Precise Design of TiO2@CoOx Heterostructure via Atomic Layer Deposition for Synergistic Sono-Chemodynamic Oncotherapy.

Sonodynamic therapy (SDT), a tumor treatment modality with high tissue penetration and low side effects, is able to selectively kill tumor cells by producing cytotoxic reactive oxygen species (ROS) with ultrasound-triggered sonosensitizers. N-type inorganic semiconductor TiO2 has low ROS quantum yields under ultrasound irradiation and inadequate anti-tumor activity. Herein, by using atomic layer deposition (ALD) to create a heterojunction between porous TiO2 and CoOx, the sonodynamic therapy efficiency of TiO2 can be improved. Compared to conventional techniques, the high controllability of ALD allows for the delicate loading of CoOx nanoparticles into TiO2 pores, resulting in the precise tuning of the interfaces and energy band structures and ultimately optimal SDT properties. In addition, CoOx exhibits a cascade of H2O2→O2→·O2 - in response to the tumor microenvironment, which not only mitigates hypoxia during the SDT process, but also contributes to the effect of chemodynamic therapy (CDT). Correspondingly, the synergistic CDT/SDT treatment is successful in inhibiting tumor growth. Thus, ALD provides new avenues for catalytic tumor therapy and other pharmaceutical applications.

Structural basis of increased binding affinities of spikes from SARS-CoV-2 Omicron variants to rabbit and hare ACE2s reveals the expanding host tendency.

The potential host range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been expanding alongside its evolution during the pandemic, with rabbits and hares being considered important potential hosts, supported by a report of rabbit sero-prevalence in nature. We measured the binding affinities of rabbit and hare angiotensin-converting enzyme 2 (ACE2) with receptor-binding domains (RBDs) from SARS-CoV, SARS-CoV-2, and its variants and found that rabbit and hare ACE2s had broad variant tropism, with significantly enhanced affinities to Omicron BA.4/5 and its subsequent-emerged sub-variants (>10 fold). The structures of rabbit ACE2 complexed with either SARS-CoV-2 prototype (PT) or Omicron BA.4/5 spike (S) proteins were determined, thereby unveiling the importance of rabbit ACE2 Q34 in RBD-interaction and elucidating the molecular basis of the enhanced binding with Omicron BA.4/5 RBD. These results address the highly enhanced risk of rabbits infecting SARS-CoV-2 Omicron sub-variants and the importance of constant surveillance.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the globe and caused immense health and economic damage. SARS-CoV-2 has demonstrated a broad host range, indicating a high risk of interspecies transmission and adaptive mutation. Therefore, constant monitoring for potential hosts is of immense importance. In this study, we found that Omicron BA.4/5 and subsequent-emerged sub-variants exhibited enhanced binding to both rabbit and hare angiotensin-converting enzyme 2 (ACE2), and we elucidated the structural mechanism of their recognition. From the structure, we found that Q34, a unique residue of rabbit ACE2 compared to other ACE2 orthologs, plays an important role in ACE2 recognition. These results address the probability of rabbits/hares being potential hosts of SARS-CoV-2 and broaden our knowledge regarding the molecular mechanism of SARS-CoV-2 interspecies transmission.

Genomic analysis of almost 8,000 Salmonella genomes reveals drivers and landscape of antimicrobial resistance in China.

IMPORTANCE: We established the largest Salmonella genome database from China and presented the landscape and spatiotemporal dynamics of antimicrobial resistance genes. We also found that economic, climatic, and social factors can drive the rise of antimicrobial resistance. The Chinese local Salmonella genome database version 2 was released as an open-access database (https://nmdc.cn/clsgdbv2) and thus can assist surveillance studies across the globe. This database will help inform interventions for AMR, food safety, and public health.

Multimodal imaging and photothermal/chemodynamic therapy of cervical cancer using GSH-responsive MoS2@MnO2 theranostic nanoparticles.

