Drivers of infection with Toxoplasma gondii genotype type II in Eurasian red squirrels (Sciurus vulgaris).

BACKGROUND: In September 2014, there was sudden upsurge in the number of Eurasian red squirrels (Sciurus vulgaris) found dead in the Netherlands. High infection levels with the parasite Toxoplasma gondii were demonstrated, but it was unclear what had caused this increase in cases of fatal toxoplasmosis. In the present study, we aimed to gain more knowledge on the pathology and prevalence of T. gondii infections in Eurasian red squirrels in the Netherlands, on the T. gondii genotypes present, and on the determinants of the spatiotemporal variability in these T. gondii infections. The presence of the closely related parasite Hammondia hammondi was also determined. METHODS: Eurasian red squirrels that were found dead in the wild or that had died in wildlife rescue centres in the Netherlands over a period of seven years (2014-2020) were examined. Quantitative real-time polymerase chain reaction was conducted to analyse tissue samples for the presence of T. gondii and H. hammondi DNA. Toxoplasma gondii-positive samples were subjected to microsatellite typing and cluster analysis. A mixed logistic regression was used to identify climatic and other environmental predictors of T. gondii infection in the squirrels. RESULTS: A total of 178 squirrels were examined (49/178 T. gondii positive, 5/178 H. hammondi positive). Inflammation of multiple organs was the cause of death in 29 squirrels, of which 24 were also T. gondii polymerase chain reaction positive. Toxoplasma gondii infection was positively associated with pneumonia and hepatitis. Microsatellite typing revealed only T. gondii type II alleles. Toxoplasma gondii infection rates showed a positive correlation with the number of days of heavy rainfall in the previous 12 months. Conversely, they showed a negative association with the number of hot days within the 2-week period preceding the sampling date, as well as with the percentage of deciduous forest cover at the sampling site. CONCLUSIONS: Toxoplasma gondii infection in the squirrels appeared to pose a significant risk of acute mortality. The T. gondii genotype detected in this study is commonly found across Europe. The reasons for the unusually high infection rates and severe symptoms of these squirrels from the Netherlands remain unclear. The prevalence of T. gondii in the deceased squirrels was linked to specific environmental factors. However, whether the increase in the number of dead squirrels indicated a higher environmental contamination with T. gondii oocysts has yet to be established.

Genetic Determinants for Virulence and Transmission of the Panzootic Avian Influenza Virus H5N8 Clade 2.3.4.4 in Pekin Ducks.

Waterfowl is the natural reservoir for avian influenza viruses (AIV), where the infection is mostly asymptomatic. In 2016, the panzootic high pathogenicity (HP) AIV H5N8 of clade 2.3.4.4B (designated H5N8-B) caused significant mortality in wild and domestic ducks, in stark contrast to the predecessor 2.3.4.4A virus from 2014 (designated H5N8-A). Here, we studied the genetic determinants for virulence and transmission of H5N8 clade 2.3.4.4 in Pekin ducks. While ducks inoculated with recombinant H5N8-A did not develop any clinical signs, H5N8-B-inoculated and cohoused ducks died after showing neurological signs. Swapping of the HA gene segments did not increase virulence of H5N8-A but abolished virulence and reduced systemic replication of H5N8-B. Only H5N8-A carrying H5N8-B HA, NP, and NS with or without NA exhibited high virulence in inoculated and contact ducks, similar to H5N8-B. Compared to H5N8-A, HA, NA, NS, and NP proteins of H5N8-B possess peculiar differences, which conferred increased receptor binding affinity, neuraminidase activity, efficiency to inhibit interferon-alpha induction, and replication in vitro, respectively. Taken together, this comprehensive study showed that HA is not the only virulence determinant of the panzootic H5N8-B in Pekin ducks, but NP, NS, and to a lesser extent NA were also necessary for the exhibition of high virulence in vivo. These proteins acted synergistically to increase receptor binding affinity, sialidase activity, interferon antagonism, and replication. This is the first ad-hoc study to investigate the mechanism underlying the high virulence of HPAIV in Pekin ducks. IMPORTANCE Since 2014, several waves of avian influenza virus (AIV) H5N8 of clade 2.3.4.4 occurred globally on unprecedented levels. Unlike viruses in the first wave in 2014-2015 (H5N8-A), viruses in 2015-2016 (H5N8-B) exhibited unusually high pathogenicity (HP) in wild and domestic ducks. Here, we found that the high virulence of H5N8-B in Pekin ducks could be attributed to multiple factors in combination, namely, hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and nonstructural protein 1 (NS1). Compared to H5N8-A, H5N8-B possesses distinct genetic and biological properties including increased HA receptor-binding affinity and neuraminidase activity. Likewise, H5N8-B NS1 and NP were more efficient to inhibit interferon induction and enhance replication in primary duck cells, respectively. These results indicate the polygenic trait of virulence of HPAIV in domestic ducks and the altered biological properties of the HPAIV H5N8 clade 2.3.4.4B. These findings may explain the unusual high mortality in Pekin ducks during the panzootic H5N8 outbreaks.

