Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

Efficiency Comparison of a Novel E2 Subunit Vaccine and a Classic C-Strain Vaccine against Classical Swine Fever

Classical swine fever (CSF) is one of the most important viral diseases in swine, causing severe economic losses in the swine industry. In China, CSF is one of the key diseases that needs to be controlled; the government has implemented control measures, and vaccination with C-strain vaccines (C-vacs) has been compulsory since the 1950s. C-vacs do not allow the differentiation of field virus-infected and vaccinated animals (DIVA). In 2012, China proposed a goal of eradicating CSF. Additionally, a baculovirus-expressed E2 subunit vaccine (E2-vac) was licensed in 2018. However, the C-vac and E2-vac characteristics have not been compared. Here, we demonstrate that both the C-vac and E2-vac provide complete protection against CSF in pigs. The E2-vac allows DIVA, and the E2 antibody responses of stimulated pigs are developed earlier and are stronger than the C-vac antibody responses. Therefore, the E2-vac is a new candidate licensed vaccine to completely eradicate CSF on pig farms.

Molecular Analysis of East African Lumpy Skin Disease Viruses Reveals a Mixed Isolate with Features of Both Vaccine and Field Isolates

Lumpy skin disease (LSD), an economically significant disease in cattle caused by lumpy skin disease virus (LSDV), is endemic to nearly all of Africa. Since 2012, LSDV has emerged as a significant epizootic pathogen given its rapid spread into new geographical locations outside Africa, including the Middle East, Eastern Europe, and Asia. To assess the genetic diversity of LSDVs in East Africa, we sequenced and analyzed the RPO30 and GPCR genes of LSDV in twenty-two archive samples collected in Ethiopia, Kenya, and Sudan before the appearance of LSD in the Middle East and its incursion into Europe. We compared them to publicly available sequences of LSDVs from the same region and those collected elsewhere. The results showed that the East African field isolates in this study were remarkably similar to each other and to previously sequenced field isolates of LSDV for the RPO30 and GPCR genes. The only exception was LSDV Embu/B338/2011, a field virus collected in Kenya, which displayed mixed features between the LSDV Neethling vaccine and field isolates. LSDV Embu/B338/2011 had the same 12-nucleotide insertion found in LSDV Neethling and KS-1 vaccines. Further analysis of the partial EEV glycoprotein, B22R, RNA helicase, virion core protein, NTPase, and N1R/p28-like protein genes showed that LSDV Embu/B338/2011 differs from previously described LSDV variants carrying the 12-nucleotide insertion in the GPCR gene. These findings highlight the importance of the constant monitoring of genetic variation among LSDV isolates.

Immunoinformatics analysis and antibody epitope comparison of Newcastle disease virus classical vaccines with a virus involved in the fifth NDV panzootic

Newcastle disease virus (NDV) has negatively affected the poultry industry worldwide. Given that the antigenic similarity of a vaccine strain to a field virus is effective in protection, an immunoinformatics study was performed to examine the similarity between antibody epitopes of classical vaccines and a sub-genotype VII.2 NDV (VII.2 NDV). Considering the role of fusion (F) and hemagglutinin-neuraminidase (HN) proteins in the induction of neutralizing antibodies, the 3D structure of HN and F proteins of the VII.2 NDV and nine vaccine strains were predicted, refined, and validated. Using these structures, linear and conformational antibody epitopes were mapped. Epitope analysis showed distinct results from the evolutionary distance and protein identity analysis and it was found that the range of difference in the number of identical epitopes in relation to F is wider than HN protein. LaSota and B1 vaccine strains showed the least epitope identity to the VII.2 NDV. The V4 and I-2 vaccine strains showed the highest epitope identity with the VII.2 NDV especially in F protein which is important in virus cell-to-cell transmission. In conclusion, excellence of the LaSota vaccine under field condition shows that protection is not just about epitope similarities and especially in the case of live vaccines, the vaccine-induced damage, replicative capacity and tropism of the vaccine strain are important. The prediction of this study may be useful for inactivated vaccines in which the amount of antigen is all that matters.