The development of nanoparticles capable of inducing reactive oxygen species (ROS) formation has become an important strategy for cancer therapy. Simultaneously, the preparation of multifunctional nanoparticles that respond to the tumor microenvironment is crucial for the diagnosis and treatment of tumors. In this study, we designed a Molybdenum disulfide (MoS2) core coated with Manganese dioxide (MnO2), which possessed a good photothermal effect and could produce Fenton-like Mn2+ in response to highly expressed glutathione (GSH) in the tumor microenvironment, thereby generating a chemodynamic therapy (CDT). The nanoparticles were further modified with Methoxypoly(Ethylene Glycol) 2000 (mPEG-NH2) to improve their biocompatibility, resulting in the formation of MoS2@MnO2-PEG. These nanoparticles were shown to possess significant Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) imaging capabilities, making them useful in tumor diagnosis. In vitro and in vivo experiments demonstrated the antitumor ability of MoS2@MnO2-PEG, with a significant killing effect on tumor cells under combined treatment. These nanoparticles hold great potential for CDT/photothermal therapy (PTT) combined antitumor therapy and could be further explored in biomedical research.

Hemicyanine-based sensor for mitochondrial viscosity imaging in BV2 cells.

Mitochondrial viscosity is a critical factor affecting numerous physiological processes, including phagocytosis. Abnormal viscosity in mitochondria is related to some pathological activities and diseases. Evaluating and detecting the changes in mitochondrial viscosity in vivo is crucial. Thus, a mitochondria-targeted red-emitting fluorescent probe (VP) was prepared, and can be used to detect viscosity with high selectivity and sensitivity. The synthesis of probe VP was as simple as two steps and the cost was low. In addition, the fluorescence intensity (log I615) exhibited an excellent relationship with viscosity (log η) in the range of 0.5 - 2.5 (R2 = 0.9985) in water/glycerol mixture. It is noteworthy that the probe VP displayed the highest signal-to-noise ratio (about 50-fold) for viscosity in water and glycerol system. The probe VP can visualize the mitochondrial viscosity change in living cells. More importantly, phagocytic test for BV2 cells further demonstrated that phagocytosis decreased with increased viscosity. Furthermore, VP was successfully used for monitoring the mitophagy process induced by starvation, and mitochondrial viscosity exhibited enhancement during mitophagy. The probe was a potential tool for studying viscosity and phagocytosis.

The temporal dynamics of antimicrobial-resistant Salmonella enterica and predominant serovars in China.

Salmonella enterica is one of the most common bacterial pathogens in humans and animals. Systematic studies on the trends and geographical distribution of antimicrobial-resistant Salmonella and dominant serovars have been well studied in European and American countries while not in China. Here, taking the One-Health strategy, we used >35 000 Salmonella enterica isolates to explore the temporal and spatial dynamics of dominant serovars in China. We found that Salmonella Typhimurium was the dominant serovar causing human infection in China, which was consistent with Australia but inconsistent with North American and European countries. The proportion of Salmonella serovars Typhimurium, London, Rissen, Corvallis, Meleagridis, Kentucky, and Goldcoast showed an increasing trend during 2006-2019. We randomly selected 1962 isolates for comparative genomics and antimicrobial resistance studies and found that the number of antibiotic resistance genes (ARGs) per isolate increased 1.84 and 2.69 times of human and non-human origins, respectively, spanning 14 years. The proportion of antimicrobial-resistant Salmonella isolates had an increasing trend during 2006-2019, especially beta-lactam, quinolone, tetracycline, and rifampicin resistance. Moreover, we found that higher diversity of sequence types (STs) in S. Typhimurium than in other serovars, ST34 from pig and ST19 from chicken origin, were mainly associated with isolates causing child and adult gastro-infection, respectively. Our results fill in the data gap on the trends of dominant serovars and antimicrobial resistance of Salmonella enterica in China. These data provide useful information for public health decision-makers prioritizing interventions for foodborne diseases and food safety.

A neutralizing-protective supersite of human monoclonal antibodies for yellow fever virus.

The yellow fever virus (YFV) is a life-threatening human pathogen. Owing to the lack of available therapeutics, non-vaccinated individuals are at risk. Here, we isolated eight human monoclonal antibodies that neutralize YFV infection. Five recognized overlapping epitopes and exhibited potent neutralizing activity. Two (YD6 and YD73) were ultra-potent and conferred complete protection against the lethal challenge of YFV as both prophylactics and therapeutics in a mouse model. Crystal structures revealed that YD6 engaged the YFV envelope protein in both pre- and post-fusion states, suggesting viral inhibition by a "double-lock" mechanism. The recognition determinants for YD6 and YD73 are clustered at the premembrane (prM)-binding site. Notably, antibodies targeting this site were present in minute traces in YFV-infected individuals but contributed significantly to neutralization, suggesting a vulnerable supersite of YFV. We provide two promising candidates for immunotherapy against YFV, and the supersite represents an ideal target for epitope-based vaccine design.

