ABSTRACT
Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.
Subject(s)
Infectious bronchitis virus , Reverse Genetics , Infectious bronchitis virus/genetics , Reverse Genetics/methods , Animals , Genome, Viral , Coronavirus Infections/virology , Coronavirus Infections/veterinary , Plasmids/genetics , Poultry Diseases/virology , Escherichia coli/geneticsSubject(s)
COVID-19 , Vaccines , Humans , COVID-19/epidemiology , COVID-19/prevention & control , United Kingdom/epidemiology , VaccinationABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) human adaptation resulted in distinct lineages with enhanced transmissibility called variants of concern (VOCs). Omicron is the first VOC to evolve distinct globally dominant subvariants. Here we compared their replication in human cell lines and primary airway cultures and measured host responses to infection. We discovered that subvariants BA.4 and BA.5 have improved their suppression of innate immunity when compared with earlier subvariants BA.1 and BA.2. Similarly, more recent subvariants (BA.2.75 and XBB lineages) also triggered reduced innate immune activation. This correlated with increased expression of viral innate antagonists Orf6 and nucleocapsid, reminiscent of VOCs Alpha to Delta. Increased Orf6 levels suppressed host innate responses to infection by decreasing IRF3 and STAT1 signalling measured by transcription factor phosphorylation and nuclear translocation. Our data suggest that convergent evolution of enhanced innate immune antagonist expression is a common pathway of human adaptation and link Omicron subvariant dominance to improved innate immune evasion.
Subject(s)
COVID-19 , Humans , SARS-CoV-2 , Cell Line , Immune Evasion , Immunity, InnateABSTRACT
SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19/virology , Immunity, Innate/genetics , Pandemics , SARS-CoV-2/geneticsSubject(s)
Antibodies, Viral , COVID-19 Vaccines , COVID-19 , SARS-CoV-2 , Vaccines, Combined , Adult , Humans , Antibodies, Neutralizing , COVID-19/immunologyABSTRACT
BACKGROUND: Patients with antibody deficiency respond poorly to coronavirus disease 2019 (COVID-19) vaccination and are at risk of severe or prolonged infection. They are given long-term immunoglobulin replacement therapy (IRT) prepared from healthy donor plasma to confer passive immunity against infection. Following widespread COVID-19 vaccination alongside natural exposure, we hypothesized that immunoglobulin preparations will now contain neutralizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antibodies, which confer protection against COVID-19 disease and may help to treat chronic infection. METHODS: We evaluated anti-SARS-CoV-2 spike antibody in a cohort of patients before and after immunoglobulin infusion. Neutralizing capacity of patient samples and immunoglobulin products was assessed using in vitro pseudovirus and live-virus neutralization assays, the latter investigating multiple batches against current circulating Omicron variants. We describe the clinical course of 9 patients started on IRT during treatment of COVID-19. RESULTS: In 35 individuals with antibody deficiency established on IRT, median anti-spike antibody titer increased from 2123 to 10 600 U/mL postinfusion, with corresponding increase in pseudovirus neutralization titers to levels comparable to healthy donors. Testing immunoglobulin products directly in the live-virus assay confirmed neutralization, including of BQ1.1 and XBB variants, but with variation between immunoglobulin products and batches.Initiation of IRT alongside remdesivir in patients with antibody deficiency and prolonged COVID-19 infection (median 189 days, maximum >900 days with an ancestral viral strain) resulted in clearance of SARS-CoV-2 at a median of 20 days. CONCLUSIONS: Immunoglobulin preparations now contain neutralizing anti-SARS-CoV-2 antibodies that are transmitted to patients and help to treat COVID-19 in individuals with failure of humoral immunity.
Subject(s)
Antibodies, Neutralizing , COVID-19 , Humans , Spike Glycoprotein, Coronavirus , COVID-19 Vaccines , SARS-CoV-2 , Antibodies, ViralABSTRACT
Wu et al. report that patients with hematologic malignancies have reduced immunity against SARS-CoV-2 Omicron subvariants and Sotrovimab retains neutralizing capacity against all tested Omicron subvariants.