Inhibition of interferon I induction by non-structural protein NSs of Puumala virus and other vole-associated orthohantaviruses: phenotypic plasticity of the protein and potential functional domains.

The orthohantavirus Puumala virus (PUUV), which is transmitted by bank voles (Clethrionomys glareolus), and other vole-borne hantaviruses contain in their small (S) genome segment two overlapping open reading frames, coding for the nucleocapsid protein and the non-structural protein NSs, a putative type I interferon (IFN-I) antagonist. To investigate the role of NSs of PUUV and other orthohantaviruses, the expression pattern of recombinant NSs constructs and their ability to inhibit human IFN-I promoter activity were investigated. The NSs proteins of PUUV and related cricetid-borne orthohantaviruses showed strong inhibition of IFN-I promoter induction. We identified protein products originating from three and two methionine initiation codons in the NSs ORF of PUUV during transfection and infection, respectively. The three putative start codons are conserved in all PUUV strains analysed. Translation initiation at these start codons influenced the inhibitory activity of the NSs products, with the wild-type (wt) construct expressing two proteins starting at the first and second methionine and showing strong inhibition activity. Analysis of in vitro-generated variants and naturally occurring PUUV NSs proteins indicated that amino acid variation in the NSs protein is well tolerated, suggesting its phenotypic plasticity. The N-terminal 20-amino-acid region of the NSs protein was found to be associated with strong inhibition and to be highly vulnerable to amino acid exchanges and tag fusions. Infection studies using human, bank vole, and Vero E6 cells did not show obvious differences in the replication capacity of PUUV Sotkamo wt and a strain with a truncated NSs protein (NSs21Stop), showing that the lack of a full-length NSs might be compensated by its N-terminal peptide, as seen in transfection experiments. These results contribute to our understanding of virus-host interactions and highlight the importance of future innate immunity studies in reservoir hosts.

The C-terminus of non-structural protein 1 (NS1) in H5N8 clade 2.3.4.4 avian influenza virus affects virus fitness in human cells and virulence in mice.

Avian influenza viruses (AIV) H5N8 clade 2.3.4.4 pose a public health threat but the viral factors relevant for its potential adaptation to mammals are largely unknown. The non-structural protein 1 (NS1) of influenza viruses is an essential interferon antagonist. It commonly consists of 230 amino acids, but variations in the disordered C-terminus resulted in truncation or extension of NS1 with a possible impact on virus fitness in mammals. Here, we analysed NS1 sequences from 1902 to 2020 representing human influenza viruses (hIAV) as well as AIV in birds, humans and other mammals and with an emphasis on the panzootic AIV subtype H5N8 clade 2.3.4.4A (H5N8-A) from 2013 to 2015 and clade 2.3.4.4B (H5N8-B) since 2016. We found a high degree of prevalence for short NS1 sequences among hIAV, zoonotic AIV and H5N8-B, while AIV and H5N8-A had longer NS1 sequences. We assessed the fitness of recombinant H5N8-A and H5N8-B viruses carrying NS1 proteins with different lengths in human cells and in mice. H5N8-B with a short NS1, similar to hIAV or AIV from a human or other mammal-origins, was more efficient at blocking apoptosis and interferon-induction without a significant impact on virus replication in human cells. In mice, shortening of the NS1 of H5N8-A increased virus virulence, while the extension of NS1 of H5N8-B reduced virus virulence and replication. Taken together, we have described the biological impact of variation in the NS1 C-terminus in hIAV and AIV and shown that this affects virus fitness in vitro and in vivo.

Interaction of Pestiviral E1 and E2 Sequences in Dimer Formation and Intracellular Retention.

