Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

Genetic regulation of carnitine metabolism controls lipid damage repair and aging RBC hemolysis in vivo and in vitro.

ABSTRACT: Recent large-scale multiomics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on 2 separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured l-carnitine and acyl-carnitines in 13 091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying 2 alleles of the rs12210538 SLC22A16 single-nucleotide polymorphism exhibited the lowest l-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice, and Percoll density gradients of human RBCs, showed age-dependent depletions of l-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

SARS-CoV-2 antibody levels and long COVID occurrence in blood donors.

BACKGROUND: Long COVID is a common condition lacking consensus definition; determinants remain incompletely understood. Characterizing immune profiles associated with long COVID could support the development of preventive and therapeutic strategies. METHODS: We used a survey to investigate blood donors' infection/vaccination history and acute/persistent symptoms following COVID-19. The prevalence of long COVID was evaluated using self-report and an adapted definition from the RECOVER study. We evaluated factors associated with long COVID, focusing on anti-spike and anti-nucleocapsid SARS-CoV-2 antibodies. Lastly, we investigated long COVID clinical subphenotypes using hierarchical clustering. RESULTS: Of 33,610 participants, 16,003 (48%) reported having had COVID-19; 1853 (12%) had self-reported long COVID, 685 (4%) met an adapted RECOVER definition, and 2050 (13%) met at least one definition. Higher anti-nucleocapsid levels measured 12-24 weeks post-infection were associated with higher risk of self-reported and RECOVER long COVID. Higher anti-spike IgG levels measured 12-24 weeks post-infection were associated with lower risk of self-reported long COVID. Higher total anti-spike measured 24-48 weeks post-infection was associated with lower risk of RECOVER long COVID. Cluster analysis identified four clinical subphenotypes; patterns included neurological and psychiatric for cluster 1; neurological and respiratory for cluster 2; multi-systemic for cluster 3; and neurological for cluster 4. DISCUSSION: Long COVID prevalence in blood donors varies depending on the adopted definition. Anti-SARS-CoV-2 antibodies were time-dependently associated with long COVID; higher anti-nucleocapsid levels were associated with higher risk; and higher anti-spike levels were associated with lower risk of long COVID. Different underlying pathophysiologic mechanisms may be associated with distinct clinical subphenotypes.

Spike and nucleocapsid antibody dynamics following SARS-CoV-2 infection and vaccination: Implications for sourcing COVID-19 convalescent plasma from routinely collected blood donations.

BACKGROUND: COVID-19 convalescent plasma (CCP) remains a treatment option for immunocompromised patients; however, the current FDA qualification threshold of ≥200 BAU/mL of spike antibody appears to be relatively low. We evaluated the levels of binding (bAb) and neutralizing antibodies (nAb) on serial samples from repeat blood donors who were vaccinated and/or infected to inform criteria for qualifying CCP from routinely collected plasma components. METHODS: Donors were categorized into four groups: (1) infected, then vaccinated, (2) vaccinated then infected during the delta, or (3) omicron waves, (4) vaccinated without infection. IgG Spike and total Nuclecapsid bAb were measured, along with S variants and nAb titers using reporter viral particle neutralization. RESULTS: Mean S IgG bAb peaks after infection alone were lower than after primary and booster vaccinations, and higher after delta and omicron infection in previously vaccinated donors. Half-lives for S IgG ranged from 34 to 66 days after first infection/vaccination events and up to 108 days after second events. The levels of S IgG bAb and nAb were similar across different variants, except for omicron, which were lower. Better correlations of nAb with bAb were observed at higher levels (hybrid immunity) than at the current FDA CCP qualifying threshold. DISCUSSION: Routine plasma donations from donors with hybrid immunity had high S bAb and potent neutralizing activity for 3-6 months after infection. In donations with high (>4000 BAU/mL) S IgG, >95% had high nAb titers (>500) against ancestral and variant S, regardless of COVID-19 symptoms. These findings provide the basis for test-based criteria for qualifying CCP from routine blood donations.

Characterization of Phietavirus Henu 2 in the virome of individuals with acute gastroenteritis.

