Werner Herbst

Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child

The coronavirus strain HECV‐4408 was isolated from diarrhea fluid of a 6‐year‐old child with acute diarrhea and propagated in human rectal tumor (HRT‐18) cells. Electron microscopy revealed coronavirus particles in the diarrhea fluid sample and the infected HRT‐18 cell cultures. This virus possessed hemagglutinating and acetylesterase activities and caused cytopathic effects in HRT‐18 cells but not in MDBK, GBK and FE cells. One of four S‐specific monoclonal antibodies reacted in Western blots with HECV‐4408, BCV‐L9 and BCV‐LY138 but not with HCV‐OC43, and two reacted with BCV‐L9 but not with HECV‐4408, BCV‐LY138 and HCV‐OC43. One S‐specific and two N‐specific monoclonal antibodies reacted with all of these strains. cDNA encompassing the 3′ 8.5 kb of the viral RNA genome was isolated by reverse transcription followed by polymerase chain reaction amplification had size and restriction endonuclease patterns similar to those of BCV‐L9 and BCV‐LY138. In contrast, the M gene of HCV‐OC43 differed in restriction patterns from HECV‐4408 and BCV. A genomic deletion located between the S and M within the nonstructural genes of HCV‐OC43 was not detected in HECV‐4408. DNA sequence analyses of the S and HE genes revealed more than 99% nucleotide and deduced amino acid homologies between HECV‐4408 and the virulent wild‐type BCV. Forty‐nine nucleotide and 22 amino acid differences were found between the HE genes of HECV‐4408 and HCV‐OC43, while only 16 nucleotide and 3 amino acid differences occurred between the HE genes of HECV‐4408 and BCVLY138. We thus conclude that the strain HECV‐4408 is a hemagglutinating enteric coronavirus that is biologically, antigenically and genomically more closely related to the virulent BCVLY138 than to HCV‐OC43.

Studies of Epidemiology and Seroprevalence of Bovine Noroviruses in Germany

ABSTRACT Jena virus (JV) is a bovine enteric calicivirus that causes diarrhea in calves. The virus is approximately 30 nm in diameter and has a surface morphology similar to the human Norwalk virus. The genome sequence of JV was recently described, and the virus has been assigned to the genus Norovirus of the family Caliciviridae. In the present study, the JV capsid gene encoded by open reading frame 2 was cloned into the baculovirus transfer vector pFastBac 1, and this was used to transform Escherichia coli to generate a recombinant bacmid. Transfection of insect cells with the recombinant baculovirus DNA resulted in expression of the JV capsid protein. The recombinant JV capsid protein undergoes self-assembly into virus-like particles (VLPs) similar to JV virions in size and appearance. JV VLPs were released into the cell culture supernatant, concentrated, and then purified by CsCl equilibrium gradient centrifugation. Purified JV VLPs were used to hyperimmunize laboratory animals. An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed and characterized initially with clinical specimens containing defined human noroviruses and bovine diarrheal samples from calves experimentally infected with JV; the ELISA was specific only for JV. The ELISA was used to screen 381 diarrheal samples collected from dairy herds in Thuringia, Hesse, and Bavaria, Germany, from 1999 to 2002; 34 of these samples (8.9%) were positive for JV infection. The unexpectedly high prevalence of JV was confirmed in a seroepidemiological study using 824 serum or plasma samples screened using an anti-JV ELISA, which showed that 99.1% of cattle from Thuringia have antibodies to JV.

Demonstration of group C rotaviruses in fecal samples of diarrheic dogs in Germany

Summary Three out of 9 fecal samples from diarrheic dogs, which were positive for rotavirus by electron microscopy, revealed RNA-migration patterns identical to those of porcine group C rotavirus when studied by polyacrylamide gel electrophoresis (PAGE). The results were further confirmed by solid phase immuno electron microscopy (SPIEM). This is the first report of the occurrence of group C rotaviruses in dogs.

Characterization of a reovirus isolate from a rattle snake,Crotalus viridis, with neurological dysfunction

SummaryA virus isolate from the brain of a rattle snake with central nervous system (CNS) symptoms was identified as a reovirus. The isolate did not agglutinate pig erythrocytes and was not neutralized by antisera against avian reovirus S1133 and mammalian reovirus type 3. The cytopathic effect in Vero cells was characterized by formation of syncytial giant cells. Electrophoretic analysis of the genome revealed 10 segments of dsRNA. The isolate displayed characteristics distinct from avian and mammalian reoviruses.

