Fungal and ciliate protozoa are the main rumen microbes associated with methane emissions in dairy cattle.

BACKGROUND: Mitigating the effects of global warming has become the main challenge for humanity in recent decades. Livestock farming contributes to greenhouse gas emissions, with an important output of methane from enteric fermentation processes, mostly in ruminants. Because ruminal microbiota is directly involved in digestive fermentation processes and methane biosynthesis, understanding the ecological relationships between rumen microorganisms and their active metabolic pathways is essential for reducing emissions. This study analysed whole rumen metagenome using long reads and considering its compositional nature in order to disentangle the role of rumen microbes in methane emissions. RESULTS: The ß-diversity analyses suggested a subtle association between methane production and overall microbiota composition (0.01 < R2 < 0.02). Differential abundance analysis identified 36 genera and 279 KEGGs as significantly associated with methane production (Padj < 0.05). Those genera associated with high methane production were Eukaryota from Alveolata and Fungi clades, while Bacteria were associated with low methane emissions. The genus-level association network showed 2 clusters grouping Eukaryota and Bacteria, respectively. Regarding microbial gene functions, 41 KEGGs were found to be differentially abundant between low- and high-emission animals and were mainly involved in metabolic pathways. No KEGGs included in the methane metabolism pathway (ko00680) were detected as associated with high methane emissions. The KEGG network showed 3 clusters grouping KEGGs associated with high emissions, low emissions, and not differentially abundant in either. A deeper analysis of the differentially abundant KEGGs revealed that genes related with anaerobic respiration through nitrate degradation were more abundant in low-emission animals. CONCLUSIONS: Methane emissions are largely associated with the relative abundance of ciliates and fungi. The role of nitrate electron acceptors can be particularly important because this respiration mechanism directly competes with methanogenesis. Whole metagenome sequencing is necessary to jointly consider the relative abundance of Bacteria, Archaea, and Eukaryota in the statistical analyses. Nutritional and genetic strategies to reduce CH4 emissions should focus on reducing the relative abundance of Alveolata and Fungi in the rumen. This experiment has generated the largest ONT ruminal metagenomic dataset currently available.

Characterisation of the rumen resistome in Spanish dairy cattle.

BACKGROUND: Rumen microorganisms carry antimicrobial resistance genes which pose a threaten to animals and humans in a One Health context. In order to tackle the emergence of antimicrobial resistance it is vital to understand how they appear, their relationship with the host, how they behave as a whole in the ruminal ecosystem or how they spread to the environment or humans. We sequenced ruminal samples from 416 Holstein dairy cows in 14 Spanish farms using nanopore technology, to uncover the presence of resistance genes and their potential effect on human, animal and environmental health. RESULTS: We found 998 antimicrobial resistance genes (ARGs) in the cow rumen and studied the 25 most prevalent genes in the 14 dairy cattle farms. The most abundant ARGs were related to the use of antibiotics to treat mastitis, metritis and lameness, the most common diseases in dairy cattle. The relative abundance (RA) of bacteriophages was positively correlated to the ARGs RA. The heritability of the RA of the more abundant ARGs ranged between 0.10 (mupA) and 0.49 (tetW), similar to the heritability of the RA of microbes that carried those ARGs. Even though these genes are carried by the microorganisms, the host is partially controlling their RA by having a more suitable rumen pH, folds, or other physiological traits that promote the growth of those microorganisms. CONCLUSIONS: We were able to determine the most prevalent ARGs (macB, msbA, parY, rpoB2, tetQ and TaeA) in the ruminal bacteria ecosystem. The rumen is a reservoir of ARGs, and strategies to reduce the ARG load from livestock must be pursued.

Sequencing of SARS-CoV-2 genome using different nanopore chemistries.

Nanopore sequencing has emerged as a rapid and cost-efficient tool for diagnostic and epidemiological surveillance of SARS-CoV-2 during the COVID-19 pandemic. This study compared the results from sequencing the SARS-CoV-2 genome using R9 vs R10 flow cells and a Rapid Barcoding Kit (RBK) vs a Ligation Sequencing Kit (LSK). The R9 chemistry provided a lower error rate (3.5%) than R10 chemistry (7%). The SARS-CoV-2 genome includes few homopolymeric regions. Longest homopolymers were composed of 7 (TTTTTTT) and 6 (AAAAAA) nucleotides. The R10 chemistry resulted in a lower rate of deletions in thymine and adenine homopolymeric regions than the R9, at the expenses of a larger rate (~10%) of mismatches in these regions. The LSK had a larger yield than the RBK, and provided longer reads than the RBK. It also resulted in a larger percentage of aligned reads (99 vs 93%) and also in a complete consensus genome. The results from this study suggest that the LSK preparation library provided longer DNA fragments which contributed to a better assembly of the SARS-CoV-2, despite an impaired detection of variants in a R10 flow cell. Nanopore sequencing could be used in epidemiological surveillance of SARS-CoV-2. KEY POINTS: • Sequencing SARS-CoV-2 genome is of great importance for the pandemic surveillance. • Nanopore offers a low cost and accurate method to sequence SARS-CoV-2 genome. • Ligation sequencing is preferred rather than the rapid kit using transposases.

A dimensional reduction approach to modulate the core ruminal microbiome associated with methane emissions via selective breeding.

The rumen is a complex microbial system of substantial importance in terms of greenhouse gas emissions and feed efficiency. This study proposes combining metagenomic and host genomic data for selective breeding of the cow hologenome toward reduced methane emissions. We analyzed nanopore long reads from the rumen metagenome of 437 Holstein cows from 14 commercial herds in 4 northern regions in Spain. After filtering, data were treated as compositional. The large complexity of the rumen microbiota was aggregated, through principal component analysis (PCA), into few principal components (PC) that were used as proxies of the core metagenome. The PCA allowed us to condense the huge and fuzzy taxonomical and functional information from the metagenome into a few PC. Bivariate animal models were applied using these PC and methane production as phenotypes. The variability condensed in these PC is controlled by the cow genome, with heritability estimates for the first PC of ~0.30 at all taxonomic levels, with a large probability (>83%) of the posterior distribution being >0.20 and with the 95% highest posterior density interval (95%HPD) not containing zero. Most genetic correlation estimates between PC1 and methane were large (≥0.70), with most of the posterior distribution (>82%) being >0.50 and with its 95%HPD not containing zero. Enteric methane production was positively associated with relative abundance of eukaryotes (protozoa and fungi) through the first component of the PCA at phylum, class, order, family, and genus. Nanopore long reads allowed the characterization of the core rumen metagenome using whole-metagenome sequencing, and the purposed aggregated variables could be used in animal breeding programs to reduce methane emissions in future generations.

Structural equation models to disentangle the biological relationship between microbiota and complex traits: Methane production in dairy cattle as a case of study.

The advent of metagenomics in animal breeding poses the challenge of statistically modelling the relationship between the microbiome, the host genetics and relevant complex traits. A set of structural equation models (SEMs) of a recursive type within a Markov chain Monte Carlo (MCMC) framework was proposed here to jointly analyse the host-metagenome-phenotype relationship. A non-recursive bivariate model was set as benchmark to compare the recursive model. The relative abundance of rumen microbes (RA), methane concentration (CH4 ) and the host genetics was used as a case of study. Data were from 337 Holstein cows from 12 herds in the north and north-west of Spain. Microbial composition from each cow was obtained from whole metagenome sequencing of ruminal content samples using a MinION device from Oxford Nanopore Technologies. Methane concentration was measured with Guardian® NG infrared gas monitor from Edinburgh Sensors during cow's visits to the milking automated system. A quarterly average from the methane eructation peaks for each cow was computed and used as phenotype for CH4 . Heritability of CH4 was estimated at 0.12 ± 0.01 in both the recursive and bivariate models. Likewise, heritability estimates for the relative abundance of the taxa overlapped between models and ranged between 0.08 and 0.48. Genetic correlations between the microbial composition and CH4 ranged from -0.76 to 0.65 in the non-recursive bivariate model and from -0.68 to 0.69 in the recursive model. Regardless of the statistical model used, positive genetic correlations with methane were estimated consistently for the seven genera pertaining to the Ciliophora phylum, as well as for those genera belonging to the Euryarchaeota (Methanobrevibacter sp.), Chytridiomycota (Neocallimastix sp.) and Fibrobacteres (Fibrobacter sp.) phyla. These results suggest that rumen's whole metagenome recursively regulates methane emissions in dairy cows and that both CH4 and the microbiota compositions are partially controlled by the host genotype.