Targeted chemo-photodynamic therapy toward esophageal cancer by GSH-sensitive theranostic nanoplatform.

As the sixth leading cause of cancer death, esophageal cancer is threatening the life of people worldwide. Traditional treatments, such as surgery, chemotherapy, radiotherapy, are facing always augmented challenges including invasion, multidrug resistance (MDR), off-target toxicity. Chemo & Photodynamic synergistic therapy represents one promising strategy for improved treatment efficiency. But it is still hindered by the lack of tumor targeting, deleterious side effects, and unfavorable microenvironment for photodynamic therapy (PDT). To overcome those obstacles, one theranostic nano-assambly drug, GCDs-Ce6/Pt-EGF, was designed and fabricated. Green fluorescence carbon dots (GCDs) with the excellent optical properties, modifiability and low toxicity were prepared as drug carrier. Epidermal growth factor (EGF) was conjugated to the nano-assembly to realize tumor specific targeting. Chlorin e6 (Ce6) in the presence of laser irradiation achieved PDT by generating proapoptosis reactive oxygen species (ROS). Moreover, Ce6 incorporated into GCDs endowed the nano-assambly imaging ability and facilitate image-guided therapy. Pt(IV), cisplatin prodrug, in the nano-assambly depleted the glutathione (GSH) of tumor microenvironment when it was reduced to cytotoxicity Pt(II). Compared with single treatment, GCDs-Ce6/Pt-EGF exhibited enhanced tumor cell killing capacity and better biosafety in vitro and in vivo, especially for EGFR bearing tumor. It paved ways for developing novel theranostic agent to be potentially applied in clinic.

The molecular basis for SARS-CoV-2 binding to dog ACE2.

SARS-CoV-2 can infect many domestic animals, including dogs. Herein, we show that dog angiotensin-converting enzyme 2 (dACE2) can bind to the SARS-CoV-2 spike (S) protein receptor binding domain (RBD), and that both pseudotyped and authentic SARS-CoV-2 can infect dACE2-expressing cells. We solved the crystal structure of RBD in complex with dACE2 and found that the total number of contact residues, contact atoms, hydrogen bonds and salt bridges at the binding interface in this complex are slightly fewer than those in the complex of the RBD and human ACE2 (hACE2). This result is consistent with the fact that the binding affinity of RBD to dACE2 is lower than that of hACE2. We further show that a few important mutations in the RBD binding interface play a pivotal role in the binding affinity of RBD to both dACE2 and hACE2. Our work reveals a molecular basis for cross-species transmission and potential animal spread of SARS-CoV-2, and provides new clues to block the potential transmission chains of this virus.

More diversified antibiotic resistance genes in chickens and workers of the live poultry markets.

BACKGROUND: Poultry farms and LPMs are a reservoir of antimicrobial resistant bacteria and resistance genes from feces. The LPM is an important interface between humans, farm animals, and environments in a typical urban environment, and it is considered a reservoir for ARGs and viruses. However, the antibiotic resistomes shared between chicken farms and LPMs, and that of LPM workers and people who have no contact with the LPMs remains unknown. METHODS: We characterized the resistome and bacterial microbiome of farm chickens and LPMs and LPM workers and control subjects. The mobile ARGs identified in chickens and the distribution of the mcr-family genes in publicly bacterial genomes and chicken gut metagenomes was analyzed, respectively. In addition, the prevalence of mcr-1 in LPMs following the ban on colistin-positive additives in China was explored. RESULTS: By profiling the microbiomes and resistomes in chicken farms, LPMs, LPM workers, and LPM environments, we found that the bacterial community composition and resistomes were significantly different between the farms and the LPMs, and the LPM samples possessed more diversified ARGs (59 types) than the farms. Some mobile ARGs, such as mcr-1 and tet(X3), identified in chicken farms, LPMs, LPM workers, and LPM environments were also harbored by human clinical pathogens. Moreover, we found that the resistomes were significantly different between the LPM workers and those who have no contact with the LPMs, and more diversified ARGs (188 types) were observed in the LPM workers. It is also worth noting that mcr-10 was identified in both human (5.2%, 96/1,859) and chicken (1.5%, 14/910) gut microbiomes. Although mcr-1 prevalence decreased significantly in the LPMs across the eight provinces in China, from 190/333 (57.1%) samples in September 2016-March 2017 to 208/544 (38.2%) samples in August 2018-May 2019, it is widespread and continuous in the LPMs. CONCLUSION: Live poultry trade has a significant effect on the diversity of ARGs in LPM workers, chickens, and environments in China, driven by human selection with the live poultry trade. Our findings highlight the live poultry trade as ARG disseminators into LPMs, which serve as an interface of LPM environments even LPM workers, and that could urge Government to have better control of LPMs in China. Further studies on the factors that promote antibiotic resistance exchange between LPM environments, human commensals, and pathogens, are warranted.

