Qiang Shao

Zika virus infection disrupts neurovascular development and results in postnatal microcephaly with brain damage

Zika virus (ZIKV) infection of pregnant women can result in fetal brain abnormalities. It has been established that ZIKV disrupts neural progenitor cells (NPCs) and leads to embryonic microcephaly. However, the fate of other cell types in the developing brain and their contributions to ZIKV-associated brain abnormalities remain largely unknown. Using intracerebral inoculation of embryonic mouse brains, we found that ZIKV infection leads to postnatal growth restriction including microcephaly. In addition to cell cycle arrest and apoptosis of NPCs, ZIKV infection causes massive neuronal death and axonal rarefaction, which phenocopy fetal brain abnormalities in humans. Importantly, ZIKV infection leads to abnormal vascular density and diameter in the developing brain, resulting in a leaky blood–brain barrier (BBB). Massive neuronal death and BBB leakage indicate brain damage, which is further supported by extensive microglial activation and astrogliosis in virally infected brains. Global gene analyses reveal dysregulation of genes associated with immune responses in virus-infected brains. Thus, our data suggest that ZIKV triggers a strong immune response and disrupts neurovascular development, resulting in postnatal microcephaly with extensive brain damage. Highlighted article: A postnatal model for ZIKV infection reveals blood-brain barrier leakage, neuronal death, apoptosis and cell cycle arrest of NPCs, leading to microcephaly with brain damage in ZIKV-infected pups.

Function of duck RIG-I in induction of antiviral response against IBDV and avian influenza virus on chicken cells.

The avian influenza (AI) H9N2 virus and IBDV are two major problems in the poultry industry. They have been prevalent among domestic poultry in Asia for many years and have caused considerable economic losses. Retinoic-acid-induced gene I (RIG-I) is a cytoplasmic sensor of dsRNA and ssRNA. It can detect Encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) in human cells, influenza virus in duck leads to production of IFN-β and IFN-stimulated antiviral genes and reductions in the replication of RNA virus. Chickens, which lack RIG-I, are more sensitive to influenza virus than ducks. However, little is known about the roles of duck RIG-I (dRIG-I) in the detection of IBDV and AI H9N2 in chicken cells DF-1. The purpose of this study was to examine the function of dRIG-I in the recognition of IBDV Ts strain and H9N2 A/Chicken/Shandong/ZB/2007(ZB07) and in the induction of antiviral gene expression to gain an understanding of antiviral ability of dRIG-I in chicken cells against dsRNA virus IBDV and ssRNA virus ZB07. After challenge with the IBDV Ts strain and ZB07 the expression levels of Type I IFN (IFN-β and IFN-α) and IFN-induced antiviral genes (Mx and PKR) were significantly up-regulated in dRIG-I-transfected DF-1cells compared with the empty-vector-transfected control. dRIG-I knockdown experiments further proved that dRIG-I is essential to sensing IBDV and ZB07 in duck embryo fibroblasts (DEF). Growth curves showed that dRIG-I repressed the replication of IBDV and almost blunted the growth of ZB07 in DF-1. Apoptosis analysis revealed that dRIG-I increase the number of the survival cells after IBDV Ts strain or ZB07 infection relative to the empty-vector-transfected control. These results indicate that dRIG-I can up-regulates type I IFN and reduce viral gene expression and viral replication and protect chicken cells from virus-induced apoptosis during ZB07 and IBDV infection.

Transgenic quail production by microinjection of lentiviral vector into the early embryo blood vessels.