Subject(s)
COVID-19 , Hematologic Neoplasms , Neoplasms , Humans , SARS-CoV-2 , Hematologic Neoplasms/drug therapyABSTRACT
Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.
Subject(s)
Infectious bronchitis virus , Mamastrovirus , Mutagenesis, Insertional , Animals , Humans , 3' Untranslated Regions/genetics , Chickens/virology , Infectious bronchitis virus/genetics , Mamastrovirus/genetics , Mutagenesis, Insertional/genetics , Poultry Diseases/virology , RNA, Viral/genetics , Virus Replication/genetics , RNA Stability/genetics , Sequence Deletion/geneticsABSTRACT
Avian coronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute highly contagious economically relevant respiratory disease of poultry. Vaccination is used to control IBV infections, with live-attenuated vaccines generated via serial passage of a virulent field isolate through embryonated hens' eggs. A fine balance must be achieved between attenuation and the retention of immunogenicity. The exact molecular mechanism of attenuation is unknown, and vaccines produced in this manner present a risk of reversion to virulence as few consensus level changes are acquired. Our previous research resulted in the generation of a recombinant IBV (rIBV) known as M41-R, based on a pathogenic strain M41-CK. M41-R was attenuated in vivo by two amino acid changes, Nsp10-Pro85Leu and Nsp14-Val393Leu; however, the mechanism of attenuation was not determined. Pro85 and Val393 were found to be conserved among not only IBV strains but members of the wider coronavirus family. This study demonstrates that the same changes are associated with a temperature-sensitive (ts) replication phenotype at 41°C in vitro, suggesting that the two phenotypes may be linked. Vaccination of specific-pathogen-free chickens with M41-R induced 100% protection against clinical disease, tracheal ciliary damage, and challenge virus replication following homologous challenge with virulent M41-CK. Temperature sensitivity has been used to rationally attenuate other viral pathogens, including influenza, and the identification of amino acid changes that impart both a ts and an attenuated phenotype may therefore offer an avenue for future coronavirus vaccine development. IMPORTANCE Infectious bronchitis virus is a pathogen of economic and welfare concern for the global poultry industry. Live-attenuated vaccines against are generated by serial passage of a virulent isolate in embryonated eggs until attenuation is achieved. The exact mechanisms of attenuation are unknown, and vaccines produced have a risk of reversion to virulence. Reverse genetics provides a method to generate vaccines that are rationally attenuated and are more stable with respect to back selection due to their clonal origin. Genetic populations resulting from molecular clones are more homogeneous and lack the presence of parental pathogenic viruses, which generation by multiple passage does not. In this study, we identified two amino acids that impart a temperature-sensitive replication phenotype. Immunogenicity is retained and vaccination results in 100% protection against homologous challenge. Temperature sensitivity, used for the development of vaccines against other viruses, presents a method for the development of coronavirus vaccines.
Subject(s)
Coronavirus Infections , Infectious bronchitis virus , Poultry Diseases , Viral Vaccines , Amino Acids , Animals , Chickens , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Poultry , Poultry Diseases/prevention & control , Poultry Diseases/virology , Temperature , Vaccines, Attenuated , Viral Vaccines/geneticsABSTRACT
In the light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have developed a porcine respiratory coronavirus (PRCV) model for in depth mechanistic evaluation of the pathogenesis, virology and immune responses of this important family of viruses. Pigs are a large animal with similar physiology and immunology to humans and are a natural host for PRCV. Four PRCV strains were investigated and shown to induce different degrees of lung pathology. Importantly, although all four strains replicated equally well in porcine cell lines in vitro and in the upper respiratory tract in vivo, PRCV strains causing more severe lung pathology were also able to replicate in ex vivo tracheal organ cultures as well as in vivo in the trachea and lung. The time course of infection of PRCV 135, which caused the most severe pulmonary pathology, was investigated. Virus was shed from the upper respiratory tract until day 10 post infection, with infection of the respiratory mucosa, as well as olfactory and sustentacular cells, providing an excellent model to study upper respiratory tract disease in addition to the commonly known lower respiratory tract disease from PRCV. Infected animals made antibody and T cell responses that cross reacted with the four PRCV strains and Transmissible Gastroenteritis Virus. The antibody response was reproduced in vitro in organ cultures. Comparison of mechanisms of infection and immune control in pigs infected with PRCVs of differing pathogenicity with human data from SARS-CoV-2 infection and from our in vitro organ cultures, will enable key events in coronavirus infection and disease pathogenesis to be identified.
