The relentless march of a highly pathogenic avian influenza virus(HPAIV)strain,known as H5N1,to become an unprecedented panzootic continues unchecked.The leap of H5N1 clade 2.3.4.4b from Eurasia and Africa to North Am...The relentless march of a highly pathogenic avian influenza virus(HPAIV)strain,known as H5N1,to become an unprecedented panzootic continues unchecked.The leap of H5N1 clade 2.3.4.4b from Eurasia and Africa to North America in 2021 and its further spread to South America and the Antarctic have exposed new avian and mammalian populations to the virus and led to outbreaks on an unrivaled scale.The virus has infected wild birds across vast geographic regions and caused wildlife deaths in some of the world's most biodiverse ecosystems.展开更多
[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the s...[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007(H9N2) (A3 for short), A/Duck/FJ/301/2007 (H9N2) (C1 for short), A/Duck/NH/306/2007(H9N2) ( D6 for short), A/duck/SS/402/2007(H9N2) ( E2 for short), and a strain named A/duck/ZC/2007(H9N2) (L1 for short) from a muscovy duck died of avian influenza virus (AIV), were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 (chicken, 2003). By comparison with the NS1 gene sequences of L1, AF523514 (duck), AY664743 (chicken) and EF155262.1 (quail) using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 ( R→Q), 70, 71 ( KE→EG), 86 ( A→S), 124 (V→M) and 225 ( S→N), and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase (PKR) binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.展开更多
All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortalit...All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortality rates. More importantly, influenza A viruses cause respiratory disease in humans with potentially fatal outcome. Local or global outbreaks in humans are typically characterized by excess hospitalizations and deaths. In 1997, highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Hong Kong that transmitted to humans, resulting in the first documented cases of human death by avian influenza virus infection. A new outbreak started in July 2003 in poultry in Vietnam, Indonesia, and Thailand, and highly pathogenic avian H5N1 influenza viruses have since spread throughout Asia and into Europe and Africa. These viruses continue to infect humans with a high mortality rate and cause worldwide concern of a looming pandemic. Moreover, H5N1 virus outbreaks have had devastating effects on the poultry industries throughout Asia. Since H5N1 virus outbreaks appear to originate from Southern China, we here examine H5N1 influenza viruses in China, with an emphasis on their biological properties.展开更多
Since the first human case of H5N1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. The ability of H5N1 t...Since the first human case of H5N1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. The ability of H5N1 to cross species boundaries, and the presence of polymorphisms that enhance virulence, present challenges to developing clear strategies to prevent the pandemic spread of this highly pathogenic avian influenza (HPAI) virus. This review summarizes the current understanding of, and recent research on, the avian influenza H5N1 virus, including transmission, virulence, pathogenesis, clinical characteristics, treatment and prevention.展开更多
In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mu...In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.展开更多
Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-Hke H1N1 viruses, which were initially detected in European pig populations in...Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-Hke H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3' end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.展开更多
Objective: To study the antiviral properties of the five Asian medicinal plants against in vitro infection by the highly pathogenic avian influenza virus(H5N1).Methods: Crude extracts of Andrographis paniculata, Curcu...Objective: To study the antiviral properties of the five Asian medicinal plants against in vitro infection by the highly pathogenic avian influenza virus(H5N1).Methods: Crude extracts of Andrographis paniculata, Curcuma longa(C. longa),Gynostemma pentaphyllum, Kaempferia parviflora(K. parviflora), and Psidium guajava obtained by both water and ethanol extractions were investigated for their cytotoxicity in the Madin–Darby canine kidney cells. Thereafter, they were investigated in vitro for antiviral activity and cytokine response upon H5N1 virus infection.Results: The results revealed that both water and ethanol extracts of all the five studied plants showed significant antiviral activity against H5N1 virus. Among these plants,C. longa and K. parviflora showed strong anti-H5N1 activity. Thus, they were selected for further studies on their cytokine response upon virus infection. It was found that ethanol and water crude extracts of C. longa and K. parviflora induced significant upregulation of TNF-a and IFN-b m RNA expressions, suggesting their roles in the inhibition of H5N1 virus replication.Conclusions: To the best of the authors' knowledge, this study is among the earliest reports to illustrate the antiviral property of these Asian medicinal plants against the highly pathogenic avian H5N1 influenza virus. The results of this study shed light on alternative therapeutic sources for treatment of H5N1 influenza virus infection in the future.展开更多
