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The virus has been shown to have a worldwide distribution and was observed primarily in the winter season in temperate climates. On the other hand, countries with extreme weather, like Canada, have also shown virus activity around January to March, although milder symptoms were reported. Interestingly, seasonal variations have been reported in China where infection with HCoV-NL63 appeared mainly in spring and summer. Also, a recent study of coronaviruses in Thailand did not show any seasonal predilection, while Wu et al. (2007) reported that the virus is detected during the autumn season in Taiwan. It is evident that the virus has no predilection to a particular season and is not affected by temperature variations as infections can occur throughout the year (Table 1).
Many groups have reported that the occurrence of co-infections with HCoV-NL63 and other respiratory viruses, including other human coronaviruses, influenza A virus, respiratory syncytial virus (RSV), parainfluenza virus and human metapneumovirus (hMPV), are common [26, 30, 46, 47, 51, 53, 54, 60, 61]. Also, co-infected patients are more likely to be hospitalized, indicating the severity of this kind of superinfection. In a study from Germany, RSV-A and HCoV-NL63 was the most common co-infection indentified in children less than three years of age. This is probably due to the high incidence of RSV-A in winter and the overlap in seasonality of the viruses. Also, in Italy, HCoV-NL63 circulates as a mixture of variant strains and is often associated with other viral infections. In South Africa, co-infection of patients with HCoV-NL63 and bocavirus in hospitalized children is reported. Nasopharyngeal and bronchoalveolar lavage samples from 341 patients were screened for common respiratory viruses, and the co-presence of HCoV-NL63 and bocavirus in at least one sample was reported.
Interestingly, the viral load of HCoV-NL63 is lower in co-infected patients than in patients infected with HCOV-NL63 only. There are various possible explanations for this phenomenon:
The high prevalence of co-infections of HCoV-NL63 and other respiratory viruses increases the chances of genetic recombination with these human or zoonotically transmitted viruses. In fact, Pryc et al. (2006) states HCoV-NL63 resulted from a recombination event between PEDV and an ancestral HCoV-NL63 strain. Theoretically, these types of recombination events could enable highly pathogenic virus variants to arise.
Since the identification of the first coronavirus – infectious bronchitis virus (IBV) isolated from birds – many coronaviruses have been discovered from such animals as bats, camels, cats, dogs, pigs, and whales. They may cause respiratory, enteric, hepatic, or neurologic diseases with different levels of severity in a variety of hosts, including humans. Coronaviruses have positive-sense single-stranded RNAs, their genomic size are 26 to 32 kilobases, the largest for an RNA virus. And the viruses themselves appear crown-shaped under electron microscopy. Coronaviruses belong to the subfamily Coronavirinae in the family Coronaviridae in the order Nidovirales. Coronavirinae is further divided into four genera, Alpha-, Beta-, Gamma-, and Deltacoronavirus, based on their phylogenetic relationships and genomic structures.
Coronaviruses occasionally jump across host barriers, often with lethal consequences. The alpha- and betacoronaviruses only infect mammals and usually cause respiratory illness in humans and gastroenteritis in animals. Gamma- and deltacoronaviruses mainly infect birds, and no human infection has been reported. Six coronaviruses known to infect humans are 229E, NL63 (genus Alpha-), OC43, HKU1, SARS-CoV, and MERS-CoV (Beta-), whereas only SARS- and MERS-CoV have caused large worldwide outbreaks with fatality, others usually cause mild upper-respiratory tract illnesses. A novel coronavirus was identified in a pneumonia patient in Wuhan on January 9 of this year represents the seventh human-infecting coronaviruses.
Severe acute respiratory syndrome (SARS, induced by SARS-CoV) first emerged in Guangdong province, China in 2002 and quickly spread around the world, with more than 8000 people infected and nearly 800 died. The MERS-CoV is a new member of Betacoronavirus and caused the first confirmed case of Middle East Respiratory Syndrome (MERS) in Saudi Arabia in 2012. Over 2000 MERS-related infections have been reported as of 2019 with a ∼34% fatality rate (https://www.who.int/).
Bats are notorious for carrying many emerging or re-emerging viruses. Epidemiological investigations have shown that almost all SARS patients have a history of animal exposure prior to the disease. SARS-CoV and anti-SARS-CoV antibodies were first found in the masked palm civet (Paguma larvata). However, coronaviruses related to human SARS-CoV were found in horseshoe bats (genus Rhinolophus) in 2005, pointing to a bat origin of SARS-CoV. This virus was named as SARS-related coronavirus (SARSr-CoV). Later, scientists reported two bat SARSr-CoVs could bind to both human and civet ACE2 receptors, suggesting that the Chinese horseshoe bats could serve as the natural reservoir of SARS-CoVs. Additionally a five-year surveillance found highly diverse SARSr-CoVs in bats in one cave of Yunnan province, China, and these viral strains in this location contain all genetic building blocks needed to form a human SARS-CoV, further supporting a direct bat origin for human SARS-CoVs.
Viruses isolated from MERS patients were found to have close contact with dromedary camels, suggesting the animal origin. Phylogenetic analysis indicated humans and camels were infected with the same source of MERS-CoV within a short time period. More than 10 bat species have been found to harbor MERS-related coronaviruses (MERSr-CoVs), but structural divergence in viral proteins (such as spike proteins) exists between bat MERSr-CoVs and human/camel MERS-CoVs, with MERSr-CoVs at the base of phylogenetic tree, suggesting MERS-CoVs likely to originate from bats.
It is worthy of notice that in 2016 swine acute diarrhea syndrome was found in Guangdong province, China, with a mortality up to 90% for piglets. The causative pathogen was identified as a new coronavirus, called swine acute diarrhea syndrome coronavirus (SADS-CoV), shared 95% genomic identity with bat alphacoronavirus HKU2, suggesting a bat spillover into pigs.
Respiratory diseases are quite a common problem in many collections of boid snakes. Viral agents like paramyxoviruses, arenaviruses and others are able to produce respiratory symptoms. However, in many collections, respiratory disease with high morbidity and mortality was found which was not caused by one of the well-known viruses. In the last years, with the discovery of snake nidoviruses the knowledge about pneumonia in boid snakes improved. These viruses were detected after different pythons succumbed to disease after a few months [4–6]. In our case, the first nidovirus detection occurred in a breeding stock of green tree pythons in which several animals showed severe respiratory signs, purulent stomatitis, poor or non-existing appetite, and weight loss. Mortality rates were high despite supportive treatment and care. Unbiased deep sequencing showed reads of a nidovirus and from two deceased animals full-length sequences could be assembled. These sequences are a little bit shorter than the other published full-length sequences of snakes, but belong still to the longest RNA genomes. The sequence identity to the other published genomes is rather low (< 66.9% on nucleotide sequence) with the highest similarity to the virus described in green tree pythons from Switzerland (Table 3), whereas the three sequences published in 2014 are more similar to each other. Nevertheless, all reptile nidoviruses cluster together within the genus Pregotovirus (Fig. 1). Besides the snake nidovirus, the metagenomics analysis showed reads of a snake retrovirus. This retrovirus could be found in control animals showing no signs of respiratory disease and it is probably an already known endogenous retrovirus without a link to pneumonia [1, 3, 31]. The bacterial findings were not consistent and were probably a matter of secondary infections. No evidence for other pathogens could be found. With a newly developed RT-qPCR different tissues from nine deceased green tree pythons were tested to further investigate the tissue tropism. Thereby, a connection between the degree of histological changes and viral RNA detection was indicated (Tables 1 & 4). The highest viral loads were detected in the lung, whereas the other tested organs showed inconsistent viral RNA amounts. This indicates the respiratory tract as primary location of virus replication, makes the transmission by respiratory secretions possible and further strengthens the usefulness of oral or tracheal swabs as in-vivo sampling method. We used the RT-qPCR for an initial screening for further snake nidovirus infected animals, including some animals deceased from other diseases or even apparently healthy (Table 5). To exclude unspecific amplification and laboratory contaminations, we generated partial sequences of the highly conserved ORF1B. Through this approach, 36 partial nidovirus sequences were obtained. Samples with very low viral loads did not result in a suitable sequence. Sequence comparison showed an identity between 99.89 and 79.4% indicating multiple virus strains. No direct relationship between collections, species or severity of disease is visible (Fig. 2). The host range of these viruses is not known and further virus strains not detectable by the used primer pairs could be possible.
Further screening of a total of 1554 animals resulted in 439 nidovirus RNA positive animals (Table 2). From 377 (for which information about the disease status was available) nidovirus RNA positive animals 285 showed no respiratory disease (Table 6). In addition to the species described in previous reports, we could prove the infection in Python brongersmai, Bothrochilus albertisii, Brothrochilus boa, Morelia boeleni, Aspidites melanocephalus as well as Papua pythons (Apodora papuana, data not shown, 2019) further expanding the viral host range. Approximately 31% of all tested pythons were positive. In spite of this, only one boa out of 128 animals revealed the presence of nidovirus genome. This is in concordance to one published study. Unfortunately, no material for sequencing was available from the infected boa. This roughly confirms the 27% positive pythons. At least in our study, the detection of viral RNA correlates not always with clinical signs.
Interestingly, in pythons originating from the asian continent, the prevalence of nidovirus was much higher than in other pythons, for example ball pythons (Africa). A total of 41% of the investigated Green Tree pythons were positive for the virus; in Carpet pythons 24% and in Ball pythons 22% were positive, respectively.
Hoon-Hanks et al. fulfilled the Koch’s postulates by experimental infection of ball pythons. Therefore, the detection of nidovirus RNA in apparently healthy individuals may reflect testing during the incubation period or a previous nidovirus infection, because some animals stayed positive in oral swab samples over several months (data not shown). Whether it is infectious virus, or rather a form of RNA persistence is unclear. Other animals from infected collections never turned positive, suggesting a non-airborne transmission. Co-infections or non-pathogenic causes like e.g. stress through newly purchased animals may play a crucial role in the development of clinical disease. No specific treatment is available, infected snakes should be isolated and the testing for nidovirus included in standard diagnostic workup.
Because of its frequent detection and the potential severe complications associated with CoV infection,, new diagnostic methods to rapidly identify these infections are needed. With the newly developed CoV antigen assay, we successfully monitored six CoV-positive patients. We showed that CoV infections are clinically diverse and, as also has been shown by earlier studies, cannot be diagnosed on the basis of clinical symptoms. Our results suggest that the assay could potentially identify patients in whom CoV is the real cause of the infection because it measures the virus itself, and the antigen level needed for detection is achieved only during the acute phase of the infection, as has also been the case with influenza. However, larger studies with more patients are needed to confirm these findings and to further determine the full diagnostic accuracy of the new assay.
The young age, the more severe illness episode and the long virus positivity time suggests that patient 6 probably had a primary infection. On the basis of patient age and data from seroprevalence studies, the other cases were likely secondary infections. Interestingly, patient 3 was diagnosed as CoV-OC43 positive again 20 months after the infection described above (data not shown), which confirms the widespread prevalence, the possibility of reinfection and the apparent lack of protecting immunity against the same subtype of CoV.
Monitoring the antigen concentrations suggested that virus load peaked around the third and fourth day after symptom onset, which confirms the findings in the experimental study by Adney et al.. Sampling should therefore be done within the first 4 days of symptom onset in order to ensure maximum sensitivity of antigen detection testing. The patient with a likely primary infection showed antigen positivity for 13 days, which is about 1 week longer than the positivity times in adults and what is usually observed for other viruses. Prompt testing and diagnosis maximize the potential to affect treatment decisions, such as prescribing virus-specific drugs, predicting the clinical course and withholding prescription of antibiotics. The new rapid test might therefore be a valuable contribution to patient care.
PHEV should be considered a major source of economic loss because of the high mortality on farms with high gilt replacement rates, specific pathogen-free animals, and gnotobiotic swine herds. Swine-breeding herds with low biosecurity or high pathogen loads may also be at risk of high piglet mortality because of PHEV. A better understanding of the mechanisms of viral infection and replication would assist in the development of better measures of prevention and treatment.
It has been proposed but not yet fully demonstrated that the transference of lactogenic passive immunity might protect piglets from PHEV infection during the first few weeks of life. Previous in vivo studies demonstrated that animals with high hemagglutination inhibition (HI) antibody titers were not susceptible to PHEV infection (55). Pigs develop a detectable circulating antibody response to PHEV between 7 and 10 days after exposure. The immune response against PHEV has been recently characterized in grow-finisher pigs under experimental conditions (75). In this study, the isotype-specific antibody responses in serum showed a strong IgM response at 7 days post-inoculation (DPI) that declined after 14 DPI. A strong IgA and IgG responses were detected by 10 DPI, peaked at 28 DPI, and declined gradually thereafter. Increasing levels of systemic INF-α (DPI 3), TNF-α (DPI 10-17), and IL-8 (DPI 14) were detected by multiplex microbead-based immunoassay (Luminex®) over the course of the infection. In addition, flow cytometry analysis revealed an increase in both monocytes (DPI 10) and cytotoxic T cell (DPI 21) populations in response to PHEV infection (75). The duration of PHEV-specific antibodies has not been determined under field conditions. Sows that were exposed to PHEV rapidly developed detectable levels of antibodies (55). The duration of anti-PHEV immunity is not a critical factor as piglets become resistant to PHEV infection with age. Neonatal pigs born from immune dams, previously exposed to PHEV, are fully protected by maternally-derived antibodies that persist until the age of 4–18 weeks (119). More recent field studies carried out in Argentina demonstrated the presence of antibodies in grower/finisher pigs, suggesting that colostral antibodies may persist for more than 6 weeks (67).
In rats, the intravenous or intraperitoneal administration of PHEV antiserum provided partial protection against PHEV infection, evidenced by the absence of viral detection in the brain and spinal cord and the absence of PHEV-related neurological clinical signs (120).
Coronaviruses (CoVs) are large, enveloped, single-stranded, positive-sense RNA viruses that belong to the Coronaviridae family. Although the first two human CoVs—CoV-229E and CoV-OC43—had already been discovered in the 1960s, no special attention was given to them because infections were primarily self-limiting and were only associated with symptoms of the mild common cold. Since 2000, several new CoV types have emerged. In 2003, the World Health Organization issued a global alert about a deadly new infectious disease, severe acute respiratory syndrome, which turned out to be caused by a CoV. In late 2004 a novel CoV, NL63, was isolated from two children with respiratory symptoms in the Netherlands, followed by the discovery of CoV-HKU1 in a patient with pneumonia. In 2012, the Middle East respiratory syndrome CoV was identified and was acknowledged to be one of the most dangerous respiratory viruses for humans,.
As a result, CoVs are increasingly recognized as important pathogens associated with a broad range of clinical diseases. Previous studies have reported CoV-OC43 to be the most prevalent CoV in many countries,. Virus isolation in cell culture and more recently molecular techniques, specifically PCR, have been the method of choice for diagnosing CoV infections, but they have several disadvantages,. Commercial PCR-based methods are often relatively expensive, they require technical expertise and the presence of viral RNA or DNA does not always reflect acute disease. Moreover, using PCR, CoVs are frequently codetected with other respiratory viruses, and the contribution of positive CoV PCR results to disease severity is not always clear,.
Despite the high morbidity and mortality associated with infections caused by some specific CoVs and the frequent detection of CoV in patients with respiratory infections, there is no rapid method available that can detect clinically relevant CoVs in humans. The aim of this study was to increase our insight in clinically relevant CoV infections by monitoring antigen concentrations in confirmed CoV patients using a newly developed assay for the rapid detection of CoV-OC43 infections.
An assay to detect species-specific CoV-OC43 nucleoprotein antigens was introduced to the mariPOC respi test in 2017. mariPOC (ArcDia Int. Ltd., Turku, Finland) is an automated and multianalyte antigen detection test system that enables rapid detection of acute infections,,. Besides the recently added CoV-OC43, the mariPOC respi test is able to detect nine respiratory viruses (influenza A and B viruses, respiratory syncytial virus, adenovirus, human metapneumovirus, parainfluenzavirus type 1–3, human bocavirus) and Streptococcus pneumoniae from one nasopharyngeal sample at the point of care. The new CoV antigen test has an analytical sensitivity of 2 ng/mL for OC43 recombinant antigen. The test cross-reacts with neither HKU1, NL63, and 229E nor with other common respiratory pathogens or microbiota. It is therefore unlikely to cross-react either with Middle East respiratory syndrome CoV or severe acute respiratory syndrome CoV. According to the manufacturers' specification, the clinical specificity of the test is 99.4% (n = 160) compared to PCR.
For this study, we used the semiquantitative property of the mariPOC analysis to obtain CoV antigen levels by extrapolation from a standard concentration curve. For verification of the results, samples were sent to two laboratories (Laboratory of Clinical Virology, Academic Medical Center (AMC), The Netherlands; and the National Institute for Health and Welfare (THL), Finland) for PCR testing with a multiplex RT-PCR and a CoV-species–specific RT-PCR, respectively.
For many years, FCoVs have been classified into different biotypes on the basis of their pathobiology. Avirulent strains, which usually induce mild or subclinical symptoms, are referred to as feline enteric coronavirus (FECV). Virulent strains cause feline infectious peritonitis and are called feline infectious peritonitis viruses (FIPV). Until 2005, CCoVs were considered to be mild enteropathogens. In 2005, a virulent variant causing systemic disease in pups and mortality was first recognized in Italy. This virulent biotype has been named canine pantropic coronavirus in reference to its systemic distribution in internal organs [39, 40]. Interestingly, ferret coronaviruses are also classified according to their virulence. The ferret enteric coronavirus (FRECV), which is widely distributed, causes an enteric disease called epizootic catarrhal enteritis, whose overall mortality rate is low. By contrast, the highly pathogenic ferret systemic coronavirus (FRSCV) induces FIP-like disease [42, 43].
Both feline genotypes may be responsible for mild enteric or FIP diseases. FIP remains a rare event, and only a minority of FCoV-infected cats (up to 10%) develop the illness [24, 44]. Two forms of FIP are recognized: the wet/effusive form with accumulation of a characteristic viscous yellow fluid in body cavities and the dry/noneffusive form with pyogranulomatous lesions affecting several organs. Both forms are progressive and ultimately fatal. FIP is often observed in young cats [47, 48]. In ferrets infected with FRSCV, the gross lesions resemble those described in cats with the dry form of FIP. Again, histologic lesions are characterized by severe pyogranulomas commonly observed in the mesentery and the peritoneal surface.
Both canine genotypes have been associated with enteric CCoV. By contrast, pantropic CCoVs identified so far all belong to the CCoV-IIa genetic cluster. Enteric CCoV infection does not prevent subsequent infection with the pantropic variant. Dogs seropositive for enteric CCoVs are still susceptible to pantropic viruses, but the clinical signs are moderate by comparison with those in seronegative dogs, probably owing to partial cross-protection induced by antibodies against enteric CCoV. During infection with the enteric CCoV, the virus remains restricted to the gastrointestinal tract. Conversely, the highly virulent pantropic CCoV is detected at high titres in lungs, spleen, liver, kidney, and brain. Clinical signs consist of fever, lethargy, haemorrhagic diarrhoea, severe lymphopenia, and neurological signs followed by death [39, 49]. The prevalence of the canine pantropic coronavirus is yet unknown, and further epidemiological studies are required to determine its distribution in dog populations. A pantropic strain (CB/05) has been successfully isolated from the lungs of a dead pup. CB/05 has subsequently been used to reproduce the disease experimentally, thereby improving understanding of this new illness. Infection with the CB/05 strain has demonstrated that disease outcome depends on the age at infection. Puppies over 6 months old may recover, whereas younger puppies (2-3 months) develop the most severe symptoms. Lymphopenia is one of the main features of pantropic CCoV infection under natural and experimental infections. While a transient reduction in T and B cell populations is observed during the first week after infection, the CD4+ T cell population remains depleted for 30 days postinfection, which could cause dysfunction of the immune system and favour opportunistic infections.
The results showed that 12 flocks (48.00%) were positive for IBV (Fig. 1). The nucleotide sequences of four isolates were submitted to the GenBank sequence database and were given the accession numbers IRIBVb: KP751243, IRIBVc: KP751244, IRIBVe: KP751245 and IRIBVf: KP751246. A phylogenetic tree (Fig. 2), based on the hypervariable region of S1 gene sequences of four IBV isolates from the present study and other strains of IBV retrieved from GenBank, was generated. Based on the Phylogenetic analysis, these four isolates were clustered with QX-like viruses. The results demonstrated the occurrence of QX-like serotype/genotype in Ahvaz, Iran. The IBV isolates were closely correlated to PCRLab/06/ 2012 (Iran), QX, HC9, HC10, CK/CH/GX/NN11-1, CK/CH/ JS/YC11-1, CK/CH/JS/2010/13, CK /CH/JS/2011/2 (China), QX/SGK-21, QX/SGK-11 (Iraq) with nucleotide homology up to 99.00%.
Avian infectious bronchitis virus (IBV) belongs to the genus Gammacoronavirus of the Coronaviridae family and is the etiologic agent of infectious bronchitis (IB), which is a major, highly complex infectious disease of poultry caused by multiple serotypes of IBV (1). IBV possesses a single-stranded positive-sense RNA genome (approximately 27.6 kb) encoding 4 structure proteins (phosphorylated nucleocapsid (N) protein, small envelope protein (E), integral membrane glycoprotein (M), and spike glycoprotein (S)) in the order of 5′-Pol-S-3a-3b-E-M-5a-5b-N-UTR-3′ (2). The S glycoprotein is cleaved into S1 and S2 subunits posttranslationally. S1 protein involves in infectivity, contains serotype-specific sequences, hemagglutinin activity, and virus neutralizing epitopes. The mutations, deletions, insertions, and recombination events that have been observed in multiple structural genes, especially in the S1 gene, of IBV isolates recovered from natural infections have been considered to contribute to the genetic diversity and evolution of IBV, and consequently, to the development of a number of IBV serotypes (3, 4). IB affects chickens of all ages, and IBV replicates primarily in the respiratory tract and in some epithelial cells of the kidney, gut and oviduct, resulting in reduced performance, reduced egg quality and quantity, increased susceptibility to infections with other pathogens, and condemnations at processing. IBV is a major poultry pathogen that is endemic worldwide and leads to serious economic losses (5, 6). IB has been reported in peafowl, teal, partridge, turkey, pheasant, racing pigeon and guinea fowl (7). Therefore, serological and molecular characterization of the field isolates of the IBV is highly important. IB was firstly described in North Dakota, USA, in 1930 (8). The first isolation of IBV in Iran was reported by Aghakhan et al. in 1994. The isolate showed the antigenic relationship to the mass serotype (9). IB is still a serious problem in Iran. Some newly emerging IBV isolates have recently been found. Backyard chicken is considered an important source of spread and persistence of different diseases (IB, Newcastle disease and avian influenza) among the chickens in poultry farms, playing a major role in the epidemiology of avian infectious diseases. Most household flocks are small and of mixed age and feed mainly by scavenging. Chickens from different households may mix, potentially exposing them to different diseases.
Moreover, no preventive and controlling strategy has been undertaken against IB in backyard chickens in Iran (10–12).
Our results show a nationwide distribution of nidoviruses in Germany with possible many existing strains. In total 439 of 1554 tested snakes were positive for nidovirus but only a few of them revealed clinical signs like stomatitis or severe respiratory disease. Therefore, no obvious correlation between virus and clinical disease could be established. Some of the positive results may be due to testing during the incubation period or samples may have been taken during reconvalescence of a nidovirus infection. Results indicate that a nidovirus infection in pythons may cause no to severe disease possibly depending on the snake species, immune status of the snake, pathogenic potential of the virus strain or other unknown factors. Our investigations show new aspects of a nidovirus infection in pythons and contribute to the understanding of the biology of snake nidoviruses.
In general, the APN receptor is used by alphacoronaviruses in a species-specific manner, that is, human APN is the cellular receptor for HCoV-229E, but not for the porcine coronaviruses, and conversely, porcine APN serves as a receptor for the porcine coronaviruses, but not for HCoV-229, FCoV, or CCoV [104, 105]. However, feline APN is a functional receptor for many alphacoronaviruses, including feline (FECV and FIPV), human (HCoV-229E), porcine (TGEV), and canine coronaviruses. Human, feline, and porcine APN show strong amino acid conservation and display about 78% identity. Yet, species-specific tropism is influenced by minor differences in certain regions of APN. Chimeras of mouse-feline APN were used by Tusell et al. to identify the three small, discontinuous regions in feline APN that are critical determinants for the host range of these coronaviruses. Amino acids (aa) 288 to 290 are essential for the entry of HCoV-229E, particularly the presence of an N-glycosylation sequon prevents virus infection. TGEV requires the region corresponding to aa 732 to 746 of feline APN, while FCoV and CCoV necessitate both aa 732 to 746 and aa 764 to 788 for entry. The entry of all of these viruses is blocked by the same monoclonal antibody directed against feline APN, suggesting that these three regions are closely link together in the three dimensional structure of feline APN. HCoV-229E, FCoV, TGEV, and CCoV probably evolved from the same ancestral alphacoronavirus, which may have infected cats using feline APN. The selection of mutations in the S protein may then have led to the appearance of viruses able to infect other host species by means of their cognate APN proteins, although all of them retained their capacity to use feline APN as a receptor in vitro.
Mazandaran province is one of the 31 provinces of Iran and is located along the Caspian Sea in Iran’s Region 3, just east of Gilan province, west of Golestan province, and north of Tehran and Semnan provinces (36.5656°N 53.0588°E). Mazandaran is a major producer of poultry, and poultry farmers in this region provide an important economic addition to the traditional dominance of agriculture. For serology, we collected 460 sera from backyard chickens (9 cities, Table 1) during October to December 2014; and for molecular detection and characterization, we collected cecal tonsils from 75 chickens.
Interest in the virome, or the entire population of viruses present in a biological sample, has increased recently due to improved availability of high throughput sequencing or next generation sequencing (NGS) technologies, and improved metagenomic analytical methods [1, 2]. The virome comprises all types of viruses, including those that infect prokaryotic and eukaryotic organisms, DNA or RNA viruses, and viruses that cause acute or chronic infections. Many of these viruses are difficult or impossible to propagate in cell culture, and molecular detection is difficult as no common gene such as the ribosomal 16S gene that is present in bacterial species exists in viruses. These limitations have hindered the identification and characterisation of uncultured viruses [3, 4]. Recently, due to the advent of molecular enrichment protocols, high throughput sequencing and new metagenomic analytical methods we are now able to explore, identify and characterise viruses from different biological and environmental samples with a greater capacity [2, 5–11]
In studies of human faeces, the virome has been shown to include viruses that infect eukaryotic organisms and viruses that infect prokaryotes (bacteriophages) [2, 5, 12–18]. Bacteriophages have been reported in many studies to be the most frequently detected viral constituent in the gut of humans [1, 2, 5, 8, 16, 19, 20]. The faecal virome has been characterised for several animal species including pigs, bats, cats, pigeons, horses and ferrets [2, 6, 7, 9–11, 21–31]. In dogs, the presence of enteric viral pathogens such as canine parvovirus, coronavirus, rotavirus and distemper virus (Paramyxoviridae) have been identified only through targeted studies [32–35]. To date, only one published study has used high throughput sequencing to investigate the faecal viral population in diarrhoeic dogs. These investigators analysed faeces from dogs with acute diarrhoea and detected two new virus species, canine sapovirus and canine kobuvirus; known canine enteric viruses such as canine coronavirus, canine parvovirus, canine rotavirus as well as plant and insect viruses were also reported.
The aim of this study was to describe the faecal virome of samples collected from healthy dogs, and compare these findings to the faecal virome of dogs with acute diarrhoea in Australia, using an Illumina MiSeq shotgun metagenomic sequencing approach.
Infectious bronchitis (IB) is an acute contagious viral infection with low mortality and a significant reduction in performance in chicken. The causative agent of IB is infectious bronchitis virus (IBV) which is an enveloped, single-stranded, positive sense and RNA virus belonging to the family Coronaviridae, the genus gamma Coronavirus. The virus consists of three important structural proteins: the nucleocapsid (N), the membrane (M), and the spike (S1 and S2) glycoproteins. The nucleotide sequence of the S1 gene is highly variable, which makes it prone to mutation and emergence of new variants of the virus. For this reason, the molecular classification of IBVs is based on the investigation of the S1 gene.1 Vaccination is one of the best ways of immunization of susceptible birds. However, vaccinated flocks may experience disease involvement because there is almost no cross-protection among serotypes.2 Consequently, the viral strains that exist in a different geographical area must first be identified and their pathogenicity should be determined in order to select an appropriate virus strain for vaccination.
In Iran, Aghakhan et al. identified IBV by virus isolation and serological techniques. This isolate belonged to the Mass serotype.3 In previous studies conducted to identify IBV serotypes in Iran, the presence of 4/91 and Massachusetts serotypes have been reported.4,5 Recently, a new isolate of IBV (IRFIBV32) was identified by Boroomand et al. which had 95.00% similarity to 793/B strains.5 The other IBV serotypes also exist in Iran neighboring countries such as Dutch strains in Pakistan.6
In Ahvaz (southwest Iran), IBV vaccines including H120 and 4/91 are used in the vaccination program in broiler chicken flocks. However, IB continues to be responsible for the economic losses of the poultry industry in the region. The purpose of this study was to evaluate the role of IBV in broiler chicken respiratory complexes in Ahvaz, and also to study the partial S1 sequences among field isolates of IBVs.
The factors regulating the course of the natural diseases caused by enteric CCoVs are not well understood. CCoVs are responsible for enteritis in dogs, and signs of infections may vary from mild to moderate, but they are more severe in young pups or in combination with other pathogens. Common signs include soft faeces or fluid diarrhoea, vomiting, dehydration, loss of appetite, and, occasionally, death. Dual infections by CCoV and canine parvovirus type 2 (CPV2) are especially severe when infections occur simultaneously, but CCoVs can also enhance the severity of a sequential CPV2 infection.
The natural route of transmission is faecal-oral, and virus in faeces is the major source of infection. In neonatal dogs, the virus appears to replicate primarily in the villus tips of the enterocytes of the small intestine causing a lytic infection followed by desquamation and shortening of the villi and resulting in diarrhoea 18–72 h post infection. Production of local IgAs restricts the spread of the virus within the intestine and arrests the progress of the infection. Therefore, infected dogs may shed virus for as long as 6 months after clinical signs have ceased [29, 38].
Recent extensive biomolecular analysis of faecal samples collected from infected dogs in Italy revealed that CCoVs infection is widespread and often characterized by the occurrence of both genotypes simultaneously [39, 40]. CCoVs type 1 and type 2 were found to be common in an Australian animal shelter with CCoV type 1 being prevalent. CCoVs have also been found in Western European dog populations. They have been detected in all European countries examined, and, except for the UK, the prevalence of CCoV type 1 was lower than for CCoV type 2. Reports of widespread CCoVs have come from Sweden and China. Soma et al. reported that CCoVs are also circulating in Japan, and the detection rate for dogs aged under 1 year was 66.3%, with a simultaneous detection rate of both types up to 40%.
These data raise several questions, and more indepth investigations into the pathobiology of CCoVs type 1 and type 2 are required. Therefore, failure to isolate CCoV type 1 in vitro hinders the acquisition of key information on the pathogenetic role of CCoV type 1 in dogs and prevents an authentic evaluation of the immunological characteristics of this new genotype.
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.
There is a new Coronavirus causing significant to severe respiratory infections in humans, causing human misery and death. This Coronavirus, SARS-CoV-2 is alarming health organisations around the world and has already caused significant social and economic losses to China and the Asian Pacific Region, and increasingly, the Globe. The virus portends to cause the loss of trillions of dollars through direct and indirect effects. This harmful viral-driven-economic damage, which has primarily hit Wuhan, