Abstract: A computer-based codon-pairs deoptimization technology, synthetic attenuated virus engineering (SAVE), is utilized to lower virulence of viruses (Coleman et al, 2008; Martrus et al, 2013). The rapid viral attenuation concept by the codon pair deoptimization is to decrease codon pair bias (CPB), which demonstrated the potential usefulness as a technology to develop live attenuated vaccine (Ni et al, 2014; Gao et al, 2015).
For this, we produced field PRRSVs attenuated by codon-pair deoptimization. The affection of SAVE on virulence of the attenuated strains was confirmed in pigs
Type 2 field porcine reproductive and respiratory syndrome viruses (PRRSV) isolated in the Republic of Korea were attenuated by de-optimization of codon pair bias in NSP 1. The attenuated viruses showed significantly lowered replication ability than the parental viruses in porcine alveolar macrophages, while comparable to the parental viruses in Marc-145. In 3 weeks-old pigs infection, the attenuated viruses showed significantly lowered replication ability than the parental viruses without distinct clinical sign and pathological lesions, which were observed in pig infected with the parental viruses. This study suggested that the attenuated PRRSV may be highly useful as a vaccine strain in the future.
Find out the reason why 2017 flu season is severe.
a.Plaque reduction assay (PRA)
b. Neuraminidase (NA) inhibition assay.
c. Hemolysis assays.
d.Cell-to-cell spreading assay
2017 H3N2 and 2014 H3N2
In the present study, 2017 H3N2showed reduction in NAIs susceptibility when compared with the 2014 H3N2 in plaque reduction assay (PRA). Further analysis revealed changes in HA1 and HA2 in the 2017 strain that might have caused the low anti-viral susceptibility. Firstly, in contrast to 2014 H3N2, the interaction between HA1 of 2017 H3N2 and its receptor could not be blocked by zanamivir. Secondly, HA2 of 2017 H3N2 had exhibited higher hemolytic activity than 2014 H3N2. Furthermore, 2017 strain spread faster than the 2014 strain in MDCK cells after blocking NA activity.
The characteristics of low drug susceptibility due to high hemolytic activity and slightly stronger acid stability in the 2017 strain might help explain the H3N2, 2017 H3N2strain which caused a severe influenza outbreak in summer of 2017 in Hong Kong
The changes in HA contribute to the severe flu season in Hong Kong during 2017.
Biography: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Abstract: Replication-limited influenza vaccine viruses, such as interferon (IFN)-sensitive and replication-incompetent influenza viruses, are likely to be the alternatives to inactivated and attenuated virus vaccines. Some IFN-sensitive influenza vaccine candidates with modified non-structural protein 1 (NS1) are highly attenuated in IFN-competent hosts but induce robust antiviral immune responses. However, little research has been done on the manufacturability of these IFN-sensitive vaccine viruses.
In this study, RIG-I-knockout 293T cells were used to package the IFN-sensitive influenza A/WSN/33 (H1N1) virus expressing the mutant NS1 R38A/K41A. We found that the packaging efficiency of the NS1 R38A/K41A virus in RIG-I-knockout 293T cells was much higher than that in 293T cells. Moreover, the NS1 R38A/K41A virus almost lost its IFN antagonist activity and could no longer replicate in A549, MDCK, and Vero cells after 3-6 passages. This indicated that the replication of NS1 R38A/K41A virus is limited in conventional cells. Therefore, we further established a stable Vero cell line expressing the wild-type (WT) NS1 of the WSN virus, based on the Tet-On 3G system. The NS1 R38A/K41A virus was able to steadily propagate in this IFN-deficient cell line for at least 20 passages. In a mouse model, the NS1 R38A/K41A virus showed more than a 4-log reduction in lung virus titers compared to the WT virus at 3 and 5 days post infection. Furthermore, we observed that the NS1 R38A/K41A virus triggered high-level of IFN-α/β production in lung tissues and was eliminated from the host in a relatively short period of time. Additionally, this virus induced high-titer neutralizing antibodies and provided 100% protection against the WT WSN virus.