Objective:To investigate the effects of influenza A virus H1N1 infection on the proliferation and apoptosis of mouse astrocytes cells and its protein expression.Methods:After mouse astrocytes was infected with purifie...Objective:To investigate the effects of influenza A virus H1N1 infection on the proliferation and apoptosis of mouse astrocytes cells and its protein expression.Methods:After mouse astrocytes was infected with purified influenza A virus H1N1 in vitro,viral integration and replication status of the cells were detected by RT-PCR assay,cell proliferation and apoptosis was determined by MTT method and flow cytometry,respectively.Associated protein expression was delected by Western blotting.Results:Agarose gel electrophoresis showed H1N1 virus can infect astrocytes and can be copied.MTT staining showed H1N1 virus infection can inhibit the proliferation of mouse astrocytes,which makes cell viability decreased significantly.Flow cytometry showed that the proportion of Annein V staining positive vascular endothelial cells in the influenza A virus group was significantly higher than that in the control group.Western blot analysis showed after24 h and 32 h of infection,there were cells caspase-3 protein and the expression of its active form(lysed caspase-3 protein)increased.The proportion of Bax/Bcl-2 also increased.Conclusions:Influenza A virus can infect human vascular endothelial cells and proliferation and it can induce apoptosis of endothelial cells.展开更多
Three polysaccharides (EW, EH and EA) were prepared from a red alga Eucheuma denticulatum by sequential extraction with cold water, hot water and sodium hydroxide water solution. Their monosaccharide compositions, r...Three polysaccharides (EW, EH and EA) were prepared from a red alga Eucheuma denticulatum by sequential extraction with cold water, hot water and sodium hydroxide water solution. Their monosaccharide compositions, relative molecular mass and structural characterization were determined by gas chromatography, high performance liquid chromatography, fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy methods. EW was hybrid l/k/v-carrageenan (701/17k/13v-car- rabiose), EH was mainly t-carrageenan, and EA was mainly α-1,4-Glucan (88%) but mixed with small amount of t-carrageenan (12%). The relative molecular mass ofEW, EH and EA was 480, 580 and 510kDa, respectively. The anti-influenza A (H1N1) virus activity of these three polysaccharides was evaluated using the Madin-Darby canine kidney cells model. EW showed good anti-H1N1 virus activity, its ICso was 276.5 μg mL-1, and the inhibition rate to H1N1 virus was 52% when its concentration was 250 μgmL-1. The ICs0 of t-carrageenan EH was 366.4 μgmL1, whereas EA showed lower anti-H1N1 virus activity (IC50〉430μgmL-1). Available data obtained give positive evidence that the hybrid carrageenan EW from Eueheuma denticulatum can be used as potential anti-H1N1 virus inhibitor in future.展开更多
Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of e...Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.展开更多
Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A (H1N1) influenza viruses. Methods A549 cells ...Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A (H1N1) influenza viruses. Methods A549 cells were infected with A/California/07/09 (H1N1) and A/GuangdongBaoan/51/08 (H1N1) respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection (p.i.). Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Results Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. Conclusion The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition .展开更多
The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evoluti...The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evolutionary constraints, host distinction, and co-mutations of influenza. Most previous studies of overlapping genes focused on their unique evolutionary constraints, and very little was achieved to assess the potential impact of the overlap on other biological aspects of influenza. In this study, our aim was to explore the mutual dependence in host differentiation and co-mutations in M, NS, and PB1 of avian, human, 2009 H1N1, and swine viruses, with Random Forests, information entropy, and mutual information. The host markers and highly co-mutated individual sites and site pairs (P values < 0.035) in the three gene segments were identified with their relative significance between the overlapping genes calculated. Further, Random Forests predicted that among the three stop codons in the current PB1-F2 gene of 2009 H1N1, the significance of a mutation at these sites for host differentiation was, in order from most to least, that at 12, 58, and 88, i.e., the closer to the start of the gene the more important the mutation was. Finally, our sequence analysis surprisingly revealed that the full-length PB1-F2, if the three stop codons were all mutated, would function more as a swine protein than a human protein, although the PB1 of 2009 H1N1 was derived from human H3N2.展开更多
