Gain without pain: adaptation and increased virulence of Zika virus in vertebrate host without fitness cost in mosquito vector.

IMPORTANCE: Previously, we modeled direct transmission chains of Zika virus (ZIKV) by serially passaging ZIKV in mice and mosquitoes and found that direct mouse transmission chains selected for viruses with increased virulence in mice and the acquisition of non-synonymous amino acid substitutions. Here, we show that these same mouse-passaged viruses also maintain fitness and transmission capacity in mosquitoes. We used infectious clone-derived viruses to demonstrate that the substitution in nonstructural protein 4A contributes to increased virulence in mice.

Assessment of Genetic Stability During Serial In Vitro Passage and In Vivo Carriage.

In this study, our objective was to evaluate the genetic stability of foodborne bacterial pathogens during serial passage in vitro and persistent in vivo carriage. Six strains of Listeria, Campylobacter, Escherichia, Salmonella, and Vibrio were serially passaged 20 times. Three colonies were picked for whole-genome sequencing (WGS) from passes P0, P5, P10, P15, and P20. In addition, isolates of Salmonella and Escherichia from three patients with persistent infections were sequenced. Genetic stability was evaluated in terms of variations detected in high-quality single-nucleotide polymorphism (hqSNP), core genome multilocus sequence typing (cgMLST), seven-gene MLST, and determinants encoding serotype, antimicrobial resistance (AMR), and virulence. During serial passage, increasing diversity was observed in Listeria, Salmonella, and Vibrio as measured by hqSNPs (from median of 0 SNPs to median of 3-5 SNPs, depending on the organism) and to a lesser extent with cgMLST (from median of 0 alleles to median of 0-5 alleles), while Escherichia and Campylobacter genomes showed minimal variation. The serotype, AMR, and virulence markers remained stable in all organisms. Isolates from persistent infections lasting up to 10 weeks remained genetically stable. However, isolates from a persistent Salmonella enterica ser. Montevideo infection spanning 9 years showed early heterogeneity leading to the emergence of one predominant genotype that continued to evolve over the years, including gains and losses of AMR markers. While the hqSNP and cgMLST variation observed during the serial passage was minimal, culture passages should be limited to as few times as possible before WGS. Our WGS data show that in vivo carriage lasting for a few weeks did not appear to alter the genotype. Longer persistent infections spanning for years, particularly in the presence of selective pressure, may cause changes in the genotype making it challenging to differentiate persistent infections from reinfections.

Assessment of Microbial Community Structure and Function in Serially Passaged Wastewater Electro-Bioreactor Sludge: An Approach to Enhance Sludge Settleability.

Several studies have been carried out to understand bulking phenomena and the importance of environmental factors on sludge settling characteristics. The main objective of this study was to carry out functional characterization of microbial community structure of wastewater electro-bioreactor sludge as it undergoes serial passaging in the presence or absence of a current density over 15 days. Illumina MiSeq sequencing and QIIME were used to assess sludge microbial community shifts over time. (α) and (ß) diversity analysis were conducted to assess the microbial diversity in electro-bioreactors. A phylogeny-based weighted UniFrac distance analysis was used to compare between bacterial communities while BIO-ENV trend and Spearman's rank correlation analysis were performed to investigate how reactor operational parameters correlated with bacterial community diversity. Results showed that the removal efficiency of soluble chemical oxygen demand (sCOD) ranged from 91-97%, while phosphorous (PO43--P) removal was approximately 99%. Phylogenetic analysis revealed stark differences in the development of sludge microbial communities in the control and treatment reactor. There was no mention of any studies aimed at characterizing functional microbial communities under electric field and the results communicated here are the first, to our knowledge, that address this gap in the literature.

Mechanisms and fitness costs of tigecycline resistance in Escherichia coli.