Comparison of the S genes and the biological properties of respiratory and enteropathogenic bovine coronaviruses

SummaryThe nucleotide sequence of the S gene of the bovine respiratory coronavirus (BRCV) strain G95, which was isolated from nasal swabs of a calf suffering from respiratory disorders, was determined and compared with the S gene of the enteropathogenic bovine coronavirus (BECV) strain LY138. Sequence analysis revealed 98.7% nucleotide and 98.3% deduced amino acid identities between the S genes of BRCV-G95 and BECV-LY138 without any deletions or insertions. Nucleotide substitutions were distributed randomly throughout the gene. Five monoclonal antibodies specific for the S protein distinguished BRCV-G95 from BECV-L9, but failed to differentiate it from BECV-LY138 in Western blots under denatured and native conditions. BRCV-G95 induced cytopathic changes in cell cultures that were similar to BECV-LY138 but different from BECV-L9. These results suggest that strain BRCV-G95 is more closely related to the virulent strain BECV-LY138 than to the avirulent, cell culture-adapted strain BECV-L9.

Isolation of a cytopathogenic virus from a case of porcine reproductive and respiratory syndrome (PRRS) and its characterization as parainfluenza virus type 2.

SummaryFrom a lung of a fetus of a breeding sow showing PRRS-like symptoms a viral agent could be isolated. It was characterized as an enveloped, hemag-glutinating RNA virus. Ultrastructural examination of purified virus revealed paramyxovirus-like pleomorphic virions of approx. 200 nm in diameter. The helical nucleocapsids were about 18 nm in diameter. The virus was found to be antigenically related to simian virus 5 (SV5) a prototype strain of parainfluenza virus type 2, but not to bovine respiratory syncytial virus, parainfluenza virus type 1, parainfluenza virus type 3, and Newcastle disease virus as determined by western blot analysis.

Demonstration of genes encoding virulence and virulence life-style factors in Brachyspira spp. isolates from pigs.

The distribution of many genes encoding virulence and virulence life-style (VL-S) factors in Brachyspira (B.) hyodysenteriae and other Brachyspira species are largely unknown. Their knowledge is essential e.g. for the improvement of diagnostic methods targeting the detection and differentiation of the species. Thus 121 German Brachyspira field isolates from diarrhoeic pigs were characterized down to the species level by restriction fragment length polymorphism analysis of the nox gene and subsequently subjected to polymerase chain reaction detecting VL-S genes for inner (clpX) and outer membrane proteins (OMPs: bhlp16, bhlp17.6, bhlp29.7, bhmp39f, bhmp39h), hemolysins (hlyA/ACP, tlyA), iron metabolism (ftnA, bitC), and aerotolerance (nox). For comparison, B. hyodysenteriae reference strains from the USA (n=7) and Australia (2) were used. Of all genes tested only nox was detected in all isolates. The simultaneous presence of both the tlyA and hlyA/ACP was restricted to the species B. hyodysenteriae. The hlyA infrequently occurred also in weakly hemolytic Brachyspira. Similarly to tlyA and hlyA all B. hyodysenteriae strains contained the ferritin gene ftnA which was also found in two Brachyspira intermedia isolates. OMP encoding genes were present in B. hyodysenteriae field isolates in rates of 0% (bhlp17.6, bhmp39h), 58.1% (bhlp29.7), and 97.3% (bhmp39f). Since the study revealed a high genetic heterogeneity among German B. hyodysenteriae field isolates differentiating them from USA as well as Australian strains, targets for diagnostic PCR were limited to the nox gene (genus specific PCR) as well as to the species specific nox(hyo) gene and the combination of hlyA and tlyA which allow to specifically detect B. hyodysenteriae.

Analysis of Multiple Brachyspira hyodysenteriae Genomes Confirms That the Species Is Relatively Conserved but Has Potentially Important Strain Variation.

