Recombinant Newcastle disease virus expressing African swine fever virus protein 72 is safe and immunogenic in mice

African swine fever(ASF) is a lethal hemorrhagic disease that affects wild and domestic swine. The etiological agent of ASF is African swine fever virus(ASFV). Since the first case was described in Kenya in 1921, the disease has spread to many other countries. No commercial vaccines are available to prevent ASF. In this study, we generated a recombinant Newcastle disease virus(r NDV) expressing ASFV protein 72(p72) by reverse genetics and evaluated its humoral and cellular immunogenicity in a mouse model. The recombinant virus, r NDV/p72, replicated well in embryonated chicken eggs and was safe to use in chicks and mice. The p72 gene in r NDV/p72 was stably maintained through ten passages. Mice immunized with r NDV/p72 developed high titers of ASFV p72 specific Ig G antibody, and had higher levels of Ig G1 than IgG2 a. Immunization also elicited T-cell proliferation and secretion of IFN-γ and IL-4. Taken together, these results indicate that r NDV expressing ASFV p72 might be a potential vaccine candidate for preventing ASF.     

Towards a safe, effective vaccine for Rift Valley fever virus

Rift Valley fever virus (RVFV) is an important animal and human threat and leads to longstanding morbidity and mortality in susceptible hosts. Since no therapies currently exist to treat Rift Valley fever, it remains a public and animal health priority to develop safe, effective RVFV vaccines (whether for animals, humans, or both) that provide long-term protective immunity. In the evaluated article, Bhardwaj and colleagues describe the creation and testing of two successful vaccine strategies against RVFV, a DNA plasmid vaccine expressing Gn coupled to C3d, and an alpha-virus replicon vaccine expressing Gn protein. Both vaccines elicited strong neutralizing antibody responses, prevented morbidity and mortality in RVFV-challenged mice, and enabled protection of naive mice via passive antibody transfer from vaccinated mice. Both DNA and replicon RVFV vaccines have previously been shown to protect against RVFV challenge, but these results allow for direct comparison of the two methods and evaluation of a combined prime-boost method. The results also highlight the specific humoral and cell-mediated immune responses to vaccination.     

非洲猪瘟病毒的生物学特性与疫苗研制的难点

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种猪烈性传染病,是全球养猪业的"头号杀手",强毒株引发的超急性和急性感染死率高达100%。2018年8月ASF首次传入我国,截止2019年6月6日,已有32个省份累计暴发137起疫情,给我国社会、经济构成巨大威胁。ASF疫苗的研制始于20世纪60年代,但均以失败而告终,其主要原因是对ASFV生物学特性缺乏深入的研究。有效控制当前ASF疫情扩散、研制安全有效的疫苗将是我国面临的巨大挑战。本文对ASFV形态与基本结构、传播途径、致病机制、基因组及编码蛋白、入侵机制、免疫逃逸等生物学特性进行了概述,并分析了当前疫苗研制面临的难点,以期为我国有效控制ASF疫情及病原研究提供参考。     

An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants

We developed an AIDS vaccine based on attenuated VSV vectors expressing env and gag genes and tested it in rhesus monkeys. Boosting was accomplished using vectors with glycoproteins from different VSV serotypes. Animals were challenged with a pathogenic AIDS virus (SHIV89.6P). Control monkeys showed a severe loss of CD4+ T cells and high viral loads, and 7/8 progressed to AIDS with an average time of 148 days. All seven vaccinees were initially infected with SHIV89.6P but have remained healthy for up to 14 months after challenge with low or undetectable viral loads. Protection from AIDS was highly significant (p = 0.001). VSV vectors are promising candidates for human AIDS vaccine trials because they propagate to high titers and can be delivered without injection.     

Glomerular pathology in surviving pigs experimentally infected with African swine fever virus.

Twelve miniature pigs were inoculated with an attenuated African swine fever virus to study glomerular involvement in surviving pigs. In acute phase, kidneys were severely affected and displayed a glomerular capillary thrombosis with fibrin deposition in vascular lumen, detected by immunofluorescence. Fibrin-positive deposits were progressively cleared between one to three months after infection in surviving pigs. The histological picture in kidneys of surviving pigs, up to one post-infection year, showed a focal and segmental glomerulonephritis with hyalinosis, and IgM and C3 deposition was detected by immunofluorescence. Its pathogeny as an evolutive stage of acute glomerular injury is pointed out.     

