Compositional features analysis by machine learning in genome represents linear adaptation of monkeypox virus.

Introduction: The global headlines have been dominated by the sudden and widespread outbreak of monkeypox, a rare and endemic zoonotic disease caused by the monkeypox virus (MPXV). Genomic composition based machine learning (ML) methods have recently shown promise in identifying host adaptability and evolutionary patterns of virus. Our study aimed to analyze the genomic characteristics and evolutionary patterns of MPXV using ML methods. Methods: The open reading frame (ORF) regions of full-length MPXV genomes were filtered and 165 ORFs were selected as clusters with the highest homology. Unsupervised machine learning methods of t-distributed stochastic neighbor embedding (t-SNE), Principal Component Analysis (PCA), and hierarchical clustering were performed to observe the DCR characteristics of the selected ORF clusters. Results: The results showed that MPXV sequences post-2022 showed an obvious linear adaptive evolution, indicating that it has become more adapted to the human host after accumulating mutations. For further accurate analysis, the ORF regions with larger variations were filtered out based on the ranking of homology difference to narrow down the key ORF clusters, which drew the same conclusion of linear adaptability. Then key differential protein structures were predicted by AlphaFold 2, which meant that difference in main domains might be one of the internal reasons for linear adaptive evolution. Discussion: Understanding the process of linear adaptation is critical in the constant evolutionary struggle between viruses and their hosts, playing a significant role in crafting effective measures to tackle viral diseases. Therefore, the present study provides valuable insights into the evolutionary patterns of the MPXV in 2022 from the perspective of genomic composition characteristics analysis through ML methods.

Genomic representation predicts an asymptotic host adaptation of bat coronaviruses using deep learning.

Introduction: Coronaviruses (CoVs) are naturally found in bats and can occasionally cause infection and transmission in humans and other mammals. Our study aimed to build a deep learning (DL) method to predict the adaptation of bat CoVs to other mammals. Methods: The CoV genome was represented with a method of dinucleotide composition representation (DCR) for the two main viral genes, ORF1ab and Spike. DCR features were first analyzed for their distribution among adaptive hosts and then trained with a DL classifier of convolutional neural networks (CNN) to predict the adaptation of bat CoVs. Results and discussion: The results demonstrated inter-host separation and intra-host clustering of DCR-represented CoVs for six host types: Artiodactyla, Carnivora, Chiroptera, Primates, Rodentia/Lagomorpha, and Suiformes. The DCR-based CNN with five host labels (without Chiroptera) predicted a dominant adaptation of bat CoVs to Artiodactyla hosts, then to Carnivora and Rodentia/Lagomorpha mammals, and later to primates. Moreover, a linear asymptotic adaptation of all CoVs (except Suiformes) from Artiodactyla to Carnivora and Rodentia/Lagomorpha and then to Primates indicates an asymptotic bats-other mammals-human adaptation. Conclusion: Genomic dinucleotides represented as DCR indicate a host-specific separation, and clustering predicts a linear asymptotic adaptation shift of bat CoVs from other mammals to humans via deep learning.

Convolutional Neural Networks Based on Sequential Spike Predict the High Human Adaptation of SARS-CoV-2 Omicron Variants.

The COVID-19 pandemic has frequently produced more highly transmissible SARS-CoV-2 variants, such as Omicron, which has produced sublineages. It is a challenge to tell apart high-risk Omicron sublineages and other lineages of SARS-CoV-2 variants. We aimed to build a fine-grained deep learning (DL) model to assess SARS-CoV-2 transmissibility, updating our former coarse-grained model, with the training/validating data of early-stage SARS-CoV-2 variants and based on sequential Spike samples. Sequential amino acid (AA) frequency was decomposed into serially and slidingly windowed fragments in Spike. Unsupervised machine learning approaches were performed to observe the distribution in sequential AA frequency and then a supervised Convolutional Neural Network (CNN) was built with three adaptation labels to predict the human adaptation of Omicron variants in sublineages. Results indicated clear inter-lineage separation and intra-lineage clustering for SARS-CoV-2 variants in the decomposed sequential AAs. Accurate classification by the predictor was validated for the variants with different adaptations. Higher adaptation for the BA.2 sublineage and middle-level adaptation for the BA.1/BA.1.1 sublineages were predicted for Omicron variants. Summarily, the Omicron BA.2 sublineage is more adaptive than BA.1/BA.1.1 and has spread more rapidly, particularly in Europe. The fine-grained adaptation DL model works well for the timely assessment of the transmissibility of SARS-CoV-2 variants, facilitating the control of emerging SARS-CoV-2 variants.

