CpG dinucleotide enrichment in the influenza A virus genome as a live attenuated vaccine development strategy.

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens' eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines.

1 Cellular protein TTC4 and its cofactor HSP90 are pro-viral for bovine herpesvirus 1.

Bovine Herpesvirus Type 1 (BoHV-1) infection causes infectious bovine rhinotracheitis and genital disease in cattle, with significant economic and welfare impacts. However, the role of cellular host factors during viral replication remains poorly characterised. A previously performed genome-wide CRISPR knockout screen identified pro- and antiviral host factors acting during BoHV-1 replication. Herein we validate a pro-viral role for a candidate from this screen: the cellular protein tetracopeptide repeat protein 4 (TTC4). We show that TTC4 transcript production is upregulated during BoHV-1 infection. Depletion of TTC4 protein impairs BoHV-1 protein production but does not reduce production of infectious virions, whereas overexpression of exogenous TTC4 results in a significant increase in production of infectious BoHV-1 virions. TTC4 itself is poorly characterized (especially in the context of virus infection), but is a known co-chaperone of heat shock protein 90 (HSP90). HSP90 has a well-characterized pro-viral role during the replication of diverse herpesviruses, and we therefore hypothesized that HSP90 is also pro-viral for BoHV-1. Drug-mediated inhibition of HSP90 using geldanamycin at sub-cytotoxic concentrations inhibited both BoHV-1 protein production and viral genome replication, indicating a pro-viral role for HSP90 during BoHV-1 infection. Our data demonstrates pro-viral roles for both TTC4 and HSP90 during BoHV-1 replication; possibly, interactions between these two proteins are required for optimal BoHV-1 replication, or the two proteins may have independent pro-viral roles.

Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes.

Species A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

Neck swelling and airway narrowing as an initial manifestation of giant cell arteritis.

Giant cell arteritis can result in a wide range of symptoms due to the extensive distribution of the external carotid artery. Face and neck swelling and trismus are under-recognised features of giant cell arteritis and can present as the initial symptom prior to the development of classical temporal tenderness and jaw claudication. The lack of awareness of the less common symptoms may result in a late diagnosis of giant cell arteritis, leading to irreversible vision loss. In this paper, we present a case of neck swelling and airway narrowing as the initial manifestation of giant cell arteritis.

Intra-genome variability in the dinucleotide composition of SARS-CoV-2.

CpG dinucleotides are under-represented in the genomes of single-stranded RNA viruses, and SARS-CoV-2 is no exception to this. Artificial modification of CpG frequency is a valid approach for live attenuated vaccine development; if this is to be applied to SARS-CoV-2, we must first understand the role CpG motifs play in regulating SARS-CoV-2 replication. Accordingly, the CpG composition of the SARS-CoV-2 genome was characterised. CpG suppression among coronaviruses does not differ between virus genera but does vary with host species and primary replication site (a proxy for tissue tropism), supporting the hypothesis that viral CpG content may influence cross-species transmission. Although SARS-CoV-2 exhibits overall strong CpG suppression, this varies considerably across the genome, and the Envelope (E) open reading frame (ORF) and ORF10 demonstrate an absence of CpG suppression. Across the Coronaviridae, E genes display remarkably high variation in CpG composition, with those of SARS and SARS-CoV-2 having much higher CpG content than other coronaviruses isolated from humans. This is an ancestrally derived trait reflecting their bat origins. Conservation of CpG motifs in these regions suggests that they have a functionality which over-rides the need to suppress CpG; an observation relevant to future strategies towards a rationally attenuated SARS-CoV-2 vaccine.

Oxford Screening CSF and Respiratory samples ('OSCAR'): results of a pilot study to screen clinical samples from a diagnostic microbiology laboratory for viruses using Illumina next generation sequencing.

