Immunogenicity and Efficacy Evaluation of Subunit Astrovirus Vaccines.

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 CPΔN (spanning amino acids 161-775) and CPΔC (spanning amino acids 1-621) 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 CPΔN. 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 CPΔN. 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.

Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus.

Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease.

Molecular investigations on the prevalence and viral load of enteric viruses in pigs from five European countries.

Enteric viral infections in pigs may cause diarrhea resulting in ill-thrift and substantial economic losses. This study reports the enteric infections with porcine astrovirus type 4 (PAstV4), porcine group A rotavirus (GARV), porcine group C rotavirus (GCRV), porcine circovirus type 2 (PCV2) and porcine kobuvirus (PKoV) in 419 pigs, comprising both healthy and diarrheic animals, from 49 farms in five European countries (Austria, Germany, Hungary, Spain and Sweden). Real-time RT-PCR assays were developed to test fecal samples and to compare the prevalence and viral load in relation to health status, farms of origin and age groups. The results showed that PAstV4 (70.4%) was the dominant virus species, followed by PKoV (56.7%), PCV2 (42.2%), GCRV (3%) and GARV (0.9%). Diarrheic pigs had a higher viral load of PAstV4 in the nursery and growing-finishing groups. Rotaviruses were mainly detected in diarrheic pigs, whereas PCV2 was more often detected in clinically healthy than in diarrheic pigs, suggesting that most PCV2 infections were subclinical. PAstV4, PCV2 and PKoV were considered ubiquitous in the European pig livestock and co-infections among them were frequent, independently of the disease status, in contrast to a low prevalence of classical rotavirus infections.

Establishment of stably transfected cells constitutively expressing the full-length and truncated antigenic proteins of two genetically distinct mink astroviruses.

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.

Equine arteritis virus induced cell death is associated with activation of the intrinsic apoptotic signalling pathway.

Equine arteritis virus (EAV) causes a respiratory and reproductive disease in horses, equine viral arteritis. Though cell death in infection with EAV is considered to occur by apoptosis, the underlying molecular mechanism has not been extensively elucidated. We investigated the expression of mRNA of pro-apoptotic and caspase genes during EAV infection in BHK21 cells, a well-established cell type for EAV replication. Using a SYBR Green real-time PCR, mRNA of p53, Bax, caspase 3 and caspase 9 were found up-regulated in a time dependent manner in EAV infected cells. Western blot analysis for caspase 3 and caspase 9 showed expression of cleaved forms of these proteins during EAV infection. In addition, a luminescence-based cell assay for caspase 3/7 activation as a hallmark in apoptosis confirmed apoptotic cell death. The findings demonstrate that cell death in EAV infected BHK21 cells results from apoptosis mediated through the intrinsic signalling pathway.

NF-κB activation by equine arteritis virus is MyD88 dependent and promotes viral replication.

NF-κB, a family of transcription factors involved in different cell functions and immune responses is targeted by viruses. The mechanism of NF-κB signalling and its role in replication of EAV have not been investigated. We demonstrate that EAV infection in BHK-21 cells activates NF-κB, and this activation was found to be mediated through the MyD88 pathway. Infection of IKKß(-/-) murine embryo fibroblasts (MEFs), which are deficient in NF-κB signalling, resulted in lower virus titre, less cytopathic effect, and reduced expression of viral proteins. These findings implicate the MyD88 pathway in EAV-induced NF-κB activation and suggest that NF-κB activation is essential for efficient replication of EAV.

Bioinformatics and evolutionary insight on the spike glycoprotein gene of QX-like and Massachusetts strains of infectious bronchitis virus.

BACKGROUND: Infectious bronchitis virus (IBV) is a Gammacoronavirus of the family Coronaviridae and is a causative agent of an economically important disease in poultry. The spike glycoprotein of IBV is essential for host cell attachment, neutralization, and is involved in the induction of protective immunity. Previously obtained sequence data of the spike gene of IBV QX-like and Massachusetts strains were subjected to bioinformatics analysis. FINDINGS: On analysis of potential phosphorylation sites, the Ser542 and Ser563 sites were not present in Massachusetts strains, while QX-like isolates did not have the Ser534 site. Massachusetts and QX-like strains showed different cleavage site motifs. The N-glycosylation sites ASN-XAA-SER/THR-55, 147, 200 and 545 were additionally present in QX-like strains. The leucine-rich repeat regions in Massachusetts strains consisted of stretches of 63 to 69 amino acids, while in the QX-like strains they contained 59 amino acids in length. An additional palmitoylation site was observed in CK/SWE/082066/2010 a QX-like strain. Primary structure data showed difference in the physical properties and hydrophobic nature of both genotypes. The comparison of secondary structures revealed no new structural domains in the genotypic variants. The phylogenetic analyses based on avian and mammalian coronaviruses showed the analysed IBV as closely related to turkey coronaviruses and distantly related to thrush and munia coronaviruses. CONCLUSION: The study demonstrated that spike glycoprotein of the Massachusetts and the QX-like variants of IBV are molecularly distinct and that this may reflect in differences in the behavior of these viruses in vivo.

