How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3- to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth.

Bacteriophage T7 gene 17.5 coding for the only known holin is one of the components of its lysis system, but the holin activity in T7 is more complex than a single gene, and evidence points to the existence of additional T7 genes with holin activity. In this study, a T7 phage with a gene 17.5 deletion (T7-△holin) was rescued and its biological characteristics and effect on cell lysis were determined. Furthermore, the genomic evolution of mutant phage T7-△holin during serial passage was assessed by whole-genome sequencing analysis. It was observed that deletion of gene 17.5 from phage T7 delays lysis time and enlarges the phage burst size; however, this biological characteristic recovered to normal lysis levels during serial passage. Scanning electron microscopy showed that the two opposite ends of E. coli BL21 cells swell post-T7-△holin infection rather than drilling holes on cell membrane when compared with T7 wild-type infection. No visible progeny phage particle accumulation was observed inside the E. coli BL21 cells by transmission electron microscopy. Following serial passage of T7-△holin from the 1st to 20th generations, the mRNA levels of gene 3.5 and gene 19.5 were upregulated and several mutation sites were discovered, especially two missense mutations in gene 19.5, which indicate a potential contribution to the evolution of the T7-△holin. Although the burst size of T7-△holin increased, high titer cultivation of T7-△holin was not achieved by optimizing the culture process. Accordingly, these results suggest that gene 19.5 is a potential lysis-related component that needs to be studied further and that the T7-△holin strain with its gene 17.5 deletion is not adequate to establish the high-titer phage cultivation process.

Influenza B viruses cause seasonal epidemics and are a considerable burden to public health. To understand their adaptation capability, we examined the genetic changes that occurred following 15 serial passages of two influenza B viruses, B/Brisbane/60/2008 and B/Victoria/504/2000, in human epithelial cells. Thirteen distinct amino acid mutations were found in the PB1, PA, hemagglutinin (HA), neuraminidase (NA), and M proteins after serial passage in the human lung epithelial cell line, Calu-3, and normal human bronchial epithelial (NHBE) cells. These changes were associated with significantly decreased viral replication levels. Our results demonstrate that adaptation of influenza B viruses for growth in human airway epithelial cells is partially conferred by selection of HA1, NA, and polymerase mutations that regulate receptor specificity, functional compatibility with the HA protein, and polymerase activity, respectively.

Cefiderocol is a siderophore cephalosporin active against MDR Gram-negatives including Stenotrophomonas maltophilia. Cefiderocol resistance remains uncommon and incompletely understood. We selected for cefiderocol-resistant S. maltophilia in vitro and characterized the genetic mechanisms and potential for cross-resistance to other antimicrobials.

Porcine enteric alphacoronavirus (PEAV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhea in newborn piglets. In this study, an original, highly virulent PEAV strain GDS04 was serially passaged in Vero cells. The virus titers and sizes of syncytia increased gradually with the cell passages. Newborn piglets were orally inoculated with PEAV P15, P67 and P100. Compared with P15 and P67, P100 resulted in only mild clinical signs and intestinal lesions in piglets. The virus shedding in feces and viral antigens in intestinal tract were markedly reduced in P100-inoculated piglets. Importantly, all P100-inoculated newborn piglets survived, indicating that P100 was an attenuated variant. Sequence analysis revealed that the virulent strain GDS04 had four, one, six and eleven amino acid differences in membrane, nucleocapsid, spike and ORF1ab proteins, respectively, from P100. Furthermore, more differences in the predicted three-dimensional structure of S protein between GDS04 and P100 were observed, indicating that these differences might be associated with the pathogenicity of PEAV. Collectively, our research successfully prepared a PEAV attenuated variant which might serve as a live attenuated vaccine candidate against PEAV infection.

Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

Highly virulent porcine epidemic diarrhea virus (PEDV) strains re-emerged and circulated in China at the end of 2010, causing significant economic losses in the pork industry worldwide. To understand the genetic dynamics of PEDV during its passage in vitro, the PEDV G2 strain FJzz1 was serially propagated in Vero cells for up to 200 passages. The susceptibility and adaptability of the FJzz1 strain increased gradually as it was serially passaged in vitro. Sequence analysis revealed that amino acid (aa) changes were mainly concentrated in the S glycoprotein, which accounted for 72.22%-85.71% of all aa changes. A continuous aa deletion (55I56G57E → 55K56Δ57Δ) occurred in the N-terminal domain of S1 (S1-NTD). To examine how the aa changes affected its virulence, FJzz1-F20 and FJzz1-F200 were selected to simultaneously evaluate their pathogenicity in suckling piglets. All the piglets in the FJzz1-F20-infected group showed typical diarrhea at 24 h postinfection, and the piglets died successively by 48 h postinfection. However, the clinical signs of the piglets in the FJzz1-F200-infected group were significantly weaker, and no deaths occurred. The FJzz1-F200-infected group also showed a lower level of fecal viral shedding and lower viral loads in the intestinal tissues, and no obvious histopathological lesions. Type I and III interferon were induced in the FJzz1-F200 infection group, together with pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-8. These results indicate that the identified genetic changes may contribute to the attenuation of FJzz1 strain, and the attenuated FJzz1-F200 may have the potential for developing PEDV live-attenuated vaccines.

Passage in mice of opportunistic pathogens such as Cryptococcus neoformans is known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype A C. neoformans identified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of a FRE3-encoded iron reductase in the two strains but not in a control strain that did not demonstrate increased virulence during mouse passage. FRE3 was shown to express an iron reductase activity and to play a role in iron-dependent growth of C. neoformans. Overexpression of FRE3 in the two original environmental strains increased growth in the macrophage cell line J774.16 and increased virulence. These data demonstrate a role for FRE3 in the virulence of C. neoformans and demonstrate how the increased expression of such a "virulence acquisition gene" during the environment-to-mammal transition, can optimize the virulence of environmental strains in mammalian hosts. IMPORTANCE Cryptococcus neoformans is a significant global fungal pathogen that also resides in the environment. Recent studies have suggested that the organism may undergo microevolution in the host. However, little is known about the permitted genetic changes facilitating the adaptation of environmental strains to mammalian hosts. The present studies subjected environmental strains isolated from several metropolitan areas of the United States to serial passages in mice. Transcriptome sequencing expression studies identified the increased expression of an iron reductase gene, FRE3, in two strains that adapted in mice to become highly virulent, and overexpression of FRE3 recapitulated the increased virulence after mouse passage. Iron reductase in yeast is important to iron uptake in a large number of microbial pathogens. These studies demonstrate the capacity of C. neoformans to show reproducible changes in the expression levels of small numbers of genes termed "virulence adaptation genes" to effectively increase pathogenicity during the environment-to-mammal transition.

Experimental evolution of bacterial populations in the laboratory has led to identification of several themes, including parallel evolution of populations adapting to carbon starvation, heat stress, and pH stress. However, most of these experiments study growth in defined and/or constant environments. We hypothesized that while there would likely continue to be parallelism in more complex and changing environments, there would also be more variation in what types of mutations would benefit the cells. In order to test our hypothesis, we serially passaged Escherichia coli in a complex medium (Luria-Bertani broth) throughout the five phases of bacterial growth. This passaging scheme allowed cells to experience a wide variety of stresses, including nutrient limitation, oxidative stress, and pH variation, and therefore allowed them to adapt to several conditions. After every ~30 generations of growth, for a total of ~300 generations, we compared both the growth phenotypes and genotypes of aged populations to the parent population. After as few as 30 generations, populations exhibit changes in growth phenotype and accumulate potentially adaptive mutations. There were many genes with mutant alleles in different populations, indicating potential parallel evolution. We examined 8 of these alleles by constructing the point mutations in the parental genetic background and competed those cells with the parent population; five of these alleles were found to be adaptive. The variety and swiftness of adaptive mutations arising in the populations indicate that the cells are adapting to a complex set of stresses, while the parallel nature of several of the mutations indicates that this behavior may be generalized to bacterial evolution. IMPORTANCE With a growing body of work directed toward understanding the mechanisms of evolution using experimental systems, it is crucial to decipher what effects the experimental setup has on the outcome. If the goal of experimental laboratory evolution is to elucidate underlying evolutionary mechanisms and trends, these must be demonstrated in a variety of systems and environments. Here, we perform experimental evolution in a complex medium allowing the cells to transition through all five phases of growth, including death phase and long-term stationary phase. We show that the swiftness of selection and the specific targets of adaptive evolution are different in this system compared to others. We also observe parallel evolution where different mutations in the same genes are under positive natural selection. Together, these data show that while some outcomes of microbial evolution experiments may be generalizable, many outcomes will be environment or system specific.

