Plasma Virus Research Articles

Administration of anti-HIV-1 broadly neutralizing monoclonal antibodies with increased affinity to Fcγ receptors during acute SHIVAD8-EO infection

Anti-HIV-1 broadly neutralizing antibodies (bNAbs) have the dual potential of mediating virus neutralization and antiviral effector functions through their Fab and Fc domains, respectively. So far, bNAbs with enhanced Fc effector functions in vitro have only been tested in NHPs during chronic simian-HIV (SHIV) infection. Here, we investigate the effects of administering in acute SHIVAD8-EO infection either wild-type (WT) bNAbs or bNAbs carrying the S239D/I332E/A330L (DEL) mutation, which increases binding to FcγRs. Emergence of virus in plasma and lymph nodes (LNs) was delayed by bNAb treatment and occurred earlier in monkeys given DEL bNAbs than in those given WT bNAbs, consistent with faster clearance of DEL bNAbs from plasma. DEL bNAb-treated monkeys had higher levels of circulating virus-specific IFNγ single-producing CD8+ CD69+ T cells than the other groups. In LNs, WT bNAbs were evenly distributed between follicular and extrafollicular areas, but DEL bNAbs predominated in the latter. At week 8 post-challenge, LN monocytes and NK cells from DEL bNAb-treated monkeys upregulated proinflammatory signaling pathways and LN T cells downregulated TNF signaling via NF-κB. Overall, bNAbs with increased affinity to FcγRs shape innate and adaptive cellular immunity, which may be important to consider in future strategies of passive bNAb therapy.

Stability of generalized models for HIV-1 dynamics with impaired CTL immunity and three pathways of infection

Highly active antiretroviral therapy (HAART) stands out as the most effective treatment for human immunodeficiency virus type-1 (HIV-1). While total eradication is still difficult, HAART can dramatically lower the virus's plasma viral level below the detection threshold. The activation of latently infected cells is considered to be the main obstacle to the total eradication of HIV-1 infection. This study investigates the dynamic characteristics of two generalized HIV-1 infection models taking into account the impairment of cytotoxic T lymphocytes (CTLs). These models include CD4+T cells that are latently infected and equipped with the capability to engage in cell-to-cell infection and elude immune responses. We introduce models featuring three infection pathways: virus-to-cell (VTC), latent cell-to-cell (L-CTC), and active cell-to-cell (A-CTC). The three pathways' infection rates are characterized by general functions, which cover the many types of infection rates documented in the literature. The second model integrates three distinct types of distributed-time delays. We demonstrate the validity of the suggested models, through their well-posedness. We determine the basic reproduction ratio (ℜ0) of the systems. Lyapunov functions and LaSalle's invariance principle are employed to verify that the global stability of both the virus-free steady state (O0) and the virus-persistence steady state (O1). More precisely, O0 achieves global asymptotic stability when ℜ0 ≤ 1, whereas O1 attains global asymptotic stability when ℜ0 > 1. To demonstrate the impact of the parameter values on ℜ0, we examine the sensitivity analysis. It is illustrated that ℜ0 comprises three components, namely ℜ0E, ℜ0P, and ℜ0K, corresponding to the transmissions of VTC, L-CTC, and A-CTC, respectively. Thus, if the L-CTC pathway is disregarded in the HIV-1 infection model, ℜ0 may be underestimated, which could lead to inadequate or erroneous medication therapy focused on eradicating HIV-1 within the body. To demonstrate the associated mathematical outcomes, we conduct numerical simulations through an illustrative example. Specifically, we delve into how the dynamics of HIV-1 are influenced by both immune impairment and time delay. Our findings suggest a significant role of reduced immunity in the progression of the infection. Furthermore, time delays possess the potential to markedly reduce ℜ0, thereby impeding the replication of HIV-1.

Quantitative risk assessment model of the presence of porcine epidemic diarrhea and African swine fever viruses in spray-dried porcine plasma.