Deep learning based on biologically interpretable genome representation predicts two types of human adaptation of SARS-CoV-2 variants.

Explosively emerging SARS-CoV-2 variants challenge current nomenclature schemes based on genetic diversity and biological significance. Genomic composition-based machine learning methods have recently performed well in identifying phenotype-genotype relationships. We introduced a framework involving dinucleotide (DNT) composition representation (DCR) to parse the general human adaptation of RNA viruses and applied a three-dimensional convolutional neural network (3D CNN) analysis to learn the human adaptation of other existing coronaviruses (CoVs) and predict the adaptation of SARS-CoV-2 variants of concern (VOCs). A markedly separable, linear DCR distribution was observed in two major genes-receptor-binding glycoprotein and RNA-dependent RNA polymerase (RdRp)-of six families of single-stranded (ssRNA) viruses. Additionally, there was a general host-specific distribution of both the spike proteins and RdRps of CoVs. The 3D CNN based on spike DCR predicted a dominant type II adaptation of most Beta, Delta and Omicron VOCs, with high transmissibility and low pathogenicity. Type I adaptation with opposite transmissibility and pathogenicity was predicted for SARS-CoV-2 Alpha VOCs (77%) and Kappa variants of interest (58%). The identified adaptive determinants included D1118H and A570D mutations and local DNTs. Thus, the 3D CNN model based on DCR features predicts SARS-CoV-2, a major type II human adaptation and is qualified to predict variant adaptation in real time, facilitating the risk-assessment of emerging SARS-CoV-2 variants and COVID-19 control.

Dual R108K and G189D Mutations in the NS1 Protein of A/H1N1 Influenza Virus Counteract Host Innate Immune Responses.

Influenza A viruses (IAV) modulate host antiviral responses to promote growth and pathogenicity. Here, we examined the multifunctional IAV nonstructural protein 1 (NS1) of influenza A virus to better understand factors that contribute to viral replication efficiency or pathogenicity. In 2009, a pandemic H1N1 IAV (A/California/07/2009 pH1N1) emerged in the human population from swine. Seasonal variants of this virus are still circulating in humans. Here, we compared the sequence of a seasonal variant of this H1N1 influenza virus (A/Urumqi/XJ49/2018(H1N1), first isolated in 2018) with the pandemic strain A/California/07/2009. The 2018 virus harbored amino acid mutations (I123V and N205S) in important functional sites; however, 108R and 189G were highly conserved between A/California/07/2009 and the 2018 variant. To better understand interactions between influenza viruses and the human innate immune system, we generated and rescued seasonal 2009 H1N1 IAV mutants expressing an NS1 protein harboring a dual mutation (R108K/G189D) at these conserved residues and then analyzed its biological characteristics. We found that the mutated NS1 protein exhibited systematic and selective inhibition of cytokine responses via a mechanism that may not involve binding to cleavage and polyadenylation specificity factor 30 (CPSF30). These results highlight the complexity underlying host-influenza NS1 protein interactions.

Machine Learning Methods for Predicting Human-Adaptive Influenza A Viruses Based on Viral Nucleotide Compositions.

