Modularity and evolutionary constraints in a baculovirus gene regulatory network

Abstract\ud \ud \ud \ud Background\ud The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression.\ud \ud \ud \ud Results\ud We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization.\ud \ud \ud \ud Conclusions\ud Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates that modularity may be a general feature of biological gene regulatory networks.JVCO, CTB and AI hold FAPESP scholarships (04/12456-0, 09/16740-8 and 12/04818-5), AFB and CCMF hold CAPES-MSc and PhD scholarships and PMAZ holds a CNPq-PQ scholarship. This work was supported financially by FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo, process: 2007/55282-0)

High Prevalence of Multidrug-Resistant Klebsiella pneumoniae Harboring Several Virulence and β-Lactamase Encoding Genes in a Brazilian Intensive Care Unit

Klebsiella pneumoniae is an important opportunistic pathogen that commonly causes nosocomial infections and contributes to substantial morbidity and mortality. We sought to investigate the antibiotic resistance profile, pathogenic potential and the clonal relationships between K. pneumoniae (n = 25) isolated from patients and sources at a tertiary care hospital’s intensive care units (ICUs) in the northern region of Brazil. Most of K. pneumoniae isolates (n = 21, 84%) were classified as multidrug resistant (MDR) with high-level resistance to β-lactams, aminoglycosides, quinolones, tigecycline, and colistin. All the 25 isolates presented extended-spectrum beta-lactamase-producing (ESBL), including carbapenemase producers, and carried the blaKPC (100%), blaTEM (100%), blaSHV variants (n = 24, 96%), blaOXA-1 group (n = 21, 84%) and blaCTX-M-1 group (n = 18, 72%) genes. The K2 serotype was found in 4% (n = 1) of the isolates, and the K1 was not detected. The virulence-associated genes found among the 25 isolates were mrkD (n = 24, 96%), fimH-1 (n = 22, 88%), entB (100%), iutA (n = 10, 40%), ybtS (n = 15, 60%). The genes related with efflux pumps and outer membrane porins found were AcrAB (100%), tolC (n = 24, 96%), mdtK (n = 22, 88%), OmpK35 (n = 15, 60%), and OmpK36 (n = 7, 28%). ERIC-PCR was employed to determine the clonal relationship between the different isolated strains. The obtained ERIC-PCR patterns revealed that the similarity between isolates was above 70%. To determine the sequence types (STs) a multilocus sequence typing (MLST) assay was used. The results indicated the presence of high-risk international clones among the isolates. In our study, the wide variety of MDR K. pneumoniae harboring β-lactams and virulence genes strongly suggest a necessity for the implementation of effective strategies to prevent and control the spread of antibiotic resistant infections

Whole genome sequencing of the multidrug-resistant Chryseobacterium indologenes isolated from a patient in Brazil

Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including β-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to β-lactamases (blaIND–13, blaCIA–3, blaTEM–116, blaOXA–209, blaVEB–15), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings

Characterization of evolutionary process of RNA viruses from patterns in viral phylogenies

No presente trabalho, investigamos a filodinâmica de três modelos virais diferentes, utilizando técnicas baseadas em verossimilhança e inferência bayesiana. Dois desses são flavivírus com genoma de RNA fita simples e senso positivo. O terceiro é um bunyavírus com genoma tri-segmentado de RNA fita simples com senso negativo. Estes diferentes modelos permitiram estudar diferentes mecanismos promotores de diversidade viral, reagrupamento de segmentos genômicos (shift) e mutação (drift), que atuam em diferentes granularidades. Descrevemos pela primeira vez o espalhamento geográfico das linhagens de vírus Zika (ZIKV) em um nível continental, assim como ocorrência de recombinação e associação entre padrões de glicosilação e vetores. Para o flavivírus da encefalite transmitida por carrapatos (TBEV), investigamos seu espalhamento e encontramos evidências que corroboram a hipótese de circulação viral restrita a focos na Europa central. As análises sobre o vírus da Febre da Grande Fenda Africana (RVFV) apontaram a ocorrência de reagrupamento de segmentos genômicos e também ajudaram a elucidar sua dispersão do leste do continente africano para o oeste, encontrando-se diversas introduções no Senegal e Mauritânia. Aparentemente, este vírus teve a entrada facilitada nesses países por uma região que funciona como um centro de dispersão (hub) por ser encontro de rotas migratórias de animais. Ademais, investigamos a ocorrência de rearranjos de segmentos genômicos de RVFV e também estudamos as diferenças nas dinâmicas evolutivas de cada segmento.In this study, we investigated the phylodynamics of three different viral models, using techniques based on maximum likelihood and Bayesian inference methods. Two of these viruses are flaviviruses, whose genomes are formed by a single-stranded positive-sense RNA molecule. The third is a Bunyavirus with tri-segmented single-stranded RNA genome with negative sense. These different models allowed us to investigate two different mechanisms to promote viral diversity, (i) recombination of genomic segments (\"shift\") and (ii) mutation (\"drift\"), therefore exploring different levels of granularity of evolutionary process. We described for the first time the geographic spread of Zika virus (ZIKV) strains in a continental level, as well as, the occurrence of recombination and association between glycosylation patterns and vectors. For the other Flavivirus, tick-borne encephalitis virus (TBEV), we investigated its spreading and found evidences to support the hypothesis that viral circulation is very constrained by the foci in central Europe. The analyses about the Rift Valley Fever Virus (RVFV) revealed the occurrence of reassortment of genomic segments and their dispersal from eastern Africa to the west, with several introductions to Senegal and Mauritania. Apparently, the entry of RVFV in these countries was facilitated by the region of Kedougou, where several migratory routes of animals converge. This place maybe works as a hub to spread RVFV for West Africa. Moreover, we also investigated the differences in evolutionary dynamics of each genomic segment of RVFV