There are no microbiological regulatory limits for viruses in animal feed and feed ingredients. A performance objective (PO) was proposed in this study to manufacture a spray-dried porcine plasma (SDPP) batch absent of any infectious viral particles. The PO levels of -7.0, -7.2, and -7.3 log TCID50/g in SDPP were estimated for three batch sizes (10, 15, and 20 tons). A baseline survey on the presence of porcine epidemic diarrhea virus (PEDV) in raw porcine plasma revealed a concentration of -1.0 ± 0.6 log TCID50/mL as calculated using a TCID50-qPCR derived standard curve. The mean African swine fever virus (ASFV) concentration in raw plasma was estimated to be 0.6 log HAD50/mL (0.1-1.4, 95% CI) during a pre-clinical scenario (collected from asymptomatic and undetected viremic pigs). Different processing scenarios (baseline: spray-drying + extended storage) and baseline + ultraviolet (UV) radiation were evaluated to meet the PO levels proposed in this study. The baseline and baseline + UV processing scenarios were >95 and 100% effective in achieving the PO for PEDV by using different batch sizes. For the ASFV in SDPP during a pre-clinical scenario, the PO compliance was 100% for all processing scenarios evaluated. Further research is needed to determine the underlying mechanisms of virus inactivation in feed storage to further advance the implementation of feed safety risk management efforts globally.

Synergistic pathogenicity of vertically transmitted chicken infectious anemia virus and avian leukosis virus subgroup J coinfection in chickens

Avian leukemia virus subgroup J (ALV-J) and chicken infectious anemia virus (CIAV) can be vertically transmitted; however, the pathogenicity of vertically transmitted coinfection with these 2 pathogens has not been studied. In this study, we created a model of chick morbidity in which chicks carried either ALV-J, CIAV, or both viruses via embryo inoculation. Thereafter, we analyzed the effects of vertically transmitted coinfection with CIAV and ALV-J on the pathogenicity of ALV-J and performed a purification assay based on hatching, mortality viremia positivity, and detection of fecal ALV-p27 antigen rates, and body weight. The hatching rate of the ALV-J+CIAV group was 68.57%, lower than those of the single infection and control groups. The survival curve showed that the mortality rates of the CIAV and ALV-J coinfection groups were higher than those of the single infection and control groups. Body weight statistics showed that coinfection aggravated the 7-d growth inhibition effect. The results of ALV-p27 antigen detection in cell culture supernatants showed that the positivity rates of the ALV-J and ALV-J+CIAV groups were 100% at all ages and 0% in the control group. The results of ALV-p27 antigen detection by anal swabs showed that the positivity rates of the ALV-J group were 92.86, 90.90, 88.89, and 93.33% at all ages, and that the ALV-J p27 positivity detection rate of anal swabs was lower than that of plasma virus isolation. The immune organ index of the ALV-J+CIAV group was significantly or very significantly lower than those of the single infection and control groups. The immune organ viral load showed that coinfection with CIAV and ALV-J promoted the proliferation of ALV-J and CIAV in immune organs. Coinfection with ALV-J and CIAV reduced chicken embryo hatchability and increased chick mortality and growth inhibition relative to their respective single infections. Additionally, coinfection with ALV-J + CIAV was even more detrimental in inducing immune organ atrophy (e.g., the thymus, spleen, and bursa), and promoted individual virus replication during coinfection.

Autologous neutralizing antibody responses after antiretroviral therapy in acute and early HIV-1.

BACKGROUNDEarly antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAbs) after acute/early ARTi is relevant but is poorly understood.METHODSWe characterized antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after acquisition) and early HIV (60-128 days after acquisition).RESULTSPlasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 envelopes representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In 2 of the 3 acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter.CONCLUSIONResults indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants.TRIAL REGISTRATIONClinicalTrials.gov NCT02656511.FUNDINGNIH grants U01AI169767, R01AI162646, UM1AI164570, UM1AI164560, U19AI096109, K23GM112526, T32AI118684, P30AI045008, P30AI027763, R24AI067039; Gilead Sciences grant INUS2361354; Viiv Healthcare grant A126326.

Plasma Virome of HIV-infected Subjects on Suppressive Antiretroviral Therapy Reveals Association of Differentially Abundant Viruses with Distinct T-cell Phenotypes and Inflammation.