Tracing the transmission of bovine coronavirus infections in cattle herds based on S gene diversity.

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.

Characterization and analysis of the full-length genome of a strain of the European QX-like genotype of infectious bronchitis virus.

In recent years, strains of infectious bronchitis virus belonging to the QX-like genotype have been causing huge economic losses in commercial chicken flocks in different countries in Europe. In order to expand the knowledge of the molecular features of these viruses, we have sequenced and characterized the complete genome of European QX-like IBV strain CK/SWE/0658946/10, which was isolated in 2010 in Sweden. The genome is 27664 nucleotides in length, comprising six genes and 5' and 3' untranslated regions. The ORF1a, spike and nucleocapsid genes were under strong positive selective pressure that resulted in genetic diversity in relation to classical IBV isolates. The full-length genome of the CK/SWE/0658946/10 strain has the highest nucleotide sequence identity (93.18%) to ITA/90254/2005 and the lowest nucleotide identity (89.10%) to strain CQ04-1. Phylogenetic analysis of partial S1 gene sequences of IBV strains showed that the European QX-like genotype comprises strains that have been predominantly circulating in this continent for the past decade.

Biological characterization and phylogenetic analysis of a novel genetic group of Newcastle disease virus isolated from outbreaks in commercial poultry and from backyard poultry flocks in Pakistan.

Newcastle disease (ND) is a contagious viral disease of many avian species particularly domestic poultry, and is responsible for devastating outbreaks in the poultry industries around the globe. In spite of its importance and endemicity in Southern Asia, data on the genetic nature of the viruses and epizootiological information of the disease is scarce. In this study, six isolates from an emerging wave of ND outbreaks in the north of Pakistan and two isolates from healthy poultry flocks were biologically and genetically characterized. Based on pathogenicity indices such as intracerebral pathogenicity index (ICPI), mean death time (MDT) and cleavage motifs in the fusion protein, all these isolates were classified as virulent. Phylogenetic analysis of the fusion (F), hemagglutinin-neuraminidase (HN) and matrix (M) genes indicated the emergence of a novel genetic group within lineage 5, distinct from isolates previously reported in the region. Several mutations in the neutralizing epitopes and functionally important motifs of the F and HN genes pose a need for re-evaluation of the currently used vaccine and vaccination practices. The characteristics of Newcastle disease virus (NDV) as virulent (F protein cleavage site, ICPI and MDT) in apparently healthy backyard poultry (BYP) explain that BYP can play crucial role in the epizootiology and spread of the disease. The present investigation provides essential information on the genetic nature of NDV circulating in Pakistan and its implication on disease diagnosis and control. Furthermore, these investigations emphasize the importance of continuous surveillance of ND in developing countries.

Phylogenetic analysis of bovine respiratory syncytial viruses from recent outbreaks in feedlot and dairy cattle herds.

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.

Emergence of novel strains of avian infectious bronchitis virus in Sweden.

Infectious bronchitis virus (IBV) causes avian infectious bronchitis, an important disease that produces severe economic losses in the poultry industry worldwide. Recent IBV infections in Sweden have been associated with poor growth in broilers, drop in egg production and thin egg shells in layers. The complete spike gene of selected isolates from IBV cases was amplified and sequenced using conventional RT-PCR. Nucleotide and amino acid sequence comparisons have shown that the recent isolates bear 98.97% genetic similarity with strains of the QX-like genotype. The phylogenetic analysis revealed that strains predominant in the nineties, which were of the Massachusetts type, have been replaced by D388/QX-like strains, however the evolutionary link could not be established. The homology between the two genotypes was 79 and 81%. Remarkably, a strong positive selection pressure was determined, mostly involving the S1 subunit of the S gene. This strong selective pressure resulted in recombination events, insertions and deletions in the S gene. Two new isolates generated from recombination were found with nucleotide sequence diverging 1.7-2.4% from the D388/QX-like branch, indicating the emergence of a new lineage. The study demonstrates a constant evolution of IBV that might be in relation to increased poultry farming, trade and vaccine pressure. The findings underscore the importance of continuous monitoring to control spread of infections, as well as to timely adjust diagnostic methods, molecular epidemiological studies, development and use of vaccines that are adapted to the changing disease scenario.

Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells.

Non-structural protein 1 (NS1) counteracts the production of host type I interferons (IFN-α/ß) for the efficient replication and pathogenicity of influenza A viruses. Here, we reveal another dimension of the NS1 protein of avian influenza A viruses in suppressing IFN-ß production in cultured cell lines. We found that allele A NS1 proteins of H6N8 and H4N6 have a strong capacity to inhibit the activation of IFN-ß production, compared with allele B from corresponding subtypes, as measured by IFN stimulatory response element (ISRE) promoter activation, IFN-ß mRNA transcription and IFN-ß protein expression. Furthermore, the ability to suppress IFN-ß promoter activation was mapped to the C-terminal effector domain (ED), while the RNA-binding domain (RBD) alone was unable to suppress IFN-ß promoter activation. Chimeric studies indicated that when the RBD of allele A was fused to the ED of allele B, it was a strong inhibitor of IFN-ß promoter activity. This shows that well-matched ED and RBD are crucial for the function of the NS1 protein and that the RBD could be one possible cause for this differential IFN-ß inhibition. Notably, mutagenesis studies indicated that the F103Y and Y103F substitutions in alleles A and B, respectively, do not influence the ISRE promoter activation. Apart from dsRNA signalling, differences were observed in the expression pattern of NS1 in transfected human and mink lung cells. This study therefore expands the versatile nature of the NS1 protein in inhibiting IFN responses at multiple levels, by demonstrating for the first time that it occurs in a manner dependent on allele type.

Whole genome sequencing and characterization of a virulent Newcastle disease virus isolated from an outbreak in Sweden.

In this study, the complete genome sequence of a Newcastle disease virus (NDV) isolate collected from an outbreak in 1995 in chickens was fully characterized and compared with other NDV sequences. The genome was found to be 15,192 nucleotides long and to consist of six genes in the order 3'-NP-P-M-F-HN-L-5', similar to other avian paramyxoviruses type-I. However, a six-nucleotide insertion was observed in the 5' non-coding regions of the nucleoprotein (NP) gene, a feature that is unique to some NDV isolates. The isolate shows the amino acid sequence (112)RRQKRF(117) at the cleavage site of the F protein, which is identical to a known motif for virulent pathotypes of NDV. The phylogenetic analysis of the coding region of the F gene indicated that this isolate belongs to genotype VI, more specifically to genotype VId, along with isolates from the other European countries (Denmark, Switzerland and Austria). The same genotype caused outbreaks in the Middle East and Greece in the late 1960s, and in Hungary, in the early 1980s, suggesting a common source for these outbreaks.

Complete genome characterisation of a Newcastle disease virus isolated during an outbreak in Sweden in 1997.

The complete genome sequence of a Newcastle disease virus (NDV) isolated from a chicken in Sweden was determined and compared with other NDV sequences. The isolate was shown to belong to genotype VIIb, which arose in the Far East and spread around the world during the 1990s. It had a length of 15,192 bases and consisted of six genes in the order 3'-NP-P-M-F-HN-L-5'. The F protein cleavage site was 112-RRQRRF-117, corresponding to that of a virulent pathotype.

Complete genome analysis of an avian paramyxovirus type 1 strain isolated in 1994 from an asymptomatic black-headed gull (Larus ridibundus) in southern Sweden.

The complete genome sequence of an avian paramyxovirus serotype 1 (APMV-1) isolated from a black-headed gull (Larus ridibundus) in Sweden was determined and compared with other APMV-1 sequences. Sequence analyses showed that this isolate consists of six genes in the order 3'-NP-P-M-F-HN-L-5', is 15,186 nucleotides long, and contains a typical, avirulent fusion protein cleavage site. It was also shown to have a hemagglutinin-neuraminidase protein with a length of 585 amino acids (aa) instead of the expected 616 aa. Phylogenetic analyses showed that the isolate belongs to genotype I, and the relationship with some other, known APMV-1 virus sequences was revealed. Waterfowl have been considered to act as a reservoir for APMV-1 and, therefore, it is important to broaden the knowledge of viruses circulating within this population.

Effects of methodology and analysis strategy on robustness of pestivirus phylogeny.