Polypeptides of African swine fever virus

The published data on the characteristics and properties of structural and nonstructural polypeptides of the African porcine virus are reviewed. Localization of the viral proteins in virions and infected cells, kinetics of biosynthesis, glycosylation, phosphorylation and the antigenicity of the proteins are discussed.     

Genetically engineered live attenuated influenza A virus vaccine candidates.

We have generated new influenza A virus live attenuated vaccine candidates by site-directed mutagenesis and reverse genetics. By mutating specific amino acids in the PB2 polymerase subunit, two temperature-sensitive (ts) attenuated viruses were obtained. Both candidates have 38 degrees C shutoff temperatures in MDCK cells, are attenuated in the respiratory tracts of mice and ferrets, and have very low reactogenicity in ferrets. Infection of mice or ferrets with either mutant conferred significant protection from challenge with the homologous wild-type virus. Three tests for genetic stability were used to assess the propensity for reversion to virulence: 14 days of replication in nude mice, growth at 37 degrees C in tissue culture, and serial passage in ferrets. One candidate, which contains mutations intended to reduce the ability of PB2 to bind to cap structures, was stable in all three assays, whereas the second candidate, which contains mutations found only in other ts strains of influenza virus, lost its ts phenotype in the last two assays. This approach has therefore enabled the creation of live attenuated influenza A virus vaccine candidates suitable for human testing.     

African swine fever virus interaction with microtubules

The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles. The results show that microtubules are involved in the transport of African swine fever virus particles from the assembly site to the cell surface and in the movement and fusion of the virus inclusions.     

Nanopore sequencing of African swine fever virus

正Dear Editor,African swine fever (ASF) is one of the most pathogenic viral diseases in pigs caused by African swine fever virus(ASFV). The fatality rate is almost 100%, which brings huge economic losses to the hog industry in countries with epi-     

Purification and properties of African swine fever virus

We describe a method for African swine fever (ASF) virus purification based on equilibrium centrifugation in Percoll density gradients of extracellular virions produced in infected VERO cells that yielded about 15 +/- 9% recovery of the starting infectious virus particles. The purified virus preparations were essentially free of a host membrane fraction (vesicles) that could not be separated from the virus by previously described purification methods. The purified virus sedimented as a single component in sucrose velocity gradients with a sedimentation coefficient of 3,500 +/- 300S, showed a DNA-protein ratio of 0.18 +/- 0.02 and a specific infectivity of 2.7 X 10(7) PFU/micrograms of protein, and remained fully infectious after storage at -70 degrees C for at least 7 months. The relative molecular weights of the 34 polypeptides detected in purified virus particles ranged from 10,000 to 150,000. Some of these proteins were probably cellular components that might account for the reactivity of purified virus with antiserum against VERO cells.     

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.     

A reassortment-incompetent live attenuated influenza virus vaccine for protection against pandemic virus strains

Although live-attenuated influenza vaccines (LAIV) are safe for use in protection against seasonal influenza strains, concerns regarding their potential to reassort with wild-type virus strains have been voiced. LAIVs have been demonstrated to induce enhanced mucosal and cell-mediated immunity better than inactivated vaccines while also requiring a smaller dose to achieve a protective immune response. To address the need for a reassortment-incompetent live influenza A virus vaccine, we have designed a chimeric virus that takes advantage of the fact that influenza A and B viruses do not reassort. Our novel vaccine prototype uses an attenuated influenza B virus that has been manipulated to express the ectodomain of the influenza A hemagglutinin protein, the major target for eliciting neutralizing antibodies. The hemagglutinin RNA segment is modified such that it contains influenza B packaging signals, and therefore it cannot be incorporated into a wild-type influenza A virus. We have applied our strategy to different influenza A virus subtypes and generated chimeric B/PR8 HA (H1), HK68 (H3), and VN (H5) viruses. All recombinant viruses were attenuated both in vitro and in vivo, and immunization with these recombinant viruses protected mice against lethal influenza A virus infection. Overall, our data indicate that the chimeric live-attenuated influenza B viruses expressing the modified influenza A hemagglutinin are effective LAIVs.     