Subject(s)
COVID-19 , Porcine Respiratory Coronavirus , Swine Diseases , Transmissible gastroenteritis virus , Animals , SARS-CoV-2 , SwineABSTRACT
The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens' eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo. Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens' eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo. Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application.
Subject(s)
Amino Acids , Coronavirus Infections , Infectious bronchitis virus , Poultry Diseases , Viral Nonstructural Proteins , Viral Vaccines , Amino Acids/chemistry , Amino Acids/genetics , Animals , Chick Embryo , Chickens , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Female , Infectious bronchitis virus/genetics , Poultry Diseases/prevention & control , Poultry Diseases/virology , Vaccines, Attenuated/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Vaccines/geneticsABSTRACT
Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Murine hepatitis virus/drug effects , Unfolded Protein Response/drug effects , Activating Transcription Factor 6/metabolism , Animals , Antiviral Agents/therapeutic use , Cell Line , Chlorocebus aethiops , Drug Delivery Systems , Endoribonucleases/metabolism , HEK293 Cells , Humans , Mice , Protein Serine-Threonine Kinases/metabolism , RNA-Seq , Vero Cells , Viral Proteins/metabolism , Virus Replication/drug effectsABSTRACT
Chicken tracheal organ cultures (TOCs) provide a simple ex vivo system that makes use of transverse section of tracheal rings extracted from embryos or adult birds to perform classical virological techniques for virus isolation, propagation and titrations, alongside with gene-expression analysis and virus-host interaction studies. Most IBV strains replicate well in TOCs, thus conveniently allowing growth kinetics analysis. Viral replication is revealed by observation of ciliostasis as marker of infection in tracheas extracted from birds ex vivo, as well as in vitro analysis providing a reliable infection model and a useful tool for titration.
Subject(s)
Organ Culture Techniques/methods , Trachea/metabolism , Virus Cultivation/methods , Animals , Cell Culture Techniques/methods , Cells, Cultured , Chickens , Epithelial Cells/metabolism , Epithelial Cells/virology , Infectious bronchitis virus/isolation & purification , Kinetics , Primary Cell Culture/methods , Trachea/virology , Virus Replication/physiologyABSTRACT
Several techniques are currently available to quickly and accurately quantify the number of virus particles in a sample, taking advantage of advanced technologies improving old techniques or generating new ones, generally relying on partial detection methods or structural analysis. Therefore, characterization of virus infectivity in a sample is often essential, and classical virological methods are extremely powerful in providing accurate results even in an old-fashioned way. In this chapter, we describe in detail the techniques routinely used to estimate the number of viable infectious coronavirus particles in a given sample. All these techniques are serial dilution assays, also known as titrations or end-point dilution assays (EPDA).
Subject(s)
Coronavirus/pathogenicity , Viral Plaque Assay/methods , Animals , Cells, Cultured , Coronavirus/growth & development , Infectious bronchitis virus/growth & development , Infectious bronchitis virus/pathogenicity , Trachea/cytologyABSTRACT
BACKGROUND: Bartonella henselae, Bartonella clarridgeiae and the rare Bartonella koehlerae are zoonotic pathogens, with cats being regarded as the main reservoir hosts. The spread of the infection among cats occurs mainly via fleas and specific preventive measures need to be implemented. The effectiveness of a 10% imidacloprid/4.5% flumethrin polymer matrix collar (Seresto®, Bayer Animal Health), registered to prevent flea and tick infestations, in reducing the risk of Bartonella spp. infection in privately owned cats, was assessed in a prospective longitudinal study. METHODS: In March-May 2015 [Day 0 (D0)], 204 privately-owned cats from the Aeolian Islands (Sicily) were collared (G1, n = 104) or left as controls (G2, n = 100). The bacteraemia of Bartonella spp. was assessed at enrolment (D0) and study closure (D360) by PCR and DNA sequencing both prior to and after an enrichment step, using Bartonella alpha proteobacteria growth medium (BAPGM). RESULTS: A total of 152 cats completed the study with 3 in G1 and 10 in G2 being positive for Bartonella spp. Bartonella henselae genotype I ZF1 (1.35%) and genotype II Fizz/Cal-1 (6.76%) as well as B. clarridgeiae (5.41%) were detected in cats of G2. Bartonella clarridgeiae was the only species detected in G1. Based on the yearly crude incidence of Bartonella spp. infection (i.e. 3.85% in G1 and 13.51% in G2; P = 0.03) the Seresto® collar achieved a preventative efficacy of 71.54%. The incidence of Bartonella spp. infection was more frequent in flea-infested cats (6/33, 18.18%) than in uninfested ones (7/112, 5.88%) (P = 0.036). CONCLUSIONS: Cats living in the Aeolian Islands are exposed to B. henselae and B. clarridgeiae. The Seresto® collar provided significant risk reduction against Bartonella spp. infection in outdoor cats under field conditions. Such a preventative tool could be a key contribution for decreasing the risk of Bartonella spp. infection in cats and thus ultimately to humans.
Subject(s)
Bartonella Infections/veterinary , Cat Diseases/epidemiology , Cat Diseases/prevention & control , Flea Infestations/veterinary , Insecticides/administration & dosage , Neonicotinoids/administration & dosage , Nitro Compounds/administration & dosage , Pyrethrins/administration & dosage , Animals , Bacteremia/epidemiology , Bacteremia/prevention & control , Bacteremia/veterinary , Bacteriological Techniques , Bartonella/genetics , Bartonella/isolation & purification , Bartonella Infections/epidemiology , Bartonella Infections/prevention & control , Cats , Flea Infestations/epidemiology , Flea Infestations/prevention & control , Incidence , Longitudinal Studies , Polymerase Chain Reaction , Prospective Studies , Sequence Analysis, DNA , Sicily/epidemiologyABSTRACT
Intra-vitam diagnosis of feline infectious peritonitis (FIP) is a challenge for veterinary diagnosticians, since there are no highly specific and sensitive assays currently available. With the aim to contribute to fill this diagnostic gap, a total of 61 effusions from cats with suspected effusive FIP were collected intra-vitam for detection of feline coronavirus (FCoV) antibodies and RNA by means of indirect immunofluorescence (IIF) assay and real-time RT-PCR (qRT-PCR), respectively. In 5 effusions there was no evidence for either FCoV RNA or antibodies, 51 and 52 specimens tested positive by IIF and qRT-PCR, respectively, although antibody titres≥1:1600, which are considered highly suggestive of FIP, were detected only in 37 effusions. Three samples with high antibody levels tested negative by qRT-PCR, whereas 18 qRT-PCR positive effusions contained no or low-titre antibodies. qRT-PCR positive samples with low antibody titres mostly contained low FCoV RNA loads, although the highest antibody titres were detected in effusions with CT values>30. In conclusion, combining the two methods, i.e., antibody and RNA detection would help improving the intra-vitam diagnosis of effusive FIP.
Subject(s)
Antibodies, Viral/chemistry , Ascitic Fluid/virology , Coronavirus, Feline/immunology , Feline Infectious Peritonitis/virology , RNA, Viral/chemistry , Animals , Ascitic Fluid/chemistry , Cats , Coronavirus, Feline/genetics , Feline Infectious Peritonitis/immunology , Real-Time Polymerase Chain Reaction/veterinary , Reverse Transcriptase Polymerase Chain Reaction/veterinaryABSTRACT
We report a case in an Italian wolf ( Canis lupus italicus) of pantropic canine coronavirus infection, which has previously been detected only in dogs. The wolf was coinfected by canine parvovirus type 2b and canine adenovirus type 2, which highlighted the crucial role of epidemiologic surveys in European wild carnivores.