Pestiviruses contain three envelope proteins: Erns, E1, and E2. Expression of HA-tagged E1 or mutants thereof showed that E1 forms homodimers and -trimers. C123 and, to a lesser extent, C171, affected the oligomerization of E1 with a double mutant C123S/C171S preventing oligomerization completely. E1 also establishes disulfide linked heterodimers with E2, which are crucial for the recovery of infectious viruses. Co-expression analyses with the HA-tagged E1 wt/E1 mutants and E2 wt/E2 mutants demonstrated that C123 in E1 and C295 in E2 are the critical sites for E1/E2 heterodimer formation. Introduction of mutations preventing E1/E2 heterodimer formation into the full-length infectious clone of BVDV CP7 prevented the recovery of infectious viruses, proving that C123 in E1 and C295 in E2 play an essential role in the BVDV life cycle, and further support the conclusion that heterodimer formation is the crucial step. Interestingly, we found that the retention signal of E1 is mandatory for intracellular localization of the heterodimer, so that absence of the E1 retention signal directs the heterodimer to the cell surface even though the E2 retention signal is still present. The covalent linkage between E1 and E2 plays an essential role for this process.

Spatial distance between sites of sampling associated with genetic variation among Neospora caninum in aborted bovine foetuses from northern Italy.

BACKGROUND: Neospora caninum, a coccidian protozoan, represents an important cause of bovine abortion. Available N. caninum strains show considerable variation in vitro and in vivo, including different virulence in cattle. To which extent sexual recombination, which is possible in the intestines of domestic dogs and closely related carnivores as definitive hosts, contributes to this variation is not clear yet. METHODS: Aborted bovine foetuses were collected between 2015 and early 2019 from Italian Holstein Friesian dairy herds suffering from reproductive problems. A total of 198 samples were collected from 165 intensive farms located in Lombardy, northern Italy. N. caninum samples were subjected to multilocus-microsatellite genotyping using ten previously established microsatellite markers. In addition to our own data, those from a recent study providing data on five markers from other northern Italian regions were included and analysed. RESULTS: Of the 55 samples finally subjected to genotyping, 35 were typed at all or 9 out of 10 loci and their individual multilocus-microsatellite genotype (MLMG) determined. Linear regression revealed a statistically significant association between the spatial distance of the sampling sites with the genetic distance of N. caninum MLMGs (P < 0.001). Including data from this and a previous North Italian study into eBURST analysis revealed that several of N. caninum MLMGs from northern Italy separate into four groups; most of the samples from Lombardy clustered in one of these groups. Principle component analysis revealed similar clusters and confirmed MLMG groups identified by eBURST. Variations observed between MLMGs were not equally distributed over all loci, but predominantly observed in MS7, MS6A, or MS10. CONCLUSIONS: Our findings confirm the concept of local N. caninum subpopulations. The geographic distance of sampling was associated with the genetic distance as determined by microsatellite typing. Results suggest that multi-parental recombination in N. caninum is a rare event, but does not exclude uniparental mating. More comprehensive studies on microsatellites in N. caninum and related species like Toxoplasma gondii should be undertaken, not only to improve genotyping capabilities, but also to understand possible functions of these regions in the genomes of these parasites.

Respiratory disease due to mixed viral infections in poultry flocks in Egypt between 2017 and 2018: Upsurge of highly pathogenic avian influenza virus subtype H5N8 since 2018.

For several years, poultry production in Egypt has been suffering from co-circulation of multiple respiratory viruses including highly pathogenic avian influenza virus (HPAIV) H5N1 (clade 2.2.1.2) and low pathogenic H9N2 (clade G1-B). Incursion of HPAIV H5N8 (clade 2.3.4.4b) to Egypt in November 2016 via wild birds followed by spread into commercial poultry flocks further complicated the situation. Current analyses focussed on 39 poultry farms suffering from respiratory manifestation and high mortality in six Egyptian governorates during 2017-2018. Real-time RT-PCR (RT-qPCR) substantiated the co-presence of at least two respiratory virus species in more than 80% of the investigated flocks. The percentage of HPAIV H5N1-positive holdings was fairly stable in 2017 (12.8%) and 2018 (10.2%), while the percentage of HPAIV H5N8-positive holdings increased from 23% in 2017 to 66.6% during 2018. The proportion of H9N2-positive samples was constantly high (2017:100% and 2018:63%), and H9N2 co-circulated with HPAIV H5N8 in 22 out of 39 (56.8%) flocks. Analyses of 26 H5, 18 H9 and 4 N2 new sequences confirmed continuous genetic diversification. In silico analysis revealed numerous amino acid substitutions in the HA and NA proteins suggestive of increased adaptation to mammalian hosts and putative antigenic variation. For sensitive detection of H9N2 viruses by RT-qPCR, an update of primers and probe sequences was crucial. Reasons for the relative increase of HPAIV H5N8 infections versus H5N1 remained unclear, but lack of suitable vaccines against clade 2.3.4.4b cannot be excluded. A reconsideration of surveillance and control measures should include updating of diagnostic tools and vaccination strategies.