Genetic variation in CCR2 and CXCL12 genes impacts on CD4 restoration in patients initiating cART with advanced immunesupression.

OBJECTIVE: We investigated the association of genetic polymorphisms in chemokine and chemokine receptor genes with poor immunological recovery in HIV patients starting combined antiretroviral therapy (cART) with low CD4 T-cell counts. METHODS: A case-control study was conducted in 412 HIV-infected patients starting cART with CD4 T-cell count <200 cells/µL and successful viral control for two years. CD4 count increase below 200 cells/µL after two years on cART was used to define INR (immunological non-responder) patients. Polymorphisms in CXCL12, CCL5 and CCR2 genes were genotyped using sequenom's MassARRAY platform. RESULTS: Thirty two percent (134/412) of patients were classified as INR. After adjusting by age, route of HIV infection, length of infection before cART and viral hepatitis coinfection, CCR2 rs1799864-AG genotype was significantly associated with INR status (OR [95% CI]: 1.80 [1.04-3.11]; p = 0.04), and CXCL12 rs1801157-TT genotype showed a trend (OR [95% CI]: 2.47 [0.96-6.35]; p = 0.06). CONCLUSIONS: CCR2 rs1799864-AG or CXCL12 rs1801157-TT genotypes influence on the probability of poor CD4 recovery in the population of HIV patients starting cART with low CD4 counts. Genotyping of these polymorphisms could be used to estimate the risk of poor CD4 restoration, mainly in patients who are diagnosed late in the course of infection.

Genetic variants upstream of TNFAIP3 in the 6q23 region are associated with liver disease severity in HIV/HCV-coinfected patients: A cross-sectional study.

BACKGROUND: TNFAIP3 is a crucial hepatoprotective factor due to its anti-inflammatory, anti-apoptotic, anti-oxidant and pro-regenerative functions. The aim of this study was to analyze the associations between genetic variants upstream of TNFAIP3 (rs675520, rs9376293 and rs6920220) and liver fibrosis severity and inflammation in HIV/HCV-coinfected patients. METHODS: A cross-sectional study was carried out in 215 HIV/HCV-coinfected patients, who underwent a liver biopsy. TNFAIP3 polymorphisms were genotyped using GoldenGate® assay. Outcome variables were: a) liver fibrosis (Metavir score) [fibrosis stage (F0, F1, F2, F3 and F4) and advanced fibrosis and cirrhosis (F ≥ 3 and F4, respectively)]; b) non-invasive indexes [FIB-4, APRI, and their cut-offs (FIB-4 ≥ 3.25 and APRI≥1.5)]; c) inflammation-related biomarkers (leptin, HGF, NGF, sFasL, sFas, MIF, HA, Ang-2, TIMP1, MMP1 and MMP2). RESULTS: Patients with rs675520 AG/GG genotypes had decreased odds of having cirrhosis (F4) and advanced fibrosis (FIB-4 ≥ 3.25 and APRI≥1.5) [adjusted Odd Ratio (aOR) = 0.30 (p = 0.025), aOR = 0.20 (p = 0.014), and aOR = 0.34 (p = 0.017), respectively] and lower levels of FIB-4 and APRI [adjusted arithmetic mean ratio (aAMR) = 0.76 (p = 0.003) and aAMR = 0.72 (p = 0.006), respectively]. Patients with rs9376293 CT/CC genotypes had decreased odds of APRI≥1.5 [aOR = 0.39 (p = 0.030)] and lower levels of APRI [aAMR = 0.77 (p = 0.018)]. Patients with rs6920220 AG/AA genotypes had higher odds of having FIB-4 ≥ 3.25 [aOR = 3.72 (p = 0.043)]. Moreover, rs675520 AG/GG genotypes, compared to AA genotype, were associated with lower levels of leptin and NGF (p = 0.002 and p = 0.001, respectively) and higher levels of sFas, MIF, TIMP1 and MMP2 (p = 0.004, p = 0.007, p = 0.020 and p = 0.036, respectively). Also, rs9376293 CT/CC genotypes were related to lower leptin levels (p = 0.026) and higher sFas, MIF, TIMP1 and MMP2 levels (p = 0.029, p = 0.040, p = 0.022 and p = 0.024, respectively). CONCLUSIONS: Genetic variants upstream of TNFAIP3 were associated with the liver fibrosis severity and inflammation in HIV/HCV-coinfected patients.