Each influenza pandemic was caused at least partly by avian- and/or swine-origin influenza A viruses (IAVs). The timing of and the potential IAVs involved in the next pandemic are currently unpredictable. We aim to build machine learning (ML) models to predict human-adaptive IAV nucleotide composition. A total of 217,549 IAV full-length coding sequences of the PB2 (polymerase basic protein-2), PB1, PA (polymerase acidic protein), HA (hemagglutinin), NP (nucleoprotein), and NA (neuraminidase) segments were decomposed for their codon position-based mononucleotides (12 nts) and dinucleotides (48 dnts). A total of 68,742 human sequences and 68,739 avian sequences (1:1) were resampled to characterize the human adaptation-associated (d)nts with principal component analysis (PCA) and other ML models. Then, the human adaptation of IAV sequences was predicted based on the characterized (d)nts. Respectively, 9, 12, 11, 13, 10 and 9 human-adaptive (d)nts were optimized for the six segments. PCA and hierarchical clustering analysis revealed the linear separability of the optimized (d)nts between the human-adaptive and avian-adaptive sets. The results of the confusion matrix and the area under the receiver operating characteristic curve indicated a high performance of the ML models to predict human adaptation of IAVs. Our model performed well in predicting the human adaptation of the swine/avian IAVs before and after the 2009 H1N1 pandemic. In conclusion, we identified the human adaptation-associated genomic composition of IAV segments. ML models for IAV human adaptation prediction using large IAV genomic data sets can facilitate the identification of key viral factors that affect virus transmission/pathogenicity. Most importantly, it allows the prediction of pandemic influenza.

A Sensitive Nano Luciferase Immune Complex Assay System for Highly Sensitive and Specific Detection of Antibodies Against Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) can cause fever, headache, neurological disorders, and/or peripheral flaccid paralysis; therefore, it is a major threat to public health. A rapid, sensitive, and simple method for detecting anti-TBEV antibodies is needed urgently to determine infection and for vaccine evaluation. Here, a luciferase-based immunocomplex assay system (Luc-IC) was developed to detect TBEV antibodies. The system is based on a reporter Nano luciferase (NLuc) that is co-expressed as a fusion protein with viral envelope domain III (ED3) in COS7 cells. The cell supernatant was used directly to detect antigen without the need for a purification step. This simple procedure effectively improved the sensitivity of the assay. Sera from 50 patients with an acute tick-borne encephalitis infection were tested to determine the sensitivity of the NLuc-IC assay. Furthermore, 62 sera from individuals infected with Japanese encephalitis virus, West Nile virus, yellow fever virus, dengue virus, or Zika virus were also tested to determine specificity. The results demonstrated that the assay was 100% sensitive and 100% specific for TBEV antibodies. Thus, this very simple NLuc-IC assay is potentially useful for rapid and accurate diagnosis of TBEV infection in both humans and animals.

H5N1 influenza A virus with K193E and G225E double mutations in haemagglutinin is attenuated and immunogenic in mice.

Live-attenuated influenza vaccines (LAIVs) are now available for the prevention of influenza, with LAIV strains generally derived from serial passage in cultures or by reverse genetics (RG). The receptor-binding domain (RBD) in haemagglutinin (HA) of influenza virus is responsible for viral binding to the avian-type 2,3-α-linked or human-type 2,6-α-linked sialic acid receptor; however, the virulence determinants in the RBD of H5N1 virus remain largely unknown. In the present study, serial passage of H5N1 virus A/Vietnam/1194/2004 in Madin-Darby canine kidney cells resulted in the generation of adapted variants with large-plaque morphology, and genomic sequencing of selected variants revealed two specific amino acid substitutions (K193E and G225E) in the RBD. RG was used to generate H5N1 viruses containing either single or double substitutions in HA. The RG virus containing K193E and G225E mutations (rVN-K193E/G225E) demonstrated large-plaque morphology, enhanced replication and genetic stability after serial passage, without changing the receptor-binding preference. Importantly, in vivo virulence assessment demonstrated that rVN-K193E/G225E was significantly attenuated in mice. Microneutralization and haemagglutination inhibition assays demonstrated that immunization with rVN-K193E/G225E efficiently induced a robust antibody response against WT H5N1 virus in mice. Taken together, our experiments demonstrated that K193E and G225E mutations synergistically attenuated H5N1 virus without enhancing the receptor-binding avidity, and that the RG virus rVN-K193E/G225E represents a potential H5N1 LAIV strategy that deserves further development. These findings identify the RBD as a novel attenuation target for live vaccine development and highlight the complexity of RBD interactions.

The first complete genomic characterization of an Amur virus isolate from China.