Wolbachia is an intracellular endosymbiont of insects that inhibits the replication of a range of pathogens in its arthropod hosts. The release of Wolbachia into wild populations of mosquitoes is an innovative biocontrol effort to suppress the transmission of arthropod-borne viruses (arboviruses) to humans, most notably dengue virus. The success of the Wolbachia-based approach hinges upon the stable persistence of the 'pathogen blocking' effect, whose mechanistic basis is poorly understood. Evidence suggests that Wolbachia may affect viral replication via a combination of competition for host resources and activation of host immunity. The evolution of resistance against Wolbachia and pathogen blocking in the mosquito or the virus could reduce the public health impact of the symbiont releases. Here, we investigate if dengue 3 virus (DENV-3) is capable of accumulating adaptive mutations that improve its replicative capacity during serial passage in Wolbachia wMel-infected cells. During the passaging regime, viral isolates in Wolbachia-infected cells exhibited greater variation in viral loads compared to controls. The viral loads of these isolates declined rapidly during passaging due to the blocking effects of Wolbachia carriage, with several being lost all together and the remainder recovering to low but stable levels. We attempted to sequence the genomes of the surviving passaged isolates but, given their low abundance, were unable to obtain sufficient depth of coverage for evolutionary analysis. In contrast, viral loads in Wolbachia-free control cells were consistently high during passaging. The surviving isolates passaged in the presence of Wolbachia exhibited a reduced ability to replicate even in Wolbachia-free cells. These experiments demonstrate the challenge for dengue in evolving resistance to Wolbachia-mediated blocking.

Analyses of viral inter- and intra-host mutations could better guide the prevention and control of infectious diseases. For a long time, studies on viral evolution have focused on viral inter-host variations. Next-generation sequencing has accelerated the investigations of viral intra-host diversity. However, the theoretical basis and dynamic characteristics of viral intra-host mutations remain unknown. Here, using serial passages of the SA14-14-2 vaccine strain of Japanese encephalitis virus (JEV) as the in vitro model, the distribution characteristics of 1,788 detected intra-host single-nucleotide variations (iSNVs) and their mutated frequencies from 477 deep-sequenced samples were analyzed. Our results revealed that in adaptive (baby hamster kidney (BHK)) cells, JEV is under a nearly neutral selection pressure, and both non-synonymous and synonymous mutations represent an S-shaped growth trend over time. A higher positive selection pressure was observed in the nonadaptive (C6/36) cells, and logarithmic growth in non-synonymous iSNVs and linear growth in synonymous iSNVs were observed over time. Moreover, the mutation rates of the NS4B protein and the untranslated region (UTR) of the JEV are significantly different between BHK and C6/36 cells, suggesting that viral selection pressure is regulated by different cellular environments. In addition, no significant difference was detected in the distribution of mutated frequencies of iSNVs between BHK and C6/36 cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) is the etiologic agent of a highly lethal pneumonia. Here, we report the full-genome sequence of the Jordan-N3/2012 strain after serial passage in two distinct mammalian cell lines. The genome exhibits noteworthy stability, which may inform the development of vaccines and therapeutics used to treat infection with this virus.

Although porcine epidemic diarrhea (PED) has caused huge economic losses in the pork industry worldwide, an effective live, attenuated vaccine is lacking. In this study, an original US, highly virulent PED virus (PEDV) strain PC22A was serially passaged in Vero CCL81 and Vero BI cells. The virus growth kinetics in cell culture, virulence in neonatal pigs and the whole genomic sequences of selected passages were examined. Increased virus titers and sizes of syncytia were observed at the 65th passage level (P65) and P120, respectively. Based on the severity of clinical signs, histopathological lesions and the distribution of PEDV antigens in the gut, the virulence of P100 and above, but not P95C13 (CCL81), was markedly reduced in 4-day-old, caesarian-derived, colostrum-deprived piglets. Subsequently, the attenuation of P120 and P160 was confirmed in 4-day-old, conventional suckling piglets. Compared with P120, P160 replicated less efficiently in the intestine of pigs and induced a lower rate of protection after challenge. Sequence analysis revealed that the virulent viruses [P3 and P95C13 (CCL81)] had one, one, sixteen (including an early termination of nine amino acids) and two amino acid differences in non-structure protein 1 (nsp1), nsp4, spike and membrane proteins, respectively, from the fully attenuated P160. However, the overall pattern of attenuation-related genetic changes in PC22A differed from those of the other four pairs of PEDV wild type strains and their attenuated derivatives. These results suggest that PEDV attenuation can occur through multiple molecular mechanisms. The knowledge provides insights into potential molecular mechanisms of PEDV attenuation.

Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti.

Duck Tembusu virus (DTMUV) is a new pathogen that produces an acute and potent disease in ducks which has caused serious economic losses in China. In this study, a virulent strain of DTMUV, designated as ZJSBL01, was attenuated by serial passages in BHK-21 cells supplied with 5-Fluorouracil (5-FU) for 50 passages to induce mutation and attenuation. Growth kinetics of different passages of ZJSBL01 strain in BHK-21 cells show that these viruses have similar replication characteristics. The virus was highly attenuated after 40 passages in BHK-21 cells supplied with 5-FU, based on mortality, morbidity, and viral load in inoculated Sheldrake ducklings. In addition, all of the ducklings immunized with ZJSBL01-P40, the virus obtained at passage 40 of ZJSBL01, showed seroconversion on day 14 post inoculation. Moreover, P40 did not cause clinical symptom for layding ducks. Immunization with ZJSBL01-P40 could provide effective protection against the virulent parental ZJSBL01 strain. Seventeen amino acid substitutions were observed in the polyprotein of ZJSBL01-P40 compared with parental ZJSBL01. These results indicate that ZJSBL01-P40 may be a live vaccine candidate for prevention of DTMUV-disease.

Mesenchymal stem cells (MSCs) are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n = 3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule (HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105) and pluripotent marker (Oct3/4) decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.

Viridans group streptococci (VGS), especially the Streptococcus mitis-oralis subgroup, are pivotal pathogens in a variety of invasive endovascular infections, including "toxic shock" in neutropenic cancer patients and infective endocarditis (IE). Previously, we showed that the serial in vitro passage of S. mitis-oralis strains in sublethal daptomycin (DAP) resulted in rapid, high-level and stable DAP-resistance (DAP-R), which is accompanied by distinct changes in several genotypic and phenotypic signatures: (1) the disappearance of two key membrane phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL); (2) increased membrane fluidity; (3) increased positive surface charge; (4) single nucleotide polymorphisms (SNPs) in two loci involved in CL biosynthesis (pgsA; cdsA); and (5) DAP hyperaccumulation. The current study examined these same metrics following in vitro serial DAP passages of a separate well-characterized S. mitis-oralis bloodstream isolate (SF100). Although some metrics seen in prior DAP post-passage strains were recapitulated with SF100 (e.g., pgsA SNPs, enhanced membrane fluidity), we observed the following major differences (comparing the parental versus post-passage variant): (1) no change in PG content; (2) reduced, but not absent, CL, with enhancement in phosphatidic acid (PA) content; (3) an unusual pattern of CL localization; (4) significantly decreased positive surface charge; (5) no difference in DAP accumulation; and (6) no cdsA SNPs. Thus, S. mitis-oralis strains are not "pre-programmed" phenotypically and/or genotypically to adapt in an identical manner during the evolution of the DAP-R.

An infectious cDNA clone of Triticum mosaic virus (TriMV) (genus Poacevirus; family Potyviridae) was used to establish three independent lineages in wheat to examine intra-host population diversity levels within protein 1 (P1) and coat protein (CP) cistrons over time. Genetic variation was assessed at passages 9, 18 and 24 by single-strand conformation polymorphism, followed by nucleotide sequencing. The founding P1 region genotype was retained at high frequencies in most lineage/passage populations, while the founding CP genotype disappeared after passage 18 in two lineages. We found that rare TriMV genotypes were present only transiently and lineages followed independent evolutionary trajectories, suggesting that genetic drift dominates TriMV evolution. These results further suggest that experimental populations of TriMV exhibit lower mutant frequencies than that of Wheat streak mosaic virus (genus Tritimovirus; family Potyviridae) in wheat. Nevertheless, there was evidence for parallel evolution at a synonymous site in the TriMV CP cistron.

