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Astroviruses (AstVs) are non-enveloped, positive-sense, single-stranded RNA viruses belonging to the Astroviridae family. Currently, two genera: namely Mamastrovirus and Avastrovirus are distinguished within this family. The genus Mamastrovirus includes astrovirus species isolated from humans and a number of mammals. Isolates originated from avian species, such as turkey, chickens, ducks, and other birds are classified into the genus Avastrovirus1, 2. AstVs have been detected in humans and a variety of animal species, including non-human primates, other mammals and avian species3–5. Their genomes are 6.8–7.9 kb in length, consisting of a 5′-untranslated region (UTR), three open reading frames (ORFs), a 3′-UTR and a poly (A) tail6. The high degree of genetic diversity among AstVs and their recombination potential signify their capacity to cause a broad spectrum of diseases in multiple host species3, 7, 8. Human classical AstVs are a frequent cause of acute gastroenteritis in young children and the elderly, occasionally with encephalitis8.
In poultry, AstV infections have been found to be associated with multiple diseases, such as poult enteritis mortality syndrome, runting-stunting syndrome of broilers, white chick syndrome, kidney and visceral gout in broilers and fatal hepatitis of ducklings, leading to substantial economic losses9–16. Increasing evidence indicates that there is a high degree of cross species transmission of AstVs between domestic birds, and even the potential to infect humans17. By comparison, fewer AstV infection cases have been described in domestic goose flocks. Bidin et al.18 reported the detection of avian nephritis virus infection in Croatian goose flocks and provided evidence that this AstV was associated with stunting and pre-hatching mortality of goose embryos. Studies to detect AstV genomes from the clinical samples of geese suggested that these viruses might distribute widely among goose flocks, as seen in other poultry flocks19, 20. In February 2017, an outbreak of disease was reported in a goose farm in Weifang, Shandong Province, China. Affected flocks (containing 2000–3000 goslings) experienced continuous mortality rates ranging from 20 to 30% during the first 2 weeks of the outbreak despite antibiotic and supportive treatment. We conducted a systematic investigation to identify the causative agent of this disease and report here the isolation and characterization of a genetically distinct avian AstV. The pathogenicity of this virus was evaluated by experimental infection of goslings.
Pet dogs play an important role in humans’ daily lives. Recently, the emergence of new pathogens and the continuous circulation of common etiological agents in dog populations have complicated canine diseases. Among these diseases, canine infectious respiratory diseases (CIRD) and viral enteritis pose notable threats to dog health.
CIRD are complex and include canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine herpesvirus (CHV), canine reovirus, Bordetella bronchiseptica and other pathogenic agents [2–4]. Among these, CAV-2, CDV or CPIV have frequently been detected in dogs with CIRD, according to previous studies [5, 6]. Avian-origin H3N2 CIV has been detected in domestic dogs in South Korea and China since 2007 [7, 8]. H3N2 CIV is now circulating in dog populations in China, South Korea, Thailand, and even the United States [9–11]. Distinguishing these pathogens can be challenging, because dogs often show similar clinical signs of infection with these viruses, such as low-grade fever, nasal discharge and cough. These respiratory symptoms are flu-like and difficult to diagnose.
Canine viral enteritis is common in dogs with acute vomiting and diarrhea. Canine parvovirus (CPV) is one of the major viruses leading to acute gastroenteritis in dogs; CPV infection is characterized by fever, severe diarrhea and vomiting, with high morbidity. Puppies tend to be intolerant of CPV infection and have higher mortality than adult dogs because of myocarditis and dehydration [14, 15]. Canine coronavirus (CCoV) is characterized by high morbidity and low mortality. Dogs infected with CCoV alone are likely to have mild diarrhea, whereas the disease may be fatal when coinfection by CCoV and CPV, CDV or canine adenovirus type 1 (CAV-1) occurs [16, 17]. CAV-2 is associated with mild respiratory infection and episodic enteritis [18, 19]. Canine circovirus (CanineCV), a newly discovered mammalian circovirus, was first reported by Kapoor et al. in 2012. CanineCV has been detected in dogs with severe hemorrhagic diarrhea, and it is more common in puppies than in adults [21, 22]. Coinfection of CanineCV with other intestinal pathogens (CPV or CCoV) is closely related to the occurrence of intestinal diseases [23, 24]. Dogs with intestinal diseases are often infected with one or more viruses, and their clinical symptoms are similar [17, 25, 26], making clinical differential diagnosis difficult. To date, no multiplex PCR (mPCR) method has been developed to detect CanineCV and other enteropathogens.
An effective diagnostic tool is important for the prevention, control and treatment of CIRD and viral-enteritis-related viral diseases. Although many methods exist to detect CIRD and canine viral enteritis, most can detect only 2 or 3 pathogens, and the current lack of systematic and comprehensive detection methods makes diagnosis impractical and time consuming [4, 27, 28]. Because mPCR can simultaneously detect multiple pathogens in a timely and inexpensive manner, this technique has become increasingly popular. Therefore, in this study, two new mPCR methods were developed for the detection of canine respiratory viruses (CRV, including CAV-2, CDV, CIV and CPIV) and canine enteric viruses (CEV, including CAV-2, CanineCV, CCoV and CPV), and we indicated that the mPCR methods established here are simple and effective tools for detecting the viruses of interest.
Many emerging infectious diseases are caused by zoonotic transmission, and the consequence is often unpredictable. Zoonoses have been well represented with the 2003 outbreak of severe acute respiratory syndrome (SARS) due to a novel coronavirus. Bats are associated with an increasing number of emerging and reemerging viruses, many of which pose major threats to public health, in part because they are mammals which roost together in large populations and can fly over vast geographical distances. Many distinct viruses have been isolated or detected (molecular) from bats including representatives from families Rhabdoviridae, Paramyxoviridae, Coronaviridae, Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, Arenaviridae, Herpesviridae, Picornaviridae, Filoviridae, Hepadnaviridae and Orthomyxoviridae.
