A case of severe Plasmodium ovale malaria with acute respiratory distress syndrome and splenic infarction in a male traveller presenting in Italy.

BACKGROUND: Plasmodium ovale malaria is usually considered a tropical infectious disease associated with low morbidity and mortality. However, severe disease and death have previously been reported. CASE PRESENTATION: A case of severe P. ovale malaria in a healthy Caucasian man with a triangle splenic infarction and clinical progression towards Acute Respiratory Distress Syndrome was reported despite a rapid response to oral chloroquine treatment with 24-h parasitaemia clearance. CONCLUSION: Plasmodium ovale malaria is generally considered as a benign disease, with low parasitaemia. However, severe disease and death have occasionally been reported. It is important to be aware that occasionally it can progress to serious illness and death even in immunocompetent individuals.

Emerging viral infections in immunocompromised patients: A great challenge to better define the role of immune response.

The immune response to invading pathogens is characterized by the rapid establishment of a complex network of cellular interactions and soluble signals. The correct balancing of activating and regulating pathways and tissue-homing signals determines its effectiveness and persistence over time. Emerging viral pathogens have always represented a great challenge to the immune system and an often uncontrolled/imbalanced immune response has been described (e.g. cytokine storm, immune paralysis), contributing to the severity of the disease. Several immune biomarkers and cell subsets have been identified as major players in the cascade of events leading to severe diseases, highlighting the rationale for host-directed intervention strategy. There are millions of immunocompromised pediatric and adult patients worldwide (e.g. transplant recipients, hematologic patients, subjects with primary immune-deficiencies), experiencing an impaired immune reactivity, due to diseases and/or to the medical treatments. The reduced immune reactivity could have two paradoxical non-exclusive effects: a weak protective immunity on one hand, and a reduced contribution to immune-mediated pathogenetic processes on the other hand. In these sensitive contexts, the impact of emerging infections represents a still open issue to be explored with several challenges for immunologists, virologists, physicians and epidemiologists. In this review, we will address emerging infections in immunocompromised hosts, to summarize the available data concerning the immune response profile, its influence on the clinical presentation, the possible contribution of persistent viral shedding in generating new viral variants with improved immune escape features, and the key role of vaccination.

Genetic predisposition to lung adenocarcinoma outcome is a feature already present in patients' noninvolved lung tissue.

Emerging evidence suggests that the prognosis of patients with lung adenocarcinoma can be determined from germline variants and transcript levels in nontumoral lung tissue. Gene expression data from noninvolved lung tissue of 483 lung adenocarcinoma patients were tested for correlation with overall survival using multivariable Cox proportional hazard and multivariate machine learning models. For genes whose transcript levels are associated with survival, we used genotype data from 414 patients to identify germline variants acting as cis-expression quantitative trait loci (eQTLs). Associations of eQTL variant genotypes with gene expression and survival were tested. Levels of four transcripts were inversely associated with survival by Cox analysis (CLCF1, hazard ratio [HR] = 1.53; CNTNAP1, HR = 2.17; DUSP14, HR = 1.78; and MT1F: HR = 1.40). Machine learning analysis identified a signature of transcripts associated with lung adenocarcinoma outcome that was largely overlapping with the transcripts identified by Cox analysis, including the three most significant genes (CLCF1, CNTNAP1, and DUSP14). Pathway analysis indicated that the signature is enriched for ECM components. We identified 32 cis-eQTLs for CNTNAP1, including 6 with an inverse correlation and 26 with a direct correlation between the number of minor alleles and transcript levels. Of these, all but one were prognostic: the six with an inverse correlation were associated with better prognosis (HR < 1) while the others were associated with worse prognosis. Our findings provide supportive evidence that genetic predisposition to lung adenocarcinoma outcome is a feature already present in patients' noninvolved lung tissue.

The natural antisense transcript HAS2-AS1 regulates breast cancer cells aggressiveness independently from hyaluronan metabolism.

Hyaluronan (HA) is a ubiquitous extracellular matrix component playing a crucial role in the regulation of cell behaviors, including cancer. Aggressive breast cancer cells tend to proliferate, migrate and metastatize. Notably, triple-negative breast cancer cells lacking the expression of estrogen receptor (ER) as well as progesterone receptor and HER2 are more aggressive than ER-positive ones. As currently no targeted therapy is available for triple-negative breast cancer, the identification of novel therapeutic targets has a high clinical priority. In ER-negative cells, tumoral behavior can be reduced by inhibiting HA synthesis or silencing the enzymes involved in its metabolism, such as HA synthase 2 (HAS2). HAS2-AS1 is a long non-coding RNA belonging to the natural antisense transcript family which is known to favor HAS2 gene expression and HA synthesis, thus bolstering malignant progression in brain, ovary, and lung tumors. As the role of HAS2-AS1 has not yet been investigated in breast cancer, in this work we report that ER-positive breast cancers had lower HAS2-AS1 expression compared to ER-negative tumors. Moreover, the survival of patients with ER-negative tumors was higher when the expression of HAS2-AS1 was elevated. Experiments with ER-negative cell lines as MDA-MB-231 and Hs 578T revealed that the overexpression of either the full-length HAS2-AS1 or its exon 2 long or short isoforms alone, strongly reduced cell viability, migration, and invasion, whereas HAS2-AS1 silencing increased cell aggressiveness. Unexpectedly, in these ER-negative cell lines, HAS2-AS1 is involved neither in the regulation of HAS2 nor in HA deposition. Finally, transcriptome analysis revealed that HAS2-AS1 modulation affected several pathways, including apoptosis, proliferation, motility, adhesion, epithelial to mesenchymal transition, and signaling, describing this long non-coding RNA as an important regulator of breast cancer cells aggressiveness.

The Easy-to-Use SARS-CoV-2 Assembler for Genome Sequencing: Development Study.

Background: Early sequencing and quick analysis of the SARS-CoV-2 genome have contributed to the understanding of the dynamics of COVID-19 epidemics and in designing countermeasures at a global level. Objective: Amplicon-based next-generation sequencing (NGS) methods are widely used to sequence the SARS-CoV-2 genome and to identify novel variants that are emerging in rapid succession as well as harboring multiple deletions and amino acid-changing mutations. Methods: To facilitate the analysis of NGS sequencing data obtained from amplicon-based sequencing methods, here, we propose an easy-to-use SARS-CoV-2 genome assembler: the Easy-to-use SARS-CoV-2 Assembler (ESCA) pipeline. Results: Our results have shown that ESCA could perform high-quality genome assembly from Ion Torrent and Illumina raw data and help the user in easily correct low-coverage regions. Moreover, ESCA includes the possibility of comparing assembled genomes of multisample runs through an easy table format. Conclusions: In conclusion, ESCA automatically furnished a variant table output file, fundamental to rapidly recognizing variants of interest. Our pipeline could be a useful method for obtaining a complete, rapid, and accurate analysis even with minimal knowledge in bioinformatics.

The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk.

Interaction between cancer cells and their microenvironment is central in defining the fate of cancer development. Tumour cells secrete signals (cytokines, chemokines, growth factors) that modify the surrounding area, while the niche supplies structures and activities necessary for tumour maintenance and growth. Hyaluronan (HA) is a glycosaminoglycan that constitute cancer cell niche and is known to influence tumour functions such as proliferation, migration and neoangiogenesis. The knowledge of the factors regulating HA synthesis and size is crucial in understanding the mechanisms sustaining tumour development. Here we show that a yet uncharacterized protein secreted by breast tumour cell lines, named c10orf118 (accession number NM_018017 in NCBI/BLAST, and Q7z3E2 according to the Uniprot identifier), with a predicted length of 898 amino acids, can induce the secretion of HA by stromal fibroblasts through the up-regulation of the hyaluronan synthase 2 gene (HAS2). Intracellularly, this protein is localized in the Golgi apparatus with a possible role in vesicle maturation and transport. The expression of c10orf118 was verified in breast cancer patient specimens and was found to be associated with the presence of estrogen receptor that characterizes a good patient survival. We suggest c10orf118 as a new player that influences the HA amount in breast cancer microenvironment and is associated with low aggressiveness of cancer.

Investigation of Nasal/Oropharyngeal Microbial Community of COVID-19 Patients by 16S rDNA Sequencing.

