Factors associated with viral rebound among COVID-19 patients receiving oral antivirals.

BACKGROUND: COVID-19 rebound is usually reported among patients experiencing concurrent symptomatic and viral rebound. But longitudinal viral RT-PCR results from early stage to rebound of COVID-19 was less characterized. Further, identifying the factors associated with viral rebound after nirmatrelvir-ritonavir (NMV/r) and molnupiravir may expand understanding of COVID-19 rebound. METHODS: We retrospectively analyzed clinical data and sequential viral RT-PCR results from COVID-19 patients receiving oral antivirals between April and May, 2022. Viral rebound was defined by the degree of viral load increase (ΔCt ≥ 5 units). RESULTS: A total of 58 and 27 COVID-19 patients taking NMV/r and molnupiravir, respectively, were enrolled. Patients receiving NMV/r were younger, had fewer risk factors for disease progression and faster viral clearance rate compared to those receiving molnupiravr (All P < 0.05). The overall proportion of viral rebound (n = 11) was 12.9%, which was more common among patients receiving NMV/r (10 [17.2%] vs. 1 [3.7%], P = 0.16). Of them, 5 patients experienced symptomatic rebound, suggesting the proportion of COVID-19 rebound was 5.9%. The median interval to viral rebound was 5.0 (interquartile range, 2.0-8.0) days after completion of antivirals. Initial lymphopenia (<0.8 × 109/L) was associated with viral rebound among overall population (adjusted odds ratio [aOR], 5.34; 95% confidence interval [CI], 1.33-21.71), and remained significant (aOR, 4.50; 95% CI, 1.05-19.25) even when patients receiving NMV/r were considered. CONCLUSION: Our data suggest viral rebound after oral antivirals may be more commonly observed among lymphopenic individuals in the context of SARS-CoV-2 Omicron BA.2 variant.

Contrasting Patterns in the Early Stage of SARS-CoV-2 Evolution between Humans and Minks.

One of the unique features of SARS-CoV-2 is its apparent neutral evolution during the early pandemic (before February 2020). This contrasts with the preceding SARS-CoV epidemics, where viruses evolved adaptively. SARS-CoV-2 may exhibit a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes before the onset of the current pandemic. To test the scenarios above, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parental humans. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratio per site in the spike protein is 2.93, indicating a selection process favoring adaptive amino acid changes. Mutations in the spike protein were concentrated within its receptor-binding domain and receptor-binding motif. An excess of high-frequency derived variants produced by genetic hitchhiking was found during the middle (June to July 2020) and late phase I (August to September 2020) of the mink epidemic. In contrast, the site frequency spectra of early SARS-CoV-2 in humans only show an excess of low-frequency mutations, consistent with the recent outbreak of the virus. Strong positive selection in the mink SARS-CoV-2 implies that the virus may not be preadapted to a wide range of hosts and illustrates how a virus evolves to establish a continuous infection in a new host. Therefore, the lack of positive selection signal during the early pandemic in humans deserves further investigation.

Evolution under Spatially Heterogeneous Selection in Solid Tumors.

Spatial genetic and phenotypic diversity within solid tumors has been well documented. Nevertheless, how this heterogeneity affects temporal dynamics of tumorigenesis has not been rigorously examined because solid tumors do not evolve as the standard population genetic model due to the spatial constraint. We therefore, propose a neutral spatial (NS) model whereby the mutation accumulation increases toward the periphery; the genealogical relationship is spatially determined and the selection efficacy is blunted (due to kin competition). In this model, neutral mutations are accrued and spatially distributed in manners different from those of advantageous mutations. Importantly, the distinctions could be blurred in the conventional model. To test the NS model, we performed a three-dimensional multiple microsampling of two hepatocellular carcinomas. Whole-genome sequencing (WGS) revealed a 2-fold increase in mutations going from the center to the periphery. The operation of natural selection can then be tested by examining the spatially determined clonal relationships and the clonal sizes. Due to limited migration, only the expansion of highly advantageous clones can sweep through a large part of the tumor to reveal the selective advantages. Hence, even multiregional sampling can only reveal a fraction of fitness differences in solid tumors. Our results suggest that the NS patterns are crucial for testing the influence of natural selection during tumorigenesis, especially for small solid tumors.