The intestinal spirochete Brachyspira hyodysenteriae is an important pathogen in swine, causing mucohemorrhagic colitis in a disease known as swine dysentery. Based on the detection of significant linkage disequilibrium in multilocus sequence data, the species is considered to be clonal. An analysis of the genome sequence of Western Australian B. hyodysenteriae strain WA1 has been published, and in the current study 19 further strains from countries around the world were sequenced with Illumina technology. The genomes were assembled and aligned to over 97.5% of the reference WA1 genome at a percentage sequence identity better than 80%. Strain regions not aligned to the reference ranged between 0.2 and 2.5%. Clustering of the strain genes found on average 2,354 (88%) core genes, 255 (8.6%) ancillary genes and 77 (2.9%) unique genes per strain. Depending on the strain the proportion of genes with 100% sequence identity to WA1 ranged from 85% to 20%. The result is a global comparative genomic analysis of B. hyodysenteriae genomes revealing potential differential phenotypic markers for numerous strains. Despite the differences found, the genomes were less varied than those of the related pathogenic species Brachyspira pilosicoli, and the analysis supports the clonal nature of the species. From this study, a public genome resource has been created that will serve as a repository for further genetic and phenotypic studies of these important porcine bacteria. This is the first intra-species B. hyodysenteriae comparative genomic analysis.

Genomic relationship of porcine hemagglutinating encephalomyelitis virus to bovine coronavirus and human coronavirus OC43 as studied by the use of bovine coronavirus S gene-specific probes

SummaryThe genomic relationship of porcine hemagglutinating encephalomyelitis virus (HEV) to bovine coronavirus (BCV) and human coronavirus (HCV) strain OC43 was examined by dot blot hybridization assays. Two BCV S gene-specific probes were generated by polymerase chain reaction from the avirulent L9-strain of BCV. Probes were located in the S1 and the S2 region of the peplomeric (S) glycoprotein gene. The S1 probe (726 bp) hybridized with BCV and HCV-OC43, but not with HEV under moderate stringency hybridization conditions (50 °C). Only slight signals were present with mouse hepatitis virus (MHV) and no signals were observed with feline infectious peritonitis virus (FIPV) or canine coronavirus (CCV). At high stringency conditions (60 °C) the S1 probe hybridized with BCV only. Using the S2 probe (680 bp) under moderate strin-gency conditions, hybridization signals were obtained with BCV, HCV-OC43 and HEV (strains 67N, NT9, VW572). The signals obtained by the three HEV strains were altogether weaker than with BCV and HCV-OC43. The S2 probe did not react with MHV, FIPV and CCV. At high stringency the S2-specific probe hybridized with BCV and HCV-OC43 but did not hybridize with HEV. Nucleotide sequence analysis of the region covering the S2 probe in HEV revealed 92.6% nucleotide sequence homology to BCV and 91.9% to HCV-OC43. In contrast, the region covering the S1 probe in HEV could not be amplified using the BCV S1-specific primers. The hybridization and sequencing results thus indicate a closer genomic relationship between BCV and HCV-OC43 than there is between HEV and BCV or HCV-OC43, respectively.

Antimicrobial susceptibility of Brachyspira hyodysenteriae determined by a broth microdilution method

Swine dysentery (SD) is an economically important disease of pigs worldwide, which results from infection with the anaerobic spirochaete Brachyspira hyodysenteriae (Taylor and Alexander 1971). Clinically the disease is characterised by a mucohaemorrhagic diarrhoea. Due to its severity and lack of vaccination antibiotics such as pleuromutilins, macrolides and lincosamides were widely used in the control of SD. However, treatment is increasingly hampered by a lowered susceptibility of field strains to antibiotics (Lobova and others 2004, Hidalgo and others 2011). Thus, antimicrobial susceptibility testing is a desirable step towards an improved treatment of SD. Unfortunately, there is no approved protocol provided by the Clinical Laboratory Standards Institute (CLSI). Alternatively, the agar dilution method that was mainly used to determine antimicrobial susceptibility of B hyodysenteriae isolates can be used as a reference method (Taylor 1976, Ronne and Szancer 1990, Karlsson and others 2002, 2003). Moreover, despite the fastidious and anaerobic character of B hyodysenteriae , a broth dilution assay was established, which makes a valid minimum inhibitory concentration (MIC) identification possible (Karlsson and others 2002). However, to date, no broth microdilution assay has been reported. Such a test, geared as closely as possible towards international standards such as CLSI, could be applied for valid MIC analyses and also …