Molecular characterization of duck plague virus from selected Haor areas of Bangladesh

Background: Duck viral enteritis, commonly known as duck plague (DP), is an acute and contagious fatal disease in ducks, geese, and swans caused by the DP virus (DPV). It poses a serious threat to the growth of duck farming in the Haor (wetland) areas of Bangladesh. Aim: This study aimed to detect the circulating DPV by molecular characterization, followed by phylogenetic analysis, targeting the UL30 gene in infected ducks from five Haor districts in Bangladesh and to observe the variation in the genome sequence between the field virus and vaccine strain of DPV. Methods: A total of 150 samples (liver, 50; intestine, 50; and oropharyngeal tissue, 50) were collected from DP-suspected sick/dead ducks from 50 affected farms in Kishoreganj, Netrokona, B. Baria, Habiganj, and Sunamganj districts in Bangladesh. For the identification of DPV in collected samples, polymerase chain reaction (PCR) was utilized. Nucleotide sequences of the amplified UL30 gene were compared with those of other DPV strains available in GenBank. Results: Of the 150 samples, 90 (60%) were found to be positive for DPV, as confirmed by PCR. Organ-wise prevalence was higher in the liver (72%), followed by the intestine (64%) and oropharyngeal tissue (44%). Regarding areas, the highest and lowest prevalence in the liver and intestine was observed in Habiganj and B. Baria, respectively, whereas the highest and lowest prevalence in the oropharyngeal tissue was observed in B. Baria and Habiganj, respectively. Two isolates, BAU/KA/DPV(B1)/2014 from Kishoreganj and BAU/KA/DPV(B4)/2014 from Sunamganj were sequenced, and phylogenetic analysis revealed that these isolates are evolutionarily closely related to Chinese isolates of DPV. Additionally, the isolates of DPV BAU/KA/DPV(B1)/2014 and BAU/KA/DPV(B4)/2014 showed the highest (98%) similarity to each other. The nucleotide sequence of the isolate BAU/KA/DPV(B1)/2014 exhibited higher nucleotide variability (246 nucleotides) than that of the vaccine strain (accession no. EU082088), which may affect protein function and additional drug sensitivity. Conclusion: Based on the findings of the molecular study, it can be assumed that the Bangladeshi isolates and all Chinese isolates of DPV may have a common ancestry.

The Isolation and Replication of African Swine Fever Virus in Primary Renal-Derived Swine Macrophages

African swine fever virus (ASFV) causes hemorrhagic disease in domestic pigs by replicating mainly in monocyte/macrophage lineages. Various primary cells including pulmonary alveolar macrophages have been used for the propagation of ASFV on this account. However, ethical constraints and consistency problems exist as it is necessary to harvest same phenotype of primary cells in order to continue a study. We suggested renal-derived swine macrophages as a novel primary cell candidate to address these issues. These primary cells proved to be permissive to both cell adapted ASFV and a wild-type ASFV. Compared to the commercial cell line MA-104, the renal-derived macrophages were more suitable to isolate the field virus. The consistent molecular characteristics of the renal-derived macrophages were demonstrated by immunocytochemistry with antibodies against macrophage cell surface markers including CD163, CD172a, and Iba-1. Viral protein p30 and p72 expression in ASFV infected macrophages was confirmed by immunocytochemistry by use of specific monoclonal antibodies. We observed increase of cell-free viral DNA and infectious virus titer in infected cell supernatant in successive days-post-infection. These results demonstrated that primary renal-derived swine macrophages are useful for ASFV isolation and propagation in terms of cell phenotypes, susceptibility to the virus, and virus production.

Commercial PRRS Modified-Live Virus Vaccines

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) presents one of the challenging viral pathogens in the global pork industry. PRRS is characterized by two distinct clinical presentations; reproductive failure in breeding animals (gilts, sows, and boars), and respiratory disease in growing pigs. PRRSV is further divided into two species: PRRSV-1 (formerly known as the European genotype 1) and PRRSV-2 (formerly known as the North American genotype 2). A PRRSV-2 modified-live virus (MLV) vaccine was first introduced in North America in 1994, and, six years later, a PRRSV-1 MLV vaccine was also introduced in Europe. Since then, MLV vaccination is the principal strategy used to control PRRSV infection. Despite the fact that MLV vaccines have shown some efficacy, they were problematic as the efficacy of vaccine was often unpredictable and depended highly on the field virus. This paper focused on the efficacy of commercially available MLV vaccines at a global level based on respiratory disease in growing pigs, and maternal and paternal reproductive failure in breeding animals.