Dihydropyridine-coumarin-based fluorescent probe for imaging nitric oxide in living cells.

Nitric oxide (NO) is a messenger molecule in organisms, participating in the regulation of many biological processes. The abnormal expression of NO is often observed in a variety of diseases, including cerebral ischemia, atherosclerosis, and cancer. However, a suitable tool that can directly and sensitively detect NO in vitro and in vivo is important for understanding its various biological functions. In this report, a new fluorescent probe for nitric oxide, DHP-4, was prepared, based on dihydropyridine-coumarin. DHP-4 was able to greatly enhance the fluorescence of NO, but did not affect the fluorescence emissions of other reactive oxygen species and nitrogen species, demonstrating its highly selective and sensitive response to NO. The probe generated stable optical signals in a buffer solution at pH values ranging from 3 to 10. In addition, DHP-4 could detect NO directly, showed low cellular toxicity, and was successfully applied to determine NO in Raw 264.7 cells, indicating its great potential as a tool for investigating the biological roles of NO in vivo.

Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance.

BACKGROUND: During the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site. Moreover, a neuraminidase inhibitor (NAI) resistance (R292K in NA) mutation was found in H7N9 isolates from humans, poultry and the environment. In this study, we set out to develop and validate a multiplex quantitative reverse transcript polymerase chain reaction (qRT-PCR) to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations. METHODS: A quadruple qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations was designed based on the analyses of the HA and NA genes of H7N9. This assay was further tested for specificity and sensitivity, and validated using clinical samples. RESULTS: The assay was highly specific and able to detect low pathogenic (LP)- or HP-H7N9 with/without the NAI-resistance mutation. The detection limit of the assay was determined to be 50 genome-equivalent copies and 2.8 × 10- 3 50% tissue culture infectious doses (TCID50) of live H7N9 per reaction. Clinical validation was confirmed by commercial kits and Sanger sequencing with ten clinical samples. CONCLUSIONS: We developed and validated a rapid, single-reaction, one-step, quadruple real-time qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP- and NAI-resistance strains with excellent performance in specificity and sensitivity. This assay could be used to monitor the evolution of H7N9 viruses in the laboratory, field and the clinic for early-warning and the prevention of H7N9 infections.

New Threats from H7N9 Influenza Virus: Spread and Evolution of High- and Low-Pathogenicity Variants with High Genomic Diversity in Wave Five.