In conclusion, we reavealed that the replication of the NS1 R38A/K41A virus was limited in IFN-competent cells and mice, but this replication-limited virus could trigger strong antiviral immune responses. We also presented a promising IFN-deficient system, involving a RIG-I-knockout 293T cell line to package the IFN-sensitive vaccine virus and a stable Vero cell line expressing NS1 to propagate the IFN-sensitive vaccine virus. The IFN-deficient system is applicable for the manufacture of IFN-sensitive and replication-limited vaccine virus.
Vaccines & Immunotherapy
Control of Viral Infections and Infectious Diseases
Anti-Viral Agents and Vaccines
Biography: Mohammad Hossein Yazdi got his PhD in the field of Pharmaceutical Biotechnology by 2014 from Tehran University of Medical Sciences, School of Pharmacy. His PhD work was about cancer treatment in particular by immunotherapy. He is now Assistant Professor at Biotechnology Research Center and Recombinant Vaccine Research Center of Tehran University of Medical Sciences and pursues his interest in both vaccine and immunotherapy of cancer and infectious diseases. He has published more than 40 papers in reputed journals and has been serving as senior lecturer of
advanced immunology and immunotherapy at Tehran University of Medical Sciences
Abstract: In this work, the effect of oral administration of Aloe vera extracted polysaccharides was investigated on the immunization profile of E7d Vaccine.Mannose containing polysaccharide in Aloe Vera mucilage absorbed after oral feeding of mice and remained unchanged in the blood stream. Subsequently it can actas as an immunomodulator which is induced both TH1 and TH2 immune response 60 C57BL/6 mice were divided into six groups as follow: Group 1, E7d-ALUM-GEL; Group 2, E7d-ALUM; Group 3, E7d-MONTANID –GEL; Group 4, E7d-MONTANID; Group 5 and Group 6, CONTROL-GEL and PBS respectively. The oral feeding of aloe vera gel with 0.05% poly saccharide was done for a period of 30 days. Mice were immunized with E7d vaccine three times on day 0, 14 and 28th. Then tumor was implanted subcoutaneosly and 2 weeks later, the serum samples of the mice were collected to evaluate the cytokines and antibodies levels by ELISA assay.Immunization of mice with E7d-ALUM-GEL significantly increased the IFN-γ and IL-4 immune response levels in comparison to other vaccinated groups. Meanwhile, analysis of humoral immune responses represented a not worthy increasment of IgG1 level in E7d-MONTANID-Gel group in comparison to E7d-montanid group. It is important to mentioned that control gel group showed an increase in the ratio of IFN γ/ IL-4 in comparison to other groups. The group which was received just aloe vera gel demonstrated the greatest reduction in proportion of tumor growth. Result of survival rate was best in E7d –ALUM-GEL and CONTROL GEL groups.Whereasmannose containing plant compounds like acemannan and glucomannan induces both TH1 and TH2 immune responses, perhaps it can be used as an adjuvant element without serious concern about the toxicity.
Abstract: Background:Recent evidence has demonstrated that dengue virus 2 requires active filopodia formation for successful infection. However, their precise role has never been fully elucidated.
Methodology:Filopodia formation was induced using bradykinin, and combination of virus overlay protein binding assay (VOPBA) and LC-MS/MS was used to identify dengue virus 2 binding protein from the plasma membrane fraction of filopodia induced cells.siRNA-mediated gene silencing and antibody-mediated infection inhibition assay, was employed to ascertain the role of this protein in dengue virus infection. Colocalization analysis, co-immunoprecipitation assay and molecular dynamic simulation was undertaken to ascertain the interaction of annexin II with dengue virus 2 E glycoprotein.