Analysis of Amino Acid Mutations of the Foot-and-Mouth Disease Virus Serotype O Using both Heparan Sulfate and JMJD6 Receptors

Foot-and-mouth disease (FMD) is an economically devastating animal disease. Adapting the field virus to cells is critical to the vaccine production of FMD viruses (FMDV), and heparan sulfate (HS) and Jumonji C-domain-containing protein 6 (JMJD6) are alternative receptors of cell-adapted FMDV. We performed serial passages of FMDV O/SKR/Andong/2010, classified as the O/Mya-98 topotype/lineage and known as a highly virulent strain, to develop a vaccine seed virus. We traced changes in the amino acid sequences of the P1 region, plaque phenotypes, and the receptor usage of the viruses, and then structurally analyzed the mutations. VP3 H56R and D60G mutations were observed in viruses using the HS receptor and led to changes in the hydrogen bonding between VP3 56 and 60. A VP1 P208L mutation was observed in the virus using the JMJD6 receptor during cell adaptation, enabling the interaction with JMJD6 through the formation of a new hydrogen bond with JMJD6 residue 300. Furthermore, VP1 208 was near the VP1 95/96 amino acids, previously reported as critical mutations for JMJD6 receptor interactions. Thus, the mutation at VP1 208 could be critical for cell adaptation related to the JMJD6 receptor and may serve as a basis for mechanism studies on FMDV cell adaptation.

MODERN APPROACHES TO THE PREVENTION OF REO-VIRUS BIRD INFECTION

Currently, reo-virus infection causes significant economic damage to industrial poultry worldwide. Losses as a result of the disease consist of: 1) direct losses - death of chickens (5-30 %), increased culling due to lameness, low weight gain (up to 40 %), reduction of carcass categories, reduction of laying by 6-20 %; in breeding farms, a decrease in the sexual activity of poultry is observed, which is the reason for the decrease in fertilization - derivability; 2) indirect losses associated with the cost of eliminating the effects of primary losses, as well as the immune-suppressive effect of reo-viruses, which increase the risk of other infections and inhibit the formation of post-vaccine immunity. Reo-virus has an immune-suppressive effect on the sick bird, which may be the cause of such diseases as small absorption syndrome and syndrome of increased bird death. Small absorption syndrome, which can be caused by reo-virus, is characterized by slowing growth, low feed conversion index and damage to the bones of the bird, which becomes apparent until 2 weeks of age and later. The syndrome of absorption is characterized by an increase in glandular stomach, sometimes with necrosis and signs of catarrhal hemorrhagic enteritis. The incidence is on average 5 to 15 %, but can reach up to 40 %. In studying the pathogenesis of reo-virus infection, it was found that the intestine is the main target of the virus, regardless of the method of its introduction. After oral or aerogenic infection, the pathogen enters the bloodstream and, as a result of viremia, is rapidly spread to various organs and tissues. In particular, the virus can be isolated from the intestines, bursa of Fabricius, liver, pancreas, heart, kidneys, joints and tendons. When conducting serological studies in ELISA (test system company IDvet, France), the presence of antibodies in the blood of broilers to reo-virus level ranged from 40-52 days in the range from 1125 to 5138 indicates the circulation of the field virus of reo-virus infection. In the study of broiler sera for 2, 3, 7 days, the cultivation of specific antibodies to reo-virus was not detected. Starting from the 14th day, the mean titer of AO to reo-virus was 1989 and their levels ranged from 3478 to 4825 at 21, 28, 35 and 45 days. 2.3 times (35 days) and 45 days 2.4 times. These data indicate the circulation of a field strain of reo-virus infection. It was inserted that at completion of broiler fattening with reo-virus lesion, body weight was lower than the technological norm by 9.8%. When determining specific BP for reo-virus in sera from different age groups of broilers of 11 batches, including from poultry: 9 batches of non-vaccinated against reo-virus infection and 2 batches of vaccinated poultry have determined the number of BPs in serum among the 9 batches received from the unvaccinated parent stock, there is evidence of field strain circulation. The percentage of specific AOs in 2 batches received from parental herds vaccinated against reo-virus infection ranged from 87.5-100, indicating the effectiveness of the vaccination. To control reo-virus infection, serological monitoring in poultry farms should be carried out.