Several strategies have been used to generate transgenic birds. The most successful method so far has been the injection of lentiviral vectors into the subgerminal cavity of a newly laid egg. We report here a new, easy and effective way to produce transgenic quails through direct injection of a lentiviral vector, containing an enhanced-green fluorescent protein (eGFP) transgene, into the blood vessels of quail embryos at Hamburger-Hamilton stage 13–15 (HH13–15). A total of 80 embryos were injected and 48 G0 chimeras (60%) were hatched. Most injected embryo organs and tissues of hatched quails were positive for eGFP. In five out of 21 mature G0 male quails, the semen was eGFP-positive, as detected by polymerase chain reaction (PCR), indicating transgenic germ line chimeras. Testcross and genetic analyses revealed that the G0 quail produced transgenic G1 offspring; of 46 G1 hatchlings, 6 were transgenic (6/46, 13.0%). We also compared this new method with the conventional transgenesis using stage X subgerminal cavity injection. Total 240 quail embryos were injected by subgerminal cavity injection, of which 34 (14.1%) were hatched, significantly lower than the new method. From these hatched quails semen samples were collected from 19 sexually matured males and tested for the transgene by PCR. The transgene was present in three G0 male quails and only 4/236 G1 offspring (1.7%) were transgenic. In conclusion, we developed a novel bird transgenic method by injection of lentiviral vector into embryonic blood vessel at HH 13–15 stage, which result in significant higher transgenic efficiency than the conventional subgerminal cavity injection.

The African Zika virus MR-766 is more virulent and causes more severe brain damage than current Asian lineage and dengue virus

The Zika virus (ZIKV) has two lineages, Asian and African, and their impact on developing brains has not been compared. Dengue virus (DENV) is a close family member of ZIKV and co-circulates with ZIKV. Here, we performed intracerebral inoculation of embryonic mouse brains with dengue virus 2 (DENV2), and found that DENV2 is sufficient to cause smaller brain size due to increased cell death in neural progenitor cells (NPCs) and neurons. Compared with the currently circulating Asian lineage of ZIKV (MEX1-44), DENV2 grows slower, causes less neuronal death and fails to cause postnatal animal death. Surprisingly, our side-by-side comparison uncovered that the African ZIKV isolate (MR-766) is more potent at causing brain damage and postnatal lethality than MEX1-44. In comparison with MEX1-44, MR-766 grows faster in NPCs and in the developing brain, and causes more pronounced cell death in NPCs and neurons, resulting in more severe neuronal loss. Together, these results reveal that DENV2 is sufficient to cause smaller brain sizes, and suggest that the ZIKV African lineage is more toxic and causes more potent brain damage than the Asian lineage. Highlighted Article: African Zika virus MR-766 causes more cell death in NPCs and neurons in the developing brain, leading to more potent brain damage than Asian strain MEX1-44 and dengue virus.

Changes of CD4+CD25+ cells ratio in immune organs from chickens challenged with infectious bursal disease virus strains with varying virulences.

In the current study, we investigate changes in CD4+CD25+ cells in chickens during infectious bursal disease virus (IBDV) infection. The percentage of CD4+CD25+ cells in lymph organs, e.g., the thymus, spleen, bursa of Fabricius and peripheral blood, during the first 1–5 days post infection (dpi) was assessed by flow cytometry. The data revealed a remarkable decrease in the percentage of CD4+CD25+ cells in the thymus from 1 to 5 dpi and in the spleen during early infection. An increase of the percentage of CD4+CD25+ cells among peripheral blood lymphocytes was observed during the first two days of IBDV infection. Additionally, CD4+CD25+ cells infiltrated the bursa along with CD4+ cells after IBDV infection. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to measure the mRNA levels of immune-related cytokines in IBDV-infected thymus and bursa of Fabricius tissues. The data revealed that IBDV caused a significant increase in interleukin (IL)-10 mRNA levels, with the Harbin-1 strain (vvIBDV) inducing higher IL-10 expression than the Ts strain. Taken together, our data suggest that chicken CD4+CD25+ cells may participate in IBDV pathogenicity by migrating from their sites of origin and storage, the thymus and spleen, to the virally targeted bursa of Fabricius during IBDV infection.

