Saffold virus infection in elderly people with acute gastroenteritis in Sweden.

Viral gastroenteritis is a major source of morbidity and mortality, predominantly caused by so-called NOROAD viruses (norovirus, rotavirus, and adenovirus). In approximately onethird of all cases, however, the exact etiology is unknown. The in 2007 discovered human cardiovirus Saffold virus (SAFV) may prove to be a plausible candidate to explain this diagnostic gap. This virus, a member of the Picornaviridae family which is closely related to the murine viruses Theiler's murine encephalomyelitis virus and Theravirus, is a widespread pathogen and causes infection early in life. Screening of 238 fecal or vomitus samples obtained from NOROAD-negative, elderly patients with acute gastroenteritis at the University Hospital of Linköping showed that SAFV is present in low abundance (4.6%). Phylogenetic analysis of the VP1 gene revealed a Swedish isolate belonging to the highly common and in Europe widespread SAFV-3 genotype. This genotype is also related to previously reported Asian strains. This study describes the first molecular typing of a Swedish SAFV isolate and is the first report to document the circulation of SAFV among elderly people. The pathogenicity of SAFV is, as of yet, still under debate; further studies are necessary to determine its role in the development of disease.

European wild boars and domestic pigs display different polymorphic patterns in the Toll-like receptor (TLR) 1, TLR2, and TLR6 genes.

During the last decade, the Toll-like receptors (TLRs) have been extensively studied, and their immense importance in innate immunity is now being unveiled. Here, we report pronounced differences--probably reflecting the domestication process and differences in selective pressure--between wild boars and domestic pigs regarding single nucleotide polymorphisms (SNPs) in TLR genes. The open reading frames of TLR1, TLR2, and TLR6 were sequenced in 25 wild boars, representing three populations, and in 15 unrelated domestic pigs of Hampshire, Landrace, and Large White origin. In total, 20, 27, and 26 SNPs were detected in TLR1, TLR2, and TLR6, respectively. In TLR1 and TLR2, the numbers of SNPs detected were significantly lower (P < or = 0.05, P < or = 0.01) in the wild boars than in the domestic pigs. In the wild boars, one major high frequency haplotype was found in all three genes, while the same pattern was exhibited only by TLR2 in the domestic pigs. The relative frequency of non-synonymous (dN) and synonymous (dS) SNPs was lower for the wild boars than for the domestic pigs in all three genes. In addition, differences in diversity between the genes were revealed: the mean heterozygosity at the polymorphic positions was markedly lower in TLR2 than in TLR1 and TLR6. Because of its localization--in proximity of the bound ligand--one of the non-synonymous SNPs detected in TLR6 may represent species-specific function on the protein level. Furthermore, the codon usage pattern in the genes studied deviated from the general codon usage pattern in Sus scrofa.

Characterization of polyclonal antibodies against the capsid proteins of Ljungan virus.

Ljungan virus (LV) is a suspected human pathogen isolated from voles in Sweden and North America. To enable virus detection and studies of localization and activity of virion proteins, polyclonal antibodies were produced against bacterially expressed capsid proteins of the LV strain, 87-012G. Specific detection of proteins corresponding to viral antigens in lysates of LV infected cells was demonstrated by immunoblotting using each one of the generated polyclonal antibodies. In addition, native viral antigens present in cell culture infected with LV strains 87-012G or 145SLG were detected in ELISA and by immunofluorescence using the antibodies against the VP0 and VP1 proteins. The anti-VP3 antibody did not react with native proteins of the LV virion, suggesting that the VP3 is less potent in evoking humoral response and may have a less exposed orientation in the virus capsid. No activity of the antibodies was observed against the closely related human parechovirus type 1. The polyclonal antibody against the VP1 protein was further used for detection of LV infected myocytes in a mouse model of LV-induced myocarditis. Thus, polyclonal antibodies against recombinant viral capsid proteins enabled detection of natural LV virions by several different immunological methods.

Replication of Ljungan virus in cell culture: the genomic 5'-end, infectious cDNA clones and host cell response to viral infections.

Ljungan virus (LV) is a picornavirus recently isolated from bank voles (Clethrionomys glareolus). The previously uncharacterised 5'-end sequence of the LV genome was determined. Infectious cDNA clones were constructed of the wild type LV prototype strain 87-012 and of the cytolytically replicating cell culture adapted variant 87-012G. Virus generated from cDNA clones showed identical growth characteristics as uncloned virus stocks. Cell culture adapted LV, 87-012G, showed a clear cytopathic effect (CPE) at 3-4 days post-infection (p.i.). Virus titers, determined by plaque titration, increased however only within the first 18h p.i. Replication of LV (+) strand RNA was determined by real-time PCR and corresponded in time with increasing titers. In contrast, the amounts of the replication intermediate, the (-) strand, continued to increase until the cells showed CPE. This indicates separate controlling mechanisms for replication of LV (+) and (-) genome strands. Replication was also monitored by immunofluorescence (IF) staining. IF staining of both prototype 87-012 and the CPE causing 87-012G showed groups of 5-25 infected cells at 48h p.i., suggesting a, for picornaviruses, not previously described direct cell-to-cell transmission.