As we enter the year of 2011, the 2009 H1N1 pandemic influenza virus is in the news again. At least 20 people have died of this virus in China since the beginning of 2011 and it is now the predominant flu strain in th...As we enter the year of 2011, the 2009 H1N1 pandemic influenza virus is in the news again. At least 20 people have died of this virus in China since the beginning of 2011 and it is now the predominant flu strain in the country. Although this novel virus was quite stable during its run in the flu season of 2009-2010, a genetic variant of this virus was found in Singapore in early 2010, and then in Australia and New Zealand during their 2010 winter influenza season. Several critical mutations in the HA protein of this variant were uncovered in the strains collected from January 2010 to April 2010. Moreover, a structural homology model of HA from the A/Brisbane/10/2010(H1N1) strain was made based on the structure of A/California/04/2009 (H1N1). The purpose of this study was to investigate mutations in the HA protein of 2009 H1N1 from sequence data collected worldwide from May 2010 to February 2011. A fundamental problem in bioinformatics and biology is to find the similar gene sequences for a given gene sequence of interest. Here we proposed the inverse problem, i.e., finding the exemplars from a group of related gene sequences. With a clustering algorithm affinity propagation, six exemplars of the HA sequences were identified to represent six clusters. One of the clusters contained strain A/Brisbane/12/2010(H1N1) that only differed from A/Brisbane/10/2010 in the HA sequence at position 449. Based on the sequence identity of the six exemplars, nine mutations in HA were located that could be used to distinguish these six clusters. Finally, we discovered the change of correlation patterns for the HA and NA of 2009 H1N1 as a result of the HA receptor binding specificity switch, revealing the balanced interplay between these two surface proteins of the virus.展开更多
The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analys...The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.展开更多
Swine influenza A virus(swine IAV) circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A virus...Swine influenza A virus(swine IAV) circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like(EA) H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription(RT)-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets(outer and inner) were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit(PFU) m L^(-1) which was over 10~4 PFU m L^(-1) for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.展开更多
BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppres...BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppression.There are limited reports of 2009 H1N1 influenza in liver transplant recipients,especially in China. METHODS:We present a case of a 48-year-old male liver transplant recipient with 2009 H1N1 influenza A virus.He received therapy for acute rejection after transplantation and was confirmed with H1N1 virus infection. RESULTS:The patient was started on oseltamivir(75 mg, orally twice daily)and had a benign hospital course,with defervescence and resolution of symptoms within 72 hours. The follow-up chest radiograph after discharge was normal. CONCLUSIONS:The 2009 H1N1 influenza in this hospitalized transplant recipient was relatively mild,and prolonged viral shedding was not noted.Oseltamivir can be a valid measure in immunocompromised individuals.展开更多
The pig industry in Colombia has grown 30% in the last decade achieving high levels of technology and efficiency;in spite of that, respiratory diseases remain a constraint. Since 1970, serological evidence and histolo...The pig industry in Colombia has grown 30% in the last decade achieving high levels of technology and efficiency;in spite of that, respiratory diseases remain a constraint. Since 1970, serological evidence and histological findings suggested the role of swine influenza virus (SIV) as part of the porcine respiratory disease complex;nevertheless, elusive and molecular typing isolates are missing. This study was aimed at isolating SIV from intensive pig farms and to achieve molecular characterization to determine strains circulating in the field. In order to accomplish this goal, 242 samples were taken from nasal swabs, 25 from bronchial washes and 8 from lung tissue. Samples were collected during a period of three years, between 2008 and 2010 and were originated from 78 farms of the three main pig production regions of the country. The samples were transported in BHI broth with 2% antibiotic and antimycotic solution and stored at –70?C until processed. The swabs were inoculated in 9 - 11 days old embryo chicken eggs and in MDCK (Madin Darby Canine Kidney) cell cultures with the addition of trypsin. The isolates were identified by the HA (hemoagglutination) test and by RT-PCR targeting the HA (hemagglutinin), NA (Neuraminidase) and M (Matrix) genes. Full length sequence of the HA and NA glycoproteins from four selected virus isolates was conducted (Macrogen?. USA). As a result, fifteen SIV isolates from nine farms distributed in the three regions were obtained. Twelve of the isolates are related to the swine origin H1N1 virus that caused the 2009 influenza pandemic. The remaining three viruses were related to classical swine influenza viruses.展开更多