Subject(s)
Coronavirus Infections/veterinary , Coronavirus, Canine/isolation & purification , Phylogeny , Wolves/virology , Animals , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Coronavirus, Canine/genetics , Italy/epidemiologyABSTRACT
The spike glycoprotein (S) of infectious bronchitis virus (IBV) comprises two subunits, S1 and S2. We have previously demonstrated that the S2 subunit of the avirulent Beau-R strain is responsible for its extended cellular tropism for Vero cells. Two recombinant infectious bronchitis viruses (rIBVs) have been generated; the immunogenic S1 subunit is derived from the IBV vaccine strain, H120, or the virulent field strain, QX, within the genetic background of Beau-R. The rIBVs BeauR-H120(S1) and BeauR-QX(S1) are capable of replicating in primary chicken kidney cell cultures and in Vero cells. These results demonstrate that rIBVs are able to express S1 subunits from genetically diverse strains of IBV, which will enable the rational design of a future generation of IBV vaccines that may be grown in Vero cells.
Subject(s)
Coronavirus Infections/prevention & control , Infectious bronchitis virus/growth & development , Poultry Diseases/prevention & control , Recombinant Proteins/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Viral Vaccines/immunology , Virus Replication , Animals , Cells, Cultured , Chickens , Chlorocebus aethiops , Coronavirus Infections/veterinary , Infectious bronchitis virus/genetics , Infectious bronchitis virus/immunology , Recombinant Proteins/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Attenuated/isolation & purification , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccines, Synthetic/isolation & purification , Vero Cells , Viral Vaccines/genetics , Viral Vaccines/isolation & purificationABSTRACT
We report detection and full-genome characterization of a novel orthopoxvirus (OPXV) responsible for a fatal infection in a cat. The virus induced skin lesions histologically characterized by leukocyte infiltration and eosinophilic cytoplasmic inclusions. Different PCR approaches were unable to assign the virus to a defined OPXV species. Large amounts of typical brick-shaped virions, morphologically related to OPXV, were observed by electron microscopy. This OPXV strain (Italy_09/17) was isolated on cell cultures and embryonated eggs. Phylogenetic analysis of 9 concatenated genes showed that this virus was distantly related to cowpox virus, more closely related to to ectromelia virus, and belonged to the same cluster of an OPXV recently isolated from captive macaques in Italy. Extensive epidemiologic surveillance in cats and rodents will assess whether cats are incidental hosts and rodents are the main reservoir of the virus. The zoonotic potential of this novel virus also deserves further investigation.
Subject(s)
Cat Diseases/diagnosis , Orthopoxvirus/isolation & purification , Poxviridae Infections/diagnosis , Animals , Cats , Diagnosis, Differential , Fatal Outcome , Italy , Male , Orthopoxvirus/genetics , Poxviridae Infections/virologyABSTRACT
The long-term shedding of Canine alphaherpesvirus 1 (CaHV-1) by neonatal pups with natural infection is reported. The pups belonged to a litter of 11 pointers of a breeding kennel in southern Italy, 9 of which developed a fatal form of systemic infection, as resulted by the detection of CaHV-1 in internal organs (kidney, liver, lung and brain) of one of this dogs and in the vaginal swab of their mother. The two remaining animals displayed a milder form of disease, with one pup showing ocular involvement, and underwent a progressive recovery. These pups were monitored from 11 to 36 â¯days of age, showing a long-term shedding of the virus through the nasal and ocular secretions and the faeces. CaHV-1 shedding, as assessed by means of a specific and sensitive real-time PCR assay, occurred mainly through the nasal secretions, although the pup displaying ocular disease shed the virus at high titres and for a long period even in the ocular secretions.