There is a growing interest in phages as potential biotechnological tools in human health owing to the antibacterial activity of these viruses. In this study, we characterized a new member (named PhiV_005_BRA/2016) of the recently identified phage species Phietavirus Henu 2. PhiV_005_BRA/2016 was detected through metagenomic analysis of stool samples of individuals with acute gastroenteritis. PhiV_005_BRA/2016 contains double-stranded linear DNA (dsDNA), it has a genome of 43,513 base pairs (bp), with a high identity score (99%) with phage of the genus Phietavirus, species of Phietavirus Henu 2. Life style prediction indicated that PhiV_005_BRA/2016 is a lysogenic phage whose the main host is methicillin-resistant Staphylococcus aureus (MRSA). Indeed, we found PhiV_005_BRA/2016 partially integrated in the genome of distinct MRSA strains. Our findings highlights the importance of large-scale screening of bacteriophages to better understand the emergence of multi-drug resistant bacterial.

Aedes aegypti Totivirus identified in mosquitoes in the Brazilian Amazon region.

The totiviridae family contains viruses with double-stranded RNA genomes of 4.6-7.0 kpb, which encode a capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), and they are approximately 40 nm in diameter with icosahedral symmetry. Totiviruses were first isolated from mosquitoes collected in Shaanxi Province (China). Here, we report a new Aedes aegypti Totivirus (AaTV) identified in mosquitoes from the Amazon rainforest. Mosquitoes (Diptera: Culicidae) were collected from a forest reserve belonging to the Amazon forest in the city of Macapá, Amapá state, Northern Brazil. A viral sequence with a 5748 nucleotide length that was nearly identical to Aedes aegypti Totivirus (AaTV), here named Aedes aegypti Totivirus BR59AP, was detected. A detailed molecular analysis was performed and shows that AaTV-BR59AP is highly related to the AaTV strain from the Caribbean region. We emphasize the importance of the characterization of new viruses in mosquitoes to deepen our understanding of viral diversity in insects and their potential role in disease.

Incidence and clinical features of HHV-7 detection in lower respiratory tract in patients with severe pneumonia: a multicenter, retrospective study.

PURPOSE: The significance of detecting human herpesvirus 7 (HHV-7) in the lower respiratory tract of patients with severe pneumonia is unclear. This study aims to evaluate the clinical characteristics and prognosis of detecting HHV-7 in the lower respiratory tract of patients with severe pneumonia. METHODS: Patients with severe pneumonia requiring invasive mechanical ventilation and underwent commercial metagenomic next-generation sequencing (mNGS) testing of bronchoalveolar lavage fluid from January 2019 to March 2023 were enrolled in 12 medical centers. Clinical data of patients were collected retrospectively, and propensity score matching was used for subgroup analysis and mortality assessment. RESULTS: In a total number of 721 patients, 45 cases (6.24%) were identified with HHV-7 positive in lower respiratory tract. HHV-7 positive patients were younger (59.2 vs 64.4, p = 0.032) and had a higher rate of co-detection with Cytomegalovirus (42.2% vs 20.7%, p = 0.001) and Epstein-Barr virus (35.6% vs 18.2%, p = 0.008). After propensity score matching for gender, age, SOFA score at ICU admission, and days from ICU admission to mNGS assay, there was no statistically significant difference in the 28-day mortality rate between HHV-7 positive and negative patients (46.2% vs 36.0%, p = 0.395). Multivariate Cox regression analysis adjusting for gender, age, and SOFA score showed that HHV-7 positive was not an independent risk factor for 28-day mortality (HR 1.783, 95%CI 0.936-3.400, p = 0.079). CONCLUSION: HHV-7 was detected in the lungs of 6.24% of patients with severe pneumonia. The presence of HHV-7 in patients with severe pneumonia requiring invasive mechanical ventilation is associated with a younger age and co-detected of Cytomegalovirus and Epstein-Barr virus. While HHV-7 positivity was not found to be an independent risk factor for mortality in this cohort, this result may have been influenced by the relatively small sample size of the study.

Virome of Bat Guano from Nine Northern California Roosts.

