Tackling feline infectious peritonitis via reverse genetics

Feline infectious peritonitis (FIP) is caused by feline coronaviruses (FCoVs) and represents one of the most important lethal infectious diseases of cats. To date, there is no efficacious prevention and treatment, and our limited knowledge on FIP pathogenesis is mainly based on analysis of experiments with field isolates. In a recent study, we reported a promising approach to study FIP pathogenesis using reverse genetics. We generated a set of recombinant FCoVs and investigated their pathogenicity in vivo. The set included the type I FCoV strain Black, a type I FCoV strain Black with restored accessory gene 7b, two chimeric type I/type II FCoVs and the highly pathogenic type II FCoV strain 79-1146. All recombinant FCoVs and the reference strain isolates were found to establish productive infections in cats. While none of the type I FCoVs and chimeric FCoVs induced FIP, the recombinant type II FCoV strain 79-1146 was as pathogenic as the parental isolate. Interestingly, an intact ORF 3c was confirmed to be restored in all viruses (re)isolated from FIP-diseased animals.

Hepeviridae: An expanding family of vertebrate viruses

The hepatitis E virus (HEV) was first identified in 1990, although hepatitis E-like diseases in humans have been recorded for a long time dating back to the 18th century. The HEV genotypes 1–4 have been subsequently detected in human hepatitis E cases with different geographical distribution and different modes of transmission. Genotypes 3 and 4 have been identified in parallel in pigs, wild boars and other animal species and their zoonotic potential has been confirmed. Until 2010, these genotypes along with avian HEV strains infecting chicken were the only known representatives of the family Hepeviridae. Thereafter, additional HEV-related viruses have been detected in wild boars, distinct HEV-like viruses were identified in rats, rabbit, ferret, mink, fox, bats and moose, and a distantly related agent was described from closely related salmonid fish. This review summarizes the characteristics of the so far known HEV-like viruses, their phylogenetic relationship, host association and proposed involvement in diseases. Based on the reviewed knowledge, a suggestion for a new taxonomic grouping scheme of the viruses within the family Hepeviridae is presented.

Structure–mechanics relationships in mineralized tendons

In this paper, we review the hierarchical structure and the resulting elastic properties of mineralized tendons as obtained by various multiscale experimental and computational methods spanning from nano- to macroscale. The mechanical properties of mineralized collagen fibres are important to understand the mechanics of hard tissues constituted by complex arrangements of these fibres, like in human lamellar bone. The uniaxial mineralized collagen fibre array naturally occurring in avian tendons is a well studied model tissue for investigating various stages of tissue mineralization and the corresponding elastic properties. Some avian tendons mineralize with maturation, which results in a graded structure containing two zones of distinct morphology, circumferential and interstitial. These zones exhibit different amounts of mineral, collagen, pores and a different mineral distribution between collagen fibrillar and extrafibrillar space that lead to distinct elastic properties. Mineralized tendon cells have two phenotypes: elongated tenocytes placed between fibres in the circumferential zone and cuboidal cells with lower aspect ratios in the interstitial zone. Interestingly some regions of avian tendons seem to be predestined to mineralization, which is exhibited as specific collagen cross-linking patterns as well as distribution of minor tendon constituents (like proteoglycans) and loss of collagen crimp. Results of investigations in naturally mineralizing avian tendons may be useful in understanding the pathological mineralization occurring in some human tendons.

Evidence for an Ancestral Association of Human Coronavirus 229E with Bats.

UNLABELLED We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by reverse transcription-PCR (RT-PCR). Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were 5- and 6-fold more diversified than HCoV-229E in the RNA-dependent RNA polymerase (RdRp) and spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationships. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca, and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3' end. ORF8 also existed in the 229E-related alpaca virus. Reanalysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E. IMPORTANCE The ancestral origins of major human coronaviruses (HCoVs) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a large sample of African bats and characterizing several bat viruses on a full-genome level. Our evolutionary analyses show that animal and human viruses are genetically closely related, can exchange genetic material, and form a single viral species. We show that the putative host switches leading to the formation of HCoV-229E were accompanied by major genomic changes, including deletions in the viral spike glycoprotein gene and loss of an open reading frame. We reanalyze a previously described genetically related alpaca virus and discuss the role of camelids as potential intermediate hosts between bat and human viruses. The evolutionary history of HCoV-229E likely shares important characteristics with that of the recently emerged highly pathogenic Middle East respiratory syndrome (MERS) coronavirus.