The plasma virome represents the overall composition of viral sequences present in it. Alteration in plasma virome has been reported in treatment naïve and immunocompromised (CD4 count < 200) people with HIV (PWH). However, the effect of ART on virome composition in PWH on ART with preserved CD4 counts is poorly understood. We aimed to assess the alterations in plasma virome in PWH on ART in comparison to HIV-negative uninfected controls and to further investigate possible associations of plasma viruses with inflammation and immune dysfunction, namely, immunosenescence and immune exhaustion. Plasma viral DNA from PWH on ART and controls was used for sequencing on the Illumina Nextseq500 platform, followed by the identification of viral sequences using an automated pipeline, VIROMATCH. Multiplex cytokine assay was performed to measure the concentrations of various cytokines in plasma. Immunophenotyping was performed on PBMCs to identify T cell markers of immunosenescence and immune exhaustion. In our observational, cross-sectional pilot study, chronically infected PWH on ART had significantly different viral species compositions compared to controls. The plasma virome of PWH showed a significantly high relative abundance of species Human gammaherpesvirus 4, also known as Epstein-Barr virus (EBV). Moreover, EBV emerged as a significant viral taxon differentially enriched in PWH on ART, which further correlated positively with the exhaustion phenotype of T cells and significantly increased TNF-α in PWH on ART. Additionally, a significantly increased proportion of senescent T cells and IL-8 cytokine was detected in PWH on ART. Altered plasma virome influenced the inflammatory response and T-cell phenotype in PWH on ART.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

Detection and persistence of Zika virus in body fluids and associated factors: a prospective cohort study

This study aimed to analyze the detection and duration of the Zika virus (ZIKV) in plasma, urine, saliva, sweat, rectal swabs, vaginal secretions, breast milk, and semen and to explore risk factors associated with prolonged viral persistence. A prospective cohort study of symptomatic patients and their household contacts was conducted in Brazil from July 2017 to June 2019. A total of 260 individuals (184 women and 76 men) with confirmed ZIKV infection were enrolled and followed up for 12 months. ZIKV RNA was present in all body fluid specimens and detectable for extended periods in urine, sweat, rectal swabs, and semen. The longest detection duration was found in semen, with high viral loads in the specimens. ZIKV RNA clearance was associated with several factors, including age, sex, education level, body mass index, non-purulent conjunctivitis, joint pain, and whether the participant had a history of yellow fever vaccination. The influence of each of these factors on the low or fast viral clearance varied according to the specific body fluid under investigation. Recurrent ZIKV detection events after total viral clearance were observed in the cohort. Our findings provide valuable insights into the persistence and potential recurrence of ZIKV infection, highlighting the need for continued monitoring and follow-up of individuals infected with ZIKV and for effective prevention measures to reduce the risk of transmission.

Viral and host mediators of non-suppressible HIV-1 viremia

Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as ‘producer proviruses’, and those that did not as ‘non-producer proviruses’. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4+ T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8+ T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence.

Disinfection of viruses with cold atmospheric‐pressure plasmas: Sources, mechanisms, and efficacy

AbstractIn recent years, the outbreaks of viral diseases have urgently demanded effective virus disinfection techniques. Among them, the use of cold atmospheric‐pressure plasma (CAP) is one of the most promising approaches. CAP generates electric fields, charged particles, reactive oxygen and nitrogen species, and ultraviolet. CAP can effectively inactivate various viruses in a short period, whether they are on surfaces, in water, or in aerosols, without significantly raising the substrate temperature. This review highlights that the simultaneous action of physical and chemical means is the key mechanism of CAP in virus inactivation. It systematically summarizes the characteristics of plasma devices suitable for different application scenarios and further points out the opportunities and challenges faced by plasma virus inactivation technology.

Human immunodeficiency virus 1 5'-leader mutations in plasma viruses before and after the development of reverse transcriptase inhibitor-resistance mutations.

Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) initiation depends on interaction between viral 5'-leader RNA, RT and host tRNA3Lys. Therefore, we sought to identify co-evolutionary changes between the 5'-leader and RT in viruses developing RT-inhibitor resistance mutations. We sequenced 5'-leader positions 37-356 of paired plasma virus samples from 29 individuals developing the nucleoside RT inhibitor (NRTI)-resistance mutation M184V, 19 developing a non-nucleoside RT inhibitor (NNRTI)-resistance mutation and 32 untreated controls. 5'-Leader variants were defined as positions where ≥20 % of next-generation sequencing (NGS) reads differed from the HXB2 sequence. Emergent mutations were defined as nucleotides undergoing a ≥4-fold change in proportion between baseline and follow-up. Mixtures were defined as positions containing ≥2 nucleotides each present in ≥20 % of NGS reads. Among 80 baseline sequences, 87 positions (27.2 %) contained a variant; 52 contained a mixture. Position 201 was the only position more likely to develop a mutation in the M184V (9/29 vs 0/32; P=0.0006) or NNRTI-resistance (4/19 vs 0/32; P=0.02; Fisher's exact test) groups than the control group. Mixtures at positions 200 and 201 occurred in 45.0 and 28.8 %, respectively, of baseline samples. Because of the high proportion of mixtures at these positions, we analysed 5'-leader mixture frequencies in two additional datasets: five publications reporting 294 dideoxyterminator clonal GenBank sequences from 42 individuals and six National Center for Biotechnology Information (NCBI) BioProjects reporting NGS datasets from 295 individuals. These analyses demonstrated position 200 and 201 mixtures at proportions similar to those in our samples and at frequencies several times higher than at all other 5'-leader positions. Although we did not convincingly document co-evolutionary changes between RT and 5'-leader sequences, we identified a novel phenomenon, wherein positions 200 and 201 immediately downstream of the HIV-1 primer binding site exhibited an extraordinarily high likelihood of containing a nucleotide mixture. Possible explanations for the high mixture rates are that these positions are particularly error-prone or provide a viral fitness advantage.

Quantification of Torque Teno Virus (TTV) in plasma and saliva of individuals with liver cirrhosis: a cross sectional study.

Torque teno virus (TTV) has been pointed as an endogenous marker of immune function, the objective of this study was to investigate the TTV viral load in plasma and saliva of cirrhotic individuals and correlate it with clinical characteristics. Blood, saliva, clinical data from records and laboratory tests were collected from 72 cirrhotic patients. Plasma and saliva were submitted to real-time polymerase chain reaction for quantification of TTV viral load. The majority of the patients presented decompensated cirrhosis (59.7%) and 47.2% had alterations in the white blood series. TTV was identified in 28 specimens of plasma (38.8%) and in 67 specimens of saliva (93.0%), with median values of TTV copies/mL of 90.6 in plasma and 245.14 in saliva. All the patients who were positive for TTV in plasma were also positive in saliva, with both fluids having a moderately positive correlation for the presence of TTV. There was no correlation between TTV viral load, either in plasma or in saliva, and any of the variables studied. TTV is more frequently found and in greater amount in the saliva than in the plasma of cirrhotic patients. There was no correlation between TTV viral load and clinical parameters.

Chronic opioid use is associated with higher flu vaccine induced Ab response in HIV+ and HIV− individuals.

Abstract Opioid dependence is frequent in people with HIV (PWH), but impact of opioids on immune system of PWH is unknown. This study tested the hypothesis that chronic opioid use exacerbates known impairment of flu vaccine responses in PWH. Chronic opioid users were compared to non-opioid users in HIV+ and HIV− groups termed HIV+OP+ (n=28), HIV-OP+ (n=55), HIV+OP− (n=53), HIV-OP− (controls, n=58). HIV+ individuals were on ART with plasma virus load &amp;lt;200 copies/mL. Flu antibody titers were determined at pre- (T0) and 4 weeks post (T2) seasonal quadrivalent influenza vaccinations (2020–2022) by hemagglutination inhibition (HAI) along with a plasma cytokine score at T0. Vaccine response (VR) was defined as a 4-fold change (FC) in titer from T0 to T2. In Kruskal Wallis analysis, all 4 groups had increases in HAI titer from T0 to T2 (p&amp;lt;0.01 to p&amp;lt;0.0001) for antigens (H1N1, H3N2, B Victoria) and whole vaccine. HIV+ status was associated with decreased responses to all antigens but OP+ status in HIV+ and HIV− had higher response than HIV+OP−, both in T2/T0 FC and T2 titer for one or more vaccine antigens (p&amp;lt;0.05 to p&amp;lt;0.001). In regression analysis controlling for demographics, opioid use was associated with increased response to H1N1 and B Yamagata antigens. In a random forest model, predicted probability of VR was lower for HIV+ status and higher for OP+ status. Cytokine score was highest in HIV+OP+. Results of quantitative Ab response to flu vaccine refute our hypothesis that flu vaccine response would be impaired in PWH who chronically use opioids. Qualitative assessments of Ab and mechanism of opioid intersection with B cell function are ongoing to understand if opioid use overcomes humoral immune deficits in PWH.