Highly pathogenic (HPAI) strains emerge from their low pathogenic (LPAI) precursors and cause severe disease in poultry with enormous economic losses, and zoonotic potential. Understanding the mechanisms involved in HPAI emergence is thus an important goal for risk assessments. In this study ostrich-origin H5N2 and H7N1 LPAI progenitor viruses were serially passaged seventeen times in 14-day old embryonated chicken eggs and Ion Torrent ultra-deep sequencing was used to monitor the incremental changes in the consensus genome sequences. Both virus strains increased in virulence with successive passages, but the H7N1 virus attained a virulent phenotype sooner. Mutations V63M, E228V and D272G in the HA protein, Q357K in the nucleoprotein (NP) and H155P in the neuraminidase protein correlated with the increased pathogenicity of the H5N2 virus; whereas R584H and L589I substitutions in the polymerase B2 protein, A146T and Q220E in HA plus D231N in the matrix 1 protein correlated with increased pathogenicity of the H7N1 virus in embryos. Enzymatic cleavage of HA protein is the critical virulence determinant, and HA cleavage site motifs containing multibasic amino acids were detected at the sub-consensus level. The motifs PQERRR/GLF and PQRERR/GLF were first detected in passages 11 and 15 respectively of the H5N2 virus, and in the H7N1 virus the motifs PELPKGKK/GLF and PELPKRR/GLF were detected as early as passage 7. Most significantly, a 13 nucleotide insert of unknown origin was identified at passage 6 of the H5N2 virus, and at passage 17 a 42 nucleotide insert derived from the influenza NP gene was identified. This is the first report of non-homologous recombination at the HA cleavage site in an H5 subtype virus. This study provides insights into how HPAI viruses emerge from low pathogenic precursors and demonstrated the pathogenic potential of H5N2 and H7N1 strains that have not yet been implicated in HPAI outbreaks.

RNA viruses are infamous for their high rates of mutation, which produce swarms of genetic variants within individual hosts. To date, analyses of intrahost genetic diversity have focused on the primary genome sequence. However, virus phenotypes are shaped not only by primary sequence but also by the secondary structures into which this sequence folds. Such structures enable viral replication, translation, and binding of small RNAs, yet within-host variation at the structural level has not been adequately explored. We characterized the structural diversity of the 5' untranslated region (UTR) of populations of West Nile virus (WNV) that had been subject to five serial passages in triplicate in each of three bird species. Viral genomes were sampled from host serum samples at each passage (n = 45 populations) and subjected to next-generation sequencing. For populations derived from passages 1, 3, and 5 (n = 9 populations), we predicted the impact of each mutation occurring at a frequency of ≥1% on the secondary structure of the 5' UTR. As expected, mutations in double-stranded (DS) regions of the 5' UTR stem structures caused structural changes of significantly greater magnitude than did mutations in single-stranded (SS) regions. Despite the greater impact of mutations in DS regions, mutations in DS and SS regions occurred at similar frequencies, with no evidence of enhanced selection against mutation in DS regions. In contrast, mutations in two regions that mediate genome cyclization and thereby regulate replication and translation, the 5' cyclization sequence and the UAR flanking stem (UFS), were suppressed in all three hosts.IMPORTANCE The enzymes that copy RNA genomes lack proofreading, and viruses that possess RNA genomes, such as West Nile virus, rapidly diversify into swarms of mutant lineages within a host. Intrahost variation of the primary genomic sequence of RNA viruses has been studied extensively because the extent of this variation shapes key virus phenotypes. However, RNA genomes also form complex secondary structures based on within-genome nucleotide complementarity, which are critical regulators of the cyclization of the virus genome that is necessary for efficient replication and translation. We sought to characterize variation in these secondary structures within populations of West Nile virus during serial passage in three bird species. Our study indicates that the intrahost population of West Nile virus is a diverse assortment of RNA secondary structures that should be considered in future analyses of intrahost viral diversity, but some regions that are critical for genome cyclization are conserved within hosts. Besides potential impacts on viral replication, structural diversity can influence the efficacy of small RNA antiviral therapies.