Report of the third conference on next-generation sequencing for adventitious virus detection in biologics for humans and animals.

Next-generation sequencing (NGS) has been proven to address some of the limitations of the current testing methods for adventitious virus detection in biologics. The International Alliance for Biological Standardization (IABS), the U.S. Food and Drug Administration (FDA), and the European Directorate for the Quality of Medicines and Healthcare (EDQM) co-organized the "3rd Conference on Next-generation Sequencing for Adventitious Virus Detection in Biologics for Humans and Animals", which was held on September 27-28, 2022, in Rockville, Maryland, U.S.A. The meeting gathered international representatives from regulatory and public health authorities and other government agencies, industry, contract research organizations, and academia to present the current status of NGS applications and the progress on NGS standardization and validation for detection of viral adventitious agents in biologics, including human and animal vaccines, gene therapies, and biotherapeutics. Current regulatory expectations were discussed for developing a scientific consensus regarding using NGS for detection of adventitious viruses. Although there are ongoing improvements in the NGS workflow, the development of reference materials for facilitating method qualification and validation support the current use of NGS for adventitious virus detection.

Towards rabies elimination in the Asia-Pacific region: From theory to practice.

Rabies is a major neglected zoonotic disease and causes a substantial burden in the Asian region. Currently, Pacific Oceania is free of rabies but enzootic areas throughout southeast Asia represent a major risk of disease introduction to this region. On September 25-26, 2019, researchers, government officials and related stakeholders met at an IABS conference in Bangkok, Thailand to engage on the topic of human rabies mediated by dogs. The objective of the meeting was focused upon snowballing efforts towards achieving substantial progress in rabies prevention, control and elimination within Asia by 2030, and thereby to safeguard the Pacific region. Individual sessions focused upon domestic animal, wildlife and human vaccination; the production and evaluation of quality, safety and efficacy of existing rabies biologics; and the future development of new products. Participants reviewed the progress to date in eliminating canine rabies by mass vaccination, described supportive methods to parenteral administration by oral vaccine application, considered updated global and local approaches at human prophylaxis and discussed the considerable challenges ahead. Such opportunities provide continuous engagement on disease management among professionals at a trans-disciplinary level and promote new applied research collaborations in a modern One Health context.

Molecular ecology of the yet uncultured bacterial Ct85-cluster in the mammalian gut.

In our previous studies on irritable bowel syndrome (IBS) -associated microbiota by molecular methods, we demonstrated that a particular 16S rRNA gene amplicon was more abundant in the feces of healthy subjects or mixed type IBS (IBS-M) -sufferers than in the feces of individuals with diarrhea-type IBS (IBS-D). In the current study, we demonstrated that this, so called Ct85-amplicon, consists of a cluster of very heterogeneous 16S rRNA gene sequences, and defined six 16S rRNA gene types, a to f, within this cluster, each representing a novel species-, genus- or family level taxon. We then designed specific PCR primers for these sequence types, mapped the distribution of the Ct85-cluster sequences and that of the newly defined sequence types in several animal species and compared the sequence types present in the feces of healthy individuals and IBS sufferers using two IBS study cohorts, Finnish and Dutch. Various Ct85-cluster sequence types were detected in the fecal samples of several companion and production animal species with remarkably differing prevalences and abundances. The Ct85 sequence type composition of swine closely resembled that of humans. One of the five types (d) shared between humans and swine was not present in any other animals tested, while one sequence type (b) was found only in human samples. In both IBS study cohorts, one type (e) was more prevalent in healthy individuals than in the IBS-M group. By revealing various sequence types in the widespread Ct85-cluster and their distribution, the results improve our understanding of these uncultured bacteria, which is essential for future efforts to cultivate representatives of the Ct85-cluster and reveal their roles in IBS.

Evolutionary trends of European bat lyssavirus type 2 including genetic characterization of Finnish strains of human and bat origin 24 years apart.