An attenuated strain of Akabane virus: a candidate for live virus vaccine

An attempt was made to attenuate the high virulent OBE-1 strain of Akabane virus by adaptation to low temperature. In it the virus was subjected to passage through HmLu-1 cell cultures at 30 degrees C. Cloning was carried out on the virus which had undergone 20 passages through these cultures to select a strain adapted to low temperature. Finally, ten clones were obtained. As a result, nine strains of clone in which virus replication was poor in HmLu-1 cell cultures at 40 degrees C were obtained. Of them, five strains of clone produced uniform plaques. Of these strains, one, or the TS-C2 strain, was selected. It was considerably lower both in peripheral infectivity to suckling mice and in intracerebral infectivity to 3-week-old mice than the OBE-1 strain. Calves and pregnant cows inoculated with the TS-C2 strain by the intracerebral, intravenous, or subcutaneous route were free from pyrexia, leukopenia, and viremia. Virus recovery was negative from various organs and fetuses. All the animals inoculated, however, were found to have neutralizing antibody produced. The results mentioned above suggested that the TS-C2 strain might have been so attenuated as to be available as a candidate strain for a live virus vaccine.     

Obstacles to the development of a safe and effective attenuated pre-erythrocytic stage malaria vaccine

Attenuated pre-erythrocytic stage malaria vaccines protect humans from sporozoite challenge. Current technology limits the feasibility of their commercial scale production and poses a significant risk of contamination with transmissible agents. Overcoming these barriers will be very challenging. In contrast, subunit vaccines appear quite feasible and show great promise as candidate malaria vaccines.     

Laboratory-scale inactivation of African swine fever virus and swine vesicular disease virus in pig slurry

Two methods were evaluated for the inactivation of African swine fever (ASV) and swine vesicular disease (SVD) viruses in pig slurry: chemical treatment and heat treatment. The addition of NaOH or Ca(OH)2 at different concentration/time combinations at 4 degrees C and 22 degrees C was examined, as was virus stability at different temperature/time combinations. ASF virus (ASFV) was less resistant to both methods than SVD virus (SVDV). In slurry from one source, ASFV was inactivated at 65 degrees C within 1 min, whereas SVDV required at least 2 min at 65 degrees C. However, it was found that thermal inactivation depended on the characteristics of the slurry used. Addition of 1% (w/v) of NaOH or Ca(OH)2 caused the inactivation of ASFV within 150 s at 4 degrees C; 0.5% (w/v) NaOH or Ca(OH)2 required 30 min for inactivation. NaOH or Ca(OH)2 (1% (w/v)) was not effective against SVDV at 22 degrees C after 30 min, and 1.5% (w/v) NaOH or Ca(OH)2 caused inactivation of SVDV at both 4 degrees C and 22 degrees C. At higher chemical concentrations or temperatures, ASFV and SVDV inactivation was faster in slurry than in buffered medium.     

A live attenuated vaccine for Lassa fever made by reassortment of Lassa and Mopeia viruses

Lassa virus (LASV) and Mopeia virus (MOPV) are closely related Old World arenaviruses that can exchange genomic segments (reassort) during coinfection. Clone ML29, selected from a library of MOPV/LASV (MOP/LAS) reassortants, encodes the major antigens (nucleocapsid and glycoprotein) of LASV and the RNA polymerase and zinc-binding protein of MOPV. Replication of ML29 was attenuated in guinea pigs and nonhuman primates. In murine adoptive-transfer experiments, as little as 150 PFU of ML29 induced protective cell-mediated immunity. All strain 13 guinea pigs vaccinated with clone ML29 survived at least 70 days after LASV challenge without either disease signs or histological lesions. Rhesus macaques inoculated with clone ML29 developed primary virus-specific T cells capable of secreting gamma interferon in response to homologous MOP/LAS and heterologous MOPV and lymphocytic choriomeningitis virus. Detailed examination of two rhesus macaques infected with this MOPV/LAS reassortant revealed no histological lesions or disease signs. Thus, ML29 is a promising attenuated vaccine candidate for Lassa fever.