[Show abstract][Hide abstract] ABSTRACT: Adamantanes (amantadine and rimantadine) have been used to prevent and treat influenza A virus infections for many years; however, resistance to these drugs has been widely reported in the world. To investigate the frequency and distribution of M2 gene mutations in adamantane-resistant influenza variants circulated in the world between 1902 and 2013, 31251 available M2 protein sequences from different HA-subtype influenza A viruses (H1-H17) were analyzed and adamantane resistance-associated mutations were compared (L26F, V27A, A30T, A30V, S31N, G34E, and L38F). We find that 45.2% (n = 14132) of influenza A (H1-H17) viruses circulating globally were resistant to adamantanes, and the vast majority of resistant viruses (95%) bear S31N mutations. Whereas, only about 1% have V27A mutations and other mutations (L26F, A30T, G34E, and L38F) were extremely rare (their prevalence appeared to be < 0.2%). Our results confirm that H1, H3, H5, H7, H9, and H17 subtype influenza A viruses exhibit high-level resistance to adamantanes. In contrast, the appearance of adamantane-resistant mutants in H2, H4, H6, H10, and H11 subtypes was rare. However, no adamantane resistance viruses were identified among other HA subtypes (H8, H12-H16). Our findings indicate that the frequency and distribution of adamantane-resistant influenza variants varied among different HA subtypes, host species, years of isolation, and geographical areas. This comprehensive study raises concerns about the increasing prevalence of adamantane-resistant influenza A viruses and highlights the importance of monitoring the emergence and worldwide spread of adamantane-resistant variants.
[Show abstract][Hide abstract] ABSTRACT: Objective
To assess the long-term effects of repeated controlled ovarian hyperstimulation (COH) on ovarian structure and function.
Adult female rhesus macaques.
A repeated COH rhesus macaque model (superovulation group) with spontaneously ovulating macaques used as controls (normal group) and samples of serum and ovarian tissue collected over a 5-year period.
Main Outcome Measure(s)
Steroid hormone levels, and structural, functional, and protein changes in ovaries.
The follicular histology, proportion of follicles at each developmental stage, and expression levels of oocyte-specific genes showed no statistically significant differences between the two groups. However, the superovulation group exhibited mitochondrial abnormalities in the granulosa cells and a low expression of genes involved in steroid hormone synthesis compared with the normal group. A comparison of protein expression in the ovaries of both groups using tandem mass tag labeling with mass spectrometry revealed that most of the differentially-expressed proteins were down-regulated in the superovulation group. These proteins were mainly localized in the mitochondria and cytosol, and changes in protein levels in the superovulation group mainly inhibited cell proliferation and differentiation but promoted apoptosis.
Our study indicates that repeated COH could change the expression of many proteins in the ovaries even after several years, potentially affecting the development and function of ovarian cells.
Full-text · Article · Sep 2014 · Fertility and Sterility
[Show abstract][Hide abstract] ABSTRACT: Highly pathogenic H5N1 avian influenza viruses have spread in poultry and wild birds in Asia, Europe, and Africa since 2003. To evaluate the role of quails in the evolution of influenza A virus, we characterized three H5N1 viruses isolated from quails (QA viruses) in southern China. Phylogenetic analysis indicated that three QA viruses derived from the A/goose/Guangdong/1/96-like lineage and most closely related to HA clade 4 A/chicken/Hong Kong/31.4/02-like viruses. Molecular analysis suggested that QA viruses and clade 4 H5N1 viruses carried consistent residue signatures, such as the characteristic M2 Ser31Asn amantadine-resistance mutation, implying a common origin of these viruses. As revealed by viral pathogenicity tests, these QA viruses could replicate in intranasally infected mice, but were not lethal to them, showing low pathogenicity in mammals. However, they killed all intravenously inoculated chickens, showing high pathogenicity in poultry. Results from amantadine sensitivity tests of wild-type QA viruses and their reverse genetic viruses demonstrated that all QA viruses were resistant to amantadine, and the M2 Ser31Asn mutation was determined as the most likely cause of the increased amantadine-resistance of H5N1 QA viruses. Our study confirmed experimentally that the amino acid at residue 31 in the M2 protein plays a major role in determining the amantadine-resistance phenotype of H5N1 influenza viruses. Our findings provide further evidence that quails may play important roles in the evolution of influenza A viruses, which raises concerns over possible transmissions of H5N1 viruses among poultry, wild birds, and humans.
[Show abstract][Hide abstract] ABSTRACT: Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.
[Show abstract][Hide abstract] ABSTRACT: Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.
No preview · Article · Sep 2013 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: Swine play an important role in the disease ecology of influenza. Swine may provide the potential for mixed infections and genetic reassortment between avian, human, and porcine influenza viruses. We investigated the prevalence of antibodies to swine H1 and H3 influenza viruses and avian H5 and H9 influenza viruses in feral swine in southern China. Serum samples were collected from 31 feral swine harvested in 2009 in southern China. Of 31 serum samples tested, 14 (45%) had detectable antibody to H1 influenza virus and 23 (74%) were positive for H3 subtype. The antibody prevalence against both the swine H1 virus and the swine H3 virus was 45% (14/31). Five samples were reactive with both H1 and N1 subtype viruses, suggesting exposure to H1N1 viruses. All the sera tested were negative for avian H5 and H9 influenza viruses. Further investigations of influenza virus exposure of feral swine are needed to clarify their role in influenza ecology.