Among other Lyssaviruses, Daubenton's and pond-bat-related European bat lyssavirus type 2 (EBLV-2) can cause human rabies. To investigate the diversity and evolutionary trends of EBLV-2, complete genome sequences of two Finnish isolates were analysed. One originated from a human case in 1985, and the other originated from a bat in 2009. The overall nucleotide and deduced amino acid sequence identity of the two Finnish isolates were high, as well as the similarity to fully sequenced EBLV-2 strains originating from the UK and the Netherlands. In phylogenetic analysis, the EBLV-2 strains formed a monophyletic group that was separate from other bat-type lyssaviruses, with significant support. EBLV-2 shared the most recent common ancestry with Bokeloh bat lyssavirus (BBLV) and Khujan virus (KHUV). EBLV-2 showed limited diversity compared to RABV and appears to be well adapted to its host bat species. The slow tempo of viral evolution was evident in the estimations of divergence times for EBLV-2: the current diversity was estimated to have built up during the last 2000 years, and EBLV-2 diverged from KHUV about 8000 years ago. In a phylogenetic tree of partial N gene sequences, the Finnish EBLV-2 strains clustered with strains from Central Europe, supporting the hypothesis that EBLV-2 circulating in Finland might have a Central European origin. The Finnish EBLV-2 strains and a Swiss strain were estimated to have diverged from other EBLV-2 strains during the last 1000 years, and the two Finnish strains appear to have evolved from a common ancestor during the last 200 years.

Bat rabies surveillance in Finland.

BACKGROUND: In 1985, a bat researcher in Finland died of rabies encephalitis caused by European bat lyssavirus type 2 (EBLV-2), but an epidemiological study in 1986 did not reveal EBLV-infected bats. In 2009, an EBLV-2-positive Daubenton's bat was detected. The EBLV-2 isolate from the human case in 1985 and the isolate from the bat in 2009 were genetically closely related. In order to assess the prevalence of EBLVs in Finnish bat populations and to gain a better understanding of the public health risk that EBLV-infected bats pose, a targeted active surveillance project was initiated. RESULTS: Altogether, 1156 bats of seven species were examined for lyssaviruses in Finland during a 28-year period (1985-2012), 898 in active surveillance and 258 in passive surveillance, with only one positive finding of EBLV-2 in a Daubenton's bat in 2009. In 2010-2011, saliva samples from 774 bats of seven species were analyzed for EBLV viral RNA, and sera from 423 bats were analyzed for the presence of bat lyssavirus antibodies. Antibodies were detected in Daubenton's bats in samples collected from two locations in 2010 and from one location in 2011. All seropositive locations are in close proximity to the place where the EBLV-2 positive Daubenton's bat was found in 2009. In active surveillance, no EBLV viral RNA was detected. CONCLUSIONS: These data suggest that EBLV-2 may circulate in Finland, even though the seroprevalence is low. Our results indicate that passive surveillance of dead or sick bats is a relevant means examine the occurrence of lyssavirus infection, but the number of bats submitted for laboratory analysis should be higher in order to obtain reliable information on the lyssavirus situation in the country.

Characterization of perch rhabdovirus (PRV) in farmed grayling Thymallus thymallus.

Two Finnish fish farms experienced elevated mortality rates in farmed grayling Thymallus thymallus fry during the summer months, most typically in July. The mortalities occurred during several years and were connected with a few neurological disorders and peritonitis. Virological investigation detected an infection with an unknown rhabdovirus. Based on the entire glycoprotein (G) and partial RNA polymerase (L) gene sequences, the virus was classified as a perch rhabdovirus (PRV). Pairwise comparisons of the G and L gene regions of grayling isolates revealed that all isolates were very closely related, with 99 to 100% nucleotide identity, which suggests the same origin of infection. Phylogenetic analysis demonstrated that they were closely related to the strain isolated from perch Perca fluviatilis and sea trout Salmo trutta trutta caught from the Baltic Sea. The entire G gene sequences revealed that all Finnish grayling isolates, and both the perch and sea trout isolates, were most closely related to a PRV isolated in France in 2004. According to the partial L gene sequences, all of the Finnish grayling isolates were most closely related to the Danish isolate DK5533 from pike. The genetic analysis of entire G gene and partial L gene sequences showed that the Finnish brown trout isolate ka907_87 shared only approximately 67 and 78% identity, respectively, with our grayling isolates. The grayling isolates were also analysed by an immunofluorescence antibody test. This is the first report of a PRV causing disease in grayling in Finland.

