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Background Research on the biogenesis of tick-borne encephalitis virus (TBEV) would benefit gene therapy. Due to specific arrangements of genes along the TBEV genome, its viral-like particles (VLPs) could be exploited as shuttles to deliver their replicon, which carries therapeutic genes, to immune system cells. Objective To develop a flaviviral vector for gene delivery as a part of gene therapy research that can be expressed in secretable VLP suicidal shuttles and provide abundant unique molecular and structural data supporting this gene therapy concept. Method TBEV structural gene constructs of a Swedish Toro strain were cloned into plasmids driven by the promoters CAG and CMV and then transfected into various cell lines, including COS-1 and BHK-21. Time-course sampling of the cells, culture fluid, cell lysate supernatant, and pellet specimens were performed. Western blotting and electron microscopy analyses of collected specimens were used to investigate molecular and structural processing of TBEV structural proteins. Results Western blotting analysis showed differences between promoters in directing the gene expression of the VLPs constructs. The premature flaviviral polypeptides as well as mature VLPs could be traced. Using electron microscopy, the premature and mature VLP accumulation in cellular compartments—and also endoplasmic reticulum proliferation as a virus factory platform—were observed in addition to secreted VLPs. Conclusions The abundant virologic and cellular findings in this study show the natural processing and safety of inserting flaviviral structural genes into suicidal VLP shuttles. Thus, we propose that these VLPs are a suitable gene delivering system model in gene therapy.

Zika virus (ZIKV) is a global public health issue due to its association with severe developmental disorders in infants and neurological disorders in adults. ZIKV uses glycosylation of its envelope (E) protein to interact with host cell receptors to facilitate entry, these interactions could also be important for designing therapeutics and vaccines. Due to a lack of proper information about Asn-linked (N-glycans) on ZIKV E, we analyzed ZIKV E of various strains derived from different cells. We found ZIKV E proteins being extensively modified with oligomannose, hybrid and complex N-glycans of a highly heterogeneous nature. Host cell surface glycans correlated strongly with the glycomic features of ZIKV E. Mechanistically, we observed that ZIKV N-glycans might play a role in viral pathogenesis, as mannose-specific C-type lectins DC-SIGN and L-SIGN mediate host cell entry of ZIKV. Our findings represent the first detailed mapping of N-glycans on ZIKV E of various strains and their functional significance.

A full understanding of the immune response to astrovirus (AstV) infection is required to treat and control AstV-induced gastroenteritis. Relative contributions of each arm of the immune system in restricting AstV infection remain unknown. In this study, two novel subunit AstV vaccines derived from capsid protein (CP) of mink AstV (MAstV) such as CP&Delta, N (spanning amino acids 161&ndash, 775) and CP&Delta, C (spanning amino acids 1&ndash, 621) were evaluated. Their immunogenicity and cytokine production in mice, as well as protective efficacy in mink litters via maternal immunization, were studied. Truncated CPs induced higher levels of serum anti-CP antibodies than CP, with the highest level for CP&Delta, N. No seronegativity was detected after booster immunization with either AstV CP truncates in both mice and mink. All mink moms stayed seropositive during the entire 104-day study. Furthermore, lymphoproliferation responses and Th1/Th2 cytokine induction of mice splenocytes ex vivo re-stimulated by truncated CPs were significantly higher than those by CP, with the highest level for CP&Delta, N. Immunization of mink moms with truncated CPs could suppress virus shedding and clinical signs in their litters during a 51-day study after challenge with a heterogeneous MAstV strain. Collectively, AstV truncated CPs exhibit better parameters for protection than full-length CP.

Zika virus (ZIKV) is a global public health issue due to its association with severe developmental disorders in infants and neurological disorders in adults. Because ZIKV uses glycosylation of its envelope (E) protein to interact with host cell receptors to facilitate entry, these interactions could also be important for designing therapeutics and vaccines. Due to a lack of information about Asn-linked (N-glycans) on ZIKV E, we analyzed ZIKV E of various strains derived from different cells. ZIKV E proteins are extensively modified with oligomannose-, hybrid- and complex-N-glycans of a highly heterogeneous nature. Host cell-surface glycans correlated strongly with the glycomic features of ZIKV E. Mechanistically, we discovered that ZIKV N-glycans are important in viral pathogenesis, as mannose-specific C-type lectins DC-SIGN and L-SIGN mediate cell entry of ZIKV. Our findings represent the first detailed mapping of N-glycans on ZIKV E of various strains and their functional significance.