Phylogenetic analysis of pestiviruses is a useful tool for classifying novel pestiviruses and for revealing their phylogenetic relationships. In this study, robustness of pestivirus phylogenies has been compared by analyses of the 5'UTR, and complete N(pro) and E2 gene regions separately and combined, performed by four methods: neighbour-joining (NJ), maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI). The strategy of analysing the combined sequence dataset by BI, ML, and MP methods resulted in a single, well-supported tree topology, indicating a reliable and robust pestivirus phylogeny. By contrast, the single-gene analysis strategy resulted in 12 trees of different topologies, revealing different relationships among pestiviruses. These results indicate that the strategies and methodologies are two vital aspects affecting the robustness of the pestivirus phylogeny. The strategy and methodologies outlined in this paper may have a broader application in inferring phylogeny of other RNA viruses.

Maximum likelihood and Bayesian analyses of a combined nucleotide sequence dataset for genetic characterization of a novel pestivirus, SVA/cont-08.

Bovine viral diarrhoea virus 1 (BVDV-1) and Bovine viral diarrhoea virus 2 (BVDV-2) are two recognised bovine pestivirus species of the genus Pestivirus. Recently, a pestivirus, termed SVA/cont-08, was detected in a batch of contaminated foetal calf serum originating from South America. Comparative sequence analysis showed that the SVA/cont-08 virus shares 15-28% higher sequence identity to pestivirus D32/00_'HoBi' than to members of BVDV-1 and BVDV-2. In order to reveal the phylogenetic relationship of SVA/cont-08 with other pestiviruses, a molecular dataset of 30 pestiviruses and 1,896 characters, comprising the 5'UTR, N(pro) and E2 gene regions, was analysed by two methods: maximum likelihood and Bayesian approach. An identical, well-supported tree topology was observed, where four pestiviruses (SVA/cont-08, D32/00_'HoBi', CH-KaHo/cont, and Th/04_KhonKaen) formed a monophyletic clade that is closely related to the BVDV-1 and BVDV-2 clades. The strategy applied in this study is useful for classifying novel pestiviruses in the future.

Two real-time RT-PCR assays of classical swine fever virus, developed for the genetic differentiation of naturally infected from vaccinated wild boars.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a disease notifiable to the Office International des Epizooties (OIE). A live marker vaccine would be the ultimate choice for controlling CSF, which enables serological and genetic differentiation of vaccine from wild type CSFV. Recently, a marker vaccine CP7_E2alf has been reported [Koenig, P., Lange, E., Reimann, I., Beer, M., 2007. CP7_E2alf: a safe and efficient marker vaccine strain for oral immunisation of wild boar against classical swine fever virus (CSFV). Vaccine 25, 3391-3399]. A vaccine-specific TaqMan real-time RT-PCR assay was developed and evaluated, and a second, wild type-specific assay was modified from an established one in such a way that both can be performed in two wells side-by-side in a microplate in a single run. The detection limit is 50 viral RNA copies per reaction for the vaccine-specific assay, and 20 copies per reaction for the wild type assay. The two assays have been shown to be highly specific and reproducible, with potential application for genetic differentiation of wild type CSFV from the marker vaccine CP7_E2alf in wild boar vaccination programs.

Virus recovery and full-length sequence analysis of atypical bovine pestivirus Th/04_KhonKaen.

Phylogenetic analysis of recently identified "atypical" bovine pestiviruses, performed based on different gene regions, has revealed unclear relationships with other established species, therefore, their phylogenetic position could not be determined so far. In this study, the atypical pestivirus Th/04_KhonKaen was recovered from serum of a naturally infected calf and the complete genome sequence was determined and analysed, as means to define its position. The viral genome is 12,337 nucleotides (nt) long, and comprises a 5'-UTR of 383 nt, a 3'-UTR of 254 nt and an open reading frame of 11,700 nt, without duplication of viral sequences or insertions of cellular sequences. The phylogenetic analyses of the full-length sequence, performed by Neighbor-joining, Maximum likelihood, and the Bayesian approach, unanimously placed Th/04_KhonKaen in a single lineage, distinct from the established pestivirus species, and close to bovine viral diarrhea virus types 1 and 2. Furthermore, Th/04_KhonKaen and two previously reported atypical pestiviruses D32/00_'HoBi' and CH-KaHo/cont formed a well-supported monophyletic clade in trees based on the complete N(pro) and E2 gene regions. The finding provides conclusive classification of the Th/04_KhonKaen virus and confirms the standing of the "atypical" bovine pestiviruses as a novel pestivirus species.