African swine fever (ASF) is an infectious Suidae disease caused by the ASF virus (ASFV). Adaptation to less susceptible, non-target host cells is one of the most common techniques used to attenuate virulent viruses. However, this may induce many mutations and large-scale rearrangements in the viral genome, resulting in immunostimulatory potential loss of the virus in vivo. This study continuously maintained the virulent ASFV strain, Armenia2007 (Arm07), to establish an attenuated ASFV strain with minimum genetic alteration in a susceptible host cell line, immortalized porcine kidney macrophage (IPKM). A mutant strain was successfully isolated via repeated plaque purification in combination with next-generation sequencing analysis. The isolated strain, Arm07ΔMGF, which was obtained from a viral fluid at a passage level of 20, lacked 11 genes in total in the MGF300 and MGF360 regions and showed marked reduction in virulence against pigs. Moreover, all the pigs survived the challenge with the parental strain when pigs were immunized twice with 10⁵ TCID₅₀ of Arm07ΔMGF, although viremia and fever were not completely prevented after the challenge infection. These findings suggest that this naturally attenuated, spontaneously occurring ASFV strain may provide a novel platform for ASF vaccine development. Full article

The past few decades have been marked by drastic modifications to the landscape by anthropogenic processes, leading to increased variability in the environment. For populations that thrive at their distributional boundaries, these changes can affect them drastically, as Schmalhausen’s law predicts that their dynamics are more likely to be susceptible to environmental variation. Recently, this evolutionary theory has been put to the test in vector-borne disease emergences systems, and has been demonstrated effective in predicting emergence patterns. However, it has yet to be tested in a directly transmitted pathogen. Here, we provide a preliminary test of Schmalhausen’s law using data on Marburg virus outbreaks originating from spillover events. By combining the two important aspects of Schmalhausen’s law, namely climatic anomalies and distance to species distributional edges, we show that Marburgvirus outbreaks may support an aspect of this evolutionary theory, with distance to species distributional edge having a weak influence on outbreak size. However, we failed to demonstrate any effect of climatic anomalies on Marburgvirus outbreaks, arguably related to the lack of importance of these variables in directly transmitted pathogen outbreaks. With increasing zoonotic spillover events occurring from wild species, we highlight the importance of considering ecological variability to better predict emergence patterns. Full article

Virotherapy is a promising, novel form of cancer immunotherapy currently being investigated in pre-clinical and clinical settings. While generally well-tolerated, the anti-tumor potency of oncolytic virus-based monotherapies needs to be improved further. One of the major factors limiting the replication efficiency of oncolytic viruses are the antiviral defense pathways activated by tumor cells. In this study, we have designed and validated a universal expression cassette for artificial microRNAs that can now be adapted to suppress genes of interest, including potential resistance factors. Transcripts are encoded as a primary microRNA for processing via the predominantly nuclear RNase III Drosha. We have engineered an oncolytic measles virus encoding this universal expression cassette for artificial microRNAs. Virally encoded microRNA was expressed in the range of endogenous microRNA transcripts and successfully mediated target protein suppression. However, absolute expression levels of mature microRNAs were limited when delivered by an oncolytic measles virus. We demonstrate that measles virus, in contrast to other cytosolic viruses, does not induce translocation of Drosha from the nucleus into the cytoplasm, potentially resulting in a limited processing efficiency of virus-derived, cytosolically delivered artificial microRNAs. To our knowledge, this is the first report demonstrating functional expression of microRNA from oncolytic measles viruses potentially enabling future targeted knockdown, for instance of antiviral factors specifically in tumor cells. Full article

