Long non-coding RNA NEAT1 mediates MPTP/MPP+-induced apoptosis via regulating the miR-124/KLF4 axis in Parkinson’s disease

Accumulating evidence suggests that dysregulation of long non-coding RNAs is closely associated with various human diseases, including Parkinson’s disease (PD). However, the role of nuclear-enriched abundant transcript 1 (NEAT1) in the PD process remains unclear. The number of TH+ cells was reduced, and the expression levels of NEAT1 and Krüppel-like factor 4 (KLF4) were increased in the midbrain of MPTP-HCl-treated mice. In addition, the expression of cleaved-caspase-3 (cleaved-casp-3) and Bax (apoptosis-related proteins) was increased, while the expression of Bcl-2 (anti-apoptotic protein) was reduced in MPTP-HCl-treated mice. The expression levels of NEAT1 and KLF4 were increased in MPP+-treated SH-SY5Y cells. Knockdown of NEAT1 promoted cell viability and decreased apoptosis in MPP+-treated SH-SY5Y cells, which could be reversed by upregulating KLF4. KLF4 was verified as a direct target of miR-124, and miR-124 could particularly bind to NEAT1. Downregulation of NEAT1 significantly increased cell viability and decreased apoptosis by regulating miR-124 expression in MPP+-treated SH-SY5Y cells. Additionally, interference of NEAT1 increased the number of TH+ cells and miR-124 expression, while reduced apoptosis and expression of KLF4 in vivo. NEAT1 knockdown increased cell viability and suppressed apoptosis in PD via regulating the miR-124/KLF4 axis, providing a promising avenue for the treatment of PD

Isolation of Tibet orbivirus from Culicoides and associated infections in livestock in Yunnan, China

Abstract Background Culicoides-borne orbiviruses, such as bluetongue virus (BTV) and African horse sickness virus (AHSV), are important pathogens that cause animal epidemic diseases leading to significant loss of domestic animals. This study was conducted to identify Culicoides-borne arboviruses and to investigate the associated infections in local livestock in Yunnan, China. Methods Culicoides were collected overnight in Mangshi City using light traps during August 2013. A virus was isolated from the collected Culicoides and grown using baby hamster kidney (BHK-21), Vero, Madin-Darby bovine kidney (MDBK) and Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by polyacrylamide gel (PAGE) analysis. A full-length cDNA copy of the genome was amplified and sequenced. Serological investigations were conducted in local cattle, buffalo and goat using plaque-reduction neutralization tests. Results We isolated a viral strain (DH13C120) that caused cytopathogenic effects in BHK-21, Vero, MDBK and C6/36 cells. Suckling mice inoculated intracerebrally with DH13C120 showed signs of fatal neurovirulence. PAGE analysis indicated a genome consisting of 10 segments of double-stranded RNA that demonstrated a 3–3–3–1 pattern, similar to the migrating bands of Tibet orbivirus (TIBOV). Phylogenetic analysis of the viral RNA-dependent RNA polymerase (Pol), sub-core-shell (T2, and outer core (T13) proteins revealed that DH13C120 clustered with TIBOV, and the amino acid sequences of DH13C120 virus shared more than 98% identity with TIBOV XZ0906. However, outer capsid protein VP2 and outer capsid protein VP5 shared only 43.1 and 79.3% identity, respectively, indicating that the DH13C120 virus belongs to TIBOV, and it may represent different serotypes with XZ0906. A serosurvey revealed the presence of neutralizing antibodies with 90% plaque-reduction neutralization against TIBOV DH13C120 in local cattle (44%), buffalo (20%), and goat (4%). Four-fold or higher levels of TIBOV-2-neutralizing antibody titers were detected between the convalescent and acute phases of infection in local livestock. Conclusions A new strain of TIBOV was isolated from Culicoides. This study provides the first evidence of TIBOV infection in livestock in Yunnan, China, and suggests that TIBOV could be a potential pathogen in livestock

Isolation and Genetic Characterization of Mangshi Virus: A Newly Discovered Seadornavirus of the Reoviridae Family Found in Yunnan Province, China.