Lower expression of plasma-derived exosome miR-21 levels in HIV-1 elite controllers with decreasing CD4 T cell count.

Exosome-derived miR-21 was independently associated with CD4 T cell decline in HIV-1-infected elite controllers (OR 0.369, 95% CI 0.137-0.994, p = 0.049). Also, a negative correlation between miR-21 expression and MCP-1 level was found (r = -0.649, p = 0.020), while no correlation between soluble biomarkers or cellular immune activation was found.

Mitochondrial haplogroup H is related to CD4+ T cell recovery in HIV infected patients starting combination antiretroviral therapy.

BACKGROUND: The mitochondrial DNA (mtDNA) seems to influence in a large number of diseases, including HIV infection. Moreover, there is a substantial inter-individual variability in the CD4+ recovery in HIV-infected patients on combination antiretroviral therapy (cART). Our study aimed to analyze the association between mtDNA haplogroups and CD4+ recovery in HIV-infected patients on cART. METHODS: This is a retrospective study of 324 naïve cART patients with CD4+ < 200 cells/mm3, who were followed-up during 24 months after initiating cART. All patients had undetectable HIV viral load during the follow-up. Besides, we included 141 healthy controls. MtDNA genotyping was performed by using Sequenom's MassARRAY platform. The primary outcome variable was the slope of CD4+ recovery. Patients were stratified into two groups by the median slope value of CD4+ (9.65 CD4+ cells/mm3/month). Logistic regression analyses were performed to calculate the odds of CD4+ recovery according to mtDNA haplogroups. RESULTS: Our study included European HIV-infected patients within the N macro-cluster. The baseline values of CD4+ T-cells were similar between groups of patients stratified by the P50th of the slope of CD4+ T-cells recovery. Patients in the low CD4+ T-cells recovery group were older (p = 0.001), but this variable was included in the multivariate models. When we analyzed the frequencies of mtDNA haplogroups, no significant differences between HIV-infected individuals and healthy controls were found. We did not find any significant association between mtDNA haplogroups and the slope of CD4+ T-cells recovery by linear regression analysis. However, Patients carrying haplogroup H had a higher odds of having a better CD4+ recovery (> 9.65 CD4+ cells/mm3/month) than patients without haplogroup H (p = 0.032). The adjusted logistic regression showed that patients carrying haplogroup H had a higher likelihood of achieving a CD4+ recovery > 9.65 CD4+ cells/mm3/month [adjusted odds ratio (aOR) = 1.75 (95% CI = 1.04; 2.95); p = 0.035]. CONCLUSIONS: European mitochondrial haplogroup H was associated with the improved CD4+ recovery in HIV-infected patients starting cART with CD4+ < 200 cells/mm3.

High Plasma Levels of sTNF-R1 and CCL11 Are Related to CD4+ T-Cells Fall in Human Immunodeficiency Virus Elite Controllers With a Sustained Virologic Control.