Due to the large geographical overlap of populations exposed to Zika virus (ZIKV) and human immunodeficiency virus (HIV), understanding disease pathogenesis in such coinfections is urgently needed. We used chronically infected simian immunodeficiency virus and chimeric simian human immunodeficiency virus (SIV/SHIV) macaques and inoculated with ZIKV. Plasma viral loads of both SIV/SHIV and ZIKV showed no significant changes as compared to ZIKV alone-infected animals. Tissue clearance of ZIKV was observed similarly. Furthermore, minimal changes in cytokines/chemokines were observed. Collectively, these data suggest that coinfection may not alter disease pathogenesis and warrants large HIV-ZIKV epidemiological studies to validate these findings.Author SummaryThe co-infection incidence of human immunodeficiency virus (HIV) infection and neglected tropical infectious diseases is increasing due to the large geographical overlap of populations exposed to both of these viruses. Thus, researching on such coinfection is of particular importance. In this study, we investigated HIV-ZIKV coinfection dynamics in adult non-pregnant Rhesus Macaques model chronically infected with simian immunodeficiency virus (SIV) - or chimeric simian human immunodeficiency virus (SHIV). We found that post ZIKV inoculation, plasma viral loads were similar to ZIKV alone infected animals in addition to minimal changes of cytokines. Dynamics of SIV and SHIV also did not change. Tissue clearance of ZIKV was found 67 months later. Our findings provide insights into HIV-ZKIV coinfection to determine the alteration of their pathogenesis.

Due to the large geographical overlap of populations exposed to Zika virus (ZIKV) and human immunodeficiency virus (HIV), understanding the disease pathogenesis of co-infection is urgently needed. This warrants the development of an animal model for HIV-ZIKV co-infection. In this study, we used adult non-pregnant macaques that were chronically infected with simian immunodeficiency virus/chimeric simian human immunodeficiency virus (SIV/SHIV) and then inoculated with ZIKV. Plasma viral loads of both SIV/SHIV and ZIKV co-infected animals revealed no significant changes as compared to animals that were infected with ZIKV alone or as compared to SIV/SHIV infected animals prior to ZIKV inoculation. ZIKV tissue clearance of co-infected animals was similar to animals that were infected with ZIKV alone. Furthermore, in co-infected macaques, there was no statistically significant difference in plasma cytokines/chemokines levels as compared to prior to ZIKV inoculation. Collectively, these findings suggest that co-infection may not alter disease pathogenesis, thus warranting larger HIV-ZIKV epidemiological studies in order to validate these findings., Author summary The co-infection incidence of human immunodeficiency virus (HIV) infection and neglected tropical infectious diseases such as Zika virus (ZIKV) is on the rise due to the large geographical overlap of populations exposed to both of these viruses. Thus, research on such co-infection is of particular importance. In this study, we investigated SIV/SHIV-ZIKV co-infection dynamics in adult non-pregnant Rhesus Macaques (RMs) chronically infected with simian immunodeficiency virus (SIV)—or chimeric simian human immunodeficiency virus (SHIV). We found that post ZIKV inoculation, ZIKV plasma viral loads in co-infected macaques were similar to ZIKV alone-infected animals, and minimal changes were observed in cytokines/chemokines levels. Viral levels of SIV and SHIV also did not change as compared to pre-ZIKV inoculation levels. These findings thus suggest that co-infection may not alter disease pathogenesis of either HIV or ZIKV infections.

Dissemination of vector-borne viruses, such as Zika virus (ZIKV), in tropical and sub-tropical regions has a complicated impact on the immunopathogenesis of other endemic viruses such as dengue virus (DENV), chikungunya virus (CHIKV) and human immunodeficiency virus (HIV). The consequences of the possible co-infections with these viruses have specifically shown significant impact on the treatment and vaccination strategies. ZIKV is a mosquito-borne flavivirus from African and Asian lineages that causes neurological complications in infected humans. Many of DENV and CHIKV endemic regions have been experiencing outbreaks of ZIKV infection. Intriguingly, the mosquitoes, Aedes Aegypti and Aedes Albopictus, can simultaneously transmit all the combinations of ZIKV, DENV, and CHIKV to the humans. The co-circulation of these viruses leads to a complicated immune response due to the pre-existence or co-existence of ZIKV infection with DENV and CHIKV infections. The non-vector transmission of ZIKV, especially, via sexual intercourse and placenta represents an additional burden that may hander the treatment strategies of other sexually transmitted diseases such as HIV. Collectively, ZIKV co-circulation and co-infection with other viruses have inevitable impact on the host immune response, diagnosis techniques, and vaccine development strategies for the control of these co-infections.