Seadornavirus is a genus of viruses in the family Reoviridae, which consists of Banna virus, Kadipiro virus, and Liao ning virus. Banna virus is considered a potential pathogen for zoonotic diseases. Here, we describe a newly discovered Seadornavirus isolated from mosquitos (Culex tritaeniorhynchus) in Yunnan Province, China, which is related to Banna virus, and referred to as Mangshi virus.The Mangshi virus was isolated by cell culture in Aedes albopictus C6/36 cells, in which it replicated and caused cytopathic effects, but not in mammalian BHK-21 or Vero cells. Polyacrylamide gel analysis revealed a genome consisting of 12 segments of double-stranded RNA, with a "6-4-2" pattern in which the migrating bands were different from those of the Banna virus. Complete genome sequencing was performed by full-length amplification of cDNAs. Sequence analysis showed that seven highly conserved nucleotides and three highly conserved nucleotides were present at the ends of the 5'- and 3'-UTRs in each of 12 genome segments. The amino acid identities of Mangshi virus shared with Balaton virus varied from 27.3% (VP11) to 72.3% (VP1) with Banna virus varying from 18.0% (VP11) to 63.9% (VP1). Phylogenetic analysis based on amino acid sequences demonstrated that Mangshi virus is a member of the genus Seadornavirus and is most closely related to, but distinct from, Balaton virus and Banna virus in the genus Seadornavirus of the family Reoviridae.Mangshi virus isolated from mosquitoes (C. tritaeniorhynchus) was identified as a newly discovered virus in the genus Seadornavirus and is phylogenetically close to Banna virus, suggesting that there is genetic diversity of seadornaviruses in tropical and subtropical areas of Southeast Asia

PCR Identification of DH13M041 virus in the culture supernatant of C6/36 cells using primers derived from segment 12 of BAV by 1% agarose gel electrophoresis (AGE).

<p>1: DH13M127-6; 2: DH13C233-5; 3: DH13M041. 4: Negative control.</p

Alignment of the sequence of guanylyltransferases of members of the family <i>Reoviridae</i> in the <i>Mangshi virus</i> (DH13M041) VP3 with other <i>Seadornaviruses</i>.

<p>(a) Alignments of sequences of guanylyltransferases in the vicinity of the motif Kx(I/V/L)S is shown in bold characters. Similar sequences are shaded. (b) Alignments of sequences of guanylyltransferases in the vicinity of the two histidine residues (bold) involved in the guanylyltransferase activity in BAV, KDV, and LNV. Similar sequences are shaded.</p

Electrophoretic migration patterns of the dsRNA of <i>Mangshi virus</i> (DH13M041) as determined by polyacrylamide gel electrophoresis.

<p>1: DH13M127-6 (BAV); 2:C6/36 Cells control; 3: DH13C233-5 (BAV); 4:DH13M041 (<i>Mangshi virus</i>).</p

The identity of amino acid between the Mangshi virus (DH13M041) and other <i>Seadornaviruses</i>.

<p>Note</p><p>*VP1–VP12 corresponding to Seg-1 to Seg-12 of the Mangshi virus (DH13M041).</p><p>The identity of amino acid between the Mangshi virus (DH13M041) and other <i>Seadornaviruses</i>.</p

Phylogenetic analysis of complete amino acid sequences of corresponding viral proteins (VP, see Table 1) of <i>Mangshi virus</i> (strain DH13M041) using representative members of the species <i>Balaton virus</i>, <i>Banna virus</i> (BAV), <i>Kadipiro virus</i> (KDV) and <i>Liao ning virus</i> (LNV) in the genus <i>Seadornavirus</i>.

<p>(A) RNA-dependent RNA polymerase (VP1); (B) T2 layer of core/subcore (VP2); (C) protein kinase (VP8); (D) outer-coat attachment protein (VP9) of <i>Mangshi virus</i>.</p

CPE caused by <i>Mangshi virus</i> (DH13M041) on C6/36 cells 72 h postinoculation (100×).

<p>CPE caused by <i>Mangshi virus</i> (DH13M041) on C6/36 cells 72 h postinoculation (100×).</p

Lengths of dsRNA segments 1 to 12, encoded putative proteins, 5′ and 3′ NCRs of the <i>Mangshi virus</i> (DH13M041) genome.

<p>Lengths of dsRNA segments 1 to 12, encoded putative proteins, 5′ and 3′ NCRs of the <i>Mangshi virus</i> (DH13M041) genome.</p