Since December 2019, SARS-CoV-2 infection has been still rapidly spreading, resulting in a pandemic, followed by an increasing number of cases in countries throughout the world. The severity of the disease depends on the patient's overall medical condition but no appropriate markers are available to establish the prognosis of the patients. We performed a 16S rRNA gene sequencing, revealing an altered composition of the nasal/oropharyngeal (NOP) microbiota in 21 patients affected by COVID-19, paucisymptomatic or in an Intensive Care Unit (ICU), as compared to 10 controls negative for COVID-19 or eight affected by a different Human Coronavirus (HKU, NL63 and OC43). A significant decrease in Chao1 index was observed when patients affected by COVID-19 (in ICU) were compared to paucisymptomatic. Furthermore, patients who were in ICU, paucisymptomatic or affected by other Coronaviruses all displayed a decrease in the Chao1 index when compared to controls, while Shannon index significantly decreased only in patients under ICU as compared to controls and paucisymptomatic patients. At the phylum level, Deinococcus-Thermus was present only in controls as compared to SARS-CoV-2 patients admitted to ICU, paucisymptomatic or affected by other coronaviruses. Candidatus Saccharibacteria (formerly known as TM7) was strongly increased in negative controls and SARS-CoV-2 paucisymptomatic patients as compared to SARS-CoV-2 ICU patients. Other modifications were observed at a lower taxonomy level. Complete depletion of Bifidobacterium and Clostridium was exclusively observed in ICU SARS-CoV-2 patients, which was the only group characterized by the presence of Salmonella, Scardovia, Serratia and Pectobacteriaceae. In conclusion, our preliminary results showed that nasal/oropharyngeal microbiota profiles of patients affected with SARS-CoV-2 may provide valuable information in order to facilitate the stratification of patients and may open the way to new interventional strategies in order to ameliorate the outcome of the patients.

Risk and predictive factors of prolonged viral RNA shedding in upper respiratory specimens in a large cohort of COVID-19 patients admitted to an Italian reference hospital.

BACKGROUND: Limited data are available about the predictors and outcomes associated with prolonged SARS-CoV-2 RNA shedding (VS). METHODS: A retrospective study including COVID-19 patients admitted to an Italian hospital between March 1 and July 1, 2020. Predictors of viral clearance (VC) and prolonged VS from the upper respiratory tract were assessed by Poisson regression and logistic regression analyses. The causal relation between VS and clinical outcomes was evaluated through an inverse probability weighted Cox model. RESULTS: The study included 536 subjects. The median duration of VS from symptoms onset was 18 days. The estimated 30-day probability of VC was 70.2%. Patients with comorbidities, lymphopenia at hospital admission, or moderate/severe respiratory disease had a lower chance of VC. The development of moderate/severe respiratory failure, delayed hospital admission after symptoms onset, baseline comorbidities, or D-dimer >1000ng/mL at admission independently predicted prolonged VS. The achievement of VC doubled the chance of clinical recovery and reduced the probability of death/mechanical ventilation. CONCLUSIONS: Respiratory disease severity, comorbidities, delayed hospital admission and inflammatory markers negatively predicted VC, which resulted to be associated with better clinical outcomes. These findings highlight the importance of prompt hospitalization of symptomatic patients, especially where signs of severity or comorbidities are present.

SARS-CoV-2 Early Screening at the Point of Entry: Travelers From Bangladesh to Italy-July 2020.

We report phylogenetic and mutational analysis by NGS of six SARS-CoV-2 strains from patients flying from Bangladesh to Italy (July 2020). Data suggest that no further circulation of such imported strains occurred in Italy, stating the efficacy of early screening at the point of entry and supporting the importance of molecular epidemiology in monitoring the efficacy of control measures.

16S rRNA gene sequencing of rectal swab in patients affected by COVID-19.