A long terminal repeat (LTR) plays an indispensable role in small ruminant lentivirus (SRLV) gene expression. In this study, we present the LTR sequence of Polish SRLVs representing different subtypes, and analyzed their impact on SRLV promoter activity, as measured in transient transfection assays. Although certain nucleotide motifs (AML(vis), TATA box and the polyadenylation site (AATAAA)) were conserved across sequences, numerous mutations within the LTR sequences have been identified. Single nucleotide polymorphisms (SNPs) were detected in both regulatory (AP-1, AP-4, Stat and Gas) and non-regulatory sequences, and subtype-specific genetic diversity in the LTR region of Polish SRLVs was observed. In vitro assays demonstrated subtype-specific functional differences between the LTR regions of distinct SRLV subtypes. Our results revealed that the promoter activity of Polish strains was lower (1.64–10.8-fold) than that noted for the K1514 reference strain; however, the differences in most cases were not statistically significant. The lowest promoter activity was observed for strains representing subtype A5 (mean 69.067) while the highest promoter activity was observed for strain K1514 representing subtype A1 (mean 373.48). The mean LTR activities of strains representing subtypes A12, A17, A23, A18 and A24 were 91.22, 137.21, 178.41, 187.05 and 236.836, respectively. The results of the inter-subtype difference analysis showed that the promoter activity of strains belonging to subtype A5 was significantly lower than that for subtype A12 strains (1.32-fold; p < 0.00). The promoter activities of the A5 strain were 1.98-fold and 2.58-fold less active than that of the A17 and A23 strains, and the promoter activities of A12 strains were 1.955 and 1.5 times lower than the promoter activity of A23 and A17 strains, respectively. Furthermore, the promoter activity of A17 strains was 1.3 lower than the promoter activity of A23 strains. Our findings suggest that subtype-specific genetic diversity, mainly in the transcription factor’s binding sites, has an impact on their transcriptional activity, producing a distinct activity pattern for the subtypes. This study provides new information that is important for better understanding the function of the SRLV LTR. However, further research including more strains and subtypes as well as other cell lines is needed to confirm these findings. Full article

Influenza A viruses are rarely symptomatic in wild birds, while representing a higher threat to poultry and mammals, where they can cause a variety of symptoms, including death. H5 and H7 subtypes of influenza viruses are of particular interest because of their pathogenic potential and reported capacity to spread from poultry to mammals, including humans. The identification of molecular fingerprints for pathogenicity can help surveillance and early warning systems, which are crucial to prevention and protection from such potentially pandemic agents. In the past decade, comparative analysis of the surface features of hemagglutinin, the main protein antigen in influenza viruses, identified electrostatic fingerprints in the evolution and spreading of H5 and H9 subtypes. Electrostatic variation among viruses from avian or mammalian hosts was also associated with host jump. Recent findings of fingerprints associated with low and highly pathogenic H5N1 viruses, obtained by means of comparative electrostatics and normal modes analysis, prompted us to check whether such fingerprints can also be found in the H7 subtype. Indeed, evidence presented in this work showed that also in H7N7, hemagglutinin proteins from low and highly pathogenic strains present differences in surface electrostatics, while no meaningful variation was found in normal modes. Full article

In 2021/2022, the re-emergence of highly pathogenic avian influenza (HPAI) occurred in Europe. The outbreak was seeded from two sources: resident and reintroduced viruses, which is unprecedented in the recorded history of avian influenza. The dominant subtype was H5N1, which replaced the H5N8 subtype that had predominated in previous seasons. In this study, we present a whole genome sequence and a phylogenetic analysis of 57 H5N1 HPAI and two low pathogenic avian influenza (LPAI) H5N1 strains collected in the Czech Republic during 2021/2022. Phylogenetic analysis revealed close relationships between H5N1 genomes from poultry and wild birds and secondary transmission in commercial geese. The genotyping showed considerable genetic heterogeneity among Czech H5N1 viruses, with six different HPAI genotypes, three of which were apparently unique. In addition, second-order reassortment relationships were observed with the direct involvement of co-circulating H5N1 LPAI strains. The genetic distance between Czech H5N1 HPAI and the closest LPAI segments available in the database illustrates the profound gaps in our knowledge of circulating LPAI strains. The changing dynamics of HPAI in the wild may increase the likelihood of future HPAI outbreaks and present new challenges in poultry management, biosecurity, and surveillance. Full article