The Reoviridae (respiratory enteric orphan viruses) comprise a large and diverse group of nonenveloped viruses containing a genome of segmented double-stranded RNA, and are taxonomically classified into 10 genera. Orthoreoviruses are divided into two subgroups, fusogenic and nonfusogenic, depending on their ability to cause syncytium formation in cell culture, and have been isolated from a broad range of mammalian, avian, and reptilian hosts. Members of the genus Orthoreovirus contain a genome with 10 segments of dsRNA; 3 large (L1-L3), 3 medium (M1-M3), and 4 small (S1 to S4).
The discovery of Melaka and Kampar viruses, two novel fusogenic reoviruses of bat origin, marked the emergence of orthoreoviruses capable of causing acute respiratory disease in humans. Subsequently, other related strains of bat-associated orthoreoviruses have also been reported, including Xi River virus from China. Wong et al. isolated and characterized 3 fusogenic orthoreoviruses from three travelers who had returned from Indonesia to Hong Kong during 2007–2010.
In the present study we isolated a novel reovirus from intestinal contents taken from one fruit bat ( Rousettus leschenaultia) in Yunnan province, China. In the absence of targeted sequencing protocols for a novel virus, we applied the VIDISCR (Virus-Discovery-cDNA RAPD) virus discovery strategy to confirm and identify a novel Melaka-like reovirus, the “Cangyuan virus”. To track virus evolution and to provide evidence of genetic reassortment PCR sequencing was conducted on each of the 10 genome segments, and phylogenetic analysis performed to determine genetic relatedness with other bat-borne fusogenic orthoreoviruses.
About 70% of microbial agents causing outbreaks of emerging infectious diseases in humans originate directly from animals. Among respiratory virus infections, the influenza A viruses H5N1 and H7N9 from avian species, and the severe acute respiratory syndrome coronavirus from bats have caused large epidemics–. Atypical bacterial pathogens causing community-acquired pneumonia include Chlamydophila psittaci from psittacine birds and Coxiella burnetti from livestock and other animals. However, human outbreaks due to zoonotic bacteria associated with the emergence of a novel animal virus in the animal host were not previously documented.
In November 2012, an outbreak of human psittacosis affecting six staff members occurred at the New Territories North Animal Management Centre (NTNAMC) in Hong Kong. The human outbreak was preceded by an outbreak of avian chlamydiosis among the detained Mealy Parrots (Amazona farinose). Although birds in the tropical and sub-tropical areas are commonly infected with C. psittaci, most infected birds are asymptomatic,. Large human outbreaks are rare even among bird handlers. Although co-infection of C. psittaci and viruses has been reported in outbreaks of avian species–, no virus-bacterium co-infection of implicated avian species has ever been reported in outbreaks of human psittacosis. In this study, we sought to investigate viruses that cause avian co-infection, which may have led to this outbreak of psittacosis.
Avian astroviruses belong to the genus Avastrovirus of the Astroviridae family. The viral particles are small, non-enveloped, positive sense RNA viruses, 28 to 30nm in diameter, and have a star-like morphology (21). Turkey Astrovirus type 1 (TAstV-1) was first described in 1980 by McNulty et al. (22) in the United Kingdom, and the first isolated of TAstV in the United States was identified in 1985 (15). A second TAstV type, which was antigenically and genetically distinct from the previously identified as TAstV-1, was isolated 1996 and designated as TAstV-2 (13,31,32). The entire genome sequence of the TAstV-2 isolate, NC/96, has been reported and bears many similar features to Human Astrovirus (HAstV) (15). Moreover, astroviruses are linked with enteric disease in humans and young animals such as calves, lambs, pigs, dogs, cats and minks (15,21). The most important infectious disease caused by TAstV are the enteric diseases affecting the digestive tract of commercial poults proposed to result in more economic loss than those affecting any other system reported worldwide, including Brazil (16,17,26,27,28,30,34,35). In addition, no vaccine currently exists for the enteric disease caused by astroviruses, which leads the disease to the bio-security manners to control virus infection and spread (15).
Turkey Coronavirus (TCoV) was described affecting commercial poults in Brazil, suspected of suffer from PEMS-Poult Enteric Mortality Syndrome (34,35). In addition, coronaviruses had often been described as being fastidious. This claim arose from the difficulty that virologists had experienced in finding types of cells in which grow coronaviruses in vitro (7,11). Moreover, the TCoV was confirmed as being in the UK in 2001 (6) and has being demonstrated as worldwide distribution (7).
Alternatives to diagnosis both TAstV-2 and TCoV, and also others viruses, has been applied in order to overcome virus isolation (5). In this way, molecular approaches seems to be more appropriated to direct detect viral RNA from clinical samples, by the use conventional and/or alternative reverse transcriptase polymerase chain reaction - RT-PCR (1,5,8,14,18,24,25,29). Since both viruses have been associated to PEMS outbreak in North America (1,2,13,18,22,23,24,27,29) and recently UK and Wales (9), few works describe TCoV and TAstV-2 epidemiology among Brazilian’s producers.
The aim of this study was to verify viral RNA from TCoV and TAstV-2 from 30-day-old affected poults, presenting clinical signs of PEMS. For this purpose, both simplex and multiplex RT-PCR assays were applied to detect viral RNA from different clinical samples at different year seasons.
Subject 2 (S2) is a 28 year old female Chinese medical officer who attended to the index patient in the government health clinic in Kampar on 21 August 2006. On 25 August, she developed nasal obstruction with mild runny nose. This was associated with a mild sore-throat and hoarseness of voice. There was no associated cough or breathing difficulty. Apart from the subjective feeling of mild lethargy and general unwellness, she did not experience fever, headache or myalgia. Her upper respiratory symptoms resolved within three days.
A case was defined as a staff member working at the NTNAMC who was hospitalized for respiratory tract infection between November 1 and November 30, 2012, and confirmed to have C. psittaci infection by polymerase chain reaction (PCR) and/or a four-fold rise in serum microimmunofluorescent antibody titer against C. psittaci (Focus Diagnostics, Cypress, California, USA).