Viral haemorrhagic septicaemia virus (VHSV) genotype II isolated from European river lamprey Lampetra fluviatilis in Finland during surveillance from 1999 to 2008.

We examined the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the main spawning stocks of wild European river lamprey Lampetra fluviatilis in the rivers of Finland from 1999 to 2008. Pooled samples of internal organs (kidney, liver and heart or brain) from 2621 lampreys were examined for the presence of VHSV by standard virological techniques. VHSV was isolated from 5 samples from the rivers Lestijoki and Kalajoki, which flow from Finland into the Bothnian Bay of the Baltic Sea. The presence of VHSV was confirmed by immunofluorescent antibody technique (IFAT), ELISA and RT-PCR. Phylogenetic analysis based on the full-length VHSV glycoprotein (G) gene sequence revealed that the isolates were most closely related to the VHSV strain isolated in 1996 from herring Clupea harengus and sprat Sprattus sprattus in the Eastern Gotland Basin of the Baltic Sea, and were therefore assigned to VHSV genotype II. The partial G gene sequences obtained (nt 1 to 672-1129) of all 5 lamprey VHSV isolates were identical, and so were the entire G genes (nt 1 to 1524) of 2 isolates sequenced. The virulence of one of the lamprey isolates was evaluated by an experimental infection trial in rainbow trout Oncorhynchus mykiss fry. No mortality was induced postinfection by waterborne and intraperitoneal challenge, respectively, while 2 genotype Id isolates originating from Finnish rainbow trout caused marked mortality under the same conditions. The infection in the European river lamprey is thought to be independent from the epidemic in farmed rainbow trout in Finnish brackish waters, because the isolates from rainbow trout were of a different genotype. This is the first report of VHSV found in the European river lamprey. The role of wild river lampreys in maintaining the infection in the marine environment remains unclear.

Genetic characterization of maedi-visna virus (MVV) detected in Finland.

The aim of the study was to characterize the small-ruminant lentiviruses (SRLVs) detected in Finland by defining their phylogenetic relationships and by studying the evolution of the virus based on a well-known epidemiology. The study material comprised lung tissue samples of 20 sheep from 5 different farms, a cell-cultured virus from one of the original sheep lung samples, and a blood sample of a goat. The sheep were identified as positive during seroepidemiologic screenings in 1994-1996 and the goat in 2001. Initial classification of a 251 nucleotide sequence within gag gene amplified from the uncultured samples as well as from the cell-cultured virus showed that the SRLVs were genetically close and that they were more closely related to the prototype ovine maedi-visna viruses (MVVs) than to the caprine arthritis-encephalitis virus (CAEV). The lentivirus detected from the goat aligned within the cluster of the Finnish ovine viruses, demonstrating a natural sheep-to-goat transmission. Further phylogenetic analysis of the proviral gag, pol and env sequences confirmed the initial classification and showed that they constituted a new subtype within the diverse MVV group. The sequence analyses also showed that the virus had remained genetically relatively stable, in spite of the time given for virus evolution, an estimated 20 years, and in spite of the virus crossing the host species barrier.

Isolation of surface (S) layer protein carrying Lactobacillus species from porcine intestine and faeces and characterization of their adhesion properties to different host tissues.

Surface-layer proteins (Slps) of lactobacilli have been shown to confer tissue adherence. This study aimed to isolate and identify Slps carrying Lactobacillus species from the porcine intestine and faeces and to characterize these S-layer-expressing strains for their ability to adhere to the pig and human intestinal cells and to extracellular matrix (ECM) proteins. In total 99 strains, putatively belonging to the genus Lactobacillus, were isolated as pure cultures. SDS-PAGE and a gene probe specific for the Lactobacillus brevis ATCC 8287 S-layer protein gene (slpA) were used to screen the presence of strains possessing putative Slps. Eight of the 99 pure cultures exhibited Slps according to the SDS-PAGE analyses. In these strains the presence of genes encoding Slps was confirmed by PCR and partial sequencing. Only one isolate of the 99 strains gave a positive hybridisation signal with the L. brevis slpA probe but did not appear to produce S-layer protein. Their taxonomic identification, based on phenotyping and the 16S rRNA sequences, revealed that the eight S-layer protein-producing strains were closely related to Lactobacillus amylovorus, Lactobacillus sobrius and Lactobacillus crispatus. The strain with the slpA positive hybridisation result was identified as Lactobacillus mucosae. The SDS-extractable protein profile, the size of the putative S-layer protein and binding capability of the strains varied greatly, even among the isolates belonging to the same Lactobacillus cluster. Removal of the intact Slps from the bacterial surface by extraction with guanidine hydrochloride reduced the adhesion of some strains to fibronectin and laminin, whereas, the adhesiveness to laminin increased with some strains.