A recent phylogenetic inference indicated that the 2009 pandemic H1N1 strains circulating from March 2009 to September 2009 could be divided into two closely related but distinct clusters. Cluster one contained most s...A recent phylogenetic inference indicated that the 2009 pandemic H1N1 strains circulating from March 2009 to September 2009 could be divided into two closely related but distinct clusters. Cluster one contained most strains from Mexico, Texas, and California, and cluster two had most strains from New York, both of which were reported to co-circulate in all continents. The same study further revealed nine nucleotide changes in six gene segments of the new virus specific for the two clusters. In the current study, the informational spectrum method (ISM), a bioinformatics technique, was employed to study the receptor binding patterns of the two clusters. It discovered that while both groups shared the same primary human binding affinity, their secondary binding preferences were different. Cluster one favored swine binding as its secondary binding pattern, whereas cluster two mostly exhibited the binding specificity of A/South Carolina/1/18 (H1N1) (one of the 1918 flu pandemic strains) as its secondary binding pattern. Besides all the nine nucleotide changes found in the previous study, Random Forests were applied to uncover several new nucleotide polymorphisms in 10 genes of the strains between the two clusters, and several amino acid changes in the HA protein that might be accountable for the discrepancy of the secondary receptor binding patterns of the two clusters. Finally, entropy analysis was conducted to present a global view of gene sequence variations between the two clusters, which illustrated that cluster one had much higher genetic divergence than cluster two. Furthermore, it suggested a significant overall correspondence between the nucleotide positions of high importance in differentiating the two clusters and nucleotide positions of high entropy in cluster one.展开更多
文摘The relentless march of a highly pathogenic avian influenza virus(HPAIV)strain,known as H5N1,to become an unprecedented panzootic continues unchecked.The leap of H5N1 clade 2.3.4.4b from Eurasia and Africa to North America in 2021 and its further spread to South America and the Antarctic have exposed new avian and mammalian populations to the virus and led to outbreaks on an unrivaled scale.The virus has infected wild birds across vast geographic regions and caused wildlife deaths in some of the world's most biodiverse ecosystems.
基金Supported by Key Specific Program for Science and Technology of Guangdong Province (2008B020700003 A2007A020400006)~~
文摘[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007(H9N2) (A3 for short), A/Duck/FJ/301/2007 (H9N2) (C1 for short), A/Duck/NH/306/2007(H9N2) ( D6 for short), A/duck/SS/402/2007(H9N2) ( E2 for short), and a strain named A/duck/ZC/2007(H9N2) (L1 for short) from a muscovy duck died of avian influenza virus (AIV), were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 (chicken, 2003). By comparison with the NS1 gene sequences of L1, AF523514 (duck), AY664743 (chicken) and EF155262.1 (quail) using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 ( R→Q), 70, 71 ( KE→EG), 86 ( A→S), 124 (V→M) and 225 ( S→N), and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase (PKR) binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.
基金Acknowledgments We thank Susan Watson for editing the manuscript and those in our laboratories who contributed to the data cited in this review. We also thank Ryo Takano for the preparation of figures. Research in HC's group is supported by the Ministry of Science and Technology, China (2004BA519A-57, 2006BAD06A05). Research in GFG's group is supported by the Ministry of Science and Technology, China (MOST, 2005CB523001 and 2006BAD06A01), the National Natural Science Foundation of China (NSFC, Grant #3059934, #30525010) and the US National Institutes of Health (U19 AI051915-05S1). Research in YS's group is supported by the Ministry of Science and Technology, China (MOST, 2005CB523006 and 2006BAD06A15), and the National Natural Science Foundation of China (NSFC, Grant #30599433). Research in YK's group is supported by National Institute of Allergy and Infectious Diseases Public Health Service research grants by CREST and ERATO (Japan Science and Technology Agency), and by grants-in-aid and a contract research fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
文摘All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortality rates. More importantly, influenza A viruses cause respiratory disease in humans with potentially fatal outcome. Local or global outbreaks in humans are typically characterized by excess hospitalizations and deaths. In 1997, highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Hong Kong that transmitted to humans, resulting in the first documented cases of human death by avian influenza virus infection. A new outbreak started in July 2003 in poultry in Vietnam, Indonesia, and Thailand, and highly pathogenic avian H5N1 influenza viruses have since spread throughout Asia and into Europe and Africa. These viruses continue to infect humans with a high mortality rate and cause worldwide concern of a looming pandemic. Moreover, H5N1 virus outbreaks have had devastating effects on the poultry industries throughout Asia. Since H5N1 virus outbreaks appear to originate from Southern China, we here examine H5N1 influenza viruses in China, with an emphasis on their biological properties.