A novel and rapid method to quantify cytolytic replication of picornaviruses in cell culture.

Determining viral titers is a key issue in a wide variety of studies regarding different aspects of virology. The standard methods used for determining picornavirus titers are endpoint titration assay and plaque assay, both time consuming and laborious. The method described uses the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide) that is reduced to formazane by cellular dehydrogenase, genes shown to be down-regulated during picornavirus infection. The amount formazane produced correlates with the viral titers obtained and can easily be measured using an ELISA plate reader. The colorimetric method has been evaluated using virus types from different genera of the Picornaviridae family. The MTT method reduces the time spent on determining the viral titers and still maintains a reliable accuracy.

Molecular analysis of the echovirus 18 prototype: evidence of interserotypic recombination with echovirus 9.

Echovirus 18 (EV18) is one of the echovirus serotypes associated with human diseases and in particular aseptic meningitis. To facilitate studies of the molecular epidemiology of EV18 and the evolution of enteroviruses in general, the complete nucleotide (nt) sequence was determined for the echovirus 18 prototype strain (Metcalf, EV18M). Excluding the poly A sequence, the genome consists of 7410 nt divided into a 740 nt 5' untranslated region (5' UTR), a 6567 nt long open reading frame coding for a 2189 amino acid (aa) polyprotein and a 103 nt 3' UTR. Molecular analysis of the EV18M genome showed a typical enterovirus-like organization. Phylogenetic analysis of the structural and non-structural genes revealed a pattern of different relationships to other echo- and coxsackieviruses. Similarity analysis demonstrated that the Hill strain of echovirus 9 is most likely the result of a previous recombination event between ancestors of the echovirus 9 strain Barty (5' half of the genome) and EV18M (3' half). Using a maximum likelihood approach, the recombination point was mapped to the 2C gene.

Studies of Echovirus 5 interactions with the cell surface: heparan sulfate mediates attachment to the host cell.

Infections caused by Echovirus 5 (E5), an enterovirus of the Picornaviridae family, have been associated with fever, rashes and sporadic cases of aseptic meningitis. To elucidate the receptor usage of this virus, the significance of a previously proposed integrin binding arginine-glycine-aspartic acid (RGD) motif found in the VP3 capsid protein was investigated, as well as the capacity of E5 to interact with heparan sulfate on the cell surface. Using the prototype strain E5 Noyce (E5N), an E5N mutant where the aspartic acid of the RGD motif has been substituted to a glutamic acid and clinical E5 isolates, the RGD motif of VP3 was found to be non-essential and hence not involved in integrin receptor binding. However, E5N and clinical E5 isolates interact with heparan sulfate at the cell surface, as demonstrated by virus replication inhibition assays using heparin and heparinase III, and studies of E5 interactions at the cell surface measured by real-time PCR analysis. In conclusion, E5 utilizes heparan sulfate as a cellular receptor, but the RGD motif of VP3 is not essential for E5 infectivity.

Physicochemical properties of the Ljungan virus prototype virion in different environments: inactivated by heat but resistant to acidic pH, detergents and non-physiological environments such as Virkon-containing solutions.

It is of great importance to know how a virus particle is affected by environmental conditions. Physicochemical properties of the virion will affect the virus viability in different environments, viral transmission between hosts, and will also be important for safe handling of the virus. The physicochemical properties of the Ljungan virus (LV) prototype, 87-012, adapted to grow in cell culture were evaluated using both LV in crude cell extracts and purified virions. Replication of LV was completely inhibited by heat. Titers of LV were unaffected by acidic pH, reduced but not completely abolished by alkaline pH, and unaffected by exposure to the detergents Triton X-100 and SDS. Surprisingly, viable LV was still detected after incubation in the acidic, oxidising and detergent-containing environment produced by the commonly used disinfectant Virkon. In conclusion, LV is resilient to extreme pH, detergents and also to oxidising environments, but is sensitive to heat treatment.

Cell culture propagation and biochemical analysis of the Ljungan virus prototype strain.

Ljungan virus (LV) is proposed as a potentially important rodent harbored viral human pathogen. Little is known about the biophysical nature of the virus and despite being molecularly characterized, progress in epidemiological and basic biological studies of LV has been hampered by the lack of a robust and reliable cell culture propagation system. Here we report the first description of an efficient lytic multi-cycle cell culture propagation of the LV prototype strain (87-012). Biophysical analysis of gradient purified LV virions generated by this system identified mature infectious virions to possess a sedimentation coefficient of 160S and in agreement with previous molecular prediction, polyprotein analysis suggests that the native virion is composed of only three major structural proteins. The nucleotide composition of the complete genome of the LV cell culture adapted virus was determined and compared to that of the parental prototype LV. Numerous mutations were observed scattered throughout the viral genome and particularly in VP1. The development of this cell culture system for LV should open new avenues in the study of LV biology, structure, pathogenesis, and prevalence of natural infection in the wider community.