Bats are hosts to a large variety of viruses, including many capable of cross-species transmissions to other mammals, including humans. We characterized the virome in guano from five common bat species in 9 Northern California roosts and from a pool of 5 individual bats. Genomes belonging to 14 viral families known to infect mammals and 17 viral families infecting insects or of unknown tropism were detected. Nearly complete or complete genomes of a novel parvovirus, astrovirus, nodavirus, circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses, and densoviruses, and more partial genomes of a novel alphacoronavirus and a bunyavirus were characterized. Lower numbers of reads with >90% amino acid identity to previously described calicivirus, circovirus, adenoviruses, hepatovirus, bocaparvoviruses, and polyomavirus in other bat species were also found, likely reflecting their wide distribution among different bats. Unexpectedly, a few sequence reads of canine parvovirus 2 and the recently described mouse kidney parvovirus were also detected and their presence confirmed by PCR; these possibly originated from guano contamination by carnivores and rodents. The majority of eukaryotic viral reads were highly divergent, indicating that numerous viruses still remain to be characterized, even from such a heavily investigated order as Chiroptera.IMPORTANCE Characterizing the bat virome is important for understanding viral diversity and detecting viral spillover between animal species. Using an unbiased metagenomics method, we characterize the virome in guano collected from multiple roosts of common Northern California bat species. We describe several novel viral genomes and report the detection of viruses with close relatives reported in other bat species, likely reflecting cross-species transmissions. Viral sequences from well-known carnivore and rodent parvoviruses were also detected, whose presence are likely the result of contamination from defecation and urination atop guano and which reflect the close interaction of these mammals in the wild.

Assessing the Suitability of Next-Generation Viral Outgrowth Assays to Measure Human Immunodeficiency Virus 1 Latent Reservoir Size.

BACKGROUND: Evaluations of human immunodeficiency virus (HIV) curative interventions require reliable and efficient quantification of replication-competent latent reservoirs. The "classic" quantitative viral outgrowth assay (QVOA) has been regarded as the reference standard, although prohibitively resource and labor intensive. We compared 6 "next-generation" viral outgrowth assays, using polymerase chain reaction or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA. METHODS: Next-generation QVOAs were compared with classic QVOA using single leukapheresis-derived samples from 5 antiretroviral therapy-suppressed HIV-infected participants and 1 HIV-uninfected control; each laboratory tested blinded batches of 3 frozen and 1 fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and laboratory levels. Models also estimated the effect of testing frozen versus fresh samples. RESULTS: Next-generation QVOAs had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher infectious units per million values than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95% credible interval [CI], 2.1-3.5-fold) for next-generation assays. Between-laboratory variation increased extra-Poisson variation to 3.4-fold (95% CI, 2.6-5.4-fold). Frozen storage did not substantially alter infectious units per million values (-18%; 95% CI, -52% to 39%). CONCLUSIONS: The data offer cautious support for use of next-generation QVOAs as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of latent reservoirs in eradication strategies would benefit from high throughput and scalable assays.

Viral Metagenomic Analysis of Cerebrospinal Fluid from Patients with Acute Central Nervous System Infections of Unknown Origin, Vietnam.

Central nervous system (CNS) infection is a serious neurologic condition, although the etiology remains unknown in >50% of patients. We used metagenomic next-generation sequencing to detect viruses in 204 cerebrospinal fluid (CSF) samples from patients with acute CNS infection who were enrolled from Vietnam hospitals during 2012-2016. We detected 8 viral species in 107/204 (52.4%) of CSF samples. After virus-specific PCR confirmation, the detection rate was lowered to 30/204 (14.7%). Enteroviruses were the most common viruses detected (n = 23), followed by hepatitis B virus (3), HIV (2), molluscum contagiosum virus (1), and gemycircularvirus (1). Analysis of enterovirus sequences revealed the predominance of echovirus 30 (9). Phylogenetically, the echovirus 30 strains belonged to genogroup V and VIIb. Our results expanded knowledge about the clinical burden of enterovirus in Vietnam and underscore the challenges of identifying a plausible viral pathogen in CSF of patients with CNS infections.

Seminal Plasma-Derived Extracellular-Vesicle Fractions from HIV-Infected Men Exhibit Unique MicroRNA Signatures and Induce a Proinflammatory Response in Cells Isolated from the Female Reproductive Tract.

