Complete genome analysis of African swine fever virus responsible for outbreaks in domestic pigs in 2018 in Burundi and 2019 in Malawi.

Several African swine fever (ASF) outbreaks in domestic pigs have been reported in Burundi and Malawi and whole-genome sequences of circulating outbreak viruses in these countries are limited. In the present study, complete genome sequences of ASF viruses (ASFV) that caused the 2018 outbreak in Burundi (BUR/18/Rutana) and the 2019 outbreak in Malawi (MAL/19/Karonga) were produced using Illumina next-generation sequencing (NGS) platform and compared with other previously described ASFV complete genomes. The complete nucleotide sequences of BUR/18/Rutana and MAL/19/Karonga were 176,564 and 183,325 base pairs long with GC content of 38.62 and 38.48%, respectively. The MAL/19/Karonga virus had a total of 186 open reading frames (ORFs) while the BUR/18/Rutana strain had 151 ORFs. After comparative genomic analysis, the MAL/19/Karonga virus showed greater than 99% nucleotide identity with other complete nucleotides sequences of p72 genotype II viruses previously described in Tanzania, Europe and Asia including the Georgia 2007/1 isolate. The Burundian ASFV BUR/18/Rutana exhibited 98.95 to 99.34% nucleotide identity with genotype X ASFV previously described in Kenya and in Democratic Republic of the Congo (DRC). The serotyping results classified the BUR/18/Rutana and MAL/19/Karonga ASFV strains in serogroups 7 and 8, respectively. The results of this study provide insight into the genetic structure and antigenic diversity of ASFV strains circulating in Burundi and Malawi. This is important in order to understand the transmission dynamics and genetic evolution of ASFV in eastern Africa, with an ultimate goal of designing an efficient risk management strategy against ASF transboundary spread.

Avian reovirus nonstructural protein p17-induced G(2)/M cell cycle arrest and host cellular protein translation shutoff involve activation of p53-dependent pathways.

The effects of avian reovirus (ARV) p17 protein on cell cycle progression and host cellular protein translation were studied. ARV infection and ARV p17 transfection resulted in the accumulation of infected and/or transfected cells in the G(2)/M phase of the cell cycle. The accumulation of cells in the G(2)/M phase was accompanied by upregulation and phosphorylation of the G(2)/M-phase proteins ATM, p53, p21(cip1/waf1), Cdc2, cyclin B1, Chk1, Chk2, and Cdc25C, suggesting that p17 induces a G(2)/M cell cycle arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/cyclin B1 and ATM/Chk1/Chk2/Cdc25C pathways. The G(2)/M cell cycle arrest resulted in increased virus replication. In the present study, we also provide evidence demonstrating that p17 protein is responsible for ARV-induced host cellular protein translation shutoff. Increased phosphorylation levels of the eukaryotic translation elongation factor 2 (eEF2) and initiation factor eIF2alpha and reduced phosphorylation levels of the eukaryotic translation initiation factors eIF4E, eIF4B, and eIF4G, as well as 4E-BP1 and Mnk-1 in p17-transfected cells, demonstrated that ARV p17 suppresses translation initiation factors and translation elongation factors to induce host cellular protein translation shutoff. Inhibition of mTOR by rapamycin resulted in a decrease in the levels of phosphorylated 4E-BP1, eIF4B, and eIF4G and an increase in the levels eEF2 but did not affect ARV replication, suggesting that ARV replication was not hindered by inhibition of cap-dependent translation. Taken together, our data indicate that ARV p17-induced G(2)/M arrest and host cellular translation shutoff resulted in increased ARV replication.

Recent patents on cell cycle regulatory proteins.

Cell cycle progression and cell division are driven by the sequential activation of a group of serine-threonine kinases called cyclin-dependent kinases (Cdks). Multiple Cdks control the cell cycle in mammals and have been long considered essential for normal proliferation, development and homeostasis. The importance of the Cdk-cyclin complexes in cell proliferation is underscored by the fact that deregulation of the Cdk activity is found in virtually the whole spectrum of human tumors. Advances in the cell cycle proteins in the last 25 years, since the discovery of cyclins, have been discussed and have shed even more light on this essential life sustaining process. Recent information from different models for the various cyclins and Cdks have made some of the generally accepted concepts of cell cycle regulation to be revised and new and exciting questions to be investigated. There is also increasing evidence that suggests that Cdks such as Cdc2 are also commonly targeted by viral proteins, which modulate host cell cycle machinery to benefit viral survival or replication. This review, describes some of the most recent and important US patents related to cell cycle regulation and those on viral proteins involved in cell cycle modulation particularly the G2/M phase transition and cancer therapy.

Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of Newcastle disease virus.

Due to appearance of new genotypes of Newcastle disease virus (NDV) with no cross-protection and with vaccine strains, some outbreaks have been reported in Taiwan that caused significant damage to the poultry industry. A reliable assay protocol, (RAPID)-bioactive amplification with probing (BAP), for detection of NDV that uses a nested PCR and magnetic bead-based probe to increase sensitivity and specificity, was developed. Primers and probes were designed based on the conserved region of the F protein-encoding gene sequences of all NDV Taiwan isolates. The optimal annealing temperature for nested reverse transcription-polymerase chain reaction (RT-PCR) to amplify the gene was 61 degrees C and optimal hybridization occurred when buffer 1x SSC and 0.5% SDS were used at 50 degrees C. The sensitivity of RAPID-BAP was 1 copy/microl for standard plasmids and 10 copy/mul for transcribed F protein-encoding gene of NDV with comparable linearity (R(2)=0.984 versus R(2)=0.99). This sensitivity was superior to that of other techniques currently used. The assay was also highly specific because the negative controls, including classical swine fever virus, avian influenza virus, avian reovirus, and infectious bursa disease virus could not be detected. Thirty-four field samples were tested using conventional RT-PCR, nested RT-PCR, real-time quantitative RT-PCR, and RAPID-BAP assay and the positive rates were 24%, 30%, 41%, and 53%, respectively. The developed assay allows for rapid, correct, and sensitive detection of NDV and fulfils all of the key requirements for clinical applicability. It could reliably rule out false negative results from antibody-based assays and also facilitate a rapid diagnosis in the early phase of the disease for emergency quarantine that may help prevent large-scale outbreaks.

Suppression of protein expression of three avian reovirus S-class genome segments by RNA interference.

RNA interference was used to suppress protein expression of three S-class genome segments of avian reovirus (ARV). Viral progeny titer was successfully down-regulated by RNA interference. Suppression of S1 genome segment, which has three open reading frames, not only decreased the expression level of the structural protein sigmaC but also reduced cell fusion and the level of Ser(15)-phosphorylated p53 protein caused by the nonstructural proteins p10 and p17, respectively. Suppression of S2 or S4 genome segment by RNA interference could also reduce the expression level of sigmaA or sigmaNS. Interestingly, suppression of sigmaNS resulted in down regulation of the expression of other viral products. In terms of variability of different genes among viral strains and of the impact after their suppression, it seems that the viral products involved in construction of viroplasm or core particles, like sigmaNS, are considerable choices to efficiently inhibit ARV multiplication by RNA interference. Using a GFP reporter system, it was discovered that ARV could not inhibit activated RNA interference, suggesting that RNA interference may be used in the suppression of ARV infection.

Apoptosis induction by avian reovirus through p53 and mitochondria-mediated pathway.