OBJECTIVES: There is increasing interest in the use of metagenomic (next generation sequencing, NGS) approaches for diagnosis of infection. We undertook a pilot study to screen samples submitted to a diagnostic microbiology laboratory in a UK teaching hospital using Illumina HiSeq. In the short-term, this small dataset provides insights into the virome of human respiratory and cerebrospinal fluid (CSF) samples. In the longer term, assimilating metagenomic data sets of this nature can inform optimization of laboratory and bioinformatic methods, and develop foundations for the interpretation of results in a clinical context. The project underpins a larger ongoing effort to develop NGS pipelines for diagnostic use. DATA DESCRIPTION: Our data comprise a complete metagenomic dataset from 20 independent samples (10 CSF and 10 respiratory) submitted to the clinical microbiology laboratory for a large UK teaching hospital (Oxford University Hospitals NHS Foundation Trust). Sequences have been uploaded to the European Nucleotide Archive and are also presented as Krona plots through which the data can be interactively visualized. In the longer term, further optimization is required to better define sensitivity and specificity of this approach to clinical samples.

Novel adenoviruses detected in British mustelids, including a unique Aviadenovirus in the tissues of pine martens (Martes martes).

Several adenoviruses are known to cause severe disease in veterinary species. Recent evidence suggests that canine adenovirus type 1 (CAV-1) persists in the tissues of healthy red foxes (Vulpes vulpes), which may be a source of infection for susceptible species. It was hypothesized that mustelids native to the UK, including pine martens (Martes martes) and Eurasian otters (Lutra lutra), may also be persistently infected with adenoviruses. Based on high-throughput sequencing and additional Sanger sequencing, a novel Aviadenovirus, tentatively named marten adenovirus type 1 (MAdV-1), was detected in pine marten tissues. The detection of an Aviadenovirus in mammalian tissue has not been reported previously. Two mastadenoviruses, tentatively designated marten adenovirus type 2 (MAdV-2) and lutrine adenovirus type 1 (LAdV-1), were also detected in tissues of pine martens and Eurasian otters, respectively. Apparently healthy free-ranging animals may be infected with uncharacterized adenoviruses with possible implications for translocation of wildlife.

PARV4 prevalence, phylogeny, immunology and coinfection with HIV, HBV and HCV in a multicentre African cohort.

Background: The seroprevalence of human parvovirus-4 (PARV4) varies considerably by region. In sub-Saharan Africa, seroprevalence is high in the general population, but little is known about the transmission routes or the prevalence of coinfection with blood-borne viruses, HBV, HCV and HIV.  Methods: To further explore the characteristics of PARV4 in this setting, with a particular focus on the prevalence and significance of coinfection, we screened a cohort of 695 individuals recruited from Durban and Kimberley (South Africa) and Gaborone (Botswana) for PARV4 IgG and DNA, as well as documenting HIV, HBV and HCV status.  Results: Within these cohorts, 69% of subjects were HIV-positive. We identified no cases of HCV by PCR, but 7.4% were positive for HBsAg. PARV4 IgG was positive in 42%; seroprevalence was higher in adults (69%) compared to children (21%) (p<0.0001) and in HIV-positive (52%) compared to HIV-negative individuals (24%) (p<0.0001), but there was no association with HBsAg status. We developed an on-line tool to allow visualization of coinfection data (https://purl.oclc.org/coinfection-viz). We identified five subjects who were PCR-positive for PARV4 genotype-3. Ex vivo CD8+ T cell responses spanned the entire PARV4 proteome and we propose a novel HLA-B*57:03-restricted epitope within the NS protein.  Conclusions: This characterisation of PARV4 infection provides enhanced insights into the epidemiology of infection and co-infection in African cohorts, and provides the foundations for planning further focused studies to elucidate transmission pathways, immune responses, and the clinical significance of this organism.

Human parvovirus 4 'PARV4' remains elusive despite a decade of study.

Human parvovirus 4 ('PARV4') is a small DNA tetraparvovirus, first reported in 2005. In some populations, PARV4 infection is uncommon, and evidence of exposure is found only in individuals with risk factors for parenteral infection who are infected with other blood-borne viruses. In other settings, seroprevalence studies suggest an endemic, age-associated transmission pattern, independent of any specific risk factors. The clinical impact of PARV4 infection remains uncertain, but reported disease associations include an influenza-like syndrome, encephalitis, acceleration of HIV disease, and foetal hydrops. In this review, we set out to report progress updates from the recent literature, focusing on the investigation of cohorts in different geographical settings, now including insights from Asia, the Middle East, and South America, and discussing whether attributes of viral or host populations underpin the striking differences in epidemiology. We review progress in understanding viral phylogeny and biology, approaches to diagnostics, and insights that might be gained from studies of closely related animal pathogens. Crucial questions about pathogenicity remain unanswered, but we highlight new evidence supporting a possible link between PARV4 and an encephalitis syndrome. The unequivocal evidence that PARV4 is endemic in certain populations should drive ongoing research efforts to understand risk factors and routes of transmission and to gain new insights into the impact of this virus on human health.

Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom.

Canine adenovirus type 1 (CAV-1) causes infectious canine hepatitis (ICH), a frequently fatal disease which primarily affects canids. In this study, serology (ELISA) and molecular techniques (PCR/qPCR) were utilised to investigate the exposure of free-ranging red foxes (Vulpes vulpes) to CAV-1 in the United Kingdom (UK) and to examine their role as a wildlife reservoir of infection for susceptible species. The role of canine adenovirus type 2 (CAV-2), primarily a respiratory pathogen, was also explored. In foxes with no evidence of ICH on post-mortem examination, 29 of 154 (18.8%) red foxes had inapparent infections with CAV-1, as detected by a nested PCR, in a range of samples, including liver, kidney, spleen, brain, and lung. CAV-1 was detected in the urine of three red foxes with inapparent infections. It was estimated that 302 of 469 (64.4%) red foxes were seropositive for canine adenovirus (CAV) by ELISA. CAV-2 was not detected by PCR in any red foxes examined. Additional sequence data were obtained from CAV-1 positive samples, revealing regional variations in CAV-1 sequences. It is concluded that CAV-1 is endemic in free-ranging red foxes in the UK and that many foxes have inapparent infections in a range of tissues.

Venous thromboembolism in medical patients during hospitalisation and 3†months after hospitalisation: a prospective observational study.

OBJECTIVES: This study was conducted to assess the incidence and risk factors for venous thromboembolism (VTE) in a cohort of medical patients both during the period of hospitalisation and following discharge. DESIGN: This was a prospective observational study to document the risk profile and incidence of VTE posthospitalisation among all medical patients admitted to our institution during the trial period. SETTINGS: Primary healthcare. Single tertiary referral centre, Tasmania, Australia. PARTICIPANTS: A total of 986 patients admitted to the medical ward between January 2012 and September 2012 were included in the study with male to female ratio of 497:489. The mean age of patients was 68 years (range 17-112, SD 16). RESULTS: Overall, 54/986 patients (5.5%) had a VTE during the study period. Of these, 40/54 (74.1%) occurred during hospitalisation and 14/54 (25.9%) occurred following discharge. VTE risk factors revealed in multivariate analysis to be associated with a previous diagnosis of VTE (p<0.001, OR=6.63, 95% CI 3.3 to 13.36), the occurrence of surgery within the past 30 days (p<0.001, OR=2.52, 95% CI 1.33 to 4.79) and an admission diagnosis of pulmonary disease (p<0.01, OR 3.61, 95% CI 1.49 to 8.76). Mobility within 24 hours of admission was not associated with an increased risk. There was risk of VTE when the length of stay prolonged (p=0.046, OR=1.01, 95% CI 1.00 to 1.03), however it was not sustained with multivariate modelling. VTE-specific prophylaxis was used in 53% of the studied patients. Anticoagulation including antiplatelet agents were administered in 63% of patients who developed VTE. CONCLUSIONS: This prospective observational study found that 5.5% of the studied patients developed VTE. Among those, 25.9% (14/54) of patients had a detected VTE posthospitalisation with this risk being increased if there was a history of VTE, recent surgery and pulmonary conditions. Thromboprophylaxis may be worth considering in these cohorts. Further study to confirm these findings are warranted. TRIAL REGISTRATION NUMBER: ACTRN12611001255976.

Evaluation of Viremia Frequencies of a Novel Human Pegivirus by Using Bioinformatic Screening and PCR.