Efficacy of rabies vaccines in dogs and cats and protection in a mouse model against European bat lyssavirus type 2.

BACKGROUND: Rabies is preventable by pre- and/or post-exposure prophylaxis consisting of series of rabies vaccinations and in some cases the use of immunoglobulins. The success of vaccination can be estimated either by measuring virus neutralising antibodies or by challenge experiment. Vaccines based on rabies virus offer cross-protection against other lyssaviruses closely related to rabies virus. The aim was to assess the success of rabies vaccination measured by the antibody response in dogs (n = 10,071) and cats (n = 722), as well as to investigate the factors influencing the response to vaccination when animals failed to reach a rabies antibody titre of ≥ 0.5 IU/ml. Another aim was to assess the level of protection afforded by a commercial veterinary rabies vaccine against intracerebral challenge in mice with European bat lyssavirus type 2 (EBLV-2) and classical rabies virus (RABV), and to compare this with the protection offered by a vaccine for humans. RESULTS: A significantly higher proportion of dogs (10.7%, 95% confidence interval CI 10.1-11.3) than cats (3.5%; 95% CI 2.3-5.0) had a vaccination antibody titre of < 0.5 IU/ml. In dogs, vaccination with certain vaccines, vaccination over 6 months prior the time of antibody determination and vaccination of dogs with a size of > 60 cm or larger resulted in a higher risk of failing to reach an antibody level of at least 0.5 IU/ml. When challenged with EBLV-2 and RABV, 80 and 100% of mice vaccinated with the veterinary rabies vaccine survived, respectively. When mice were vaccinated with the human rabies vaccine and challenged with EBLV-2, 75-80% survived, depending on the booster. All vaccinated mice developed sufficient to high titres of virus-neutralising antibodies (VNA) against RABV 21-22 days post-vaccination, ranging from 0.5 to 128 IU/ml. However, there was significant difference between antibody titres after vaccinating once in comparison to vaccinating twice (P < 0.05). CONCLUSIONS: There was a significant difference between dogs and cats in their ability to reach a post vaccination antibody titre of ≥ 0.5 IU/ml. Mice vaccinated with RABV-based rabies vaccines were partly cross-protected against EBLV-2, but there was no clear correlation between VNA titres and cross-protection against EBLV-2. Measurement of the RABV VNA titre can only be seen as a partial tool to estimate the cross-protection against other lyssaviruses. Booster vaccination is recommended for dogs and cats if exposed to infected bats.

Effect of Lactobacillus brevis ATCC 8287 as a feeding supplement on the performance and immune function of piglets.

Lactobacillus brevis ATCC 8287, a surface (S-layer) strain, possesses a variety of functional properties that make it both a potential probiotic and a good vaccine vector candidate. With this in mind, our aim was to study the survival of L. brevis in the porcine gut and investigate the effect of this strain on the growth and immune function of recently weaned piglets during a feeding trial. For this, 20 piglets were divided evenly into a treatment and a control group. Piglets in the treatment group were fed L. brevis cells (1×10(10)) daily for three weeks, whereas those in the control group were provided an equivalent amount of probiotic-free placebo. For assessing the impact of L. brevis supplementation during the feeding trial, health status and weight gain of the piglets were monitored, pre- and post-trial samples of serum and feces were obtained, and specimens of the small and large intestinal mucosa and digesta were collected at slaughter. The results we obtained indicated that L. brevis-supplemented feeding induced a non-significant increase in piglet body weight and caused no change in the morphology of the intestinal mucosa. L. brevis cells were found to localize mainly in the large intestine, but they could not be isolated from feces. To a lesser extent, L. brevis was detected in the small intestine, although there was no specific attachment to the Peyer's patches. Changes in total serum IgG and IgA concentrations were not caused by supplemented L. brevis and no measurable rise in L. brevis-specific IgG was observed. However, analysis of cytokine gene expression in intestinal mucosa revealed downregulation of TGF-ß1 in the ileum and upregulation of IL-6 in the cecum in the L. brevis-supplemented group. Based on the results from this study, we conclude that whereas L. brevis appears to have some intestinal immunomodulatory effects, the ability of this strain to survive and colonize within the porcine gut appears to be limited.