Although induction of apoptosis by avian reovirus has been demonstrated in primary chicken embryonic fibroblast and several cell lines, to date, the potential significance of avian reovirus (ARV)-induced apoptosis and its pathways in cultured cells are still largely unknown. We now provide the first evidence of upregulation of p53 and Bax and specifically for Bax translocation from cytosol to mitochondria following infection with a cytoplasmically replicating RNA virus. Bax translocation to the mitochondria led to the release of mitochondrial proapoptic factors cytochrome c and Smac/DIABLO from mitochondria to the cytosol, but not the release of apoptosis-inducting factor. Activation of caspases-9 and -3 which cleaves the enzyme poly(ADP-ribose) polymerase in ARV-infected BHK-21 cells was also detected. Internucleosomal DNA cleavage was prevented by caspase inhibitors, further demonstrating that ARV-induced apoptosis was executed through caspase-dependent mechanisms. Stable expression of human bcl-2 in BHK-21 cells not only blocked ARV-induced apoptosis and DNA fragmentation but also reduced the level of infectious virus production and its spread in BHK-21 cells infected with ARV at a low multiplicity of infection. All our data suggest that p53 and the mitochondria-mediated pathway played an important regulatory role in ARV-induced apoptosis in BHK-21 cells. To further study the pathogenesis of ARV infection, a dual-labeling assay was used for the simultaneous detection of cells containing viral antigen and apoptotic cells. Dual-labeling assay revealed that the majority of antigen-expressing cells were not apoptotic. Remarkably, some apoptotic but non-antigen-expressing cells were frequently located in the vicinity of antigen-expressing cells. Syncytium formation in ARV-infected BHK-21 cells undergoing apoptosis, was apparent in large syncytia at late infection times, indicating a correlation between virus replication and apoptosis in cultured cells.

Development and characterization of monoclonal antibodies against avian reovirus sigma C protein and their application in detection of avian reovirus isolates.

Avian reovirus (ARV) is a non-enveloped virus with a segmented double-stranded RNA genome surrounded by a double icosahedral capsid shell. ARVs are associated with viral arthritis, immunosuppression, and enteric diseases in poultry. The sigma C protein was involved in induction of apoptosis and neutralization antibody. In the present study, sigma C-His protein was expressed in Sf9 insect cells and purified by immobilized metal affinity chromatography. Eight monoclonal antibodies (mAbs) against sigma C-His and three mAbs against His were screened from hybridoma cells produced by fusion of splenocytes from immunized mice with NS1 myeloma cells. Among the eight mAbs against sigma C protein, all belonged to the IgG isotype except three for IgM. It was discovered that all anti-His mAbs were mixtures of IgG and IgM isotypes. mAbs reacted with sigma C-His protein in a conformation-independent manner based on dot blot and western blotting assays. The competitive binding assay indicated that all mAbs recognized the same epitope on sigma C protein that was conserved in different isolates. Compared with the commercial anti-ARV S1133 polyclonal antibody, mAb (D15) had universal reactivity to all serotypes or genotypes of ARVs tested. This monoclonal antibody may therefore be useful for the development of an antigen-capture enzyme-linked immunosorbent assay for rapid detection of field isolates.

Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system.

Baculovirus/insect cell system is used widely for recombinant protein production. The hemagglutinin (HA) gene of H5N2 avian influenza virus (AIV) 1209 strain and the enhanced green fluorescent protein (EGFP) gene were cloned into pFastBac DUAL vector that has two promoters and cloning sites, allowing simultaneous expression of these two genes. The HA protein of AIV was fused with a hexahistidine (His6) tag for purification. The coexpression of EGFP allowed identification of the recombinant baculoviruses in Sf-9 insect cells, eliminating cumbersome and time-consuming assays. A recombinant baculovirus, Bac-HA, was generated by transfecting pBac-HA to bacmid inside DH10B(AC)Escherichia coli by site-specific transposition, followed by transfection into the Sf-9 cells. Fluorescence in the insect cells was observed from 3 days post-infection. The expressed HA protein was confirmed by Western blot using an anti-HA monoclonal antibody. Also, different detergents and incubation times on ice were tested. The two-stage extraction with Triton X-100 or Tween 20 and incubation on ice for 2h exhibited high efficiency. Since purification of HA with ConA resin resulted in low protein recovery, lentil lectin affinity column was used and was useful for HA purification.

Development of a quantitative Light Cycler real-time RT-PCR for detection of avian reovirus.

A robust, ultrasensitive, and accurate quantitative assay was developed for avian reovirus (ARV) with the Light Cycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR). The assay exhibited high specificity as all negative controls and other avian pathogens, such as Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), avian influenza virus (AIV), and mycoplasma synovia (MS), failed to show any positive detection. A minimum of 39 copies/microl of ARV genomic RNA could be detected by the assay. By dilution analysis, the real-time LC RT-PCR developed in this study was 3-log more sensitive than the conventional RT-PCR for the detection of ARV. The vaccine and field isolates of ARV were detected by the real-time LC RT-PCR. As a result of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of ARV infection.