Our aim was to analyze the relationship between plasma inflammatory biomarkers and CD4+ T-cells evolution in human immunodeficiency virus (HIV) elite controllers (HIV-ECs) with a suppressed viremia. We carried out a retrospective study in 30 HIV-ECs classified into two groups: those showing no significant loss of CD4+ T-cells during the observation period (stable CD4+, n = 19) and those showing a significant decrease of CD4+ T-cells (decline CD4+, n = 11). Baseline plasma biomarkers were measured using a multiplex immunoassay: sTNF-R1, TRAIL, sFas (APO), sFasL, TNF-α, TNF-ß, IL-8, IL-18, IL-6, IL-10, IP-10, MCP-1, MIP-1α, MIP-1ß, RANTES, SDF1α, GRO-α, and CCL11. Baseline levels of sTNF-R1 and CCL11 and sTNF-R1/TNF-α ratio correlated with the slope of CD4+ T-cells (cells/µl/year) during follow-up [r = -0.370 (p = 0.043), r = -0.314 (p = 0.091), and r = -0.381 (p = 0.038); respectively]. HIV-ECs with declining CD4+ T-cells had higher baseline plasma levels of sTNF-R1 [1,500.7 (555.7; 2,060.7) pg/ml vs. 450.8 (227.9; 1,263.9) pg/ml; p = 0.018] and CCL11 [29.8 (23.5; 54.9) vs. 19.2 (17.8; 29.9) pg/ml; p = 0.041], and sTNF-R1/TNF-α ratio [84.7 (33.2; 124.2) vs. 25.9 (16.3; 75.1); p = 0.012] than HIV-1 ECs with stable CD4+ T-cells. The area under the receiver operating characteristic (ROC) curve [area under ROC curve (AUROC)] were 0.758 ± 0.093 (sTNF-R1), 0.727 ± 0.096 (CCL11), and 0.777 ± 0.087 (sTNF-R1/TNF-α). The cut-off of 75th percentile (high values) for these biomarkers had 71.4% positive predictive value and 73.9% negative predictive value for anticipating the evolution of CD4+ T-cells. In conclusion, the loss of CD4+ T-cells in HIV-ECs was associated with higher levels of two plasma inflammatory biomarkers (sTNF-R1 and CCL11), which were also reasonably accurate for the prediction of the CD4+ T-cells loss.

IL7RA polymorphisms are not associated with AIDS progression.

BACKGROUND: Our aim was to determine whether α-chain of the IL-7 receptor (IL7RA) polymorphisms (rs10491434, rs6897932 and rs987106) are associated with the clinical pattern of AIDS progression in ART-naïve HIV-infected patients. MATERIALS AND METHODS: We carried out a cross-sectional study in 673 HIV-infected patients who were classified into three groups according to the clinical pattern of AIDS progression (188 long-term nonprogressors (LTNPs), 334 moderate progressors (MPs) and 151 rapid progressors (RPs)). Additionally, 134 healthy blood donors participated as a Control-group. We selected three IL7RA polymorphisms located at three regulatory regions [rs6897932 (exon 6), rs987106 (intronic region) and rs10491434 (3'UTR)]. DNA genotyping was performed using Sequenom's MassARRAY platform. RESULTS: The Control-group and all HIV-infected patients had similar age and percentage of males. LTNP-group was older at HIV diagnosis and at the inclusion in the study and had higher percentage of intravenous drug users (IDU) (P < 0·001). Besides, LTNP-group had lower proportion of male patients and homosexual HIV transmission than MP and RP groups (P < 0·001). Moreover, similar values of allelic, genotypic and haplotype frequencies for IL7RA polymorphisms were found between healthy controls and HIV-infected patients (P > 0·05), and among different subgroups of HIV patients according to AIDS progression (LTNPs, MPs and RPs) (P > 0·05). The adjusted logistic regression did not show any significant association between IL7RA polymorphisms and AIDS progression. CONCLUSIONS: IL7RA polymorphisms (rs6897932, rs987106 and rs10491434) were not associated with AIDS progression in Spanish population. Therefore, IL7RA polymorphisms do not seem to help us to understand HIV pathogenesis in untreated HIV-infected patients with different clinical evolution.

Low frequency of NS5A relevant resistance-associated substitutions to Elbasvir among hepatitis C virus genotype 1a in Spain: a cross-sectional study.

Relevant resistance-associated substitutions (RASs) to elbasvir, the new HCV NS5A inhibitor, may limit its efficacy and lead to virological failure in HCV-GT1a-infected patients. There are few data outside clinical trials evaluating their prevalence and impact of elbasvir/grazoprevir. A multicenter cross-sectional study of 617 HCV-GT1a-infected individuals attended in 84 Spanish hospitals from the 17 Autonomous Communities and two Autonomous cities was performed. HCV population sequencing was used to identify RASs to elbasvir and the mutational pattern and drug sensitivity were confirmed by geno2pheno[HCV]. Viruses bearing RASs to elbasvir were present in 6.2% of HCV-GT1a infected patients. The most common RASs were the Y93C/H/N and Q30E/H/R (2.4% and 2.3%; respectively). Only 3.4% of patients had viruses with RASs that confer reduced susceptibility to elbasvir by geno2pheno[HCV] that identified exclusively the positions Q30H/R (n = 7) and Y93C/H/N (n = 8) as single mutations and Q30H + Y93H (n = 4) and Q30R + Y93H (n = 2) as double mutations considered as RASs to elbasvir. Lower prevalence of RASs to elbasvir in our HCV-GT1a-Spanish cohort was observed than reported previously in clinical trials. This information may be essential to guiding the implementation of elbasvir/grazoprevir in Spain, expected at the beginning of 2017 and the management of GT1a-infected patients.

NS3 Resistance-Associated Variants (RAVs) in Patients Infected with HCV Genotype 1a in Spain.

BACKGROUND: Resistance-associated variants have been related to treatment failure of hepatitis C virus (HCV) therapy with direct-acting antiviral drugs. The aim of our study was to analyze the prevalence of clinically relevant resistance-associated variants within NS3 in patients infected with HCV genotype 1a (GT1a) in Spain. METHODS: We performed a cross-sectional study on 2568 patients from 115 hospitals throughout Spain (2014-2015). The viral NS3 protease gene was amplified by nested polymerase chain reaction and sequenced by Sanger sequencing using an ABI PRISM 377 DNA sequencer. Additionally, clade information for genotype 1a was obtained by using the software geno2pheno (http://hcv.geno2pheno.org/). RESULTS: In total, 875 out of 2568 samples were from human immunodeficiency virus (HIV)/HCV-coinfected patients. Q80K was the main RAV found in our patients (11.1%) and the rest of the resistance-associated variants had a lower frequency, including S122G (6.23%), T54S (3.47%), V55A (2.61%), and V55I (2.15%), which were among the most frequent after Q80K. Overall, 286 samples had the Q80K polymorphism (11.1%) and 614 (23.9%) were GT1a clade I. HIV/HCV-coinfected patients had a higher frequency of Q80K and GT1a clade I than HCV-monoinfected patients (12.9% vs. 9.6% [p = 0.012] and 28.5% vs. 21.4% [p<0.001], respectively). Both the prevalence of Q80K and GT1a clade I were not uniform throughout the country (p<0.001), which ranged from 7.3%-22.2% and 15.7%-42.5%, respectively. The frequency of the Q80K polymorphism was far higher in patients infected with GT1a clade I than in patients infected with GT1a clade II (41.5% vs. 1.6%; p<0.001). CONCLUSIONS: The prevalence of most resistance-associated variants in NS3 was low in patients infected with HCV GT1a in Spain, except for Q80K (11.1%), which was also notably higher in HIV/HCV-coinfected patients. The vast majority of Q80K polymorphisms were detected in GT1a clade I.

Optimal vitamin D plasma levels are associated with lower bacterial DNA translocation in HIV/hepatitis c virus coinfected patients.

OBJECTIVE: Vitamin D has been linked to the immune response modulation and the integrity of the intestinal mucosal barrier. Therefore, vitamin D might be involved in bacterial translocation related to HIV infection. Our major aim was to analyze the association between plasma levels of 25-hydroxy-vitamin D [25(OH)D] and bacterial 16S ribosomal DNA (bactDNA) in 120 HIV/hepatitis c virus (HCV) coinfected patients. DESIGN: Cross-sectional study. METHODS: Plasma 25(OH)D levels were quantified by enzyme immunoassay. The vitamin D status was defined as deficient (<25 nmol/l), insufficient (25-74 nmol/l), and optimal (≥75 nmol/l) plasma levels. Plasma bactDNA levels were measured by quantitative real-time PCR. For bactDNA levels the cutoffs used were as follows: low [p75th). RESULTS: Eighteen (15%) patients had 25(OH)D deficiency, 93 (77.5%) had insufficiency and nine (7.5%) had 25(OH)D optimal values. The bactDNA levels were lower in patients with 25(OH)D at least 75 nmol/l [37 copies/µl] than in patients with 25(OH)D insufficiency [84.2 copies/µl; P = 0.042]. Conversely, low bactDNA levels (

Impact of patatin-like phospholipase domain-containing 3 gene polymorphism (rs738409) on severity of liver disease in HIV/hepatitis C virus-coinfected patients.

OBJECTIVE: To analyze the association between patatin-like phospholipase domain-containing 3 gene (PNPLA3) rs738409 polymorphism and severity of liver disease in HIV/hepatitis C virus-coinfected patients. METHODS: We performed a cross-sectional study of 215 patients who underwent a liver biopsy. PNPLA3 rs738409 polymorphism was genotyped using GoldenGate assay. The outcome variables were as follows: advanced fibrosis (F ≥3 and FIB-4 ≥3.25), rapid fibrosis progression (FPR ≥0.10 fibrosis units/year), severe activity grade (A≥3), and steatosis (fatty hepatocytes ≥10%). The genetic association analysis was carried out according to an additive genetic model through logistic regressions adjusted by the most significant covariables. RESULTS: Overall, 21.4% had F at least 3, 8.9% had FIB-4 at least 3.25, 11.4% had A at least 3, 60.6% had steatosis, and 32.5% had FPR at least 0.10. For each rs738409 G allele, we found an increased frequency of patients with advanced fibrosis (F at least 3) (0% CC, 18.5% CG, and 25.2% GG; P = 0.049) and FIB-4 at least 3.25 (0% CC, 3.8% CG, and 13.2% GG; P = 0.016). Furthermore, for each rs738409 G allele, the odds of having F at least 3 increased 2.15 times (95% confidence interval=1.07; 4.35; P = 0.029) and having FIB-4 at least 3.25 increased 8.77 times (95% of confidence interval = 1.11; 69.0; P = 0.039). Note that rs738409 G allele carriers tended to higher likelihood of having FPR at least 0.10, but statistical significance was not reached (P = 0.054). Finally, we did not find any association for A at least 3 and liver steatosis. CONCLUSION: PNPLA3 rs738409 polymorphism was associated with the severity of liver fibrosis in patients coinfected with HIV and hepatitis C virus, suggesting that this polymorphism might also play a significant role in the progression of hepatic fibrosis in this group of patients.

Evidence of ongoing replication in a human immunodeficiency virus type 1 persistently infected cell line.

Human immunodeficiency virus type 1 (HIV-1) persistently infected cell lines are characterized by the continuous viral production without cytopathic effect. However, it is not completely clear if this production is contributed only by viral transcription or also by new cycles of viral replication. We studied an HIV-1 persistently infected cell line, designated H61-D, providing evidence of new replication cycles as sustained by: (i) a decrease in viral production, measured by p24 protein, after treatment of the culture with 3'-azydo-3'-deoxythymydine; (ii) detection of new integration events in the course of cell culture, and (iii) finding of two-long-terminal repeat circles in the cells. H61-D cells were not infected by cell-free virus, but infection was possible by co-culture with another productive-infected cell line. In conclusion, ongoing viral replication is taking place in H61-D persistent cells and new infections are mediated by a cell-to-cell spread mechanism.

Mutagen-mediated enhancement of HIV-1 replication in persistently infected cells.