Longitudinal analysis of SARS-CoV-2 reinfection reveals distinct kinetics and emergence of cross-neutralizing antibodies to variants of concern.

The ongoing evolution of SARS-CoV-2 continues to raise new questions regarding the duration of immunity to reinfection with emerging variants. To address these knowledge gaps, controlled investigations in established animal models are needed to assess duration of immunity induced by each SARS-CoV-2 lineage and precisely evaluate the extent of cross-reactivity and cross-protection afforded. Using the Syrian hamster model, we specifically investigated duration of infection acquired immunity to SARS-CoV-2 ancestral Wuhan strain over 12 months. Plasma spike- and RBD-specific IgG titers against ancestral SARS-CoV-2 peaked at 4 months post-infection and showed a modest decline by 12 months. Similar kinetics were observed with plasma virus neutralizing antibody titers which peaked at 2 months post-infection and showed a modest decline by 12 months. Reinfection with ancestral SARS-CoV-2 at regular intervals demonstrated that prior infection provides long-lasting immunity as hamsters were protected against severe disease when rechallenged at 2, 4, 6, and 12 months after primary infection, and this coincided with the induction of high virus neutralizing antibody titers. Cross-neutralizing antibody titers against the B.1.617.2 variant (Delta) progressively waned in blood over 12 months, however, re-infection boosted these titers to levels equivalent to ancestral SARS-CoV-2. Conversely, cross-neutralizing antibodies to the BA.1 variant (Omicron) were virtually undetectable at all time-points after primary infection and were only detected following reinfection at 6 and 12 months. Collectively, these data demonstrate that infection with ancestral SARS-CoV-2 strains generates antibody responses that continue to evolve long after resolution of infection with distinct kinetics and emergence of cross-reactive and cross-neutralizing antibodies to Delta and Omicron variants and their specific spike antigens.

HIV-1 DNA Testing in Viremic Patients Identifies More Drug Resistance Than HIV-1 RNA Testing.

The Department of Health and Human Services HIV-1 Treatment Guidelines recommend drug resistance testing in HIV-1 RNA to guide the selection of antiretroviral therapy in patients with viremia. However, resistance-associated mutations (RAMs) in HIV-1 RNA may reflect only the patient's current regimen and can be lost during prolonged absence of therapy. We determined if HIV-1 DNA testing can provide drug resistance information beyond that identified in contemporaneous plasma virus. This was a retrospective database review of results obtained for patients with viremia for whom commercial HIV-1 RNA and HIV-1 DNA drug resistance testing was ordered on the same day. Resistance-associated mutations and drug susceptibility calls were compared between paired tests, and the effect of HIV-1 viral load (VL) on test concordance was assessed using Spearmen's rho correlation. Among 124 paired tests, more RAMs were identified in HIV-1 DNA in 63 (50.8%) cases, and in HIV-1 RNA in 11 (8.87%) cases. HIV-1 DNA testing captured all contemporaneous plasma virus RAMs in 101/117 (86.3%) cases and identified additional RAMs in 63/117 (53.8%) cases. There was a significant positive correlation between the viral load at the time of resistance testing and the percentage of plasma virus RAMs detected in HIV-1 DNA (rs = 0.317; P < .001). In 67 test pairs demonstrating pan-sensitive plasma virus, resistance in HIV-1 DNA was seen in 13 (19.4%) cases. HIV-1 DNA testing identified more resistance than HIV-1 RNA testing in most patients with viremia and may be informative in patients whose plasma virus reverts to wild-type following therapy discontinuation.