H7N9 virus has caused five infection waves since it emerged in 2013. The highest number of human cases was seen in wave 5; however, the underlying reasons have not been thoroughly elucidated. In this study, the geographical distribution, phylogeny, and genetic evolution of 240 H7N9 viruses in wave 5, including 35 new isolates from patients and poultry in nine provinces, were comprehensively analyzed together with strains from first four waves. Geographical distribution analysis indicated that the newly emerging highly pathogenic (HP) and low-pathogenicity (LP) H7N9 viruses were cocirculating, causing human and poultry infections across China. Genetic analysis indicated that dynamic reassortment of the internal genes among LP-H7N9/H9N2/H6Ny and HP-H7N9, as well as of the surface genes, between the Yangtze and Pearl River Delta lineages resulted in at least 36 genotypes, with three major genotypes (G1 [A/chicken/Jiangsu/SC537/2013-like], G3 [A/Chicken/Zhongshan/ZS/2017-like], and G11 [A/Anhui/40094/2015-like]). The HP-H7N9 genotype likely evolved from G1 LP-H7N9 by the insertion of a KRTA motif at the cleavage site (CS) and then evolved into 15 genotypes with four different CS motifs, including PKGKRTAR/G, PKGKRIAR/G, PKRKRAAR/G, and PKRKRTAR/G. Approximately 46% (28/61) of HP strains belonged to G3. Importantly, neuraminidase (NA) inhibitor (NAI) resistance (R292K in NA) and mammalian adaptation (e.g., E627K and A588V in PB2) mutations were found in a few non-human-derived HP-H7N9 strains. In summary, the enhanced prevalence and diverse genetic characteristics that occurred with mammalian-adapted and NAI-resistant mutations may have contributed to increased numbers of human infections in wave 5.IMPORTANCE The highest numbers of human H7N9 infections were observed during wave 5 from October 2016 to September 2017. Our results showed that HP-H7N9 and LP-H7N9 had spread virtually throughout China and underwent dynamic reassortment with different subtypes (H7N9/H9N2 and H6Ny) and lineages (Yangtze and Pearl River Delta lineages), resulting in totals of 36 and 3 major genotypes, respectively. Notably, the NAI drug-resistant (R292K in NA) and mammalian-adapted (e.g., E627K in PB2) mutations were found in HP-H7N9 not only from human isolates but also from poultry and environmental isolates, indicating increased risks for human infections. The broad dissemination of LP- and HP-H7N9 with high levels of genetic diversity and host adaptation and drug-resistant mutations likely accounted for the sharp increases in the number of human infections during wave 5. Therefore, more strategies are needed against the further spread and damage of H7N9 in the world.

Water-soluble Hantzsch ester as switch-on fluorescent probe for efficiently detecting nitric oxide.

A water soluble Hantzsch ester derivative of coumarin, DHPS, was synthesized and successfully applied in the fluorescent sensing nitric oxide (NO) in aqueous solution. The fluorescence of probe DHPS is extremely weak, while its fluorescence was greatly switched on upon the addition of NO solution and showed high selectivity and sensitivity to NO. The limitation of the detection was calculated to be 18nM. The NO-induced aromatization of dihydropyridine in DHPS to pyridine derivative (PYS) proved to be the switching mechanism for the fluorescent sensing process, which was confirmed through spectra characterization and computation study. Cytotoxicity assay demonstrated both DHPS and PYS are biocompatible, the DHPS was successfully applied to track the endogenously produced NO in the RAW 264.7 cells.

Discrete stacking of aromatic oligoamide macrocycles.

Unlike the precise structural control typical of closed assemblies, curbing the stacking of disc- and ring-shaped molecules is quite challenging. Here we report the discrete stacking of rigid aromatic oligoamide macrocycles 1. With increasing concentration, the aggregation of 1 quickly plateaus, forming a discrete oligomer, as suggested by 1D (1)H, 2D nuclear Overhauser effect, and diffusion-ordered NMR spectroscopy. Quantum-chemical calculations indicate that the tetramer of 1 is the most stable among oligomeric stacks. X-ray crystallography revealed a tetrameric stack containing identical molecules adopting two different conformations. With a defined length and an inner pore capable of accommodating distinctly different guests, the tetramers of 1 densely pack into 2D layers. Besides being a rare system of conformation-regulated supramolecular oligomerization, the discrete stacks of 1, along with their higher-order assemblies, may offer new nanotechnological applications.

In vitro transcriptome analysis of two Chinese isolates of Streptococcus suis serotype 2.

The Streptococcus suis serotype 2 (S. suis 2) isolates 05ZYH33 and 98HAH33 have caused severe human infections in China. Using a strand-specific RNA-seq analysis, we compared the in vitro transcriptomes of these two Chinese isolates with that of a reference strain (P1/7). In the 89K genomic island that is specific to these Chinese isolates, a toxin-antitoxin system showed relatively high levels of transcription among the S. suis. The known virulence factors with high transcriptional activity in these two highly-pathogenic strains are mainly involved in adhesion, biofilm formation, hemolysis and the synthesis and transport of the outer membrane protein. Furthermore, our analysis of novel transcripts identified over 50 protein-coding genes with one of them encoding a toxin protein. We also predicted over 30 small RNAs (sRNAs) in each strain, and most of them are involved in riboswitches. We found that six sRNA candidates that are related to bacterial virulence, including cspA and rli38, are specific to Chinese isolates. These results provide insight into the factors responsible for the difference in virulence among the different S. suis 2 isolates.