No preview · Article · Apr 2013 · Journal of wildlife diseases
[Show abstract][Hide abstract] ABSTRACT: H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies (Pica pica) in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2, PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1) PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46) discovered in our recent study. Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants. However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice. Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.
[Show abstract][Hide abstract] ABSTRACT: H9N2 influenza A viruses have become established worldwide in terrestrial poultry and wild birds, and are occasionally transmitted to mammals including humans and pigs. To comprehensively elucidate the genetic and evolutionary characteristics of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571 viral genomes from the NCBI Influenza Virus Resource Database, representing the spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses, and for describing the history of H9N2 viruses circulating in diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2 viral genomes were classified into 74 separate lineages, which had marked host and geographical differences in phylogeny. Panorama genotypical analysis also revealed that H9N2 viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series (A-G). Phylogenetic analysis of the internal genes showed that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that H9N2 viruses have undergone extensive reassortments to generate multiple reassortants and genotypes, suggesting that the continued circulation of multiple genotypical H9N2 viruses throughout the world in diverse hosts has the potential to cause future influenza outbreaks in poultry and epidemics in humans. We propose a nomenclature system for identifying and unifying all lineages and genotypes of H9N2 influenza viruses in order to facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses.
[Show abstract][Hide abstract] ABSTRACT: Highly pathogenic H5N1 avian influenza is considered an avian disease, although there is some evidence of limited human-to-human
transmission of the virus. A global effort is underway to control or eradicate the highly pathogenic H5N1 avian influenza
virus in poultry and prevent human exposure, both of which may also reduce the risk of pandemic emergence. Hemagglutinin gene
sequences from 215 human H5N1 influenza viruses were used to trace the source and dispersal pattern of human H5N1 influenza
viruses on a global scale. A mutation network and phylogenetic analyses of the hemagglutinin gene show that human H5N1 influenza
viruses can be clearly divided among 4 clusters across geographic space. On the basis of analysis of the N-glycosylation sites
at positions 100 and 170 in the hemagglutinin protein, human H5N1 influenza viruses were also divided into 3 types. When we
combined these analyses with geographic information system data analyses, we found that Southern China is often a common source
of multiple clusters of H5N1 influenza viruses and that each cluster has different dispersal patterns and individual evolutionary
features. In summary, the genetic evidence presented here provides clear evidence for multiple clusters of human H5N1 influenza
viruses that initially originated in Southern China.
Full-text · Article · Aug 2010 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome (PRRS) has now been widely recognized as an economically important disease. The objective of this study was to compare the molecular and biological characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) field isolates in China to those of the modified live virus (MLV) PRRS vaccine and its parent strain (ATCC VR2332).
Five genes (GP2, GP3, GP4, GP5 and NSP2) of seven isolates of PRRSV from China, designated LS-4, HM-1, HQ-5, HQ-6, GC-2, GCH-3 and ST-7/2008, were sequenced and analyzed. Phylogenetic analyses based on the nucleotide sequence of the ORF2-5 and NSP2 showed that the seven Chinese isolates belonged to the same genetic subgroup and were related to the North American PRRSV genotype. Comparative analysis with the relevant sequences of another Chinese isolate (BJ-4) and North American (VR2332 and MLV) viruses revealed that these isolates have 80.8-92.9% homology with VR-2332, and 81.3-98.8% identity with MLV and 80.7-92.9% with BJ-4. All Nsp2 nonstructural protein of these seven isolates exhibited variations (a 29 amino acids deletion) in comparison with other North American PRRSV isolates. Therefore, these isolates were novel strain with unique amino acid composition. However, they all share more than 97% identity with other highly pathogenic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and the Nsp2 protein when compared with the previous isolates.
These results might be useful to study the genetic diversity of PRRSV in China and to track the infection sources as well as for vaccines development.
[Show abstract][Hide abstract] ABSTRACT: Subtypes of H1N1 influenza virus can be found in humans in North America, while they are also associated with the infection
of swine. Characterization of the genotypes of viral strains in human populations is important to understand the source and
distribution of viral strains. Genomic and protein sequences of 10 isolates of the 2009 outbreak of influenza A (H1N1) virus
in North America were obtained from GenBank database. To characterize the genotypes of these viruses, phylogenetic trees of
genes PB2, PB1, PA, HA, NP, NA, NS and M were constructed by Phylip3.67 program and N-Linked glycosylation sites of HA, NA, PB2, NS1 and M2 proteins were analyzed online by NetNGlyc1.0 program. Phylogenetic
analysis indicated that these isolates are virtually identical but may be recombinant viruses because their genomic fragments
come from different viruses. The isolates also contain a characteristic lowly pathogenic amino acid motif at their HA cleavage
sites (IPSIQSR↓GL), and an E residue at position 627 of the PB2 protein which shows its high affinity to humans. The homologous
model of M proteins showed that the viruses had obtained the ability of anti-amantadine due to the mutation at the drug-sensitive
site, while sequence analysis of NA proteins indicated that the viruses are still susceptible to the neuraminidase inhibitor
drug (i.e. oseltamivir and zanamivir) because no mutations have been observed. Our results strongly suggested that the viruses
responsible for the 2009 outbreaks of influenza A (H1N1) virus have the ability to cross species barriers to infect human
and mammalian animals based on molecular analysis. These findings may further facilitate the therapy and prevention of possible
transmission from North America to other countries.
No preview · Article · Jul 2009 · Chinese Science Bulletin