基金National Natural Science Foundation of China (30979144 and 81271821)
文摘Since the first human case of H5N1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. The ability of H5N1 to cross species boundaries, and the presence of polymorphisms that enhance virulence, present challenges to developing clear strategies to prevent the pandemic spread of this highly pathogenic avian influenza (HPAI) virus. This review summarizes the current understanding of, and recent research on, the avian influenza H5N1 virus, including transmission, virulence, pathogenesis, clinical characteristics, treatment and prevention.
文摘In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.
基金Supported by the Natural Science Foundation of Jiangsu Province(BK2009434)the Innovation Platform for Public Health Emergency Preparedness and Response(NO.ZX201109)the Key Medical Talent Foundation of Jiangsu Province(RC2011084)
文摘Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-Hke H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3' end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.
基金supported by the Young Researcher Award of Chiang Mai University grant number R000009357the CMU Mid-Career Research Fellowship Program,Chiang Mai University,Chiang Mai,Thailand
文摘Objective: To study the antiviral properties of the five Asian medicinal plants against in vitro infection by the highly pathogenic avian influenza virus(H5N1).Methods: Crude extracts of Andrographis paniculata, Curcuma longa(C. longa),Gynostemma pentaphyllum, Kaempferia parviflora(K. parviflora), and Psidium guajava obtained by both water and ethanol extractions were investigated for their cytotoxicity in the Madin–Darby canine kidney cells. Thereafter, they were investigated in vitro for antiviral activity and cytokine response upon H5N1 virus infection.Results: The results revealed that both water and ethanol extracts of all the five studied plants showed significant antiviral activity against H5N1 virus. Among these plants,C. longa and K. parviflora showed strong anti-H5N1 activity. Thus, they were selected for further studies on their cytokine response upon virus infection. It was found that ethanol and water crude extracts of C. longa and K. parviflora induced significant upregulation of TNF-a and IFN-b m RNA expressions, suggesting their roles in the inhibition of H5N1 virus replication.Conclusions: To the best of the authors' knowledge, this study is among the earliest reports to illustrate the antiviral property of these Asian medicinal plants against the highly pathogenic avian H5N1 influenza virus. The results of this study shed light on alternative therapeutic sources for treatment of H5N1 influenza virus infection in the future.
文摘Objective:To investigate the effects of influenza A virus H1N1 infection on the proliferation and apoptosis of mouse astrocytes cells and its protein expression.Methods:After mouse astrocytes was infected with purified influenza A virus H1N1 in vitro,viral integration and replication status of the cells were detected by RT-PCR assay,cell proliferation and apoptosis was determined by MTT method and flow cytometry,respectively.Associated protein expression was delected by Western blotting.Results:Agarose gel electrophoresis showed H1N1 virus can infect astrocytes and can be copied.MTT staining showed H1N1 virus infection can inhibit the proliferation of mouse astrocytes,which makes cell viability decreased significantly.Flow cytometry showed that the proportion of Annein V staining positive vascular endothelial cells in the influenza A virus group was significantly higher than that in the control group.Western blot analysis showed after24 h and 32 h of infection,there were cells caspase-3 protein and the expression of its active form(lysed caspase-3 protein)increased.The proportion of Bax/Bcl-2 also increased.Conclusions:Influenza A virus can infect human vascular endothelial cells and proliferation and it can induce apoptosis of endothelial cells.