Amur virus (AMRV) is a member of the genus Hantavirus in the family Bunyaviridae. In this study, we determined for the first time the complete genome sequence of the AMRV H8205 strain, which was isolated from a patient with hemorrhagic fever with renal syndrome (HFRS) in China. The complete nucleotide sequence of the S segment of AMRV H8205 is 1699 nt long, with a 5' noncoding region (5'NC) of 36 nt, followed by a coding sequence of 1290 nt and a 3'NC of 373 nt. The complete sequence of the M segment is 3615 nt long, with a 5'NC of 40 nt, followed by a coding sequence of 3408 nt and a 3'NC of 167 nt. The complete sequence of the L segment is 6536 nt long, with a 5'NC of 37 nt, followed by a coding sequence of 6453 nt and a 3'NC of 40 nt. The major open reading frame (ORF) of each of the three segments (S, nt 37-1326; M, nt 41-3445; L, nt 38-6490) has a coding capacity of 430 aa, 1135 aa, 2151 aa, respectively. Phylogenetic analysis of the nucleotide sequences using the NJ method indicated that H8205 virus, together with the Amur strains isolated from Far-Eastern Russia and Korea, forms a well-supported lineage. Our results will provide insights into the genetic diversity of hantaviruses (HNTV).

The confirmation of three repeated sequence elements in the 3' untranslated region of Chikungunya virus.

In this study, the complete genomic nucleotide sequence of Chikungunya virus (CHIKV) strain S27 African prototype was determined and three 21 nucleotides repeated sequence elements (RSEs) at positions 11398-11418, 11533-11553, and 11620-11640 in the 3' untranslated region (3'UTR) were confirmed. In addition, the 3'UTRs of all CHIKV strains deposited in GenBank were analyzed. The results displayed that the majority of the CHIKV strains consisted of the three 21 nucleotides RSEs in the 3'UTRs, and the third RSE was the most conservative. The conservation of the three RSEs of 21 nucleotides within the 3'UTR of CHIKV genome may play an important role on the virus replication cycle.

[Correlation between feeding index and growth development of 6-36 month-old infants in two counties of western China by applying multiple correspondence analysis].

OBJECTIVE: To explore the correlation between feeding index and growth development status of infants from two counties of western China by applying the method of multiple correspondence analysis. METHODS: Two sample counties were randomly selected from the ones that satisfied the research conditions in Shaanxi province and Chongqing in western China. In the study, 472 premature/low birth weight infants (PLBW) and 461 normal term infants (NT) of 6-36 months from the two counties were investigated from September 2010 to November 2010. The SPSS 19.0 software was applied to analyze the data using general statistical analysis and multiple correspondence analysis. RESULTS: In the two counties of western China, the proportion of infants with feeding index at the medium level was the highest, which was between 50% and 60%. In the PLBW group and the NT group, the proportion of low level of feeding index among 6-9 month-old infants was the highest, and the proportion was 33.3% for the PLBW group and 29.4% for the NT group. For both the PLBW group and the NT group, the distribution of feeding index among the different age groups showed significant difference (P<0.05).Among the infants with low level of feeding index, the growth development of the PLBW lay behind that of the NT. We could see a catching-up trend of the PLBW with medium or good level of feeding index, but their growth development index was still at a lower level than that of the NT with the same level of feeding condition. Through multiple correspondence analyses, the outcomes of PLBW corresponded and strongly correlated with low level of feeding index, low level of growth development index, mother's low education degree and low annual family income. And the outcomes of NT corresponded and strongly correlated with medium/good level of feeding index, medium level of growth development status, mother's medium/high education degree and medium/high level of annual family income. CONCLUSION: There are good correspondence correlations at different hierarchical levels of the infants' group, feeding index, growth development index and family factors in the two counties of western China. Multiple correspondence analysis could directly reveal the correlation among several variables, which is a suitable method for categorical data. The result can be illustrated directly through a two-dimensional graph and could provide the suggestion of feeding practice for different infants in western rural China.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

[The analysis of follow-up results of 612 cases of cholecystolithiasis treated with the minimal invasive operation with gallbladder preserved via choledochoscopy].