D614G Substitution of SARS-CoV-2 Spike Protein Increases Syncytium Formation and Virus Titer via Enhanced Furin-Mediated Spike Cleavage.

Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 (S-G614 and S-D614, respectively). The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. IMPORTANCE Analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time has identified the D614G substitution in spike (S) as the most prevalent expanding variant worldwide, which might confer a selective advantage in transmission. Several studies showed that the D614G variant replicates and transmits more efficiently than the wild-type virus, but the mechanism is unclear. By comparing 18 virus isolates containing S with either D614 or G614, we found significantly higher virus titers in association with higher furin protease-mediated cleavage of S, an event that promotes syncytium formation and virus infectivity, in the S-G614 viruses. The effect of the D614G substitution on furin-mediated S cleavage and the resulting enhancement of the syncytium phenotype has been validated in S-expressing cells. This study suggests a possible effect of the D614G substitution on S of SARS-CoV-2; the antiviral effect through targeting furin protease is worthy of being investigated in proper animal models.

Gut microbiota in the innate immunity against hepatitis B virus - implication in age-dependent HBV clearance.

Hepatitis B virus (HBV) chronically infects 257 million people and is one of the most important liver diseases worldwide. A unique feature of HBV infection in humans is that viral clearance heavily depends on the age at exposure. Recent studies demonstrated that the virus takes advantage of immature innate immunity, especially hepatic macrophages, and not-yet-stabilized gut microbiota in early life to establish a chronic infection. The liver contains resident and infiltrating myeloid cells involved in immune responses to pathogens. They influence both innate and adaptive sectors of the immune system and their interplay with HBV has only been noticed recently. Here, we discuss how interactions between gut microbiota and hepatic macrophages influence the outcomes of HBV infection. Understanding the underlying mechanism would pave the way for the treatment of chronic HBV infection.

Whole genome deep sequencing analysis of viral quasispecies diversity and evolution in HBeAg seroconverters.

BACKGROUND & AIMS: We aimed to investigate how viral quasispecies of the HBV whole genome evolves and diversifies in response to HBeAg seroconversion and viral control utilising next-generation sequencing (NGS). METHODS: Fifty HBeAg-positive chronic hepatitis B patients, including 18 treatment-naïve and 32 interferon (IFN)-treated individuals, were recruited. Serial HBV whole genomes in serum were analysed by NGS to determine sequence characteristics and viral quasispecies. RESULTS: HBV quasispecies diversity, measured by nucleotide diversity, was negatively correlated with viral load and hepatitis activity. Spontaneous HBeAg seroconverters exhibited significantly greater viral quasispecies diversity than treatment-naïve non-seroconverters from >1 year before seroconversion (0.0112 vs. 0.0060, p <0.01) to >1 year after seroconversion (0.0103 vs. 0.0068, p <0.01). IFN-induced HBeAg seroconverters tended to have higher viral genetic diversity than non-seroconverters along with treatment. Particularly, the IFN responders, defined as IFN-induced HBeAg seroconversion with low viraemia, exhibited significantly greater genetic diversity of whole HBV genome at 6 months post-IFN treatment than IFN non-responders (0.0148 vs. 0.0106, p = 0.048). Moreover, spontaneous HBeAg seroconverters and IFN responders exhibited significantly higher evolutionary rates and more intra-host single-nucleotide variants. Interestingly, in spontaneous HBeAg seroconverters and IFN responders, there were distinct evolutionary patterns in the HBV genome. CONCLUSIONS: Higher HBV quasispecies diversity is associated with spontaneous HBeAg seroconversion and IFN-induced HBeAg seroconversion with low viraemia, conferring a favourable clinical outcome. LAY SUMMARY: HBeAg seroconversion is a landmark in the natural history of chronic HBV infection. Using next-generation sequencing, we found that the nucleotide diversity of HBV was negatively correlated with viral load and hepatitis activity. Patients undergoing HBeAg seroconversion had more diverse HBV genomes and a faster viral evolution rate. Our findings suggest HBeAg seroconversion is driven by host selection pressure, likely immune selection pressure.

The origin and past demography of murine astrovirus 1 in laboratory mice.