基金supported by International Science and Technology Collaboration Program of China (2007DFA-30980)Program for Changjiang Scholars,Innovative Research Team in University (IRT0944)+1 种基金Natural Science Foundation of China (31070724)Special Fund for Marine Scientific Research in the Public Interest (201005024)
文摘Three polysaccharides (EW, EH and EA) were prepared from a red alga Eucheuma denticulatum by sequential extraction with cold water, hot water and sodium hydroxide water solution. Their monosaccharide compositions, relative molecular mass and structural characterization were determined by gas chromatography, high performance liquid chromatography, fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy methods. EW was hybrid l/k/v-carrageenan (701/17k/13v-car- rabiose), EH was mainly t-carrageenan, and EA was mainly α-1,4-Glucan (88%) but mixed with small amount of t-carrageenan (12%). The relative molecular mass ofEW, EH and EA was 480, 580 and 510kDa, respectively. The anti-influenza A (H1N1) virus activity of these three polysaccharides was evaluated using the Madin-Darby canine kidney cells model. EW showed good anti-H1N1 virus activity, its ICso was 276.5 μg mL-1, and the inhibition rate to H1N1 virus was 52% when its concentration was 250 μgmL-1. The ICs0 of t-carrageenan EH was 366.4 μgmL1, whereas EA showed lower anti-H1N1 virus activity (IC50〉430μgmL-1). Available data obtained give positive evidence that the hybrid carrageenan EW from Eueheuma denticulatum can be used as potential anti-H1N1 virus inhibitor in future.
基金supported by the General Program of the National Natural Science Foundation of China[No.31570162]the National Key Research Program[No.2016YFC1200200].
文摘Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.
基金supported by the National Basic Research Program of China (973 program: 2010CB534001)
文摘Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A (H1N1) influenza viruses. Methods A549 cells were infected with A/California/07/09 (H1N1) and A/GuangdongBaoan/51/08 (H1N1) respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection (p.i.). Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Results Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. Conclusion The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition .
文摘The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evolutionary constraints, host distinction, and co-mutations of influenza. Most previous studies of overlapping genes focused on their unique evolutionary constraints, and very little was achieved to assess the potential impact of the overlap on other biological aspects of influenza. In this study, our aim was to explore the mutual dependence in host differentiation and co-mutations in M, NS, and PB1 of avian, human, 2009 H1N1, and swine viruses, with Random Forests, information entropy, and mutual information. The host markers and highly co-mutated individual sites and site pairs (P values < 0.035) in the three gene segments were identified with their relative significance between the overlapping genes calculated. Further, Random Forests predicted that among the three stop codons in the current PB1-F2 gene of 2009 H1N1, the significance of a mutation at these sites for host differentiation was, in order from most to least, that at 12, 58, and 88, i.e., the closer to the start of the gene the more important the mutation was. Finally, our sequence analysis surprisingly revealed that the full-length PB1-F2, if the three stop codons were all mutated, would function more as a swine protein than a human protein, although the PB1 of 2009 H1N1 was derived from human H3N2.
文摘As we enter the year of 2011, the 2009 H1N1 pandemic influenza virus is in the news again. At least 20 people have died of this virus in China since the beginning of 2011 and it is now the predominant flu strain in the country. Although this novel virus was quite stable during its run in the flu season of 2009-2010, a genetic variant of this virus was found in Singapore in early 2010, and then in Australia and New Zealand during their 2010 winter influenza season. Several critical mutations in the HA protein of this variant were uncovered in the strains collected from January 2010 to April 2010. Moreover, a structural homology model of HA from the A/Brisbane/10/2010(H1N1) strain was made based on the structure of A/California/04/2009 (H1N1). The purpose of this study was to investigate mutations in the HA protein of 2009 H1N1 from sequence data collected worldwide from May 2010 to February 2011. A fundamental problem in bioinformatics and biology is to find the similar gene sequences for a given gene sequence of interest. Here we proposed the inverse problem, i.e., finding the exemplars from a group of related gene sequences. With a clustering algorithm affinity propagation, six exemplars of the HA sequences were identified to represent six clusters. One of the clusters contained strain A/Brisbane/12/2010(H1N1) that only differed from A/Brisbane/10/2010 in the HA sequence at position 449. Based on the sequence identity of the six exemplars, nine mutations in HA were located that could be used to distinguish these six clusters. Finally, we discovered the change of correlation patterns for the HA and NA of 2009 H1N1 as a result of the HA receptor binding specificity switch, revealing the balanced interplay between these two surface proteins of the virus.