Avian infectious bronchitis virus (IBV) is a highly contagious pathogen of chickens that replicates primarily in the respiratory tract and also in some epithelial cells of the gut, kidney and oviduct. IBV is a virus member of genus Coronavirus, family Coronaviridae, order Nidovirales. The virus possesses a positive stranded RNA genome that encodes phosphorylated nucleocapsid protein (N), membrane glycoprotein (M), spike glycoprotein (S) and small membrane protein (E). The spike glycoprotein is post-translationally cleaved into two subunits, S1 and S2. The S1 protein forms the N-terminal portion of the peplomer and contains antigenic epitopes mainly within three HVRs. Neutralizing and serotype specific epitopes are associated within the defined HVRs.
Variation in S1 sequences, has been recently used for distinguishing between different IBV serotypes. Diversity in S1 probably results from mutation, recombination and strong positive selection in vivo. Antigenically different serotypes and newly emerged variants from field chicken flocks sometimes cause vaccine breaks. The generation of genetic variants is thought to be resulted from few amino acid changes in the spike (S) glycoprotein of IBV.
In Egypt, isolates related to Massachusetts, D3128, D274, D-08880, 4/91 and the novel genotype; Egypt/Beni-Suef/01 were isolated from different poultry farms. The commonly used IBV attenuated vaccine is H120 while the Mass 41 (M41) strain is commonly used in inactivated vaccines.
In the present study, Egypt/F/03 was isolated from 25-day-old broiler chickens in Fayoum Governorate, identified by Dot-ELISA, RT-PCR and sequenced to determine its serotype. Pathogenicity test to 1-day-old chickens and protection afforded by the commonly used H120 live attenuated vaccine were also performed.
Astroviruses are currently classified into two genera, Mamastroviruses (MAstVs) and Avastroviruses (AAstVs)1. MAstVs mainly infect mammals including human, ovine, bovine, porcine, feline, canine, mink, bat, deer, mouse, sea lion, dolphin etc., whereas AAstVs generally infect aviansuch as turkey, chicken, duck, pigeon, and goose1,2. Notably, genetic variation and cross-species transmission of astrovirusespose the risk for zoonotic infection2,3. Infection with astroviruses mainly cause enteric diseases such as gastroenteritis and diarrhea in human and animals as reported initially, then nephritis in chicken and pigeon, hepatitis in ducklings, and encephalitis in human, cattle, and sheep recently, which broadens the disease pattern of astroviruses and highlights its significance2,4–6. In 2015, a gout disease emerged in 1-week-old goslings in Anhui province, which had spread to most provinces of China by 2017 with high morbidity (80–90%) and mortality (20–70%), and no pathogenic bacteria could be isolated from the diseased goslings. However, little is known about the pathogen for the goose gout disease endemic in goose flocks in China. During 2011–2012, the outbreaks of gout disease were reported in broilers in India. Through virus isolation and infection study, Bulbule et al. demonstrated that a novel chicken astrovirus (CAstV) could be as one of the causative agents for the gout disease in chicken flocks in India7. To control the spread of the goose gout disease, we investigated the pathogenic agent of the disease. Through in vitro and in vivo experiments, we identified and isolated a novel goose astrovirus different from the CAstVas a causative agent of the gout disease recently circulating in gosling flocks in China.
In 2015, a gout disease emerged in 1-week-old goslings in Anhui province, which has spread to most provinces of China by 2017. The outbreak of the gosling gout disease has caused significant economic loss in goose industry. The clinical signs of the disease were characterized by white feces, leg joint enlargement with urate deposits and paralysis. At necropsy, kidney enlargement and intensive urate deposits were found in the gallbladder, knees, and ureters, and on the surfaces of cardiac, heart, liver, air sacs, trachea, and proventriculus (Fig. 1a–f). The disease lasted for 7–10 days with high morbidity and mortality. The survival goslings grew slowly and were susceptible to bacterial infection.
Viruses are obligate intracellular pathogens that require host cells in order to replicate and produce infectious progeny. Virus entry into host cells is followed by capsid uncoating, genome transcription and replication, synthesis of viral proteins, assembly of progeny virions, and egress. For most viruses, genome replication and assembly take place in specialized intracellular compartments known as viral factories or inclusions, which are often composed of membranous scaffolds, viral and cellular factors, and mitochondria. Viral inclusions (VIs) serve multiple purposes during infection, including the concentration of viral and host factors to ensure the high efficiency of replication, sequestration of viral nucleic acids and proteins from innate immune responses, and the spatial coordination of consecutive replication cycle steps. Most double-stranded RNA (dsRNA) viruses form cytoplasmic inclusions with a characteristic morphology. These neoorganelles constitute sites of genome replication and virion assembly, and contain abundant viral RNA and proteins.
The combination of ultrastructural and functional studies has enhanced our knowledge about VI biogenesis. However, for many viruses, it is still not known how these structures form and mediate functions in viral replication. Here, we describe the current understanding of the morphogenesis and function of reovirus inclusions and compare these neoorganelles with the replication factories formed by other members of the Reoviridae family.
Mammalian reoviruses (MRVs) are prototypical members of the family Reoviridae, which contains segmented double-stranded RNA (dsRNA) viruses of both medical (rotavirus) and economic (bluetongue virus) importance (reviewed in–). These viruses have a segmented dsRNA genome encoding eight structural proteins and three nonstructural proteins. Reoviruses were originally called “respiratory enteric orphan” viruses based on their repeated isolation from the respiratory and enteric tracts of children with asymptomatic illnesses. However, there have been reports describing reoviruses associated with meningitis in infants and children–, and with acute respiratory disease in adults. Despite limited reports of severe reovirus infections in humans, pulmonary infection of mice with reovirus serotype 1 strain Lang is a very clinically relevant model of infection-induced acute viral pneumonia leading to acute respiratory distress syndrome, the most severe form of acute lung injury–. In a mouse infection model, reovirus-induced apoptosis is a major determinant of virulence, causing neural and cardiac injury (reviewed in), and mice treated with inhibitors of apoptosis before infection have reduced tissue damage in the central nervous system and heart,. Recent studies have indicated that the reovirus outer capsid protein μ1 is the primary factor involved in reovirus-induced apoptosis,.