Lethal mutagenesis, a new antiviral strategy to extinguish virus through elevated mutation rates, was explored in H61-D cells an HIV-1 persistently infected lymphoid cell line. Three mutagenic agents: 5-hydroxy-2(')-deoxycytidine (5-OHdC), 5-fluorouracil (5-FU) and 2,2(')-difluoro-2(')-deoxycytidine (gemcitabine) were used. After 54 passages, treatments with 5-FU and gemcitabine reduced virus infectivity, p24 and RT activity. Treatment with the pyrimidine analog 5-OHdC resulted in increases of p24 production, RT activity and infectivity. Rise in viral replication by 5-OHdC during HIV-1 persistence is in contrast with its inhibitory effect in acute infections. Viral replication enhancement by 5-OHdC was associated with an increase in intracellular HIV-1 RNA mutations. Mechanisms of HIV-1 replication enhancement by 5-OHdC are unknown but some potential factors are discussed. Increase of HIV-1 replication by 5-OHdC cautions against the use, without previous analyses, of mutagenic nucleoside analogs for AIDS treatment.

Tolerance to mutations in the foot-and-mouth disease virus integrin-binding RGD region is different in cultured cells and in vivo and depends on the capsid sequence context.

Engineered RNAs carrying substitutions in the integrin receptor-binding Arg-Gly-Asp (RGD) region of foot-and-mouth disease virus (FMDV) were constructed (aa 141-147 of VP1 capsid protein) and their infectivity was assayed in cultured cells and suckling mice. The effect of these changes was studied in the capsid proteins of two FMDVs, C-S8c1, which enters cells through integrins, and 213hs(-), a derivative highly adapted to cell culture whose ability to infect cells using the glycosaminoglycan heparan sulfate (HS) as receptor, acquired by multiple passage on BHK-21 cells, has been abolished. The capsid sequence context determined infectivity in cultured cells and directed the selection of additional replacements in structural proteins. Interestingly, a viral population derived from a C-S8c1/L144A mutant, carrying only three substitutions in the capsid, was able to expand tropism to wild-type (wt) and mutant (mt) glycosaminoglycan-deficient CHO cells. In contrast, the 213hs(-) capsid tolerated all substitutions analysed with no additional mutations, and the viruses recovered maintained the ability of the 213hs(-) parental virus to infect wt and mt CHO cells. Viruses derived from C-S8c1 with atypical RGD regions were virulent and transmissible for mice with no other changes in the capsid. Substitution of Asp143 for Ala in the C-S8c1 capsid eliminated infectivity in cultured cells and mice. Co-inoculation with a neutralizing monoclonal antibody directed against the type C FMDV RGD region abolished infectivity of C-S8c1 virus on suckling mice, suggesting that FMDV can infect mice using integrins. Sequence requirements imposed for viral entry in vitro and in vivo are discussed.

Tryptophan scanning mutagenesis of aromatic residues within the polymerase domain of HIV-1 reverse transcriptase: critical role of Phe-130 for p51 function and second-site revertant restoring viral replication capacity.

The effects on virus viability and reverse transcriptase (RT) function of substituting Trp for Tyr or Phe residues within the polymerase domain of human immunodeficiency virus type 1 (HIV-1) RT have been analyzed with an infectious HIV-1 clone. Viruses containing mutations Y56W, F61W, F87W, F116W, Y127W, Y144W, F171W, Y181W, Y183W, Y188W, F227W, or Y232W in their RT-coding regions were viable and showed replication capacities similar or slightly reduced in comparison with the wild-type HIV-1. However, RTs bearing mutations F77W or Y146W had a dNTP-binding defect, rendering nonviable viruses. HIV-1 carrying RT mutations F124W or F130W replicated very poorly, but compensatory changes (K83R for F124W, and T58S for F130W) were selected upon passaging the virus in cell culture. The amino acid substitution F130W diminishes the stability of the 51-kDa subunit of the RT (p51) and impairs polyprotein processing in virus-infected cells, an effect that can be mitigated when T58S is found in p51.