The continuing spread of HIV/AIDS is predominantly fueled by sexual exposure to HIV-contaminated semen. Seminal plasma (SP), the liquid portion of semen, harbors a variety of factors that may favor HIV transmission by facilitating viral entry into host cells, eliciting the production of proinflammatory cytokines, and enhancing the translocation of HIV across the genital epithelium. One important and abundant class of factors in SP is extracellular vesicles (EVs), which, in general, are important intercellular signal transducers. Although numerous studies have characterized blood plasma-derived EVs from both uninfected and HIV-infected individuals, little is known about the properties of EVs from the semen of HIV-infected individuals. We report here that fractionated SP enriched for EVs from HIV-infected men induces potent transcriptional responses in epithelial and stromal cells that interface with the luminal contents of the female reproductive tract. Semen EV fractions from acutely infected individuals induced a more proinflammatory signature than those from uninfected individuals. This was not associated with any observable differences in the surface phenotypes of the vesicles. However, microRNA (miRNA) expression profiling analysis revealed that EV fractions from infected individuals exhibit a broader and more diverse profile than those from uninfected individuals. Taken together, our data suggest that SP EVs from HIV-infected individuals exhibit unique miRNA signatures and exert potent proinflammatory transcriptional changes in cells of the female reproductive tract, which may facilitate HIV transmission.IMPORTANCE Seminal plasma (SP), the major vehicle for HIV, can modulate HIV transmission risk through a variety of mechanisms. Extracellular vesicles (EVs) are extremely abundant in semen, and because they play a key role in intercellular communication pathways and immune regulation, they may impact the likelihood of HIV transmission. However, little is known about the properties and signaling effects of SP-derived EVs in the context of HIV transmission. Here, we conduct a phenotypic, transcriptomic, and functional characterization of SP and SP-derived EVs from uninfected and HIV-infected men. We find that both SP and its associated EVs elicit potent proinflammatory transcriptional responses in cells that line the genital tract. EVs from HIV-infected men exhibit a more diverse repertoire of miRNAs than EVs from uninfected men. Our findings suggest that EVs from the semen of HIV-infected men may significantly impact the likelihood of HIV transmission through multiple mechanisms.

Human astrovirus types 1, 4 and 5 circulating among children with acute gastroenteritis in a rural Brazilian state, 2010-2016.

This study combined conventional epidemiology of human astroviruses. From 2010 to 2016, 232 stool samples from children under 5 years of age were screened using NGS and conventional RT-PCR followed by genetic analysis in order to investigate the genotypic diversity of classical human astrovirus (HAstV) circulating in Tocantins State, Brazil. HAstV was detected in 16 cases (6.9%). Seven specimens (43.7%; 7/16) were positive according RT-PCR and next-generation sequencing (NGS) to investigate the molecular to both NGS and RT-PCR. NGS and RT-PCR individually revealed six (37.5%; 6/16) and three (18.8%; 3/16) additional positive samples, respectively. Sequencing of the HAstV-positive samples revealed HAstV-1a (9/16), HAstV-4c (3/16), and HAstV-5c (4/16) lineages.

Norovirus strains in patients with acute gastroenteritis in rural and low-income urban areas in northern Brazil.

From 2010-2016, a total of 251 stool samples were screened for norovirus using next-generation sequencing (NGS) followed by phylogenetic analysis to investigate the genotypic diversity of noroviruses in rural and low-income urban areas in northern Brazil. Norovirus infection was detected in 19.9% (50/251) of the samples. Eight different genotypes were identified: GII.4_Sydney[P31] (64%, 32/50), GII.6[P7] (14%, 7/50), GII.17[P17] (6%, 3/50), GII.1[P33] (6%, 3/50), GII.3[P16] (4%, 2/50), GII.2[P16] (2%, 1/50), GII.2[P2] (2%, 1/50), and GII.4_New Orleans[P4] (2%, 1/50). Distinct GII.6[P7] variants were recognized, indicating the presence of different co-circulating strains. Elucidating norovirus genetic diversity will improve our understanding of their potential health burden, in particular for the GII.4_Sydney[P31] variant.

Viral gastroenteritis in Tocantins, Brazil: characterizing the diversity of human adenovirus F through next-generation sequencing and bioinformatics.

Human enteric adenovirus species F (HAdV-F) is one of the most common pathogens responsible for acute gastroenteritis worldwide. Brazil is a country with continental dimensions where continuous multiregional surveillance is vital to establish a more complete picture of the epidemiology of HAdV-F. The aim of the current study was to investigate the molecular epidemiology of HAdV-F using full-genome data in rural and low-income urban areas in northern Brazil. This will allow a genetic comparison between Brazilian and global HAdV-F strains. The frequency of HAdV-F infections in patients with gastroenteritis and molecular typing of positive samples within this period was also analysed. A total of 251 stool samples collected between 2010 and 2016 from patients with acute gastroenteritis were screened for HAdV-F using next-generation sequencing techniques. HAdV-F infection was detected in 57.8 % (145/251) of samples. A total of 137 positive samples belonged to HAdV-F41 and 7 to HAdV-F40. HAdV-F40/41 dual infection was found in one sample. Detection rates did not vary significantly according to the year. Single HAdV-F infections were detected in 21.9 % (55/251) of samples and mixed infections in 37.4 % (94/251), with RVA/HAdV-F being the most frequent association (21.5 %; 54/251). Genetic analysis indicated that the HAdV-F strains circulating in Brazil were closely related to worldwide strains, and the existence of some temporal order was not observed. This is the first large-scale HAdV-F study in Brazil in which whole-genome data and DNA sequence analyses were used to characterize HAdV-F strains. Expanding the viral genome database could improve overall genotyping success and assist the National Center for Biotechnology Information (NCBI)/GenBank in standardizing the HAdV genome records by providing a large set of annotated HAdV-F genomes.