Astroviruses are non-enveloped, positive-sense, ssRNA viruses and often associated with gastrointestinal diseases. Murine astrovirus (MuAstV) was first confirmed in a laboratory mouse colony in 2011. Although infected mice do not present significant clinical symptoms, the virus might interfere with research results. A recent surveillance has shown that MuAstV is highly prevalent in laboratory mice. The aims of the present study were to identify and characterize MuAstV strains as well as to investigate the prevalence rate of viral RNA in laboratory mice in Taiwan, and to estimate the origin and past population demography of MuAstVs. Based on molecular surveillance, MuAstV RNA was detected in 45.7 % of laboratory mice (48/105) from seven of nine colonies. Three fully sequenced MuAstV strains, MuAstV TW1, TW2 and TW3, exhibited 89.1-94.4 % and 89.1-90.0 % nucleotide identities with the reference strains MuAstV STL1 and STL2, respectively. Phylogenetic analyses of the partial regions of the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP) genes of 18 Taiwan strains along with other astroviruses revealed that there are three distinct lineages of mouse astrovirus, MuAstV1, MuAstV2 and mouse astrovirus JF755422. The mutation rates of MuAstV1 were 2.6×10-4 and 6.2×10-4 substitutions/site/year for the RdRp and CP regions, respectively. Based on the above molecular clock, the colonization of MuAstV1 in laboratory mice was between 1897 and 1912, in good agreement with the establishment of 'modern' laboratory mouse facilities. Since its initial infection, the population size of MuAstV1 has increased 15-60-fold, probably consistent with the increased use of laboratory mice. In conclusion, MuAstV1 has been associated with modern laboratory mice since the beginning, and its influence on research results may require further investigation.

Implication of single year seasonal sampling to genetic diversity fluctuation that coordinates with oceanographic dynamics in torpedo scads near Taiwan.

Many fisheries management and conservation plans are based on the genetic structure of organisms in pelagic ecosystems; however, these structures tend to vary over time, particularly in cyclic ocean currents. We performed genetic analyses on the populations of the pelagic fish, Megalaspis cordyla (Osteichthyes: Carangidae) in the area surrounding Taiwan during 2000-2001. Genotyping was performed on M. cordyla collected seasonally around Taiwan as well as specimens collected from Singapore (Malacca strait) and Indonesia (Banda Sea). Gonadosomatic indices (GSI) revealed that M. cordyla does not spawn near Taiwan. Data related to the mitochondrial control region revealed that the samples from Singapore and Indonesia represented two distinct genetic cohorts. Genotyping revealed that during the summer (June-August 2000), the Indonesian variant was dominant in eastern Taiwan (presumably following the Kuroshio Current) and in the Penghu region (following the Kuroshio Branch Current). During the same period, the Singapore genotype was dominant along the western coast of Taiwan (presumably following the South China Sea Current); however, the number dropped during the winter (December-February 2001) under the effects of the China Coast Current. Divergence time estimates indicate that the two genetic cohorts split during the last glacial maximum. Despite the fact that these results are based on sampling from a single year, they demonstrate the importance of seasonal sampling in unravelling the genetic diversity in pelagic ecosystems.

The origin and underlying driving forces of the SARS-CoV-2 outbreak.

BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019-12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

A rapid insect species identification system using mini-barcode pyrosequencing.

BACKGROUND: Rapid and accurate species identification is not only important for biodiversity studies and pest quarantine and management, but in some cases may also influence the results of international trade negotiations. In this study, we developed a rapid species identification system for insects. RESULTS: A universal DNA mini-barcode primer pair was designed to target ∼ 120 bp of the mitochondrial 16S rDNA gene. This primer set can amplify the targeted region from all 300 species of 26 insect orders tested as well as other classes of Arthropoda. Although we found no within-species variation in this region, it provided enough information to separate closely related species or species complexes, in particular Thrips spp. and Bemisia spp. By combining a quick DNA extraction method with pyrosequencing, we were able to generate DNA sequences and complete species identification within 5 h. CONCLUSION: Mini-barcode pyrosequencing of 16S rDNA coupled with the GenBank database provides a rapid, accurate, and efficient species identification system. This system is therefore useful for biodiversity discovery, forensic identification, and quarantine control and management. © 2019 Society of Chemical Industry.

Effects of indoor residual spraying and outdoor larval control on Anopheles coluzzii from São Tomé and Príncipe, two islands with pre-eliminated malaria.