During the COVID-19 pandemic, a reduction in vaccination coverage of children and adolescents was observed in several countries. The aim of this study was to assess the impact of the pandemic, in the first two years, on human rotavirus vaccine (HRV) coverage in Brazil compared with previous years. The number of doses of HRV administered in the period from January 2015 to December 2021 and its annual vaccination coverage were analyzed. The vaccination coverage decreased to 77.3% in 2020 and to 70.4% in 2021, substantially lower than the minimum that would be expected (89.2%); the decline was more pronounced in the second year of the pandemic despite the fact that in this period, the circulation restrictions were already less tight. Of the five Brazilian macro-regions, the northeast had the largest decline, and the south had the smallest impact on coverage. At the municipal level, less than half of the Brazilian municipalities managed to achieve vaccination coverage above 90% in either pandemic year. Although there was already a downward trend in coverage in the pre-pandemic years, the present study shows that the values recorded in 2020 and 2021 were significantly lower. Monitoring of vaccination coverage in the coming years should be carried out continuously in order to avoid a possible resurgence of rotavirus-induced diarrhea. Full article

Infectious bursal disease virus is an immunosuppressive ubiquitous pathogen that causes serious economic losses in poultry production. The virus is prone to genetic changes through mutations and reassortment, which drive the emergence of new variants and lead to a change in the epidemiological situation in a field. Such a situation is currently being reported due to a large wave of IBDV A3B1 reassortant infections in northwestern Europe. On the other hand, in Poland, which is the largest producer of chicken meat in the EU, the IBDVs of genotypes A3B2 and A3B4 were circulating just before the emergence of A3B1 reassortants. The purpose of the presented study was to update the IBDV epidemiological situation. The performed molecular survey based on the sequence of both genome segments showed the presence of very virulent strains (A3B2) and reassortants of genotypes A3B4 and A3B1; moreover, two of these genotypes are newly introduced IBDV lineages. In addition, a number of amino acid substitutions were demonstrated, including within antigenic epitopes and virulence determinants. In conclusion, the results obtained indicated a dynamic epidemiological situation in Poland, which highlights the need for further monitoring studies in the region and verification of protection conferred by the vaccines used against infection with detected IBDV. Full article

Classical swine fever (CSF) is one of the most important re-emergent swine diseases worldwide. Despite concerted control efforts in the Andean countries, the disease remains endemic in several areas, limiting production and trade opportunities. In this study, we aimed to determine the risk factors and spatiotemporal implications associated with CSF in Ecuador. We analysed passive surveillance and vaccination campaign datasets from 2014 to 2020; Then, we structured a herd-level case–control study using a logistic and spatiotemporal Bayesian model. The results showed that the risk factors that increased the odds of CSF occurrence were the following: swill feeding (OR 8.53), time until notification (OR 2.44), introduction of new pigs during last month (OR 2.01) and lack of vaccination against CSF (OR 1.82). The spatiotemporal model showed that vaccination reduces the risk by 33%. According to the priority index, the intervention should focus on Morona Santiago and Los Rios provinces. In conclusion, the results highlight the complexity of the CSF control programs, the importance to improve the overall surveillance system and the need to inform decision-makers and stakeholders. Full article

Gamma delta (γδ) T cells play a significant role in the prevention of viral infection and tumor surveillance in mammals. Although the involvement of γδ T cells in Marek’s disease virus (MDV) infection has been suggested, their detailed contribution to immunity against MDV or the progression of Marek’s disease (MD) remains unknown. In the current study, T cell receptor (TCR)γδ-activated peripheral blood mononuclear cells (PBMCs) were infused into recipient chickens and their effects were examined in the context of tumor formation by MDV and immunity against MDV. We demonstrated that the adoptive transfer of TCRγδ-activated PBMCs reduced virus replication in the lungs and tumor incidence in MDV-challenged chickens. Infusion of TCRγδ-activated PBMCs induced IFN-γ-producing γδ T cells at 10 days post-infection (dpi), and degranulation activity in circulating γδ T cell and CD8α⁺ γδ T cells at 10 and 21 dpi in MDV-challenged chickens. Additionally, the upregulation of IFN-γ and granzyme A gene expression at 10 dpi was significant in the spleen of the TCRγδ-activated PBMCs-infused and MDV-challenged group compared to the control group. Taken together, our results revealed that TCRγδ stimulation promotes the effector function of chicken γδ T cells, and these effector γδ T cells may be involved in protection against MD. Full article