OBJECTIVES: To stepwise select tigecycline-resistant Escherichia coli mutants in vitro, determine the mutation rates, identify the resistance mechanisms, determine the resistance level and cross-resistance to other antibiotic classes, evaluate the fitness costs of tigecycline resistance mechanisms and investigate if the same in vitro-identified target genes were mutated in clinical isolates. METHODS: Spontaneous mutants with reduced susceptibility to tigecycline were selected on agar plates supplemented with tigecycline. Resistance levels and cross-resistance were evaluated by performing MIC assays and determining mutation rates using Luria-Delbruck fluctuation tests. Mutant fitness was estimated by measuring exponential growth rates, lag phase and total yield. Illumina whole-genome sequencing was used to identify mutations increasing MICs of tigecycline. RESULTS: Spontaneous mutants with reduced susceptibility to tigecycline were selected at a rate of ~10(-8) to 10(-6) per cell per generation; however, the clinical MIC breakpoint was not reached. The resistance level of tigecycline was low and some of the mutants had elevated MICs of hydrophobic drugs (chloramphenicol, erythromycin and novobiocin) or decreased MICs of SOS response inducers (ciprofloxacin and nitrofurantoin). Mutations were identified in efflux regulatory network genes (lon, acrR and marR) or lipopolysaccharide core biosynthesis pathway genes (lpcA, rfaE, rfaD, rfaC and rfaF). Mutations in the same target genes were found in clinical isolates. CONCLUSIONS: Tigecycline selects for low-level resistance mutations with relatively high mutation rates and the majority of them come with a substantial fitness cost. Further in vivo experiments are needed to evaluate how these mutations affect bacterial virulence and ability to establish a successful infection.

Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4 virus.

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G(2337) C (Met(457) Ile) in the E gene and A(6751) G (Lys(125) Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this article could be useful for monitoring the molecular consistency and quality control of vaccine strains.

Fitness cost and impaired survival in penicillin-resistant Streptococcus gordonii isolates selected in the laboratory.

Cycling of Streptococcus gordonii (115 times) with penicillin resulted in a MIC increase of more than 100-fold, from 0.008 to 2 microg/ml. The 2-microg/ml MIC maximum was already reached after 36 passages but resulted in impaired fitness. Although the MIC did not increase further, fitness was partially recovered during the 79 additional cycles.

Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2.

BACKGROUND: A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Delta30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. METHODS: A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Delta30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. RESULTS: The rDEN2Delta30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Delta30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Delta30, designated rDEN2Delta30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Delta30 in SCID-HuH-7 mice. CONCLUSIONS: The rDEN2Delta30 and rDEN2Delta30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.

Molecular analysis of the capsid coding region of a virulent encephalomyocarditis virus isolate after serial cell passages and assessment of its virulence.

EMCV has been recognized as a worldwide-shared pathogen in many host species with natural differences of virulence among strains. Differences of virulence were also demonstrated experimentally after serial passages of particular EMCV strains in cell culture. In order to evaluate the genetic variability of the CCR of an highly virulent Belgian EMCV isolate after 210 passages in cell culture, this region has been molecularly characterised by RT-PCR and sequencing. The analyses enlightened the stability of the isolate as only 3 mutations appeared within the CCR and this was observed between passage 200 and 210. One of the mutations is adjacent to the amino acid described as the differentiation amino acid between diabetogenic and non-diabetogenic variants of EMCV. An experimental inoculation of pigs with the passage 210 of the virus enlightened its attenuation and the challenged pigs were protected against the wild-type virus. Results confirm the ability of EMCV to become attenuated after passages in cell culture.

Effects of environment on compensatory mutations to ameliorate costs of antibiotic resistance.

Most types of antibiotic resistance impose a biological cost on bacterial fitness. These costs can be compensated, usually without loss of resistance, by second-site mutations during the evolution of the resistant bacteria in an experimental host or in a laboratory medium. Different fitness-compensating mutations were selected depending on whether the bacteria evolved through serial passage in mice or in a laboratory medium. This difference in mutation spectra was caused by either a growth condition-specific formation or selection of the compensated mutants. These results suggest that bacterial evolution to reduce the costs of antibiotic resistance can take different trajectories within and outside a host.

Detection of mutants in polio vaccine viruses using pooled antipoliovirus monoclonal antibodies.

We prepared six monoclonal antibodies (mAbs) for type 1 polioviruses, and analysed their neutralizing specificities for use in safety tests in oral poliomyelitis vaccine (OPV) production. Pools of two or more individual mAbs showed high neutralizing activity against high-titre (approximately 10(7) CCID (50)/25 microl) of Sabin type 1 virus. It was demonstrated that the pooled mAbs can be utilized effectively in detection tests of adventitious viruses, which are among the safety tests in OPV production. Moreover, some pooled mAbs were shown to be capable of detecting very small amounts of type 1 virulent viruses and mutants in high-titre Sabin type 1 virus suspensions. Neutralizing antibody titres of these pooled mAbs decreased with increasing numbers of mutants containing neurovirulent activity in high-titre Sabin type 1 viruses which were repeatedly passaged in culture. It is expected that these pooled mAbs will contribute greatly to safety tests for OPV production.