BACKGROUND: Vector control is a key component of malaria prevention. Two major vector control strategies have been implemented in São Tomé and Príncipe (STP), indoor residual spraying (IRS) and outdoor larval control using Bacillus thuringiensis israelensis (Bti). This study evaluated post-intervention effects of control strategies on vector population density, composition, and knockdown resistance mutation, and their implications for malaria epidemiology in STP. METHODS: Mosquitoes were collected by indoor and outdoor human landing catches and mosquito light traps in seven districts. Mosquito density was calculated by numbers of captured adult mosquitoes/house/working hour. Mitochondrial cytochrome c oxidase subunit I (COI) was PCR amplified and sequenced to understand the spatial-temporal population composition of malaria vector in STP. Knockdown resistance L1014F mutation was detected using allele-specific PCR. To estimate the malaria transmission risks in STP, a negative binomial regression model was constructed. The response variable was monthly incidence, and the explanatory variables were area, rainfall, entomological inoculation rate (EIR), and kdr mutation frequency. RESULTS: Malaria vector in STP is exophilic Anopheles coluzzii with significant population differentiation between Príncipe and São Tomé (mean FST = 0.16, p < 0.001). Both vector genetic diversity and knockdown resistance mutation were relatively low in Príncipe (mean of kdr frequency = 15.82%) compared to São Tomé (mean of kdr frequency = 44.77%). Annual malaria incidence rate in STP had been rapidly controlled from 37 to 2.1% by three rounds of country-wide IRS from 2004 to 2007. Long-term application of Bti since 2007 kept the mosquito density under 10 mosquitoes/house/hr/month, and malaria incidence rate under 5% after 2008, except for a rising that occurred in 2012 (incidence rate = 6.9%). Risk factors of area (São Tomé compared to Príncipe), rainfall, outdoor EIR, and kdr mutation frequency could significantly increase malaria incidence by 9.33-11.50, 1.25, 1.07, and 1.06 fold, respectively. CONCLUSIONS: Indoor residual spraying could rapidly decrease Anopheles density and malaria incidence in STP. Outdoor larval control using Bti is a sustainable approach for controlling local vector with exophilic feature and insecticide resistance problem. Vector control interventions should be intensified especially at the north-eastern part of São Tomé to minimize impacts of outbreaks.

Clonality of Fluconazole-Nonsusceptible Candida tropicalis in Bloodstream Infections, Taiwan, 2011-2017.

Candida tropicalis is the leading cause of non-C. albicans candidemia in tropical Asia and Latin America. We evaluated isolates from 344 patients with an initial episode of C. tropicalis candidemia. We found that 58 (16.9%) patients were infected by fluconazole-nonsusceptible (FNS) C. tropicalis with cross resistance to itraconazole, voriconazole, and posaconazole; 55.2% (32/58) of patients were azole-naive. Multilocus sequence typing analysis revealed FNS isolates were genetically closely related, but we did not see time- or place-clustering. Among the diploid sequence types (DSTs), we noted DST225, which has been reported from fruit in Taiwan and hospitals in Beijing, China, as well as DST376 and DST505-7, which also were reported from hospitals in Shanghai, China. Our findings suggest cross-boundary expansion of FNS C. tropicalis and highlight the importance of active surveillance of clinical isolates to detect dissemination of this pathogen and explore potential sources in the community.

Genetic Load and Potential Mutational Meltdown in Cancer Cell Populations.

Large genomes with elevated mutation rates are prone to accumulating deleterious mutations more rapidly than natural selection can purge (Muller's ratchet). As a consequence, it may lead to the extinction of small populations. Relative to most unicellular organisms, cancer cells, with large and nonrecombining genome and high mutation rate, could be particularly susceptible to such "mutational meltdown." However, the most common type of mutation in organismal evolution, namely, deleterious mutation, has received relatively little attention in the cancer biology literature. Here, by monitoring single-cell clones from HeLa cell lines, we characterize deleterious mutations that retard the rate of cell proliferation. The main mutation events are copy number variations (CNVs), which, estimated from fitness data, happen at a rate of 0.29 event per cell division on average. The mean fitness reduction, estimated reaching 18% per mutation, is very high. HeLa cell populations therefore have very substantial genetic load and, at this level, natural population would likely face mutational meltdown. We suspect that HeLa cell populations may avoid extinction only after the population size becomes large enough. Because CNVs are common in most cell lines and tumor tissues, the observations hint at cancer cells' vulnerability, which could be exploited by therapeutic strategies.