Biological and protective properties of immune sera directed to the influenza virus neuraminidase

The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain that is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably as a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, the antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in an NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activities of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spread of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high-quality NA antibodies for controlling influenza virus replication and place emphasis on NA as a vaccine antigen. IMPORTANCE The neuraminidase of influenza A viruses is a sialidase that acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera inhibited not only sialidase activity, but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtype activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next-generation influenza virus vaccines.

Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis

Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

First international external quality assessment of molecular diagnostics for Mers-CoV

BACKGROUND: Since the discovery of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, diagnostic protocols were quickly published and deployed globally. OBJECTIVES: We set out to assess the quality of MERS-CoV molecular diagnostics worldwide. STUDY DESIGN: Both sensitivity and specificity were assessed using 12 samples containing different viral loads of MERS-CoV or common coronaviruses (OC43, 229E, NL63, HKU1). RESULTS: The panel was sent to more than 106 participants, of which 99 laboratories from 6 continents returned 189 panel results.Scores ranged from 100% (84 laboratories) to 33% (1 laboratory). 15% of respondents reported quantitative results, 61% semi-quantitative (Ct-values or time to positivity) and 24% reported qualitative results. The major specific technique used was real-time RT-PCR using the WHO recommended targets upE, ORF1a and ORF1b. The evaluation confirmed that RT-PCRs targeting the ORF1b are less sensitive, and therefore not advised for primary diagnostics. CONCLUSIONS: The first external quality assessment MERS-CoV panel gives a good insight in molecular diagnostic techniques and their performances for sensitive and specific detection of MERS-CoV RNA globally. Overall, all laboratories were capable of detecting MERS-CoV with some differences in sensitivity. The observation that 8% of laboratories reported false MERS-CoV positive single assay results shows room for improvement, and the importance of using confirmatory targets.

New insights on the role of paired membrane structures in coronavirus replication.

The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the Coronaviridae family members, but across the order Nidovirales. Taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral RNA. However, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. Here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication.

One-Step Identification of Five Prominent Chicken Salmonella Serovars and Biotypes.

Based on bacterial genomic data, we developed a one-step multiplex PCR assay to identify Salmonella and simultaneously differentiate the two invasive avian-adapted S. enterica serovar Gallinarum biotypes Gallinarum and Pullorum, and the most frequent, specific, and asymptomatic colonizers of chickens, serovars Enteritidis, Heidelberg, and Kentucky.

Use of a modified Delphi panel to identify and weight criteria for prioritization of zoonotic diseases in Switzerland

Zoonotic diseases have a significant impact on public health globally. To prevent or reduce future zoonotic outbreaks, there is a constant need to invest in research and surveillance programs while updating risk management strategies. However, given the limited resources available, disease prioritization based on the need for their control and surveillance is important. This study was performed to identify and weight disease criteria for the prioritization of zoonotic diseases in Switzerland using a semi-quantitative research method based on expert opinion. Twenty-eight criteria relevant for disease control and surveillance, classified under five domains, were selected following a thorough literature review, and these were evaluated and weighted by seven experts from the Swiss Federal Veterinary Office using a modified Delphi panel. The median scores assigned to each criterion were then used to rank 16 notifiable and/or emerging zoonoses in Switzerland. The experts weighted the majority of the criteria similarly, and the top three criteria were Severity of disease in humans, incidence and prevalence of the disease in humans and treatment in humans. Based on these weightings, the three highest ranked diseases were Avian Influenza, Bovine Spongiform Encephalitis, and Bovine Tuberculosis. Overall, this study provided a preliminary list of criteria relevant for disease prioritization in Switzerland. These were further evaluated in a companion study which involved a quantitative prioritization method and multiple stakeholders.