We previously reported the isolation and partial characterization of a new reovirus strain, designated BYD1, isolated from the throat swabs of one patient in Beijing with severe acute respiratory syndrome (SARS) in Hep-2 cell cultures. SARS-coronavirus (SARS-CoV) was also isolated from the same samples in Vero-E6 cell cultures. Three other reovirus strains, designated BLD, JP, and BYL, were isolated from other SARS patients. We propose that these new reovirus strains be designated “SARS-associated mammalian reovirus” (SARS-MRV). All four SARS-MRV strains were purified by plaque assay and identified as novel members of serotype 2 by sequence analysis of their S1 segments. Genome comparisons showed that BYD1 is a reassortant virus, with its S1 gene segment derived from a previously unidentified serotype 2 isolate and its other nine segments derived from ancestors of homologous serotype 1 and serotype 3 segments. Notably, these SARS-MRVs have been attributed an important role in the etiology of SARS, based on the initial finding of high anti-BYD1 antibody titers in some SARS patients and experimental infections showing that BYD1 can cause SARS-like symptoms in macaques and guinea pigs,.
The purpose of this study was to determine whether SARS-MRV or its proteins can induce apoptosis and to characterize the pathology of the viral infection in a murine model. To this end, we found that Hep-2 cells infected with BYD1 undergo different stages of apoptosis, characterized by nuclear and cytoplasmic shrinkage, together with various forms of chromatin margination and condensation, and followed by karyorrhexis and the formation of apoptotic bodies. We show that the ectopically expressed cell-attachment protein σ1 and the outer capsid protein μ1 can independently induce infection-like pathological apoptosis in 293 T cells. We also show that suckling mice intracranially inoculated with BYD1 display signs of central nerve damage, myocarditis, and pneumonia. The potential of viral proteins σ1 and μ1 to induce apoptosis could be associated with multi-organ injuries in vivo. The findings described here provide new insight into the pathogenesis of SARS-MRV.
Infectious bronchitis (IB) is primarily a respiratory disease of chickens but with potential to cause more widespread infection in the urinary and reproductive tracts in chicken leading to significant production losses in commercial broiler and layer flocks worldwide. The causative infectious bronchitis virus (IBV) belongs to the family Coronaviridae. The disease is usually characterized by high morbidity and low mortality in mature birds, whereas in naive young birds (2–3 weeks of age), mortality up to 100% can be observed. Being an RNA virus with the ability to mutate and recombine, IBV persist as numerous serotypes and strains. The control of IB relies on vaccination. Vaccines are available for commonly occurring serotypes and strains but they are not necessarily antigenically similar to the wild-type viral strains circulating in poultry barns. Although, these vaccine strains may provide some degree of protection for some related strains known as protectotypes, the commonly available vaccines may not elicit protective immune responses in a flock if the field strains are antigenically very different from the vaccine strains. Owing to this reason, vaccination against IBV is not currently considered to be a very effective control method and other biosecurity measures are necessary to prevent the introduction of IBV into poultry production facilities.
IBV is known to replicate in the respiratory tract leading to changes in the muco-cilliary clearance mechanism, as such, expose the IBV infected birds to secondary bacterial infections. Additionally, IBV has tropisms for a variety of tissues. However, the mode of dissemination from the common route of entry, i.e. the respiratory route, to the rest of the body systems could potentially be due to the initial viremia. Once disseminated, IBV infects epithelial cells of the reproductive and urinary systems, particularly the oviduct and kidney depending on the infecting strain. Recently, it has been shown that a nephro-pathogenic strain of IBV (B1648) could replicate in peripheral blood monocytes leading to viremia. The infection of circulating monocytes could potentially disseminate IBV to the urinary tract, liver and spleen.
Macrophages play roles in innate immune responses, as well as in mounting adaptive immune responses by functioning as antigen presenting cells, as such they are critical in protecting animals from microbial infections. Although it is known that macrophage numbers are elevated in the respiratory tract in response to IBV infection, the role played by macrophages in IBV infection, particularly if they serve as a target cell for viral replication is not known. Macrophages have been implicated to play in an important role in the pathogenesis of some animal and human viruses including Marek’s disease virus in birds, feline corona virus in cats, and human immunodeficiency virus (HIV). It was also shown that coronaviruses such as severe acute respiratory syndrome (SARS)-coronavirus (CoV) can replicate within human macrophages thereby interfering with macrophage functions leading to severe pathology. However, a single report based on in vitro studies indicated that IBV, particularly nonpathogenic Beaudette and Massachusetts type 82822 strains do not replicate in avian macrophages.
Therefore, in this study we investigated the interaction of IBV with macrophages in lungs and trachea in vivo and macrophage cell cultures in vitro using two IBV strains, Connecticut A5968 (Conn A5968) and Massachusetts-type 41 (M41) which are known to induce clinical disease and pathological lesions in chickens. As implicated in some other viruses, we hypothesized that these two strains of IBV replicate within avian macrophages leading to productive replication and interfering with selected macrophage functions in the process.
Bats (order Chiroptera) are natural reservoirs for zoonotic viruses that cause some of the deadliest diseases in humans, including filoviruses (such as Ebola and Marburg viruses), lyssaviruses, severe acute respiratory syndrome (SARS)-related coronaviruses and henipaviruses (e.g. Hendra and Nipah viruses) [1–3]. Despite being hosts to such an array of pathogens, bats generally show mild or no clinical symptoms to their presence, a phenomenon that is largely a mystery and a potential biomedical treasure trove that could offer new insights into the treatment and control of such pathogens in humans and affected animals. The lack of illness does not mean that bat cells are not infected by such viruses. Bat cells are susceptible to infections with paramyxoviruses and filoviruses, and show varying degree of permissiveness to virus replication, which is a pre-requisite for the hosts to acquire carrier status. Bat lung epithelial cells (TB1-Lu) of Tadarida brasiliensis display resistance to reovirus infection; infected cells show no cytopathic effects and rapid decline in virus production; however, low virus release is maintained for at least 2 months. Murine encephalomyocarditis virus, in contrast, causes severe cytopathic damage in TB1 Lu cells, and Ebola virus shows persistent infection in such cells.