Bovine coronavirus (BCoV) is found worldwide and causes respiratory infections and diarrhoea in calves and adult cattle. In order to investigate the molecular epidemiology of BCoV, 27 reverse transcription polymerase chain reaction (RT-PCR) positive samples from 25 cattle herds in different parts of Sweden were analysed. A 1038-nucleotide fragment was PCR amplified and directly sequenced. The analysed BCoV strains showed a high sequence identity, regardless of whether they were obtained from outbreaks of respiratory disease or diarrhoea or from calves or adult cattle. Circulation of an identical BCoV strain during a 4-month period was demonstrated in calves in one dairy herd. In a regional epizootic of winter dysentery in Northern Sweden, highly similar BCoV strains were detected. In the Southern and Central regions, several genotypes of BCoV circulated contemporaneously, indicating that in these regions, which had a higher density of cattle than the Northern regions, more extensive transmission of the virus was occurring. Identical BCoV sequences supported the epidemiological data that inter-herd contact through purchased calves was important. Swedish BCoV strains unexpectedly showed a high homology with recently detected Italian strains. This study shows that molecular analysis of the spike (S) glycoprotein gene of BCoV can be a useful tool to support or rule out suspected transmission routes.

The prevalence of antibodies to bovine coronavirus (BCV) and bovine respiratory syncytial virus (BRSV) infections was studied in 20 conventional and 20 organic dairy herds. The organic farms had produced ‘certified’ milk for at least 2 years. On two occasions, with a 1-year interval, 699 serum samples from 624 peri-parturient cows were tested by ELISA for antibodies to BCV and BRSV. Accompanying data relating to the sampled animals were collected in order to identify potential factors associated with increased antibody prevalence. The antibody prevalence was high at both sampling times with approximately 85% and 80% of animals positive for antibodies to BCV and to BRSV, respectively. Conventional herds had a significantly higher mean antibody prevalence to BCV and BRSV than the organically managed herds (P

Astroviruses are becoming a growing concern in veterinary and public health. To date there are no registered vaccines against astrovirus-induced disease, mostly due to the difficulty to cultivate astroviruses to high titer for vaccine development using conventional techniques. As means to circumvent this drawback, we have developed stably transfected mink fetal cells and BHK21 cells constitutively expressing the full-length and truncated capsid proteins of two distinct genotypes of mink astrovirus. Protein expression in these stably transfected cells was demonstrated by strong signals as evaluated by in-situ PLA and IFA, and confirmed by Western blotting. The recombinant full-length and truncated proteins induced a high level of antibodies in mink, evaluated by ELISA, demonstrating their immunogenicity. In a challenge experiment in mink, a reduction in presentation clinical signs and virus shedding was observed in mink kits born from immunized females. The gene integration and protein expression were sustained through cell passage, showing that the used approach is robust and reliable for expression of functional capsid proteins for vaccine and diagnostic applications.

Bovine respiratory syncytial virus (BRSV) is one of the major causes of bovine respiratory disease worldwide. In order to study the molecular epidemiology of the virus, samples from 30 BRSV outbreaks in cattle herds located in different parts of Sweden were collected from 2007 to 2011. The samples were analyzed by PCR, and the glycoprotein (G) gene was sequenced. BRSV was detected in outbreaks of respiratory disease in both dairy and feedlot herds most often during the winter period but also during the summer months (May to August). This indicates that circulation of the virus between herds occurs throughout the year. Comparative sequence analysis revealed a high degree (more than 94.5%) of sequence identity among the collected strains. Phylogenetic analysis showed that 29 out of the 30 strains formed a unique clade. Identical sequences found in herds sampled within a few months' time suggested that these herds were part of a common transmission chain. One strain from a single outbreak in a herd in southern Sweden clustered with Danish strains and showed a distant relationship to the rest of the Swedish strains. Further studies are highly warranted to clarify the inter-herd transmission routes of BRSV. Such knowledge is essential for the control of the spread of this virus between herds, regions and even countries.