implementation of hepatitis B surface antigen (HBsAg) in the early 1970s. However, the studies quoted in this review demonstrate that transmission by blood components negative for HBsAg can still occur in the acute phase of infection during the seronegative window period, or during chronic stages of infection (i.c. "occult" HBV infection, OHB). OHB is defined as the presence of HBV DNA in blood or liver tissues in patients negative for HBsAg, with or without any HBV antibodies. Because of limitations in current blood screening practices, OHB is an overlooked source of HBV transmission. For policy development on screening for HBV infection in blood donors, it would be useful to assess the relative contribution of the above two sources of transfusion-transmitted HBV infection from HBsAg-negative donations. New screening policy should be evaluated on the basis of available data or newly designed studies. While anti-HBc screening can climinate residual risk of occult HBV transmission by transfusion in low-endemic areas, it would not be practical in most parts of the world where the prevalence of anti-HBc is >10% as too many otherwise healthy donors will be ineligible. On the contrary, studies mentioned in this paper indicate that nucleic acid amplification test (NAT) or new HBsAg tests of enhanced sensitivity would be effective in the screening of blood donors for OHB in highly endemic countries. However, the cost-effectiveness of blood screening tests is a major concern in Asia. We therefore have systemically reviewed the literature on prevalence and infectivity of OHB in Asian countries and the possible role of NAT for identifying blood donors in the pre-HBsAg window phase or in later stages of OHB.

Data regarding the occurrence of a viremia during rabies virus infections are contradictory. Here, we attempted to clarify the dissimilar results using a qualitative TaqMan PCR assay to detect viral RNA in blood of mice that had been injected intramuscularly with rabies virus. Viral RNA was detected at two different intervals. Initially, RNA was present in blood of 30/32 (94%) mice, from 1h to 2 days after injection of virus. The RNA in the blood at this time most likely resulted from trauma to blood vessels at the injection site and leakage of the inoculated virus into the circulation. Thereafter, from 3 to 30 days, viral RNA was undetectable in the blood of 37 mice that remained free of clinical disease. However, and more importantly, viral RNA was detected again in 21/25 (84%) mice that became clinically ill and were exsanguinated 2-4 days after the onset of paralysis. The presence of viral RNA in blood of the clinically ill mice might have been due to an escape of virus into the bloodstream as a result of viral replication induced injury in the central nervous system and other tissues. Anti-rabies virus neutralizing antibody was detected in sera of 11/21 (52%) clinically ill mice whose blood was positive for rabies viral RNA. The presence of viral RNA in the bloodstream of mice that developed clinical rabies suggested that a viremia might occur in rabies-infected mice. Thus, the current opinion that a viremia does not occur in experimental or natural rabies infections of other species might need to be re-evaluated.

Background: Blood transfusions carry risks of untoward reactions, including the transmission of infections, such as hepatitis B and C. Proper blood donor recruitment and selection, and adequate laboratory screening for infectious markers diminish the risk of transfusion-transmitted infections., Objectives: To estimate the potential risk of acquiring transfusion-transmitted infections by hepatitis B or hepatitis C in 24 Caribbean countries during the period of 1996 to 2003., Study Design: Official national reports for 1996, 2000-2003 of the yearly number of blood donors, screening coverage, and prevalence of serological markers for infectious diseases were used to estimate the risk of patients receiving an HBV- or HCV-positive unit of blood, and of developing an infection after receiving a positive unit. Estimates of number of infections transmitted through transfusion and number of infections prevented by screening of blood were also obtained., Results: During the period analyzed, HBV screening coverage among blood donors was 100% in all countries with the exception of Grenada (0% in 1996) and Saint Lucia (99.5% in 2002). For HCV, only 10 countries reported universal screening in 1996, while 15 did in 2003. The number of countries that did not screen any units for HCV decreased from 11 in 1996 to five in 2003. In general, high prevalence rates of HBV (10-75 per 1000 donors) and HCV (7-19.3 per 1000 donors) markers were found in the majority of countries. We estimated that 235 infections by HCV (1:12471 donations) and two infections by HBV (1:1465373) were transmitted through transfusion because of lack of screening. On the other hand, screening of blood for transfusion prevented 21 005 HCV and 22 100 HBV infections., Conclusions: Blood donor recruitment and coverage of screening for transfusion-transmitted infections, especially HCV, must be improved in the Caribbean countries.

The issue of HBV DNA screening on blood donations is controversially discussed since the economic impact of post-transfusion hepatitis B is expected to be relatively low. We report on a case of HBsAg negative unapparent acute HBV infection, which was detected by HBV NAT testing on 96-member maxi-pools with a commercially available NAT assay, which has a detection threshold of 3 IU/mL of plasma. The presence of an HBsAg escape mutant could be excluded by sequencing the amplified DNA. Follow-up testing showed the presence of an acute HBV infection (anti-HBc-IgM positive) and finally anti-HBs seroconversion. Although the reduction of the diagnostic window with NAT screening on maxi-pools may be relatively low, it may help to improve the residual risk of blood donation, especially in asymptomatic HBV infection, where the HBsAg positive period may be very short and low levels of circulating surface antigen are present. It would also permit to detect occult HBV infection in chronic carriers who are HBsAg negative. Since the viral load in chronic isolated anti-HBc positive carriers is low, there is a potential risk for failure of HBV DNA detection with pool-PCR in blood donors. Anti-HBc screening would reduce the residual risk.

We cloned all open reading frames of a Danish isolate of porcine reproductive and respiratory syndrome (PRRS) virus in DNA vaccination vectors. Pigs were vaccinated using a gene gun with each single construct (ORF1, ORF2, ORF3, ORF4, ORF5, ORF6, or ORF7) or combinations thereof. Vaccination with ORF7 consistently induced antibodies after three vaccinations, while antibodies were only sporadically detected in the remaining groups. After six vaccinations, all pigs were inoculated with PRRS virus and the post-inoculation antibody response was studied. Pigs vaccinated with ORF1 or ORF4 were primed for antibody response against NSP2 or GP4, respectively. Neutralising antibodies were detected in all pigs, with ORF5 vaccinated pigs showing the highest titres.

Bovine viral diarrhea virus (BVDV) with deletions in the 5'-nontranslated region (5'-NTR) were tested for their suitability as live BVD vaccines. Firstly, the genetic stability of the mutants was established by culturing over 15 passages in bovine cells. Secondly, two deletion mutants and the parent strain CP7-5A were characterised with respect to in vivo replication competence, attenuation and induction of protective immunity against BVDV. Naïve calves (n = 5 per group) were inoculated with mutants d2-31 and d5-57 or CP7-5A and 5 weeks later, a challenge with the BVDV type 1 strain New York was performed. The mutants were found to be genetically and phenotypically stable. Moreover, the results indicate that the mutants were attenuated with regard to effects including pyrexia and drop in leucocyte counts. Infection with the mutants induced moderate to high titers of BVDV neutralizing antibodies and completely prevented viremia after challenge infection with a heterologous BVDV strain. Taken together, the 5'-NTR deletion mutants combine a good safety profile with good efficacy and are therefore well suited as candidate live vaccines.

Background: Monitoring trends in residual risk of transfusion-transmitted viral infections is important to assess improvements in blood safety. These trends were analysed over nine overlapping periods of 3 years from 1992 to 2002. The 2000-2002 estimates were compared to the results of HIV-1 and HCV NAT implemented on all blood donations in July 2001., Method: As risk is mainly associated with the window period, residual risks were estimated by multiplying incidence rates by the durations of the window periods. For the first seven periods, incidence rates were calculated from data collected by the blood centres belonging to the Transfusion-Transmissible Agents Working Group which collect more than 50% of blood donations in France, and for the two last periods, on the overall blood supply., Results: On the 2000-2002 period, residual risks without NAT were estimated at 1 in 1,400,000 for HIV, at 1 in 1,000,000 for HCV and at 1 in 400,000 for HBV. With minipool NAT, the residual risk become nearly two times lower for HIV (1 in 2.5 million donations) and seven times lower for HCV (1 in 6.65 million donations). For HIV, of the 4.9 million donations screened with NAT between July 2001 and June 2003, two were remote thanks to the NAT, which is consistent with the NAT expected yield. Concerning HCV, one out of the four WP predicted cases was detected with NAT. Without NAT, the overall residual risk for the three viruses combined (HIV, HCV, HBV) decreased from 1 in 65,000 to 1 in 235,000 donations between 1992 and 2002. Since the implementation of NAT, the current overall residual risk is 1 in 325,000 donations (28% less than without NAT)., Conclusion: NAT results confirm the validity of residual risk estimates given by the model, and the limited benefit of genomic screening due to the very low level of residual risk at the time of its implementation.

Prevention of transfusion-transmitted hepatitis B virus (HBV) has historically relied on serological screening of blood donors using progressively more sensitive HBsAg assays; in some countries anti-HBc assays have also been employed to detect chronic carriers with low-level viremia who lack detectable HBsAg. Nucleic acid amplification testing (NAT) for HCV and HIV has been successfully introduced to screen donors in many developed countries over the past several years; for logistical and cost reasons HCV/HIV NAT screening has been applied to mini-pools (MP) of eight to 96 donor specimens, with only minimal impact of MP dilutions on clinical sensitivity for interdiction of window period (WP) donations. In several countries (e.g., Japan and Germany), HBV NAT has been added to HIV/HCV MP-NAT blood donor screening with small incremental yields of HBsAg/anti-HBc-negative donations, and the major vendors of NAT systems (Roche and Chiron/Gen-Probe) have been developing triplex assays that include HBV DNA detection capacity without compromising HIV or HCV detection. Pooled specimen HBV NAT has also become the standard of practice for screening source plasma donors, with pressure to include HBV DNA detection as a required procedure for use of recovered plasma in manufacture of fractionated derivatives. However, there is controversy over the magnitude of the incremental yield and clinical benefit of HBV MP-NAT over serological screening strategies, as well as the impact of implementation of HBV NAT on need for retention of HBsAg and anti-HBc screening. This presentation will review recent modeled and empirical data on the value of HBV MP- and individual donation (ID)-NAT for detection of (1) pre-HBsAg WP units and (2) chronic anti-HBc-reactive carriers with undetectable HBsAg. The presentation will also review policy considerations and data that address the potential for discontinuation of either HBsAg or anti-HBc following implementation of HBV NAT. Finally it will address the cost effectiveness of incorporation of HBV DNA detection into HBV screening and NAT testing algorithms.

We derived the baby hamster kidney (BHK)-21 cell culture-adapted, tick-borne encephalitis (TBE) virus mutant. To reveal the pathogenicity of the TBE virus, we compared the pathogenicity of the mutant (Oshima Cl-1) and parental (Oshima 5-10) virus in mouse model. The neurovirulence of mutant in mice was identical to that of parent. However, the level of neuroinvasiveness was higher for parent than for mutant. The degrees of viremia and virus titers in the spleen were lower in mice that were inoculated subcutaneously (s.c.) with mutant than in mice that received parent. Unlike parent, mutant was rarely detected in the brains of s.c. inoculated mice. Genetic analysis revealed that mutant had single amino acid substitutions in each of the E and NS5 proteins compared with parent. Furthermore, while mutant infection of BHK-21 cells was inhibited by glycosaminoglycans (GAGs), this was not the case for parent. In summary, the BHK-21-cell-adapted mutant virus showed reduced neuroinvasiveness in mice due to low-level induction of viremia. The attenuation process involved a single amino acid change in the E protein, which may have resulted in the rapid clearance of the virus due to its high affinity for negatively charged molecules in vivo.

Purpose: Influenza vaccination is recommended for HIV-infected patients, although the efficacy is not clear. Prior studies have yielded differing results with regard to the effects of influenza vaccination on HIV viral load and CD4 cell counts. The effects of acute influenza on HIV viral replication and CD4 cell counts have not been well described. We sought to assess the effect of influenza infection and vaccination on HIV viral load and CD4 cell counts., Subjects and Methods: All cases of influenza occurring in HIV-infected individuals over 3 years at a large county hospital were reviewed. For the year 1997-1998, data on all HIV clinic patients who were vaccinated for influenza were recorded prospectively. In order to assess the effects of influenza infection (Group I) and vaccination (Group II) on HIV viral load and CD4 cell counts, values from before and after influenza infection or vaccination were compared to each other and to a matched control group not vaccinated and without influenza infection (Group III)., Results: Forty-three cases of influenza were diagnosed. Pre- and post-influenza viral load in Group I was not significantly different: 3.34 versus 3.49 log copies/ml (P=0.36). Viral load was unchanged in 22 of 37 patients, increased in ten patients and decreased in five patients. Similarly, pre- and post-vaccination viral load in Group II was not significantly different: 3.52 versus 3.66 log copies/ml (P=0.12). Thirty-four of 47 patients who received influenza vaccine had no significant change in viral load-viral load increased in ten patients and decreased in three patients. No significant CD4 cell count changes were noted following influenza infection or vaccination. In contrast, Group III patients experienced a small decline in viral load from 4.23 to 3.39 log copies/ml, P<0.05, while there was a trend towards an increase in CD4 cell counts (P=0.06)., Conclusions: Following influenza infection or vaccination, most patients did not have a significant increase in HIV viral load or decrease in CD4 cell count.