基金supported by the major national S&T projects for infectious diseases(2018ZX10301401)the Key Research&Development Plan of Zhejiang Province(2019C04005)the National Key Research,and the Development Program of China(2018YFC2000500).
文摘The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.
基金supported by the National High-Tech R&D Program of China (2012AA101303)
文摘Swine influenza A virus(swine IAV) circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like(EA) H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription(RT)-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets(outer and inner) were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit(PFU) m L^(-1) which was over 10~4 PFU m L^(-1) for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.
基金supported by a grant from the National Key Technology R&D Program of China(2008ZX10002-26)
文摘BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppression.There are limited reports of 2009 H1N1 influenza in liver transplant recipients,especially in China. METHODS:We present a case of a 48-year-old male liver transplant recipient with 2009 H1N1 influenza A virus.He received therapy for acute rejection after transplantation and was confirmed with H1N1 virus infection. RESULTS:The patient was started on oseltamivir(75 mg, orally twice daily)and had a benign hospital course,with defervescence and resolution of symptoms within 72 hours. The follow-up chest radiograph after discharge was normal. CONCLUSIONS:The 2009 H1N1 influenza in this hospitalized transplant recipient was relatively mild,and prolonged viral shedding was not noted.Oseltamivir can be a valid measure in immunocompromised individuals.
文摘The pig industry in Colombia has grown 30% in the last decade achieving high levels of technology and efficiency;in spite of that, respiratory diseases remain a constraint. Since 1970, serological evidence and histological findings suggested the role of swine influenza virus (SIV) as part of the porcine respiratory disease complex;nevertheless, elusive and molecular typing isolates are missing. This study was aimed at isolating SIV from intensive pig farms and to achieve molecular characterization to determine strains circulating in the field. In order to accomplish this goal, 242 samples were taken from nasal swabs, 25 from bronchial washes and 8 from lung tissue. Samples were collected during a period of three years, between 2008 and 2010 and were originated from 78 farms of the three main pig production regions of the country. The samples were transported in BHI broth with 2% antibiotic and antimycotic solution and stored at –70?C until processed. The swabs were inoculated in 9 - 11 days old embryo chicken eggs and in MDCK (Madin Darby Canine Kidney) cell cultures with the addition of trypsin. The isolates were identified by the HA (hemoagglutination) test and by RT-PCR targeting the HA (hemagglutinin), NA (Neuraminidase) and M (Matrix) genes. Full length sequence of the HA and NA glycoproteins from four selected virus isolates was conducted (Macrogen?. USA). As a result, fifteen SIV isolates from nine farms distributed in the three regions were obtained. Twelve of the isolates are related to the swine origin H1N1 virus that caused the 2009 influenza pandemic. The remaining three viruses were related to classical swine influenza viruses.
文摘A recent phylogenetic inference indicated that the 2009 pandemic H1N1 strains circulating from March 2009 to September 2009 could be divided into two closely related but distinct clusters. Cluster one contained most strains from Mexico, Texas, and California, and cluster two had most strains from New York, both of which were reported to co-circulate in all continents. The same study further revealed nine nucleotide changes in six gene segments of the new virus specific for the two clusters. In the current study, the informational spectrum method (ISM), a bioinformatics technique, was employed to study the receptor binding patterns of the two clusters. It discovered that while both groups shared the same primary human binding affinity, their secondary binding preferences were different. Cluster one favored swine binding as its secondary binding pattern, whereas cluster two mostly exhibited the binding specificity of A/South Carolina/1/18 (H1N1) (one of the 1918 flu pandemic strains) as its secondary binding pattern. Besides all the nine nucleotide changes found in the previous study, Random Forests were applied to uncover several new nucleotide polymorphisms in 10 genes of the strains between the two clusters, and several amino acid changes in the HA protein that might be accountable for the discrepancy of the secondary receptor binding patterns of the two clusters. Finally, entropy analysis was conducted to present a global view of gene sequence variations between the two clusters, which illustrated that cluster one had much higher genetic divergence than cluster two. Furthermore, it suggested a significant overall correspondence between the nucleotide positions of high importance in differentiating the two clusters and nucleotide positions of high entropy in cluster one.