Evaluation of a Semiautomated System for the Quantitation of Human Adenovirus DNA from Clinical Samples.

Human adenoviruses (HAdVs) cause severe disease in immunocompromised patients. Quantitation of HAdV DNA in peripheral blood is used to assess the risk of disseminated disease and to monitor response to therapy. The lower limit of detection, precision, and linearity of the semiautomated AltoStar adenovirus quantitative PCR (qPCR) was evaluated using reference HAdV-E4 in EDTA plasma and respiratory virus matrix. Qualitative and quantitative agreement was determined using 122 clinical EDTA plasma specimens previously tested using a laboratory-developed HAdV qPCR. The 95% lower limit of detection (LLOD) was 33 IU/mL (95% confidence interval [CI], 10 to 56) for EDTA plasma and 188 IU/mL (95% CI, 145 to 304) for respiratory swab matrix. In both matrices, the AltoStar HAdV qPCR was linear from 7.0 to 2.0 log10 IU/mL. For the clinical specimens, overall agreement was 96.7% (95% CI, 91.8 to 99.1), positive percent agreement was 95.5% (95% CI, 87.6 to 98.5), and negative percent agreement was 98.2% (95% CI, 88.5 to 99.7). Passing-Bablok analysis of specimens quantifiable by both methods revealed a regression line of Y = 1.11 · X + 0.00; there was positive proportional bias (95% CI of the slope, 1.05 to 1.22) but no systematic bias (95% CI of the Y-intercept, -0.43 to 0.23) compared to the reference. The AltoStar platform provides accurate quantitation of HAdV DNA and provides a semiautomated option for the clinical monitoring of HAdV following transplantation. IMPORTANCE Accurate quantification of human adenovirus DNA in the peripheral blood plays a critical role in the management of adenovirus infections in transplant recipients. Many laboratories utilize in-house laboratory-based PCR assays for the quantification of human adenovirus, as there are few commercial options available. Here, we describe the analytical and clinical performance of the semiautomated AltoStar adenovirus quantitative PCR (Altona Diagnostics). This platform provides sensitive, precise, and accurate quantification of adenovirus DNA that is well suited for virological testing following transplantation. Prior to implementing a new quantitative test in the clinical laboratory, a rigorous evaluation is required to determine assay performance characteristics and to correlate results to current in-house methods of quantitation.

Inactivation of SARS-CoV-2 on Surfaces by Cold-Plasma-Generated Reactive Species.

A Cold Atmospheric Plasma (CAP) apparatus was designed and developed for SARS-CoV-2 killing as evaluated by pseudotyped viral infectivity assays. The reactive species generated by the plasma system was fully characterized by using Optical Emission Spectroscopy (OES) measurement under given conditions such as plasma power, flow rate, and treatment time. A variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were identified from plasma plume with energies of 15-72 eV in the frequency range between 500-1000 nm. Systematic virus killing experiments were carried out, and the efficacy of CAP treatment in reducing SARS-CoV-2 viral infectivity was significant following treatment for 8 s, with further enhancement of killing upon longer exposures of 15-120 s. We correlated killing efficacy with the reactive species in terms of type, intensity, energy, and frequency. These experimental results demonstrate effective cold plasma virus killing via ROS and RNS under ambient conditions.

Antiretroviral therapy reveals triphasic decay of intact SIV genomes and persistence of ancestral variants

The decay kinetics of HIV-1-infected cells are critical to understand virus persistence. We evaluated the frequency of simian immunodeficiency virus (SIV)-infected cells for 4 years of antiretroviral therapy (ART). The intact proviral DNA assay (IPDA) and an assay for hypermutated proviruses revealed short- and long-term infected cell dynamics in macaques starting ART ∼1 year after infection. Intact SIV genomes in circulating CD4+T cells showed triphasic decay with an initial phase slower than the decay of the plasma virus, a second phase faster than the second phase decay of intact HIV-1, and a stable third phase reached after 1.6-2.9 years. Hypermutated proviruses showed bi- or mono-phasic decay, reflecting different selective pressures. Viruses replicating at ART initiation had mutations conferring antibody escape. With time on ART, viruses with fewer mutations became more prominent, reflecting decay of variants replicating at ART initiation. Collectively, these findings confirm ART efficacy and indicate that cells enter the reservoir throughout untreated infection.