Recently, two novel influenza viruses, H17N10 and H18N11, were identified in bats by deep sequencing analyses (although live viruses have not been directly isolated) which have understandably caused much speculation about their zoonotic potential. These viruses are, however, highly divergent from conventional mammalian and avian influenza A viruses. Chimeric virus housing the six core genes from bat H17N10 virus replicated well in human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Furthermore, the chimeric bat virus failed to reassort with conventional influenza viruses in MDCK cells. Bat viral ribonucleopolymerase (vRNP) complex subunits (PB1, PB1 and PA) were not functionally interchangeable with corresponding human virus-derived vRNP subunits suggesting there is limited reassortment potential between bat and human influenza viruses. However, vRNP from bat H17N10 virus is able to drive with high efficiency the non-coding region of human H1N1 virus (A/WSN/1933) in vRNP minigenome reporter assays, highlighting the possibility of viable reassortment between bat and human influenza viruses. Although the issue of functional reassortment between native bat and conventional influenza A viruses has not been fully resolved, its likelihood is presently considered low.
Single-cycle green fluorescent protein (GFP) reporter virus (human A/WSN/33) was variably able to infect all eleven bat cell lines, derived from seven bat species. Similar number of infected cells were found among all seven bat cell lines by immunocytochemical detection of viral nucleoprotein (NP). Human virus-derived vRNP complex was shown to perform better than avian virus-derived vRNP complex in the same A/WSN/33 viral backbone at progeny virus release, based mostly on the use of TB1-Lu bat cells, which appear inherently resistant to influenza virus infection. Although there is limited potential for reassortment between human and bat influenza viruses, Pteropus alecto kidney cells were able to produce reassorted progeny from human H1N1 (A/WSN/1933) and highly pathogenic avian influenza (HPAI) H5N1 (A/Vietnam/1203/04) viruses. Collectively, these findings appear to indicate that bat cells are susceptible to infection with conventional mammalian and avian influenza viruses. However, we are unclear about the relative permissiveness of bat respiratory epithelial cells to conventional influenza viruses in the production of viable progeny. Although bats are not known to act as hosts for human and avian influenza viruses, the potential epidemiological significance of avian influenza virus infection in bats was highlighted by the recent discovery that around 30 out of 100 free ranging Eidolon helvum (fruit bats) in Ghana were serologically positive for avian H9 virus.
We report here on the relative susceptibility of lung epithelial cells from three diverse bat species, T. brasiliensis (a medium insectivorous bat), E. helvum, (a large fruit bat) and C. perspicillata (a small mainly fruit, and insect eating bat), to avian and human influenza A viruses. We found that all three species of bat cells were more resistant than control Mardin-Darby canine kidney (MDCK) cells, in terms of reduced progeny virus production and higher cell viability, which appeared not to depend on JAK/STAT signalling. Although the three species of bat cells showed variation in resistance to infection, they were relatively more permissive to avian than human influenza viruses which could be important in the ecology of avian influenza viruses.
Viruses are the smallest among all self-replicating organisms and yet they are the etiological agents of many difficult to treat diseases in human populations. There are broad types of human infections caused by viruses, such as respiratory infections (common cold, Influenza), digestive infections (viral gastroenteritis), central nervous system infections (viral meningitis, viral encephalitis), skin or mucosal infections (herpes, measles, mumps, smallpox and rubella), hepatic infections (hepatitis A, B, C, E), blood infections (acquired immunodeficiency syndrome) and hemorrhagic fever (yellow fever, Ebola hemorrhagic fever). Viruses are the most abundant and diverse biological entities on Earth and this is the reason for the high incidence of viral infections. In addition, some viruses are etiological agents in the development of human tumors, particularly cervical cancer and hepatic cancer.
The main method and most cost-effective strategy for preventing viral infections is through vaccination, which is meant to prevent outbreaks by increasing immunity. Vaccines for the prevention of several common acute viral infections, such as polio, rubella, measles, mumps, Influenza, yellow fever, encephalitis, rabies, smallpox and hepatitis B were developed during the 20th century and are available on a large scale. Efforts to develop safe and effective vaccines against viruses that cause chronic infections, such as human immunodeficiency virus or hepatitis C virus did not give the expected results.
For many viral infections, only symptomatic treatment is indicated, while it is expected the immune system to fight off the virus. However, there are high-virulence viruses that cause serious viral infections where antiviral treatment is essential for patient survival. Although great efforts have been made to find effective medication, there are still no drugs that truly cure viral infections. Moreover, due to the ability of viruses to undergo rapid mutations, the mechanisms involved in developing resistance to antiviral drugs are activated in most cases. As resistance toward antiviral drugs is becoming a global health threat, there is an intrinsic need to identify new scaffolds that are useful in discovering innovative, less toxic and highly active antiviral agents.
Enteritis is a main problem in poultry, associated to considerable direct and indirect economic losses. Several enteric viruses have been identified in commercial flocks of turkeys worldwide. Enteric diseases may be occur in all age groups, nevertheless, they are predominantly affect young birds in the three first weeks of age, where infections appear more severe (Nuñez and Piantino Ferreira, 2013; Mettifogo et al., 2014). Enteric viruses increase susceptibility of affected birds to secondary infections and others immunosuppressive diseases.
Several viruses are incriminated in the enteric diseases in commercial turkeys. Interaction between them is very complex, including many other management, feeding, and infectious factors. Because of the various etiologies, clinical signs are in general nonspecific, including diarrhea, increased mortality, and poor performances. Gross pathology showed gastrointestinal lesions, associated to liver, pancreatic, and lymphoid damage. These symptoms and lesions are considered to be the main enteric syndrome that is why laboratory investigations consist on the use of essential tool to confirm etiological agents (Alavarez et al., 2014; Mettifogo et al., 2014).
In turkeys, the most important enteric viral diseases are represented by hemorrhagic enteritis (HE), runting stunting syndrome (RSS) and PEMS (Table 1).
HE is an acute disease of turkeys caused by Siadenovirus (group II Aviadenovirus), immunosuppressive virus, which infect essentially animals at 4 weeks of age and older. Depression, bloody droppings, heterogeneity of the flock, and increased mortality characterize this disease. In field outbreaks, mortality varied from 0.1% to 60% (Gross and Moore, 1967). Virus replication occurs essentially in spleen, considered to be the major site (Saunders et al., 1993; Pierson and Fitzgerald, 2013). However, Enzyme Linked Immunosorbent Assay (ELISA), Immunofluorescent (IF), and Polymerase Chain Reaction (PCR) are used to confirm the presence of infected cells in many other tissues, such as intestine, bursa of Fabricius, caecal tonsils, thymus, liver, kidney, leukocytes, and lungs (Silim and Thorsen, 1981; Fasina and Fabricant, 1982; Fitzgerald et al., 1992; Trampel et al., 1992; Hussain et al., 1993; Suresh and Sharma, 1996). Primarily viral replication occurs in B cells and macrophages. Other cells target are represented by adherent mononuclear macrophages and non-adherent mononuclear cells (van den Hurk, 1990) bearing IgM (Suresh and Sharma, 1995; 1996).
Virulent HEV strains are capable to induce apoptosis in spleen cells, due to the induction of interleukine-6 (IL-6) secretion in the spleen (Rautenschlein et al., 2000b). Activation of macrophages leads to cytokines (IL-6, interferon type I and II, and TNF) production. Immunosuppressive is the consequence of the nitric acid production, stimulated by the interferon-II (IFN-II) (Dhama et al., 2017). Transient immunosuppression has been reported during clinical phase of the disease, with considerable depletion of IgM-bearing B cells (Rautenschlein et al., 2000b).
Vaccination failures are observed in infected turkeys. A significant decrease in hemagglutination inhibition antibody titers is detected in turkeys infected with virulent HEV. Moreover, depression in phytohemagglutinin (PHA) is also described in inoculated birds (Nagaraja et al., 1985).
Secondary bacterial infections may extend the course of illness and increase mortality for an additional 2–4 weeks (Dhama et al., 2017). Increased predisposition to enteropathogenic Escherichia coli infection (Larsen et al., 1985; van den Hurk et al., 1994; Giovanardi et al., 2014) and clostridial dermatitis (Thachil and Nagaraja, 2013) has been well documented.
Resistance of the virus outside, poor hygiene conditions, and short down time between flocks, contribute to the persistence of the HE (Pierson and Fitzgerald, 2013).
Due to hemorrhage, carcasses appear pale. Gross pathology showed hemorrhagic intestinal mucosa, with the presence of natural coagulated blood. Spleen is characteristically enlarged, marbled, and friable. In dead birds, spleen may be smaller and pale because of blood loss and subsequent splenic contraction (Gross, 1967; Carlson et al., 1974; Fujiwara et al., 1975; Itakura and Carlson, 1975). Histological findings are more apparent in lymphoreticular and gastrointestinal systems. Hyperplasia of the white pulp, lymphoid necrosis and intranuclear inclusion body within lymphoreticular body cells are the most described microscopic modifications (Saunders et al., 1993).
Histopathological changes are more evident in the duodenum, where congestion, hemorrhage, and hetetrophils infiltration and epithelium villus degeneration, consist the major observations. Less severe lesions cans be also find in the gizzard, the proventiculus, the caeca tonsils and the bursa of Fabricius (Saunders et al., 1993; Pierson and Fitzgerald, 2013). Intranuclear inclusions have been detected in many tissues, such as liver, pancreas, bone marrow, renal tubular epithelium, and lung (Gross, 1967; Carlson et al., 1974; Fujiwara et al., 1975; Itakura and Carlson, 1975; Meteyer et al., 1992; Trampel et al., 1992; Hussain et al., 1993).
Many other fowl adenoviruses (FAV) are considered as immunosuppressive agent in turkey. Adenovirus responsible of inclusion body hepatitis (IBH) can induce atrophy of the bursa, the thymus and the spleen, that occurs following challenges involving serotypes 1, 4, and 8 (Singh et al., 2006; Schonewille et al., 2008).
Virulent strains show affinity to lymphocytes and consequently cause impairment of the humoral and cellular responses. Effects on immune system are more severe when associated to aflatoxins (Shivachandra et al., 2003). Several FAV strains are capable of increasing the susceptibility of the bids to E. coli infections (Rosenberger et al., 1985). Vaccination failures again ND and avian influenza (subtype H9) is reported in animals inoculated by FAV serotype 4 (Niu et al., 2017).
Reoviruses (respiratory enteric orphan viruses), members of the family Reoviridae, are a large and diverse group of non-enveloped viruses with segmented dsRNA genomes, which are taxonomically classified into ten genera,. Members of the genus Orthoreovirus contain 10 genome segments and have been isolated from a broad range of mammalian, avian and reptilian hosts. Although orthoreoviruses have been identified as the causative agents of diseases in animals, infections in humans are generally benign with very rare cases of mild upper respiratory tract illness or enteritis in infants or children. Orthoreoviruses are divided into two subgroups, fusogenic and nonfusogenic, based on the ability of the virus to induce cell-cell fusion and syncytium formation.
A fusogenic orthoreovirus, the Melaka virus (MelV), was isolated from a human patient suffering acute upper respiratory disease. MelV was shown to be capable of human-to-human transmission and has close sequence relatedness to two bat-borne orthoreoviruses, the Nelson Bay virus (NBV) isolated from fruit bats in Australia and the Pulau virus (PulV) isolated from fruit bats in Malaysia,. Epidemiological tracing suggested that MelV originated from bats and was transmitted directly to the index case, followed by subsequent transmission to other members of the same family.
Bats have been shown to be the reservoir hosts of many recently emergent zoonotic viruses, including Hendra virus, Nipah virus, Menangle virus, and potentially SARS and Ebola viruses–. NBV was the first reovirus of bat origin, which was isolated in 1968 from the heart blood of a flying fox (Pteropus poliocephalus) in New South Wales, Australia,. NBV was also the first mammalian reovirus to display fusogenic properties, a characteristic previously only known for avian reoviruses (ARVs). In 1999, during a search for Nipah virus in pteropid bats on Tioman Island, PulV was isolated from Pteropus hypomelanus
Here, we report the discovery and characterization of Kampar virus (KamV), the fourth member in the NBV species group and its isolation from a human patient with fever and acute respiratory illness. Although there is no direct evidence to suggest that KamV originated from bats, the close relationship of KamV with other members of the NBV group and preliminary epidemiological data suggest that KamV is most likely a bat-borne orthoreovirus.
Sequencing of the amplified products revealed a high homology with TAstV-2 North Carolina Q/34/1990 strain for polymerase gene and to TCoV for the 3‘UTR of turkey\UK\412\00 strain (FJ178641 and , respectively).
PCRs testing were repeated on the 50 fruit bats original samples including the Kidney, heart, lung, liver, spleen, intestine, rectal swab sample, and brain samples. Two bat’s QPCRs results were positive. One bat’s QPCRs result was positive in the lung, intestine sample (Cangyuan virus isolated) and rectal swab sample, and the Ct (Threshold Cycle) of QPCR were 19.86 ± 0.056, 19.52 ± 0.041, 19.64 ± 0.061 respectively. The Ct of another bat’s PCR were 23.07 ± 0.253, 22.53 ± 0.171 in the intestine sample and rectal swab sample, respectively.
To establish the evolutionary relationship between Cangyuan virus and other known orthoreoviruses, Homology were compared (Table 2, Table 3 and Additional file 1: Table S1, Additional file 2: Table S2 and Additional file 3: Table S3) and phylogenetic trees were constructed based on the nucleotide sequences of the L genome segments (Figure 2), the M genome segments (Figure 3) and the S genome segments (Figure 4). The Cangyuan virus L1-L3, M1-M3 segments sequence identity were 81.6% –94.2%, 83.8%–97.9%, 85.9%–97.6% ( Additional file 1: Table S1), 82.2%–94.1%, 78. 1%–95.0%, and 83.0%–93.9% (Table 2, Additional file 2: Table 2), respectively, by alignment with Pteropine orthoreovirus (PRV) species group. The phylogenetic trees for L2, L3, M1 and M2 segments demonstrated that Cangyuan virus was most closely related to Melaka and Kampar viruses, and was placed in Pteropine orthoreovirus (PRV) species group which covers all known bat-borne orthoreoviruses together with Nelson Bay orthoreovirus.
To better understand the genetic relatedness of Cangyuan virus to other known bat-borne orthoreoviruses, the published sequences for the S genome segment of bat-borne orthoreoviruses known for causing acute respiratory disease in humans were retrieved from GenBank and used to compare homology (Table 3 and Additional file 2: Table S2) and construct phylogenetic trees (Figure 4). The Cangyuan virus S1-S4 segments sequence identity were 55.3%–94.7%, 86.2%–95.5%, 86.5%–97.9%%, and 83.5%–98.2%, respectively (Table 3 and Additional file 2: Table S2). The S1 segment demonstrated a greater heterogeneity than other S segments in Pteropine orthoreovirus (PRV) species group.
The genus Gyrovirus, a diverse group of non-enveloped icosahedral viruses containing circular single-stranded DNA, infects a wide range of hosts. They also trigger several serious diseases in animals as causative agents. In particular, chicken anaemia virus (CAV), a member of family Anelloviridae genus Gyrovirus, is the etiological agent of chicken infectious anaemia. CAV infects several bone marrow-derived cells which results in severe anaemia and immunosuppression in young chickens. In terms of older birds, CAV can jeopardize the immune responses [3, 4]. Since its first reported in 1979, CAV infection has become epidemic among chicken populations on a global scale [5–7]. CAV also has dramatic financial impact in areas of intensive chicken farming. Vaccination is generally used to contain the spread of the virus. In a recent study, a novel human gyrovirus was isolated from a skin swab and designated as human Gyrovirus (HGyV). Since Circovirus shares partial homology to CAV, the identification of HGyV signals possible threats for human pathogenesis, further investigation is yet required.
The negative-sense CAV genome consists of 2,319 nucleotides and is replicated by a rolling-circle mechanism; but the packaging and egress of viral particles are poorly characterised [1, 10]. The CAV genome encodes multiple overlapping open reading frames (ORFs) that are translated into three main distinct polypeptides: CAV viral protein 1 (VP1, 52 kDa), viral protein 2 (VP2, 24 kDa) and viral protein 3 (VP3, 16 kDa). VP1 is the major structural protein while the VP2 is a replicase with dual-specificity phosphatase activity. VP3, also named apoptin, is also a non-structural protein that mainly implicats in the induction of apoptosis and viral cytotoxicity in host cells.
In 1996, CAV was first reported from young broilers in China. 42% of overall seroprevalence was shown in farms of five Chinese provinces in a domestic poultry survey. In addition, a high prevalence of 87% resulted in studies of the virus on live bird markets in Southeast China.
In the present study, our group investigated the epidemiology of CAV in sick or dead chickens in 12 provinces throughout China from 2014 to 2015. Totally, we obtained 96 positive results for CAV infection in 722 clinical samples, 24 out of 149 in 2014, and 72 out of 573 in 2015. We analysed the infection type of CAV in association with other pathogens including Marek’s disease virus (MDV), reticuloendotheliosis virus (REV), avian leukosis virus (ALV), avian gyrovirus 2 (AGV2), and avian reovirus (ARV). We found that coinfection was the main infection type of CAV. In addition, we analysed the characteristics of the new CAV sequenced strains together with those available in GenBank. The analysis revealed that all the sequences could be clustered into four major groups. Furthermore, we compared the key amino acids in VP1 that determined the virulence of CAV, providing new insights into the epidemiology of CAV.
Avian reoviruses (ARVs) belong to the genus Orthoreovirus in the family Reoviridae, which includes mammalian reovirus (MRV), Nelson Bay reovirus (NBV), Baboon reovirus (BRV), and Reptilian reovirus (RRV). ARV is a double-stranded RNA (dsRNA) virus, and the genome consists of 10 segments packaged into a non-enveloped icosahedral double-capsid shell, with a diameter of 70–80 nm [1, 2]. The genome of ARV is comprised of three large (L1, L2, and L3), three medium (M1, M2, and M3), and four small (S1, S2, S3, and S4) segments, which encode proteins of the λ, μ, and σ classes, respectively [3–6]. The first seven bases (5′-GCUUUUU-3′) of the 5′ non-coding regions (NCRs) and the last five bases (5′-UCAUC-3′) of the 3′ NCR of each ARV genome segment are highly conserved across all ARV strains.
ARVs are important etiological agents; they can cause large economic losses in the poultry industry, as they infect a variety of domestic poultry and wild avian species, including chickens, turkeys, Muscovy ducks, Pekin ducks, geese, wild mallard ducks, pigeons, psittacine birds, and other wild birds. The signs of ARV infection in waterfowl include general weakness, diarrhea, serofibrinous pericarditis, and a swollen liver and spleen with small white necrotic foci [10, 16, 17]. Waterfowl-origin reovirus (WRV) was first identified as a pathogen in South Africa in 1950. It was subsequently isolated from Muscovy ducks in France in 1972 and was designated as classical Muscovy duck reovirus (MDRV). Classical MDRV first emerged in China in 1997. It mainly infects Muscovy ducklings at 10 days of age, and the infection persists until the ducklings are 6 weeks old. The mortality rate ranges from 10 to 30%. However, many researchers have reported that classical MDRV isolates are non-pathogenic in shelduck ducklings, Pekin ducklings, and other duckling varieties [19, 20].
In 2002, a new infectious disease emerged in Muscovy ducklings in southeast China, which is the major Muscovy duck production area in China. Unlike classical MDRV infection, this disease is mainly characterized by severe hemorrhagic, necrotic lesions in the liver and spleen, with a mortality rate ranging from 5 to 50%. The virus can infect a variety of duck species, such as Pekin, Muscovy, and domesticated wild duck. Goslings have also been infected in some parts of China [21–23]. Because of the different clinical signs and cytopathic effects compared to chicken-origin ARVs and classical MDRVs, the causative agent of this disease was named as novel Muscovy duck reovirus (N-MDRV). Phylogenetic analyses based on the amino acid sequences encoded by the S2 and S3 segments also demonstrated that N-MDRVs are significantly different from chicken-origin ARVs and classical MDRVs.
In this study, two novel field strains of duck reovirus, named MDRV-SH12 and MDRV-DH13, were isolated from two diseased Muscovy ducklings in Guangdong province, China in June 2012 and September 2013, respectively. To better understand the molecular characteristics of the reoviruses circulating in waterfowl populations, the whole genomes of these two viruses were cloned, sequenced, and analyzed. These complete genomic data may be helpful for understanding the evolutionary relationships among the WRVs and other orthoreoviruses circulating in China.
The mPCRs for detection of CRV (Fig 6A) and CEV (Fig 6B) were tested on 20 NS and 20 AS clinical samples, respectively, and the statistics are shown in Table 2.
Detection of CRV in clinical NS samples: 80% (16/20) of the samples were virus positive. Among single infections, CDV was the predominant virus, appearing in 45% (9/20) of NS. Both CAV-2 and CIV were detected in 5% (1/20) of the NS samples. Among dual infections, CDV and CPIV coinfection was identified in 5% (1/20) of NS samples, and CAV-2 and CPIV coinfection was found in 5% (1/20) of NS samples. Only one sample demonstrated triple infection (CAV-2, CPIV and CDV coinfection). Furthermore, two dogs with particularly severe respiratory symptoms were demonstrated to be infected with CAV-2, CDV, CIV and CPIV.
Detection of CEV in clinical AS samples: 85% (17/20) of the samples were virus positive. Among single infections, CPV was the most common virus, appearing in 70% (14/20) of AS, and CCoV was detected in 10% (2/20) of AS samples. Among dual infections, CPV and CCoV coinfection represented 5% (1/20) of AS samples. Notably, CAV-2 and CanineCV were not detected in any of the clinical samples.
Zoonotic transmission events play a major role in the emergence of novel diseases. While it is difficult to predict the emergence of zoonotic pathogens, wildlife screening of healthy animals for novel pathogens and evaluation of their capacity to induce disease can be a first step. Among mammals, bats have gained special interest as potential reservoirs of emerging viruses such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Ebola [2, 3]. A number of reoviruses (family Reoviridae; respiratory, enteric, orphan virus) have been described in bats in Europe and Asia, all of which belong to the genus Orthoreovirus in the Spinareovirinae subfamily (e.g. [4–9, 10, 11]). Some of these have also been isolated from humans suffering from acute respiratory or gastrointestinal illness and likely originated from bats [4–6, 12]. As part of a bigger study [13, 14] we investigated bats from Sub-Sahara Africa for the presence of pathogens with zoonotic potential.
The family Reoviridae harbors viruses with a segmented double-stranded RNA genome. It is currently divided into two subfamilies; the Sedoreovirinae comprising six, and the Spinareovirinae comprising nine genera. The genus Coltivirus in the latter subfamily is comprised of two species only: Colorado tick fever virus (CTFV) and Eyach virus (EYAV). CTFV is the etiologic agent of a febrile human disease, Colorado tick fever, occurring in the Rocky Mountains in the Western United States and Canada [16–18]. It is rarely fatal but can cause severe complications like encephalitis, haemorrhage, or pericarditis, especially in children. The related EYAV was isolated in Germany in 1976 and has been associated with human neurological disease by serological evidence [20, 21]. Animal reservoirs of coltiviruses are small mammals like rodents and lagomorphs and transmission to humans occurs by ticks of the family Ixodidae [18, 22, 23]. We describe a novel reovirus, designated Taï Forest reovirus (TFRV), which is closely related to coltiviruses, a genus not described in bats before, isolated from blood of African free-tailed bats (Chaereophon aloysiisabaudiae).
Viruses, as obliged intracellular parasites, need to take advantage of a wide variety of cellular processes to successfully produce infectious progeny. Interestingly, different viruses can exploit the same cellular process, and the biomolecules related to it, in many different ways. In recent years, increasing evidence of the importance of tight junctions (TJs) for the infection of several viruses has arisen, making it clear that studying the role of the components of this cellular pathway during viral replication is important to achieve a better understanding of how viruses make use of the cellular machinery in order to complete their infectious cycle.