Background: The role of therapeutic drug monitoring (TDM) in the routine management of HIV-infected individuals is still unclear, largely due to a lack of basic data regarding specific drug concentrations and how they correlate with maximal effect and minimal toxicity within given populations. Nevertheless, it has a potentially important role to play in the management of HIV-infected patients, with the aim of limiting toxicity, optimising antiviral effect and decreasing virological failure and emergence of viral resistance., Objectives: To measure serum concentrations of specific antiretroviral drugs in individuals changing antiretroviral therapy and assess relationship to virological response., Study Design: A prospective, non-randomised, 24-week study of 40 antiretroviral experienced HIV-infected patients. Subjects had failed their previous antiretroviral regimen and were beginning new regimens based on genotypic testing. Serum antiretroviral concentrations and virological response was measured after initiation of treatment., Results: There was a significant correlation between higher concentrations of lopinavir and efavirenz and better virological outcome. This was not seen with amprenavir., Conclusions: Use of TDM in this setting helps predict virological response to therapy. Optimal use of TDM would require dose adjustment on the basis of a TDM level. Further research is necessary to enable this practice to become routine in the management of HIV-infected patients., (Copyright 2002 Elsevier Science B.V.)

Venezuelan equine encephalitis (VEE) virus-replicon particles (VRP) were used to generate feline immunodeficiency virus (FIV) Gag- and ENV-expressing vaccine vectors. Serum and mucosal FIV-specific antibody was detected in cats immunized subcutaneously, once monthly for 5 months, with FIV-expressing VRP. Expansion of the CD8+ L-selectin negative phenotype and transient CD8+ noncytolytic suppressor activity were seen in cats immunized with FIV-expressing or control VRP. Despite induction of FIV-specific immune responses and nonspecific suppressor responses, all cats became infected following vaginal challenge with high dose, pathogenic cell-associated FIV-NCSU(1) although relative early maintenance of CD4+ cells was seen in FIV-immunized cats.

In France, screening for anti-hepatitis C virus (HCV) among blood donors has been performed since 1 March 1990. It is usually carried out using enzyme immunoassays (EIA). Positive or dubious results may be linked to false positive EIA reactions. Therefore the use of an immunoblot assay such as RIBA can be very useful to assess false positive signals. Moreover, the analysis of the different antibody reactivities makes it possible to evoke a viremic status or not. The follow-up provides essential information on recent seroconversion. However, some profiles do not allow a conclusion to be drawn between chronicity and serological sequel. In these cases, polymerase chain reaction (PCR) has to be implemented in order to conclude between the two.

Because of the HIV and HCV virus transmission by transfusion during the eighties, there has been a retrospective reflection about the non-application of the precautionary principle, which has appeared only recently in the medical world. Since it was difficult to identify the real cause of the above-cited transmission in France, mainly because of the bad selection of blood donors, we feel we are justified in applying this precautionary principle more and more, in a monopolistic way, for biological security reasons. As a result, the biological research is not limited to looking for a 'degree zero' risk. Whether it concerns the 'PCR', the research of a new potential virus, the excessive fear regarding the transfusion of the new ESB agent, the worry caused by the blood donors who lived in the British Isles, the need for security based upon the precautionary principle is increasing endlessly. It is, however, more reasonable to consider that the precautionary principle should be essence incite a multi-disciplinary reflection involving biological sciences as well as social sciences. The precautionary principle would not make sense if it were not questioned for bad estimations, its harmful influences or its opportunistic use. Transfusion security, which is so important as a goal and as a principle, cannot appeal to the precautionary principle all the time, since the excessive use of this principle would lead to the paradox of not being able to identify the issues anymore.

This article shows that policies aimed at reducing risks of infectious agents transmissible through blood unfortunately follow a law of 'diminishing returns': increasing marginal costs have to be devoted for limited reductions in the risks of contamination through blood donations. Therefore, the economic cost-effectiveness analysis is appropriate to identify screening strategies which may minimize costs to reach a certain level of safety. Moreover, economic analysis can contribute to public debates about the level of residual risk that society is willing to accept. Empirical results from French studies about screening for hepatitis C virus (HCV) in individuals who have received blood transfusions and in blood donations are presented to illustrate these points.

The five available p24 Ag/anti-HIV combined tests were compared to the six third-generation anti-HIV assays mainly used in blood transfusion centers. Among 70 selected HIV-1 positive samples (12 samples from early infected blood donors and 58 from ten commercial panels), 59 were positive with at least one assay. False negative results were observed for zero to six samples with p24 Ag/Ab assays versus seven to 19 with antibody (Ab) tests. In five cases, one or more combined assays gave a positive signal later than the most sensitive Ab screening test. One sample with a high p24 Ag titer was missed by one combined test. The mean time delay between the most sensitive test and the second one was 0.3 to 2 days. The p24 Ag limit of detection was investigated with seven dilutions of the HIV Ag reference. The threshold of the p24 Ag detection was found to be between 65 and 250 pg/mL of HIV Ag. For four of the five combined assays, p24 Ag detectability was assessed with dilutions of infected culture cell supernatants from 13 HIV-1 different genotype strains exhibiting HIV Ag titers from 300 to 450 pg/mL. One of the four combined assays gave negative results but close to the cut-off for three supernatant dilutions (1 B, 1 F, 1 HIV-1/O) and one missed the HIV-1/O dilution. The p24 Ag/Ab combined assays permit an earlier diagnosis of HIV infection than third generation assays even if the yield in terms of reduction of the window period is moderate. They are less sensitive than p24 Ag screening assays for the detection of this marker. Consequently, the p24 Ag/Ab assays have not been used for the diagnosis of a primary infection instead of p24 Ag screening tests. They must be considered only as good tools for the detection of HIV infection.

Cytomegalovirus (CMV) can be transmitted by fresh blood components containing leukocytes. Consequences of CMV infection are serious in immunocompromised patients and in neonates. Thus, prevention of transfusion-transmitted CMV is of paramount importance. The use of blood products from CMV seronegative donors has been shown effective in preventing transmission. However, it does not completely eliminate the risk of transmission. Moreover, as CMV seroprevalence reaches 50 to 100% depending on the geographical and socioeconomic conditions, the availability of CMV seronegative products is limited. Leukodepletion of cellular blood products can be achieved by various filtration techniques. A method capable of achieving a residual leukocyte count < 5 x 10(6) per unit allows for the reduction of CMV transmission to a level at least equivalent to the transfusion of seronegative blood components. Moreover, leukodepletion may reduce endogenous virus reactivation. Administration of filtered blood products from CMV seronegative donors is usually recommended for those patients at major risk of severe CMV transfusion-associated disease. The ability of the most efficient methods for blood filtration in preventing CMV transmission has to be assessed. Such methods would make it possible to avoid serological screening of blood donors.

It has been reported that hepatitis C virus (HCV) may cause oxidative stress in infected cells. Patients with chronic hepatitis C exhibit an increased production of tumor necrosis factor-alpha (TNF alpha), a cytokine that can produce oxidative stress by stimulating the generation of reactive oxygen species (ROS). Cell defense against ROS includes overexpression of Mn-superoxide dismutase (SOD), an inducible mitochondrial enzyme. To investigate cell defense against oxidative stress in HCV infection, we analyzed Mn-SOD mRNA in liver and in peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis C. Mn-SOD expression in PBMC was significantly increased in patients with HCV infection. Patients with sustained virological and biochemical response after therapy showed significantly lower Mn-SOD than patients with positive viremia. By contrast, Mn-SOD expression was not enhanced in the liver of patients with chronic hepatitis C. The values of Mn-SOD mRNA did not correlate with TNF alpha mRNA expression, viral load, or liver disease activity. Our results indicate that in HCV infection an induction of Mn-SOD was present in PBMC but absent in the liver, suggesting that this organ could be less protected against oxidative damage. Oxidative stress could participate in the pathogenesis of HCV infection.

Direct viral DNA or RNA detection by genomic amplification methods should contribute to the improvement of blood supply safety by reducing the pre-seroconversion window period. However, nucleic acid testing may not eliminate virus transmission from window period donations. Moreover, the virus subtype diversity has to be taken into account for the choice of nucleic amplification assays. Technology for nucleic acid testing has not been developed to be implemented in blood screening laboratories. The limitations of these procedures are mainly linked to the difficulties related to the automation of sample processing and to the possibilities of cross contamination of samples due to the high sensitivity of amplification methods. The extreme complexity of nucleic acid testing for single donation screening (development of an on-site automated high-throughput instrumentation, training, specialized facilities for sample processing, amplification and detection...) and its high cost mean that strategies using amplification testing of pooled samples should be developed.