Results and conclusion:An approximately 38kDa protein was detected in the plasma membrane fraction of filopodia induced cell, this was identified by LC-MS/MS as annexin II. Upon filopodia formation annexin II translocate to the extracellular leaflet of plasma membrane. Antibody-mediated infection inhibition and siRNA-mediated knockdown of annexin II expression significantly reduces dengue virus 2 infection and production level. Colocalization analysis showed extracellular and intracellular colocalization of annexin II with dengue virus 2 E glycoproteins, while their direct interaction was confirmed by molecular dynamic simulation and co-immunoprecipitation assay. Furthermore, their putative interaction sites were determined by molecular docking and molecular dynamic simulation.Collectively, we demonstrated for the first time that annexin II mediates dengue virus 2 binding and internalization into Vero cells via filopodia, this could be a potential target for the development of novel potent antivirus therapeutics
Abstract: Many viral infections causing human or animal morbidity or mortality are emerging in different regions as outbreak or even turn out as routine infections. Few of them like West Nile, Zika and Nipah infections have persistence giving further load on even blood bank system. Current methodology to contain these is based on expressed protein or DNA based vaccine. Although DNA vaccine is supposed to produce both antibody and MHC I response, it has limitation as amount of protein produced, expression in non-APC cells leading to short term or even anergy response which leads to short term memory cells. Using expressed protein capable of producing neutralizing antibody response lacks as monomer protein does not give sufficient response. It lead to development of VLP based empty virion which has few problems.
Many viruses have positive sense RNA as a genome either polyprotein or few have multiple level expression of different proteins. In viruses like rabies, all proteins are contained in virion. Usually, as virion released out of the cell, MHC I response is comparative low response. While, other non-structural (NS) proteins which remain associated with infected cells are taken up by APC through phagocytosis process or by multiplication within APCs. Many studies on delineation of peptide epitopes for CD4 and CD8 response in infected recovered individuals have shown that major these responses are within NS proteins. In many studies, longevity or recall of immune response depends on these proteins.
In many outbreakssituation, it is necessary to quickly develop successful and effective vaccine without side effects etc. Thus, inclusion of purified NS proteinsproduced simultaneous in infected cells along with inactivated virion, can give the quick effective vaccine. By using appropriate delivery system, the same can be directed to Th1/CTL response also.
Abstract: Several reports showed that photodynamic inactivation of viruses (PDI) could be an efficient alternative antiviral agent. However, unlike other antiviral drugs and disinfectants, nothing is known about possible emergence of PDI resistant virus population either due to repeated exposure to PDI conditions or no prior exposure to PDI but repeated regrowth in their host cells. In this work, we used PDI conditions of 0.5 µM TMPyP, 32 mW.cm-2 and 30 sec illumination at 32 mWcm-2 which caused reductions of 4 log PFU/ml of MS2 (initial titre 9.6 log PFU/ml) to select about 5 log PFU/ml of the MS2 population still viable.Our results showed that sensitivity of MS2 populations did not change even at the 10th cycle of PDI and or 10th MS2 passage with no prior PDI exposure even though genetic mutations were observed in both PDI exposed and none-exposed MS2. Throughout the PDI cycles, we consistently observed reductions of 4 log PFU/ml of MS2 and this indicates, that at least over the 10 cycles no resistance to PDI had emerged in the MS2 population. In total, we observed 13 mutations and 10 of these mutations were found in the MS2 A-protein encoding region. The PDI exposed MS2 population had 5 mutations (of which 2 are synonymous) on the A-protein region and the none-exposed PDI but passage population had 5 mutations (of which 1 is synonymous) as well on the same region.This confirmed that the causes and sources of viral genetic mutations are embedded in the process of growth and regrowth of viruses in their host cells and prior exposure to antiviral drugs or disinfectants might be of least importance in the rise of such mutations in viruses.
Biography: David Nadeba Bukbuk, PhD, is a Pioneer Head of Microbiology Department at the Faculty of Science, University of Maiduguri (UNIMAID) from 2006 to 2012. He is the Assistant Director of the WHO-National Polio Laboratory at the University of Maiduguri Teaching Hospital. He graduated with a BSc and MSc degree in Microbiology from the Ahmadu Bello University, Samaru, Zaria. He holds a PhD degree in Microbiology with specialty in viral haemorrhagic fevers (Virology) from the University of Maiduguri. His PhD research work is on the seroepidemiological, vector and molecular studies on selected African viral haemorrhagic fevers in Borno state, Nigeria.