Does adaptation to vertebrate codon usage relate to flavivirus emergence potential?

<div><p>Codon adaptation index (CAI) is a measure of synonymous codon usage biases given a usage reference. Through mutation, selection, and drift, viruses can optimize their replication efficiency and produce more offspring, which could increase the chance of secondary transmission. To evaluate how higher CAI towards the host has been associated with higher viral titers, we explored temporal trends of several historic and extensively sequenced zoonotic flaviviruses and relationships within the genus itself. To showcase evolutionary and epidemiological relationships associated with silent, adaptive synonymous changes of viruses, we used codon usage tables from human housekeeping and antiviral immune genes, as well as tables from arthropod vectors and vertebrate species involved in the flavivirus maintenance cycle. We argue that temporal trends of CAI changes could lead to a better understanding of zoonotic emergences, evolutionary dynamics, and host adaptation. CAI appears to help illustrate historically relevant trends of well-characterized viruses, in different viral species and genetic diversity within a single species. CAI can be a useful tool together with <i>in vivo</i> and <i>in vitro</i> kinetics, phylodynamics, and additional functional genomics studies to better understand species trafficking and viral emergence in a new host.</p></div

CAI and phylodynamics of West Nile virus lineage 2 sequences.

<p>A) Bayesian maximum clade credibility tree representing a time scaled phylogeny of a WNV lineage 2 polyprotein sequences. Bayesian posterior probabilities > 0.9 are marked with an asterisk at major nodes. Averages for the European epidemic lineage (yellow bar) and a 2^nd European lineage (highlighted in red) are shown. B) Malthusian fitness (<i>Wm</i>) was calculated from 2004–2014, and compared to LOESS trend lines generated from CAI values to the codon usages of C) human house-keeping genes, D) human immune/antiviral genes, E) pigeon (Columba livia) genes, and F) mosquito (<i>Culex pipiens</i>) genes. G) The Spearman’s rank correlation test was used to test if there were any correlations between the 2010 to 2012 CAI increase in mosquitoes and the CAI increase in vertebrate species from 2012–2014, as well as Wm. The ΔCAI, Δ<i>Wm</i> and <i>rho</i> are shown for clarity. # = for all correlations, <i>p</i>-values were < 0.05.</p

Does adaptation to vertebrate codon usage relate to flavivirus emergence potential? - Fig 3

<p><b>CAI changes across time for (A) Yellow fever virus, (B) endemic strains of Dengue 2 virus, and (C) sylvatic strains of Dengue 2 virus.</b> For each codon usage table, the CAI was normalized by length, GC% and amino acid content for each dataset. Area of plot points reflects the density of sequences at a specific coordinate. A trend line was generated using LOESS, a non-parametric regression method, with 0.95 confidence interval shading. For B), CAI data to monkeys was removed for clarity, but was positioned in between the human table trend lines.</p

The CAI of Tobacco Mosaic virus to house-keeping genes.

<p>A violin dotplot of the CAI of complete Tobacco Mosaic virus (TMV) coding sequences to the codon usages of human house-keeping genes, antiviral/immune genes, and tobacco (<i>Nicotiana tabacum</i>) house-keeping genes. Average CAI values for each group are shown.</p