The rational design of a highly sensitive and selective fluorogenic probe for detecting nitric oxide.

A rationally designed small-molecule fluorogenic probe for nitric oxide (NO) detection based on a new switching mechanism has been developed. Attaching a NO-responsive dihydropyridine pendant group to a fluorophore led to a probe that displays a very high sensitivity to NO concentrations down to the low nM range and a very high specificity to NO while being insensitive to other oxidative oxygen/nitrogen species that often interfere with the sensing of NO.

Prevalent HBV point mutations and mutation combinations at BCP/preC region and their association with liver disease progression.

BACKGROUND: Mutations in the basic core promoter (BCP) and its adjacent precore (preC) region in HBV genome are common in chronic hepatitis B patients. However, the patterns of mutation combinations in these two regions during chronic infection are less understood. This study focused on single base mutations in BCP and preC region and the multi-mutation patterns observed in chronic HBV infection patients. METHODS: Total 192 blood samples of chronic HBV infection patients were included. Direct PCR sequencing on the target region of HBV genome was successfully conducted in 157 samples. The rest 35 samples were analyzed by clone sequencing. Only the nucleotide substitutions with their frequencies no less than 10% were included in multi-mutation analysis with the exception for the polymorphic sites between genotypes B and C. RESULTS: Five high frequency mutations (≥10%) were found in BCP and preC region. Thirteen types of multi-mutations in one fragment were observed, among which 3 types were common combinations (≥5%). The top three multi-mutations were A1762T/G1764A (36%), A1762T/G1764A/G1896A (11%) and T1753(A/C)/A1762T/G1764A/G1896A (8%). Patients with multi-mutations in viral genomes (≥3) were more likely to have liver cirrhosis or hepatocellular carcinoma (OR = 3.1, 95% CI: 1.6-6.0, P = 0.001). G1896A mutation seemed to be involved in liver disease progression independent of the patient age (OR = 3.6, 95% CI: 1.5-8.6; P = 0.004). In addition, patients with more viral mutations detected (≥3) were more likely to be HBeAg negative (OR = 2.7, 95% CI: 1.1-6.4; P = 0.027). Moreover, G1776A mutation was shown to contribute to HBeAg negativity in our study (OR = 8.6, 95% CI: 1.2-44.9; P = 0.01). CONCLUSIONS: Patients with advanced liver diseases and with HBeAg negativity more likely have multi-mutations in HBV genomes but with different mutation combination patterns. G1896A mutation appears to be independent of infection history.

Dynamic changes of HBV quasispecies and deletion patterns in a chronic hepatitis B patient.

Hepatitis B virus (HBV) infection is a dynamic process during which molecular variants are selected continuously to adapt to changes. In addition to drug resistant mutations, sequential antiviral therapy may also lead to the selection of deleted mutants. To investigate this process, the following samples were collected from a patient who failed lamivudine therapy and then was switched to adefovir dipivoxil. HBV DNA was sequenced at two separate regions; a 1 kb region of reverse transcriptase (RT) and a 1.5 kb region encompassing the C gene and part of the preS gene. Sequence analysis of the RT region showed that the prevailing lamivudine resistant mutations were reduced after switching to adefovir dipivoxil, and ultimately the mutations were undetectable. Quasispecies distribution and deletion patterns in the C and preS regions were also different between the two antiviral therapies. In lamivudine-treated samples, wild-type strains (57.7%) were dominant and deletions in the preS region were observed. However, in the subsequent therapy involving adefovir dipivoxil, a virus population harboring 81 and 96 bp deletions (86%) in the C gene prevailed. Both major deletions encompassed T- and B-cell epitopes. Meanwhile, the frequencies of the preS deletions decreased significantly, except for the 129 bp deletion. Notably, the presence of 81, 96, and 129 bp deletions was always accompanied with some nucleotide substitutions. In conclusion, the prevalence of deletions at the C gene epitopes accompanied with the gradual disappearance of lamivudine resistance mutations may contribute to the survival of HBV under sequential antiviral therapy.