Abstract: Despite several studies on the seroprevalence of antibodies against CCHFV from humans and animals in Nigeria [1-3], there has not been any confirmed case of human CCHFV infection reported from any part of the Country. The use of molecular diagnostic tools has resulted in highly sensitive and specific tests for infectious organisms and genetic diseases. These tools, which include polymerase chain reaction (PCR), metagenomics and next generation molecular sequencing, have come in handy for the efficient and rapid identification and characterization of CCHFV strains and other viral Haemorrhagic fever (VHF) agents from suspected cases, vectors and reservoir hosts of these viruses [4-6].
Sera (n=380) collected as part of a serosurveillance programme for VHFs from undiagnosed febrile (fever of unknown origin) patients in Borno state were analyzed by real time RT-PCR assay for the presence of CCHFV RNA. One positive sample (N428) from a 15-year old female showed a cycle threshold (CT) value of 29.42 and a slope formation typically seen for a positive sample (figure1). The RNA from this sample was further characterized by next generation sequencing (NGS) which resulted in complete S, M, and L viral RNA segment sequences.
Phylogenetic analysis clustered the S-segment in the Africa3 clade or phylogenetic group (figure2). The S-segment open-reading frame showed close homology with a previous isolate of CCHFV from Nigeria (IbAr10200), as well as isolates from Mauritania (ArD39554) and South Africa (SPU415/85 and SPU128/61/7). The M and L clustered closely with the Sudan ABI-2009 isolate and the Nigeria IbAr10200 (figure3). The L, M, and S sequences were submitted to GenBank with accession numbers KX238956, KX238957 and KX238958 respectively .
Previous serological results by Bukbuk and co-workers in 2016  have demonstrated the circulation of CCHFV in 4 different Local Government Areas (LGAs) of Borno state, Nigeria with average positivity rates of 10.6% and 3.5% for IgG and IgM responses respectively. While, between September 2011 and February 2012, the same Author in 2014, reported a prevalence rate of 2.4% for IgG antibodies from 10 LGAs in the same state, using similar ELISA test. This indicates that the virus is actively circulating in either the arthropod vectors (Ixotid - hard ticks) or animal and human population. This current study has therefore unequivocally demonstrated the virological evidence for the presence and continued exposure to CCHF virus of the human population in Nigeria. This genomic analysis provides the first published evidence of a human case of CCHFV in Nigeria and its phylogenetic context.
Biography: Iraj Khalili has completed his PhD in field of Virology at the ANAU University. He has more than 23 years experience in vaccine industry specially in live attenuated and oily inactivated poultry vaccines.Now, he is in incharge of Virus biobank laboratory. He has published more than 15 papers in reputed journals.
Abstract: Avian influenza A subtype H9N2 virus is belong to Orthomyxoviridae family and causes low pathogenic disease avian influenza. The use of gamma irradiated viral antigens has been developed in the production of effective vaccines. In this research LPAIV H9N2 strain, A/Chicken/IRN/ Ghazvin/2001 was multiplied on SPF eggs and irradiated by a Nordian gamma cell instrument. Irradiated and non-irradiated AIV samples were titrated by EID50 method and Hem¬agglutinin antigen were analyzed by Hem¬agglutinin test as the WHO method. Infectivity of irradiated virus was determined by eggs inoculation method during four blind cultures. The results showed after increasing dose of gamma radiation, virus titer decreased gradually. D10 value and optimum dose for complete virus inactivation were calculated by Dose/response curve, 3.36 and 29.52 kGy, respectively. Also HA antigenicity of gamma irradiated virus samples from 0- 30 kGy was not changed. The results of safety test for gamma irradiated AIV samples showed complete inactivation with gamma ray doses: 30 and 35 kGy without any multiplication on eggs after four blind cultures. According to the results of HA antigen assay and safety test, the gamma irradiated and complete inactivated AIV subtype H9N2 is a good candidate as an inactivated immunogenic agent for poultry vaccination.