Lactobacillus sobrius Konstantinov et al. 2006 is a later synonym of Lactobacillus amylovorus Nakamura 1981.

While studying the taxonomy of six lactic acid bacterium isolates from Finnish porcine intestine and faeces, the taxonomic positions of Lactobacillus sobrius type strain DSM 16698T and strain AD5 based on comparative 16S rRNA sequence analysis were found to be controversial, as they showed high similarity to Lactobacillus amylovorus strains. Therefore, the taxonomy of these species was addressed in a polyphasic taxonomy study that included, in addition to re-evaluating the 16S rRNA gene sequence and DNA-DNA reassociation results, multilocus sequence analysis (MLSA) of the housekeeping genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) as well as numerical analysis of HindIII and EcoRI ribotypes. 16S rRNA gene sequence analysis demonstrated a very high similarity between the L. sobrius and L. amylovorus type and reference strains and representative Finnish porcine isolates (99.6-99.9 %). The MLSA data showed the close phylogenetic relationship of these strains; pheS and rpoA gene sequence similarities were 98.5-100 % and 99.6-99.8 %, respectively. Numerical analyses of HindIII/EcoRI ribotypes placed these strains in a single cluster by both enzymes. Finally, the DNA-DNA reassociation experiments revealed high reassociation levels (higher than 79 %) between the strains. These results indicate that DSM 16698T, AD5 and the related porcine lactobacilli strains from Finland constitute a single species, Lactobacillus amylovorus, and that the name Lactobacillus sobrius should be considered as a later synonym of Lactobacillus amylovorus.

Isolation of three new surface layer protein genes (slp) from Lactobacillus brevis ATCC 14869 and characterization of the change in their expression under aerated and anaerobic conditions.

Two new surface layer (S-layer) proteins (SlpB and SlpD) were characterized, and three slp genes (slpB, slpC, and slpD) were isolated, sequenced, and studied for their expression in Lactobacillus brevis neotype strain ATCC 14869. Under different growth conditions, L. brevis strain 14869 was found to form two colony types, smooth (S) and rough (R), and to express the S-layer proteins differently. Under aerobic conditions R-colony type cells produced SlpB and SlpD proteins, whereas under anaerobic conditions S-colony type cells synthesized essentially only SlpB. Anaerobic and aerated cultivations of ATCC 14869 cells in rich medium also resulted in S-layer protein patterns similar to those of the S- and R-colony type cells, respectively. Electron microscopy suggested the presence of only a single S-layer with an oblique structure on the cells of both colony forms. The slpB and slpC genes were located adjacent to each other, whereas the slpD gene was not closely linked to the slpB-slpC gene region. Northern analyses confirmed that both slpB and slpD formed a monocistronic transcription unit and were effectively expressed, but slpD expression was induced under aerated conditions. slpC was a silent gene under the growth conditions tested. The amino acid contents of all the L. brevis ATCC 14869 S-layer proteins were typical of S-layer proteins, whereas their sequence similarities with other S-layer proteins were negligible. The interspecies identity of the L. brevis S-layer proteins was mainly restricted to the N-terminal regions of those proteins. Furthermore, Northern analyses, expression of a PepI reporter protein under the control of the slpD promoter, and quantitative real-time PCR analysis of slpD expression under aerated and anaerobic conditions suggested that, in L. brevis ATCC 14869, the variation of S-layer protein content involves activation of transcription by a soluble factor rather than DNA rearrangements that are typical for most of the S-layer phase variation mechanisms known.