Replicate Aptima Assay for Quantifying Residual Plasma Viremia in Individuals on Antiretroviral Therapy.

Detection of residual plasma viremia in antiretroviral therapy (ART)-suppressed HIV-infected individuals is critical for characterizing the latent reservoir and evaluating the impact of cure interventions. Ultracentrifugation-based single-copy assays are sensitive but labor intensive. Fully automated replicate testing using a standard clinical viral load assay was evaluated as a high-throughput alternative for the quantification of low-level viremia. Four plasma samples from blood donors with acute HIV-1 infection and one viral culture supernatant were serially diluted into 25-ml samples to nominal viral loads ranging from 39 to <0.5 copies (cp)/ml. Each dilution was tested with 45 replicates (reps) using 0.5 ml/rep with the Aptima HIV-1 Quant assay. The nominal and estimated viral loads based on the single-hit Poisson model were compared, and a hybrid Poisson digital model for calibrated viral load estimation was derived. Testing performed using 45 reps on longitudinal plasma samples from 50 ART-suppressed individuals in the Reservoir Assay Validation and Evaluation Network (RAVEN) study cohort (range of 1 to 19 years of continuous ART suppression) showed a median viral load of 0.54 cp/ml (interquartile range [IQR], 0.22 to 1.46 cp/ml) and a 14% (95% confidence interval [CI], 9% to 19%) decline in viral load for each additional year in duration suppressed. Within the RAVEN cohort, the expected false-negative rate for detection at lower rep numbers using 9 and 18 reps was 26% and 14%, respectively. Residual plasma viremia levels positively correlated with cell-associated HIV RNA and DNA. The performance characteristics of the replicate Aptima assay support its use for quantifying residual plasma viremia to study the latent HIV reservoir and cure interventions.

Effect of Geographic Isolation on the Nasal Virome of Indigenous Children.

The influence of living in small remote villages on the diversity of viruses in the nasal mucosa was investigated in three Colombian villages with very different levels of geographic isolation. Metagenomic analysis was used to characterize viral nucleic acids in nasal swabs from 63 apparently healthy young children. Sequences from human virus members of the families Anelloviridae, Papillomaviridae, Picornaviridae, Herpesviridae, Polyomaviridae, Adenoviridae, and Paramyxoviridae were detected in decreasing proportions of children. The number of papillomavirus infections detected was greater among Hispanic children most exposed to outside contacts, while anellovirus infections were more common in the isolated indigenous villages. The diversity of the other human viruses detected did not differ among the villages. Closely related variants of rhinovirus A or B were identified in 2 to 4 children from each village, reflecting ongoing transmission clusters. Genomes of viruses not currently known to infect humans, including members of the families Parvoviridae, Partitiviridae, Dicistroviridae, and Iflaviridae and circular Rep-encoding single-stranded DNA (CRESS-DNA) virus, were also detected in nasal swabs, possibly reflecting environmental contamination from insect, fungal, or unknown sources. Despite the high levels of geographic and cultural isolation, the overall diversity of human viruses in the nasal passages of children was not reduced in highly isolated indigenous villages, indicating ongoing exposure to globally circulating viruses.IMPORTANCE Extreme geographic and cultural isolation can still be found in some indigenous South American villages. Such isolation may be expected to limit the introduction of otherwise common and widely distributed viruses. Very small population sizes may also result in rapid local viral extinction due to a lack of seronegative subjects to maintain transmission chains for rapidly cleared viruses. We compared the viruses in the nasal passages of young children in three villages with increasing levels of geographic isolation. We found that isolation did not reduce the overall diversity of viral infections. Multiple infections with nearly identical rhinoviruses could be detected within each village, likely reflecting recent viral introductions and transmission clusters among epidemiologically linked members of these very small communities. We conclude that, despite their geographic isolation, remote indigenous villages show evidence of ongoing exposure to globally circulating viruses.

Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size.

Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a "gold standard" for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates-as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir-rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.

A recombinant infectious bronchitis virus from a chicken with a spike gene closely related to that of a turkey coronavirus.

Using viral metagenomics, the complete genome sequence of an infectious bronchitis virus (IBV) strain (named ahysx-1) from a fecal sample from a healthy chicken in Anhui province, China, was determined. The genome sequence of ahysx-1 was found to be very similar to that of IBV strain ck/CH/LLN/131040 (KX252787), except for the spike gene region, which is similar to that of a turkey coronavirus strain (EU022526), suggesting that ahysx-1 is a recombinant. Recombination analysis and phylogenetic analysis based on the genomic sequences of ahysx-1 and other related strains confirmed that ahysx-1 appears to be a recombinant resulting from a recombination event that occurred between a chicken coronavirus and a turkey coronavirus. Further studies need to be performed to determine whether this recombinant IBV strain is pathogenic and whether it is transmitted between chickens and turkeys.

Viruses in Vietnamese Patients Presenting with Community-Acquired Sepsis of Unknown Cause.

Community-acquired (CA) sepsis is a major public health problem worldwide, yet the etiology remains unknown for >50% of the patients. Here we applied metagenomic next-generation sequencing (mNGS) to characterize the human virome in 492 clinical samples (384 sera, 92 pooled nasal and throat swabs, 10 stools, and 6 cerebrospinal fluid samples) from 386 patients (213 adults and 173 children) presenting with CA sepsis who were recruited from 6 hospitals across Vietnam between 2013 and 2015. Specific monoplex PCRs were used subsequently to confirm the presence of viral sequences detected by mNGS. We found sequences related to 47 viral species belonging to 21 families in 358 of 386 (93%) patients, including viruses known to cause human infections. After PCR confirmation, human viruses were found in 52 of 386 patients (13.4%); picornavirus (enteroviruses [n = 14], rhinovirus [n = 5], and parechovirus [n = 2]), hepatitis B virus (n = 10), cytomegalovirus (n = 9), Epstein-Barr virus (n = 5), and rotavirus A (n = 3) were the most common viruses detected. Recently discovered viruses were also found (gemycircularvirus [n = 5] and WU polyomavirus, Saffold virus, salivirus, cyclovirus-VN, and human pegivirus 2 [HPgV2] [n, 1 each]), adding to the growing literature about the geographic distribution of these novel viruses. Notably, sequences related to numerous viruses not previously reported in human tissues were also detected. To summarize, we identified 21 viral species known to be infectious to humans in 52 of 386 (13.4%) patients presenting with CA sepsis of unknown cause. The study, however, cannot directly impute sepsis causation to the viruses identified. The results highlight the fact that it remains a challenge to establish the causative agents in CA sepsis patients, especially in tropical settings such as Vietnam.

Early changes in pro-inflammatory cytokine levels in neonates with encephalopathy are associated with remote epilepsy.

BACKGROUND: Neonatal seizures are associated with adverse neurologic sequelae including epilepsy in childhood. Here we aim to determine whether levels of cytokines in neonates with brain injury are associated with acute symptomatic seizures or remote epilepsy. METHODS: This is a cohort study of term newborns with encephalopathy at UCSF between 10/1993 and 1/2000 who had dried blood spots. Maternal, perinatal/postnatal, neuroimaging, and epilepsy variables were abstracted by chart review. Logistic regression was used to compare levels of cytokines with acute seizures and the development of epilepsy. RESULTS: In a cohort of 26 newborns with neonatal encephalopathy at risk for hypoxic ischemic encephalopathy with blood spots for analysis, diffuse alterations in both pro- and anti-inflammatory cytokine levels were observed between those with (11/28, 39%) and without acute symptomatic seizures. Seventeen of the 26 (63%) patients had >2 years of follow-up and 4/17 (24%) developed epilepsy. Higher levels of pro-inflammatory cytokines IL-6 and TNF-α within the IL-1ß pathway were significantly associated with epilepsy. CONCLUSIONS: Elevations in pro-inflammatory cytokines in the IL-1ß pathway were associated with later onset of epilepsy. Larger cohort studies are needed to confirm the predictive value of these circulating biomarkers.