COronaVIrus Disease-2019 (COVID-19) is a pandemic respiratory infection caused by a new betacoronavirus, the Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2). Few data are reported on the gut microbiota in COVID-19 patients. 16S rRNA gene sequencing was performed to reveal an altered composition of the gut microbiota in patients with COVID-19 pneumonia admitted in intensive care unit (ICU) (i-COVID19), or in infectious disease wards (w-COVID19) as compared to controls (CTRL). i-COVID19 patients showed a decrease of Chao1 index as compared to CTRL and w-COVID19 patients indicating that patients in ICU displayed a lower microbial richness while no change was observed as for Shannon Index. At the phylum level, an increase of Proteobacteria was detected in w-COVID19 patients as compared to CTRL. A decrease of Fusobacteria and Spirochetes has been found, with the latter decreased in i-COVID19 patients as compared to CTRL. Significant changes in gut microbial communities in patients with COVID-19 pneumonia with different disease severity compared to CTRL have been identified. Our preliminary data may provide valuable information and promising biomarkers for the diagnosis of the disease and, when validated in larger cohort, it could facilitate the stratification of patients based on the microbial signature.

Virological and Serological Characterisation of SARS-CoV-2 Infections Diagnosed After mRNA BNT162b2 Vaccination Between December 2020 and March 2021.

BACKGROUND: Vaccines for coronavirus disease 2019 (COVID-19) are proving to be very effective in preventing severe illness; however, although rare, post-vaccine infections have been reported. The present study focuses on virological and serological features of 94 infections that occurred in Lazio Region (Central Italy) between 27 December 2020, and 30 March 2021, after one or two doses of mRNA BNT162b2 vaccine. METHODS: We evaluated clinical features, virological (viral load; viral infectiousness; genomic characterisation), and serological (anti-nucleoprotein Ig; anti-Spike RBD IgG; neutralising antibodies, nAb) characteristics of 94 post-vaccine infections at the time of diagnosis. Nasopharyngeal swabs (NPSs) and serum samples were collected in the framework of the surveillance activities on SARS-CoV-2 variants established in Lazio Region (Central Italy) and analysed at the National Institute for Infectious Diseases "L. Spallanzani" in Rome. RESULTS: The majority (92.6%) of the post-vaccine infections showed pauci/asymptomatic or mild clinical course, with symptoms and hospitalisation rate significantly less frequent in patients infected after full vaccination course as compared to patients who received a single dose vaccine. Although differences were not statistically significant, viral loads and isolation rates were lower in NPSs from patients infected after receiving two vaccine doses as compared to patients with one dose. Most cases (84%) had nAb in serum at the time of infection diagnosis, which is a sub-group of vaccinees, were found similarly able to neutralise Alpha and Gamma variants. Asymptomatic individuals showed higher nAb titres as compared to symptomatic cases (median titre: 1:120 vs. 1:40, respectively). Finally, the proportion of post-vaccine infections attributed either to Alpha and Gamma variants was similar to the proportion observed in the contemporary unvaccinated population in the Lazio region, and mutational analysis did not reveal enrichment of a defined set of Spike protein substitutions depending on the vaccination status. CONCLUSION: Our study conducted using real-life data, emphasised the importance of monitoring vaccine breakthrough infections, through the characterisation of virological, immunological, and clinical features associated with these events, in order to tune prevention measures in the next phase of the COVID-19 pandemic.

Chikungunya Outbreak in the Republic of the Congo, 2019-Epidemiological, Virological and Entomological Findings of a South-North Multidisciplinary Taskforce Investigation.

The Republic of Congo (RoC) declared a chikungunya (CHIK) outbreak on 9 February 2019. We conducted a ONE-Human-Animal HEALTH epidemiological, virological and entomological investigation. Methods: We collected national surveillance and epidemiological data. CHIK diagnosis was based on RT-PCR and CHIKV-specific antibodies. Full CHIKV genome sequences were obtained by Sanger and MinION approaches and Bayesian tree phylogenetic analysis was performed. Mosquito larvae and 215 adult mosquitoes were collected in different villages of Kouilou and Pointe-Noire districts and estimates of Aedes (Ae.) mosquitos' CHIKV-infectious bites obtained. We found two new CHIKV sequences of the East/Central/South African (ECSA) lineage, clustering with the recent enzootic sub-clade 2, showing the A226V mutation. The RoC 2019 CHIKV strain has two novel mutations, E2-T126M and E2-H351N. Phylogenetic suggests a common origin from 2016 Angola strain, from which it diverged around 1989 (95% HPD 1985-1994). The infectious bite pattern was similar for 2017, 2018 and early 2019. One Ae. albopictus pool was RT-PCR positive. The 2019 RoC CHIKV strain seems to be recently introduced or be endemic in sylvatic cycle. Distinct from the contemporary Indian CHIKV isolates and in contrast to the original Central-African strains (transmitted by Ae. aegypti), it carries the A226V mutation, indicating an independent adaptive mutation in response to vector replacement (Ae. albopictus vs Ae. aegypti).

Compartmentalized Replication of SARS-Cov-2 in Upper vs. Lower Respiratory Tract Assessed by Whole Genome Quasispecies Analysis.

We report whole-genome and intra-host variability of SARS-Cov-2 assessed by next generation sequencing (NGS) in upper (URT) and lower respiratory tract (LRT) from COVID-19 patients. The aim was to identify possible tissue-specific patterns and signatures of variant selection for each respiratory compartment. Six patients, admitted to the Intensive Care Unit, were included in the study. Thirteen URT and LRT were analyzed by NGS amplicon-based approach on Ion Torrent Platform. Bioinformatic analysis was performed using both realized in-house and supplied by ThermoFisher programs. Phylogenesis showed clade V clustering of the first patients diagnosed in Italy, and clade G for later strains. The presence of quasispecies was observed, with variants uniformly distributed along the genome and frequency of minority variants spanning from 1% to ~30%. For each patient, the patterns of variants in URT and LRT were profoundly different, indicating compartmentalized virus replication. No clear variant signature and no significant difference in nucleotide diversity between LRT and URT were observed. SARS-CoV-2 presents genetic heterogeneity and quasispecies compartmentalization in URT and LRT. Intra-patient diversity was low. The pattern of minority variants was highly heterogeneous and no specific district signature could be identified, nevertheless, analysis of samples, longitudinally collected in patients, supported quasispecies evolution.

SARS-CoV-2 Phylogenetic Analysis, Lazio Region, Italy, February-March 2020.

We report phylogenetic and mutational analysis of severe acute respiratory syndrome coronavirus 2 virus strains from the Lazio region of Italy and provide information about the dynamics of virus spread. Data suggest effective containment of clade V strains, but subsequently, multiple waves of clade G strains were circulating widely in Europe.

Heparan Sulfate in the Tumor Microenvironment.

The biology of tumor cells strictly depends on their microenvironment architecture and composition, which controls the availability of growth factors and signaling molecules. Thus, the network of glycosaminoglycans, proteoglycans, and proteins known as extracellular matrix (ECM) that surrounds the cells plays a central role in the regulation of tumor fate. Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are highly versatile ECM components that bind and regulate the activity of growth factors, cell membrane receptors, and other ECM molecules. These HS binding partners modulate cell adhesion, motility, and proliferation that are processes altered during tumor progression. Modification in the expression and activity of HS, HSPGs, and the respective metabolic enzymes results unavoidably in alteration of tumor cell microenvironment. In this light, the targeting of HS structure and metabolism is potentially a new tool in the treatment of different cancer types.

Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2-AS1.

Hyaluronan (HA) is one of the most prevalent glycosaminoglycans of the vascular extracellular matrix (ECM). Abnormal HA accumulation within blood vessel walls is associated with tissue inflammation and is prominent in most vascular pathological conditions such as atherosclerosis and restenosis. Hyaluronan synthase 2 (HAS2) is the main hyaluronan synthase enzyme involved in HA synthesis and uses cytosolic UDP-glucuronic acid and UDP-GlcNAc as substrates. The synthesis of UDP-glucuronic acid can alter the NAD+/NADH ratio via the enzyme UDP-glucose dehydrogenase, which oxidizes the alcohol group at C6 to the COO- group. Here, we show that HAS2 expression can be modulated by sirtuin 1 (SIRT1), the master metabolic sensor of the cell, belonging to the class of NAD+-dependent deacetylases. Our results revealed the following. 1) Treatments of human aortic smooth muscle cells (AoSMCs) with SIRT1 activators (SRT1720 and resveratrol) inhibit both HAS2 expression and accumulation of pericellular HA coats. 2) Tumor necrosis factor α (TNFα) induced HA-mediated monocyte adhesion and AoSMC migration, whereas SIRT1 activation prevented immune cell recruitment and cell motility by reducing the expression levels of the receptor for HA-mediated motility, RHAMM, and the HA-binding protein TNF-stimulated gene 6 protein (TSG6). 3) SIRT1 activation prevented nuclear translocation of NF-κB (p65), which, in turn, reduced the levels of HAS2-AS1, a long-noncoding RNA that epigenetically controls HAS2 mRNA expression. In conclusion, we demonstrate that both HAS2 expression and HA accumulation by AoSMCs are down-regulated by the metabolic sensor SIRT1.

Acute Exposure of Collecting Lymphatic Vessels to Low-Density Lipoproteins Increases Both Contraction Frequency and Lymph Flow: An In Vivo Mechanical Insight.

Background: Lymphatic vessels drain fluids and solutes from interstitial spaces and serosal cavities. Among the solutes, low-density lipoproteins (LDL) are drained and can be detected in peripheral lymph, where they have been reported to exert a modulatory action on lymphatic vessels intrinsic contraction rate. In the present work, we investigated lymphatic vessel mechanical properties (contraction frequency and amplitude) that may be modulated by LDL application and the consequence on lymph flow. Methods and Results: Human-derived LDL were resuspended in phosphate-buffered saline (PBS) and microinjected in the interstitial space surrounding spontaneously contracting lymphatic vessels of the rat diaphragm, in vivo. Vessels' contraction rate and diameter were measured in control conditions (PBS) and after LDL injection. Lymph flow (Jlymph) was computed from contraction rate and diameter change. In some animals, after the recording procedure, diaphragmatic tissue samples were excised and immunostained with antilymphatic muscle (LM) actin to investigate the correlation between LM signal level and contraction amplitude. Data indicate a positive, saturating correlation between the abundance of LM actin and contraction amplitude, and LDL microinjection caused an acute increase in contraction frequency (+126%), a reduction of contraction amplitude to 75% of that obtained after PBS injection, and a +63% increase in Jlymph. Conclusions: From our in vivo analysis of the mechanical parameters affected by LDL, Jlymph was increased by a predominant effect on the contraction rate rather than amplitude, suggesting that the still elusive messaging system might be linked to the pacemaker sites.

Revisiting the hallmarks of cancer: The role of hyaluronan.

Extracellular matrix (ECM) is a complex network of macromolecules such as proteoglycans (PGs), glycosaminoglycans (GAGs) and fibrous proteins present within all tissues and organs. The main role of ECM is not only to provide an essential mechanical scaffold for the cells but also to mediate crucial biochemical cues that are required for tissue homeostasis. Dysregulations in ECM deposition alter cell microenvironment, triggering the onset or the rapid progression of several diseases, including cancer. Hyaluronan (HA) is a ubiquitous component of ECM considered as one of the main players of cancer initiation and progression. This review discusses how HA participate in and regulate several aspects of tumorigenesis, with particular attention to the hallmarks of cancer proposed by Hanahan and Weinberg such as sustaining of the proliferative signaling, evasion of apoptosis, angiogenesis, activation of invasion and metastases, reprogramming of energy metabolism and evasion of immune response.

Geographical Variability Affects CCHFV Detection by RT-PCR: A Tool for In-Silico Evaluation of Molecular Assays.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is considered to be a major emerging infectious threat, according to the WHO R&D blueprint. A wide range of CCHFV molecular assays have been developed, employing varied primer/probe combinations. The high genetic variability of CCHFV often hampers the efficacy of available molecular tests and can affect their diagnostic potential. Recently, increasing numbers of complete CCHFV genomic sequences have become available, allowing a better appreciation of the genomic evolution of this virus. We summarized the current knowledge on molecular methods and developed a new bioinformatics tool to evaluate the existing assays for CCHFV detection, with a special focus on strains circulating in different geographical areas. Twenty-two molecular methods and 181 sequences of CCHFV were collected, respectively, from PubMed and GenBank databases. Up to 28 mismatches between primers and probes of each assay and CCHFV strains were detected through in-silico PCR analysis. Combinations of up to three molecular methods markedly decreased the number of mismatches within most geographic areas. These results supported the good practice of CCHFV detection of performing more than one assay, aimed for different sequence targets. The choice of the most appropriate tests must take into account patient's travel history and geographic distribution of the different CCHFV strains.