Pigeon RIG-I Function in Innate Immunity against H9N2 IAV and IBDV

Retinoic acid-inducible gene I (RIG-I), a cytosolic pattern recognition receptor (PRR), can sense various RNA viruses, including the avian influenza virus (AIV) and infectious bursal disease virus (IBDV), and trigger the innate immune response. Previous studies have shown that mammalian RIG-I (human and mice) and waterfowl RIG-I (ducks and geese) are essential for type I interferon (IFN) synthesis during AIV infection. Like ducks, pigeons are also susceptible to infection but are ineffective propagators and disseminators of AIVs, i.e., “dead end” hosts for AIVs and even highly pathogenic avian influenza (HPAI). Consequently, we sought to identify pigeon RIG-I and investigate its roles in the detection of A/Chicken/Shandong/ZB/2007 (H9N2) (ZB07), Gansu/Tianshui (IBDV TS) and Beijing/CJ/1980 (IBDV CJ-801) strains in chicken DF-1 fibroblasts or human 293T cells. Pigeon mRNA encoding the putative pigeon RIG-I analogs was identified. The exogenous expression of enhanced green fluorescence protein (EGFP)-tagged pigeon RIG-I and caspase activation and recruitment domains (CARDs), strongly induced antiviral gene (IFN-β, Mx, and PKR) mRNA synthesis, decreased viral gene (M gene and VP2) mRNA expression, and reduced the viral titers of ZB07 and IBDV TS/CJ-801 virus strains in chicken DF-1 cells, but not in 293T cells. We also compared the antiviral abilities of RIG-I proteins from waterfowl (duck and goose) and pigeon. Our data indicated that waterfowl RIG-I are more effective in the induction of antiviral genes and the repression of ZB07 and IBDV TS/CJ-801 strain replication than pigeon RIG-I. Furthermore, chicken melanoma differentiation associated gene 5(MDA5)/ mitochondrial antiviral signaling (MAVS) silencing combined with RIG-I transfection suggested that pigeon RIG-I can restore the antiviral response in MDA5-silenced DF-1 cells but not in MAVS-silenced DF-1 cells. In conclusion, these results demonstrated that pigeon RIG-I and CARDs have a strong antiviral ability against AIV H9N2 and IBDV in chicken DF-1 cells but not in human 293T cells.

RIG-I from waterfowl and mammals differ in their abilities to induce antiviral responses against influenza A viruses.

The retinoic acid-induced gene I (RIG-I) plays a crucial role in sensing viral RNA and IFN-β production. RIG-I varies in length and sequence between different species. We assessed the functional differences between RIG-I proteins derived from mammals and birds. The transfection of duck caspase recruitment domains (CARDs) and duck RIG-I (dCARDs and dRIG-I) and goose CARDs and goose RIG-I (gCARDs and gRIG-I) into chicken DF-1 cells increased the production of IFN-β mRNA and IFN-stimulated genes and decreased influenza A virus (IAV) replication; whereas human CARDs and RIG-I (hCARDs and hRIG-I) and mouse CARDs and RIG-I (mCARDs and mRIG-I) had no effect. In human 293T and A549 cells, hCARDs had the strongest IFN-inducing activity, followed by mCARDs, dCARDs and gCARDs. The IFN-inducing activity of hRIG-I was stronger than that of mRIG-I, dRIG-I and gRIG-I, in that order. The results showed that, although the ability of dCARDs to activate IFN was stronger than that of gCARDs in DF-1, 293T and A549 cells, dRIG-I had a weaker ability to activate IFN than gRIG-I in DF-1 cells with or without IAV infection. These data suggest that RIG-I proteins from different species have different amino acid sequences and functions. This genetic and functional diversity renders RIG-I flexible, adaptable and capable of recognizing many viruses in different species.

Specific Inhibitory Effect of κ-Carrageenan Polysaccharide on Swine Pandemic 2009 H1N1 Influenza Virus.