Pathogen reduction of monkeypox virus in plasma and whole blood using riboflavin and UV light.

Monkeypox virus has recently emerged from endemic foci in Africa and, since October 20, 2022, more than 73,000 human infections have been reported by the CDC from over 100 countries that historically have not reported monkeypox cases. The detection of virus in skin lesions, blood, semen, and saliva of infected patients with monkeypox infections raises the potential for disease transmission via routes that have not been previously documented, including by blood and plasma transfusions. Methods for protecting the blood supply against the threats of newly emerging disease agents exist and include Pathogen Reduction Technologies (PRT) which utilize photochemical treatment processes to inactivate pathogens in blood while preserving the integrity of plasma and cellular components. Such methods have been employed broadly for over 15 years, but effectiveness of these methods under routine use conditions against monkeypox virus has not been reported. Monkeypox virus (strain USA_2003) was used to inoculate plasma and whole blood units that were then treated with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of monkeypox virus in the samples before and after riboflavin + UV treatment were determined by plaque assay on Vero cells. The levels of spiked virus present in whole blood and plasma samples exceeded 103 infectious particles per dose, corresponding to greater than 105 DNA copies per mL. Treatment of whole blood and plasma units under standard operating procedures for the Mirasol PRT System resulted in complete inactivation of infectivity to the limits of detection. This is equivalent to a reduction of ≥ 2.86 +/- 0.73 log10 pfu/mL of infectivity in whole blood and ≥ 3.47 +/-0.19 log10 pfu/mL of infectivity in plasma under standard operating conditions for those products. Based on this data and corresponding studies on infectivity in patients with monkeypox infections, use of Mirasol PRT would be expected to significantly reduce the risk of transfusion transmission of monkeypox.

Clinical value of plasma and peripheral blood mononuclear cells Epstein-Barr Virus DNA dynamics on prognosis of allogeneic stem cell transplantation.

The application of intracellular and extracellular Epstein–Barr virus (EBV) DNA in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been poorly characterized. We conducted a combined prospective-retrospective study of 300 patients who underwent allo-HSCT between 2016 to 2019 in our center and monitored for EBV DNA within the first year after HSCT. Combining the optimal cut-off value of EBV DNA load (7.3×104 copies/106 cells) in peripheral blood mononuclear cells (PBMCs) and qualitative detection in plasma (400 copies/mL) allowed for the better differentiation of EBV-related posttransplant lymphoproliferative disorders (EBV-PTLD), with increased sensitivity (100%) and specificity (86%), and provided the effective risk stratification of EBV DNA level according to their impact on transplant outcomes. By multivariate analysis, patients with intermediate-level of EBV DNA load (low EBV DNA load in PBMCs or high load in PBMCs but negative in plasma) was associated with superior overall survival (HR 1.92, 95% CI 1.03-3.57, p=0.039) and lower transplant-related mortality (HR 3.35, 95% CI 1.31-8.58, p=0.012) compared to those with high-level (high load in PBMCs and positive in plasma). Notably, high EBV-level group had poor reconstitution of CD4+ and CD8+T cells, and both low and high EBV-level groups showed abnormally increase in IL-10 level within one year. Additionally, patients with peak EBV DNA load in PBMCs during 3-12 months had a higher incidence of chronic graft versus host disease (GVHD) than those within 3 months post transplantation (17.4% vs 13.7%, p=0.029). Collectively, EBV DNA in PBMCs can synergistically predict the risk of EBV-PTLD and GVHD. The intermediate-level of EBV DNA presented in plasma and PBMCs might contribute to a better reconstitution of T cells associated with favorable prognosis of allo-HSCT.