Surveillance of European Domestic Pig Populations Identifies an Emerging Reservoir of Potentially Zoonotic Swine Influenza A Viruses.

Swine influenza A viruses (swIAVs) can play a crucial role in the generation of new human pandemic viruses. In this study, in-depth passive surveillance comprising nearly 2,500 European swine holdings and more than 18,000 individual samples identified a year-round presence of up to four major swIAV lineages on more than 50% of farms surveilled. Phylogenetic analyses show that intensive reassortment with human pandemic A(H1N1)/2009 (H1pdm) virus produced an expanding and novel repertoire of at least 31 distinct swIAV genotypes and 12 distinct hemagglutinin/neuraminidase combinations with largely unknown consequences for virulence and host tropism. Several viral isolates were resistant to the human antiviral MxA protein, a prerequisite for zoonotic transmission and stable introduction into human populations. A pronounced antigenic variation was noted in swIAV, and several H1pdm lineages antigenically distinct from current seasonal human H1pdm co-circulate in swine. Thus, European swine populations represent reservoirs for emerging IAV strains with zoonotic and, possibly, pre-pandemic potential.

Major Capsid Protein Synthesis from the Genomic RNA of Feline Calicivirus.

Caliciviruses have a positive-strand RNA genome with a length of about 7.5 kb that contains 2, 3, or 4 functional open reading frames (ORFs). A subgenomic mRNA (sg-RNA) is transcribed in the infected cell, and both major capsid protein viral protein 1 (VP1) and minor capsid protein VP2 are translated from the sg-RNA. Translation of proteins from the genomic RNA (g-RNA) and from the sg-RNA is mediated by the RNA-linked viral protein VPg (virus protein, genome linked). Most of the calicivirus genera have translation mechanisms leading to VP1 expression from the g-RNA. VP1 is part of the polyprotein for sapoviruses, lagoviruses, and neboviruses, and a termination/reinitiation mechanism was described for noroviruses. Vesiviruses have no known mechanism for the expression of VP1 from the g-RNA, and the Vesivirus genus is the only genus of the Caliciviridae that generates VP1 via a precursor capsid leader protein (LC-VP1). Analyses of feline calicivirus (FCV) g-RNA translation showed a low level of VP1 expression with an initiation downstream of the original start codon of LC-VP1, leading to a smaller, truncated LC-VP1 (tLC-VP1) protein. Deletion and substitution analyses of the region surrounding the LC-VP1 start codon allowed the identification of sequences within the leader protein coding region of FCV that have an impact on VP1 translation frequency from the g-RNA. Introduction of such mutations into the virus showed an impact of strongly reduced tLC-VP1 levels translated from the g-RNA on viral replication.IMPORTANCE Caliciviruses are a cause of important diseases in humans and animals. It is crucial to understand the prerequisites of efficient replication of these viruses in order to develop strategies for prevention and treatment of these diseases. It was shown before that all caliciviruses except vesiviruses have established mechanisms to achieve major capsid protein (VP1) translation from the genomic RNA. Here, we show for the first time that a member of the genus Vesivirus also has a translation initiation mechanism by which a precursor protein of the VP1 protein is expressed from the genomic RNA. This finding clearly points at a functional role of the calicivirus VP1 capsid protein in early replication, and we provide experimental data supporting this hypothesis.

Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites.

An intravenous pathogenicity index (IVPI) of > 1.2 in chickens or, in case of subtypes H5 and H7, expression of a polybasic hemagglutinin cleavage site (HACS), signals high pathogenicity (HP). Viruses of the H9N2-G1 lineage, which spread across Asia and Africa, are classified to be of low pathogenicity although, in the field, they became associated with severe clinical signs and epizootics in chickens. Here we report on a pre-eminent trait of recent H9N2-G1 isolates from Bangladesh and India, which express a tribasic HACS (motif PAKSKR-GLF; reminiscent of an HPAIV-like polybasic HACS) and compare their features to H9Nx viruses with di- and monobasic HACS from other phylogenetic and geographic origins. In an in vitro assay, the tribasic HACS of H9N2 was processed by furin-like proteases similar to bona fide H5 HPAIV while some dibasic sites showed increased cleavability but monobasic HACS none. Yet, all viruses remained trypsin-dependent in cell culture. In ovo, only tribasic H9N2 viruses were found to replicate in a grossly extended spectrum of embryonic organs. In contrast to all subtype H5/H7 HPAI viruses, tribasic H9N2 viruses did not replicate in endothelial cells either in the chorio-allantoic membrane or in other embryonic tissues. By IVPI, all H9Nx isolates proved to be of low pathogenicity. Pathogenicity assessment of tribasic H9N2-G1 viruses remains problematic. It cannot be excluded that the formation of a third basic amino acid in the HACS forms an intermediate step towards a gain in pathogenicity. Continued observation of the evolution of these viruses in the field is recommended.

Structure-function relationship in the 'termination upstream ribosomal binding site' of the calicivirus rabbit hemorrhagic disease virus.

Caliciviruses use a termination/reinitiation mechanism for translation of their minor capsid protein VP2. A sequence element of about 80 nucleotides denoted 'termination upstream ribosomal binding site' (TURBS) is crucial for reinitiation. RNA secondary structure probing and computer aided secondary structure prediction revealed a rather low degree of secondary structure determinants for the TURBS of the rabbit hermorrhagic disease virus. Mutation analysis showed that prevention of duplex formation had major impact on the VP2 expression levels. Restoration of complementarity of the respective sequences by reciprocal mutation at least partially restored reinitiating rates. Synthetic TURBS structures preserving only the secondary structure forming sequences and the known short motifs important for TURBS function were found to drive reinitiation when the altered sequence could be predicted to allow establishment of the crucial secondary structures of the TURBS.

Feline calicivirus can tolerate gross changes of its minor capsid protein expression levels induced by changing translation reinitiation frequency or use of a separate VP2-coding mRNA.

Caliciviruses use reinitiation of translation governed by a 'termination upstream ribosomal binding site' (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication. In addition, full length constructs were established that displayed a conformation, in which VP2 expression occurred under control of a duplicated subgenomic promoter. Viable viruses recovered from such constructs revealed a rather broad range of VP2 expression levels but comparable growth kinetics showing that caliciviruses can tolerate gross changes of the VP2 expression level.

Two alternative ways of start site selection in human norovirus reinitiation of translation.

The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.

The importance of inter- and intramolecular base pairing for translation reinitiation on a eukaryotic bicistronic mRNA.

Calicivirus structure proteins are expressed from a subgenomic mRNA with two overlapping cistrons. The first ORF of this RNA codes for the viral major capsid protein VP1, and the second for the minor capsid protein VP2. Translation of VP2 is mediated by a termination/reinitiation mechanism, which depends on an upstream sequence element of approximately 70 nucleotides denoted "termination upstream ribosomal binding site" (TURBS). Two short sequence motifs within the TURBS were found to be essential for reinitiation. By a whole set of single site mutations and reciprocal base exchanges we demonstrate here for the first time conclusive evidence for the necessity of mRNA/18S rRNA hybridization for translation reinitiation in an eukaryotic system. Moreover, we show that motif 2 exhibits intramolecular hybridization with a complementary region upstream of motif 1, thus forming a secondary structure that positions post-termination ribosomes in an optimal distance to the VP2 start codon. Analysis of the essential elements of the TURBS led to a better understanding of the requirements for translation termination/reinitiation in eukaryotes.

A bipartite sequence motif induces translation reinitiation in feline calicivirus RNA.

The mechanism leading to reinitiation of translation after termination of protein synthesis in eukaryotes has not yet been resolved in detail. One open question concerns the way the post-termination ribosome is tethered to the mRNA to allow binding of the necessary initiation factors. In caliciviruses, a family of positive strand RNA viruses, the capsid protein VP2 is translated via a termination/reinitiation process. VP2 of the feline calicivirus is encoded in the 3'-terminal open reading frame 3 (ORF3) that overlaps with the preceding ORF2 by four nucleotides. In transient expression studies, the efficiency of VP2 expression was 20 times lower than that of the ORF2 proteins. The close vicinity of the ORF2 termination signal and the ORF3 AUG codon was crucial, whereas the AUG could be replaced by alternative codons. Deletion mapping revealed that the 3'-terminal 69 nucleotides of ORF2 are crucial for VP2 expression. This sequence contains two essential sequence motifs. The first motif is conserved among caliciviruses and complementary to part of the 18 S rRNA. In conclusion, VP2 is expressed in a translation termination/reinitiation process that is special because it requires a sequence element that could prevent dissociation of post-termination ribosomes via hybridization with 18 S rRNA.