OBJECTIVE: To discuss the feasibility of the operation of minimal invasive with gallbladder preserved via choledochoscopy. METHODS: From February 1992 to June 2006, there were 760 patients who underwent cholecystolithiasis treated with the minimal invasive operation with gallbladder preserved via choledochoscopy, among which there were 428 males and 332 females, aged from 18 to 81 years old. All cases were diagnosed by ultrasonography and their gallbladder functions were proved normal by the examination of oral cholecystography or ECT before operation. In the operation gallstones were removed from gallbladder completely. RESULTS: There were 612 cases who were followed up for 1-15 years and the follow-up rate was 80.5%. All patients recovered well after operation. The post-operation rate of recurrence of gallstone was 0.49%, 4.39%, 5.83%, 6.60%, 7.21% and 8.38% within the first year, the second year, the third year, the fifth year, the seventh year and the ninth year respectively, rate of recurrence of gallstone were 10.11% within both the tenth and the fifteenth year. CONCLUSIONS: The minimal invasive operation with gallbladder preserved via choledochoscopy is effective to cholecystolithiasis patients whose gallbladder function is normal. It is a feasible operation that preserves the normal functional gallbladder and improves the patients' life quality.

[A clinical study of adefovir dipivoxil, made in China, for treatment of hepatitis B e antigen-positive patients with chronic hepatitis B].

OBJECTIVE: To evaluate the efficacy and safety of a China made adefovir dipivoxil (ADV) treatment for hepatitis B e antigen-positive patients with chronic hepatitis B. METHODS: Two hundred and thirty patients with chronic hepatitis B who were positive for hepatitis B e antigen (HBeAg) were randomly put into groups A or B, and 58 patients with lamivudine-resistant chronic hepatitis B were randomly put into groups C or D. During the first 12 weeks of the trial, 112 patients in group A and 115 patients in group B received 10 mg of ADV and a placebo once a day; 28 patients in group C received 100 mg of lamivudine (LMV) and 10 mg of ADV; 29 patients in group D received 100 mg of LMV and a placebo once a day. In the second trial period, all patients received ADV for 36 weeks. The primary checking criterion was the serum HBV DNA change during the treatment. The secondary ones were alanine aminotransferase (ALT) normalization, HBeAg loss, and HBeAg seroconversion. RESULTS: At week 12, the median serum hepatitis B virus (HBV) DNA level of group A (ADV-ADV) was reduced 2.8 log10 copies/ml, significantly greater than that of group B (placebo-ADV) of 0.3 log10 copies/ml reduction (P = 0.000). At week 48, the median serum HBV DNA level of group A and group B were reduced 3.6 and 3.4 log10 copies/ml respectively. At week 12, the median serum HBV DNA level of group C (LMV+ADV) was reduced 3.0 log10 copies/ml, significantly greater than that of the group D (LMV+placebo) of 0.16 log10 copies/ml reduction (P = 0.000). At week 48, the median serum HBV DNA level of group C and group D were reduced 3.6 and 3.8 log10 copies/ml respectively. Only 5.56% (16/288) patients had adverse events that were mild to moderate. There was no significant difference in the change of serum creatinine compared with their baseline levels. CONCLUSION: In our HBeAg positive lamivudine-resistant chronic hepatitis B patients, 48 weeks of ADV treatment was safe and resulted in significant virological and biochemical improvements.

[Construction and screening of human phage antibody library against SARS virus].

AIM: To construct human phage antibody library against SARS virus. METHODS: Human Fab genes were amplified by RT-PCR from lymphocytes of a convalescent SARS patient and cloned into vector pComb3 to construct phage antibody library. Antibodies against SARS virus were screened by biopanning with immobilized virus antigen. The binding specificity of phage antibody to SARS virus was detected by ELISA. RESULTS: 13 Ig genes were obtained by PCR. A human phage antibody library consisting of 1.3 x 10(6) clones was constructed by electrotransformation and 60 percent of the clones contained Fab genes. After three rounds of panning with SARS virus antigen, phage antibodies against SARS virus were specifically enriched. ELISA detection verified 10 positive phage antibodies that were highly specific for SARS virus. CONCLUSION: Human phage antibody library against SARS virus has been constructed successfully, from which 10 anti-SARS virus antibodies were obtained.