Lymphocyte Antigen 6 Complex, Locus C+ Monocytes and Kupffer Cells Orchestrate Liver Immune Responses Against Hepatitis B Virus in Mice.

To understand the mechanism(s) of age-dependent outcomes of hepatitis B virus (HBV) infection in humans, we previously established an age-related HBV mouse model in which 6-week-old (N6W) C3H/HeN mice exhibited virus tolerance whereas 12-week-old (N12W) counterparts presented virus clearance. By investigating the hepatic myeloid cell dynamics in mice of these two ages, we aim to identify factors associated with HBV clearance. C3H/HeN mice were transfected with an HBV plasmid by hydrodynamic injection. Serum HBV markers were monitored weekly. Hepatic leucocyte populations and their cytokine/chemokine productions were examined at baseline, day 3 (D3), day 7 (D7), and day 14 after injection. C-C chemokine receptor type 2 (CCR2) antagonist and clodronate (CLD) were respectively administered to N12W and N6W mice to study the roles of lymphocyte antigen 6 complex, locus C (Ly6C)+ monocytes and Kupffer cells (KCs) in viral clearance. N12W mice had a significantly higher number of TNF-α-secreting Ly6C+ monocytes and fewer IL-10-secreting KCs at D3 in the liver than their younger N6W counterparts after HBV transfection. In addition, the elevated number of interferon-γ+ TNF-α+ CD8+ T cells at D7 was only seen in the older cohort. The enhanced Ly6C+ monocyte induction in N12W mice resulted from elevated C-C motif chemokine ligand 2 (CCL2) secretion by hepatocytes. CCR2 antagonist administration hampered Ly6C+ monocyte recruitment and degree of KC reduction and delayed HBV clearance in N12W animals. Depletion of KCs by CLD liposomes enhanced Ly6C+ monocyte recruitment and accelerated HBV clearance in N6W mice. Conclusions: Ly6C+ monocytes and KCs may, respectively, represent the resistance and tolerance arms of host defenses. These two cell types play an essential role in determining HBV clearance/tolerance. Manipulation of these cells is a promising avenue for immunotherapy of HBV-related liver diseases.

De novo assembly of highly polymorphic metagenomic data using in situ generated reference sequences and a novel BLAST-based assembly pipeline.

BACKGROUND: The accuracy of metagenomic assembly is usually compromised by high levels of polymorphism due to divergent reads from the same genomic region recognized as different loci when sequenced and assembled together. A viral quasispecies is a group of abundant and diversified genetically related viruses found in a single carrier. Current mainstream assembly methods, such as Velvet and SOAPdenovo, were not originally intended for the assembly of such metagenomics data, and therefore demands for new methods to provide accurate and informative assembly results for metagenomic data. RESULTS: In this study, we present a hybrid method for assembling highly polymorphic data combining the partial de novo-reference assembly (PDR) strategy and the BLAST-based assembly pipeline (BBAP). The PDR strategy generates in situ reference sequences through de novo assembly of a randomly extracted partial data set which is subsequently used for the reference assembly for the full data set. BBAP employs a greedy algorithm to assemble polymorphic reads. We used 12 hepatitis B virus quasispecies NGS data sets from a previous study to assess and compare the performance of both PDR and BBAP. Analyses suggest the high polymorphism of a full metagenomic data set leads to fragmentized de novo assembly results, whereas the biased or limited representation of external reference sequences included fewer reads into the assembly with lower assembly accuracy and variation sensitivity. In comparison, the PDR generated in situ reference sequence incorporated more reads into the final PDR assembly of the full metagenomics data set along with greater accuracy and higher variation sensitivity. BBAP assembly results also suggest higher assembly efficiency and accuracy compared to other assembly methods. Additionally, BBAP assembly recovered HBV structural variants that were not observed amongst assembly results of other methods. Together, PDR/BBAP assembly results were significantly better than other compared methods. CONCLUSIONS: Both PDR and BBAP independently increased the assembly efficiency and accuracy of highly polymorphic data, and assembly performances were further improved when used together. BBAP also provides nucleotide frequency information. Together, PDR and BBAP provide powerful tools for metagenomic data studies.