The 2009 influenza A H1N1 pandemic placed unprecedented demands on antiviral drug resources and the vaccine industry. Carrageenan, an extractive of red algae, has been proven to inhibit infection and multiplication of various enveloped viruses. The aim of this study was to examine the ability of κ-carrageenan to inhibit swine pandemic 2009 H1N1 influenza virus to gain an understanding of antiviral ability of κ-carrageenan. It was here demonstrated that κ-carrageenan had no cytotoxicity at concentrations below 1000 μg/ml. Hemagglutination, 50% tissue culture infectious dose (TCID50) and cytopathic effect (CPE) inhibition assays showed that κ-carrageenan inhibited A/Swine/Shandong/731/2009 H1N1 (SW731) and A/California/04/2009 H1N1 (CA04) replication in a dose-dependent fashion. Mechanism studies show that the inhibition of SW731 multiplication and mRNA expression was maximized when κ-carrageenan was added before or during adsorption. The result of Hemagglutination inhibition assay indicate that κ-carrageenan specifically targeted HA of SW731 and CA04, both of which are pandemic H1N/2009 viruses, without effect on A/Pureto Rico/8/34 H1N1 (PR8), A/WSN/1933 H1N1 (WSN), A/Swine/Beijing/26/2008 H1N1 (SW26), A/Chicken/Shandong/LY/2008 H9N2 (LY08), and A/Chicken/Shandong/ZB/2007 H9N2 (ZB07) viruses. Immunofluorescence assay and Western blot showed that κ-carrageenan also inhibited SW731 protein expression after its internalization into cells. These results suggest that κ-carrageenan can significantly inhibit SW731 replication by interfering with a few replication steps in the SW731 life cycles, including adsorption, transcription, and viral protein expression, especially interactions between HA and cells. In this way, κ-carrageenan might be a suitable alternative approach to therapy meant to address anti-IAV, which contains an HA homologous to that of SW731.

Production of chimeras between the Chinese soft-shelled turtle and Peking duck through transfer of early blastoderm cells

SUMMARY Chimeras are useful models for studies of developmental biology and cell differentiation. Intraspecies and interspecies germline chimeras have been produced in previous studies, but the feasibility of producing chimeras between animals of two different classes remains unclear. To address this issue, we attempted to produce chimeras between the Chinese soft-shelled turtle and the Peking duck by transferring stage X blastoderm cells to recipient embryos. We then examined the survival and development of the PKH26-labeled donor cells in the heterologous embryos. At early embryonic stages, both turtle and duck donor cells that were labeled with PKH26 were readily observed in the brain, neural tube, heart and gonads of the respective recipient embryos. Movement of turtle donor-derived cells was observed in the duck host embryos after 48 h of incubation. Although none of the hatchlings presented a chimeric phenotype, duck donor-derived cells were detected in a variety of organs in the hatchling turtles, particularly in the gonads. Moreover, in the hatched turtles, mRNA expression of tissue-specific duck genes MEF2a and MEF2c was detected in many tissues, including the muscle, heart, small and large intestines, stomach and kidney. Similarly, SPAG6 mRNA was detected in a subset of turtle tissues, including the gonad and the small and large intestines. These results suggest that duck donor-derived cells can survive and differentiate in recipient turtles; however, no turtle-derived cells were detected in the hatched ducks. Our findings indicate that chimeras can be produced between animals of two different classes.

Expression of stem cell pluripotency factors during regeneration in the earthworm Eisenia foetida

Stem cell pluripotency factors can induce somatic cells to form induced pluripotent stem cells, which are involved in cell reprogramming and dedifferentiation. The tissue regeneration in the earthworm Eisenia foetida may involve cell dedifferentiation. There is limited information about associations between pluripotency factors and the regeneration. In this report, cDNA sequences of pluripotency factors, oct4, nanog, sox2, c-myc and lin28 genes from the earthworm E. foetida were cloned, and quantitative PCR analysis was performed for their mRNA expressions in the head, clitellum and tail. The maximum up-regulation of oct4, nanog, sox2, c-myc and lin28 occurred at 12h, 4 days, 12h, 2 days, and 24h after amputation for 110, 178, 21, 251 and 325-fold, respectively, in comparison with the controls. The results suggest that the tissues are regenerated via cellular dedifferentiation and reprogramming.