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance. Oncolytic viruses have emerged as a new treatment approach and convey their antitumor activity through lysis of cancer cells. The therapeutic efficacy of oncolytic viruses is largely dependent on the induction of immunogenic cell death (ICD) and the subsequent antitumor immune responses. However, the concurrent generation of antiviral immune responses may also limit the a virus’ therapeutic window. GoraVir is a new oncolytic adenovirus derived from the Human Adenovirus B (HAdV-B) isolate AdV-lumc007 which was isolated from a gorilla and has demonstrated excellent lytic activity in both in vitro and in vivo models of PDAC. In this study, we characterized the immunostimulatory profile of cancer cell death induced by GoraVir and the concerted cellular antiviral responses in three conventional pancreatic cancer cell lines. While GoraVir was shown to induce late apoptotic/necrotic cell death at earlier time points post infection than the human adenovirus type 5 (HAdV-C5), similar levels of ICD markers were expressed. Moreover, GoraVir was shown to induce ICD not dependent on STING expression and regardless of subsequent antiviral responses. Together, these data demonstrate that GoraVir is an excellent candidate for use in oncolytic virotherapy. Full article

After the onset of the AIDS pandemic, HIV-1 (genus Lentivirus) became the predominant model for studying retrovirus Env glycoproteins and their role in entry. However, HIV Env is an inadequate model for understanding entry of viruses in the Alpharetrovirus, Gammaretrovirus and Deltaretrovirus genera. For example, oncogenic model system viruses such as Rous sarcoma virus (RSV, Alpharetrovirus), murine leukemia virus (MLV, Gammaretrovirus) and human T-cell leukemia viruses (HTLV-I and HTLV-II, Deltaretrovirus) encode Envs that are structurally and functionally distinct from HIV Env. We refer to these as Gamma-type Envs. Gamma-type Envs are probably the most widespread retroviral Envs in nature. They are found in exogenous and endogenous retroviruses representing a broad spectrum of vertebrate hosts including amphibians, birds, reptiles, mammals and fish. In endogenous form, gamma-type Envs have been evolutionarily coopted numerous times, most notably as placental syncytins (e.g., human SYNC1 and SYNC2). Remarkably, gamma-type Envs are also found outside of the Retroviridae. Gp2 proteins of filoviruses (e.g., Ebolavirus) and snake arenaviruses in the genus Reptarenavirus are gamma-type Env homologs, products of ancient recombination events involving viruses of different Baltimore classes. Distinctive hallmarks of gamma-type Envs include a labile disulfide bond linking the surface and transmembrane subunits, a multi-stage attachment and fusion mechanism, a highly conserved (but poorly understood) “immunosuppressive domain”, and activation by the viral protease during virion maturation. Here, we synthesize work from diverse retrovirus model systems to illustrate these distinctive properties and to highlight avenues for further exploration of gamma-type Env structure and function. Full article

H5N8 and H5N1 high pathogenicity avian influenza viruses (HPAIVs) caused outbreaks in poultry farms in Japan from November 2021 to May 2022. Hemagglutinin genes of these viruses belong to clade 2.3.4.4B and can be divided phylogenetically into the following groups: 20A, 20E, and 21E. In this study, we compared the infectivity and transmissibility of HPAIVs from three groups of chickens. Representative strains from 20A, 20E, and 21E groups are A/chicken/Akita/7C/2021(H5N8)(Akita7C), A/chicken/Kagoshima/21A6T/2021(H5N1)(Kagoshima6T), and A/chicken/Iwate/21A7T/2022(H5N1)(Iwate7T), respectively. Fifty percent lethal dose of Akita7C in chickens (10^3.83 fifty percent egg infectious dose (EID50)) was up to seven times lower than those of Kagoshima6T and Iwate7T (10^4.50 and 10^4.68 EID_50, respectively). Mean death times for Akita7C- and Kagoshima6T-infected chickens (3.45 and 3.30 days, respectively) were at least a day longer than that of Iwate7T (2.20 days). Viral titers of the trachea and cloaca of Iwate7T-infected chicken were the highest detected. The transmission rate of the Akita7C strain (100%) was markedly higher than those of the two strains (<50%). These data suggest that the infectivity and transmissibility of the Akita7C strain (H5N8) in chickens are higher than those of H5N1 viruses, providing fundamental information needed for formulating effective prevention and control strategies for HPAI outbreaks. Full article