Hydrodynamic HBV Transfection Mouse Model.

Hydrodynamic HBV transfection mouse model is an established method of the last decade where macromolecules, non-normally permeable to cell membrane, are delivered intracellular. The basic principle is that a large volume of solution, containing HBV plasmid construct, is infused rapidly in circulation to permit the preferential entrance of these macromolecules to liver parenchymal cells. The aim of this chapter is to describe the basic principles of the hydrodynamic HBV transfection in mouse models.

The Ecology and Evolution of Cancer: The Ultra-Microevolutionary Process.

Although tumorigenesis has been accepted as an evolutionary process ( 20 , 102 ), many forces may operate differently in cancers than in organisms, as they evolve at vastly different time scales. Among such forces, natural selection, here defined as differential cellular proliferation among distinct somatic cell genotypes, is particularly interesting because its action might be thwarted in multicellular organisms ( 20 , 29 ). In this review, selection is analyzed in two stages of cancer evolution: Stage I is the evolution between tumors and normal tissues, and Stage II is the evolution within tumors. The Cancer Genome Atlas (TCGA) data show a low degree of convergent evolution in Stage I, where genetic changes are not extensively shared among cases. An equally important, albeit much less highlighted, discovery using TCGA data is that there is almost no net selection in cancer evolution. Both positive and negative selection are evident but they neatly cancel each other out, rendering total selection ineffective in the absence of recombination. The efficacy of selection is even lower in Stage II, where neutral (non-Darwinian) evolution is increasingly supported by high-density sampling studies ( 81 , 123 ). Because natural selection is not a strong deterministic force, cancers usually evolve divergently even in similar tissue environments.

PATHOLOGY AND MOLECULAR DETECTION OF RABIES VIRUS IN FERRET BADGERS ASSOCIATED WITH A RABIES OUTBREAK IN TAIWAN.

Until Rabies virus (RABV) infection in Taiwan ferret badgers (TWFB; Melogale moschata subaurantiaca) was diagnosed in mid-June 2013, Taiwan had been considered rabies free for >50 yr. Although rabies has also been reported in ferret badgers in China, the pathologic changes and distribution of viral antigens of ferret badger-associated rabies have not been described. We performed a comprehensive pathologic study and molecular detection of rabies virus in three necropsied rabid TWFBs and evaluated archival paraffin-embedded tissue blocks of six other TWFBs necropsied during 2004 and 2012. As in other RABV-infected species, the characteristic pathologic changes in TWFBs were nonsuppurative meningoencephalomyelitis, ganglionitis, and the formation of typical intracytoplasmic Negri bodies, with the brain stem most affected. There was also variable spongiform degeneration, primarily in the perikaryon of neurons and neuropil, in the cerebral cortex, thalamus, and brain stem. In nonnervous system tissues, representative lesions included adrenal necrosis and lymphocytic interstitial sialadenitis. Immunohistochemical staining and fluorescent antibody test demonstrated viral antigens in the perikaryon of the neurons and axonal or dendritic processes throughout the nervous tissue and in the macrophages in various tissues. Similar to raccoons (Procyon lotor) and skunks (Mephitidae), the nervous tissue of rabid TWFBs displayed widely dispersed lesions, RABV antigens, and large numbers of Negri bodies. We traced the earliest rabid TWFB case back to 2004.

Age-related immune clearance of hepatitis B virus infection requires the establishment of gut microbiota.

A unique feature of hepatitis B virus (HBV) infection in humans is that viral clearance heavily depends on the age of exposure. However, the reason for this remains unclear. Here we show that gut microbiota contribute to the age dependence of HBV immunity in a hydrodynamic transfection mouse model. Although adult (12-wk-old) C3H/HeN mice cleared HBV within 6 wk postinjection (wpi), their young (6-wk-old) counterparts remained HBV-positive at 26 wpi. Sterilization of gut microbiota from 6 to 12 wk of age using antibiotics prevented adult mice from rapidly clearing HBV. Young mice with the Toll-like-receptor (TLR) 4 mutation (C3H/HeJ) exhibited rapid HBV clearance. The results suggest that an immuno-tolerating pathway to HBV prevailed in young mice, before the establishment of gut bacteria, through a TLR4-dependent pathway and that the maturation of gut microbiota in adult mice stimulated liver immunity, resulting in rapid HBV clearance.