Next-generation sequencing has critical applications in virus discovery, diagnostics, and environmental surveillance. We used metagenomic sequence libraries for retrospective screening of plasma samples for the recently discovered human hepegivirus 1 (HHpgV-1). From a cohort of 150 hepatitis C virus (HCV)-positive case-patients, we identified 2 persons with HHpgV-1 viremia and a high frequency of human pegivirus (HPgV) viremia (14%). Detection of HHpgV-1 and HPgV was concordant with parallel PCR-based screening using conserved primers matching groups 1 (HPgV) and 2 (HHPgV-1) nonstructural 3 region sequences. PCR identified 1 HHPgV-1-positive person with viremia from a group of 195 persons with hemophilia who had been exposed to nonvirally inactivated factor VII/IX; 18 (9%) were HPgV-positive. Relative to HCV and HPgV, active infections with HHpgV-1 were infrequently detected in blood, even in groups that had substantial parenteral exposure. Our findings are consistent with lower transmissibility or higher rates of virus clearance for HHpgV-1 than for other bloodborne human flaviviruses.

High prevalence and diversity of bovine astroviruses in the faeces of healthy and diarrhoeic calves in South West Scotland.

Astroviruses (AstV) are single-stranded, positive-sense RNA viruses and one of the major causes of infant diarrhoea worldwide. Diarrhoea is a common and important cause of morbidity and mortality in calves; therefore, we investigated whether the presence of AstV is associated with calf diarrhoea. We identified diverse AstV lineages from faecal samples of both healthy and diarrhoeic calves and healthy adult cattle in South West Scotland. AstV was common in calves (present in 74% (85/115) of samples) but uncommon in adult cattle (present in 15% (3/20) of samples). No association was found between the presence of AstV and calf diarrhoea or the presence of a specific AstV lineage and calf diarrhoea. AstV was strongly associated with the presence of rotavirus Group A (RVA), and a protective effect of age was evident for both AstV and RVA. Co-infections with multiple AstV lineages were detected in several calves and serial infection with different viruses could also be seen by longitudinal sampling of individuals. In summary, our study found genotypically diverse AstV in the faeces of calves in South West Scotland. However, no association was identified between AstV and calf diarrhoea, which suggests the virus does not play a primary role in the aetiology of calf diarrhoea in the group studied.

Complete genome sequences of novel anelloviruses from laboratory rats.

Anelloviruses are nonenveloped single-stranded DNA viruses infecting a wide range of mammals. We report three complete genomes of novel anelloviruses detected in laboratory rats. Phylogenetic analysis demonstrates that these viruses are related to but distinct from recently described rodent Torque teno viruses (RoTTVs) found in wild rodent species.

Identification of novel anelloviruses with broad diversity in UK rodents.

Anelloviruses are a family of small circular ssDNA viruses with a vast genetic diversity. Human infections with the prototype anellovirus, torque teno virus (TTV), are ubiquitous and related viruses have been described in a number of other mammalian hosts. Despite over 15 years of investigation, there is still little known about the pathogenesis and possible disease associations of anellovirus infections, arising in part due to the lack of a robust cell culture system for viral replication or tractable small-animal model. We report the identification of diverse anelloviruses in several species of wild rodents. The viruses are highly prevalent in wood mice (Apodemus sylvaticus) and field voles (Microtus agrestis), detectable at a low frequency in bank voles (Myodes glareolus), but absent from house mice (Mus musculus). The viruses identified have a genomic organization consistent with other anelloviruses, but form two clear phylogenetic groups that are as distinct from each other as from defined genera.

Hypereosinophilic syndrome associated with ulcerative colitis presenting with recurrent Loeffler's endocarditis and left ventricular thrombus treated successfully with immune suppressive therapy and anticoagulation.

We reported a case of a 28-year-old Caucasian woman with hypereosinophilic syndrome (HES) associated with ulcerative colitis who presented on separate occasions with Loeffler's endocarditis. She was admitted in 2008 with fever, headache, confusion and visual loss. Diagnostic workup uncovered an eosinophilia of 3.1×109/L and major ECG abnormalities. Subsequent echocardiography revealed left ventricular wall motion abnormalities with mural thrombus. MRI brain scan showed multiple white matter lesions consistent with acute infarcts. She recovered rapidly with corticosteroids and anticoagulation. Four years later she re-presented with headache, fatigue and an eosinophilia of 13.4×109/L. This occurred 3 months after cessation of immunosuppression and within 12 months of total colectomy for fulminant ulcerative colitis. Echocardiography was suggestive of hypereosinophilic endomyocardial fibrosis with left ventricular thrombus. Anticoagulation and corticosteroids were resumed with good effect. This report highlights the findings, treatment and outcome of ulcerative colitis-associated HES manifesting as recurrent Loeffler's endocarditis.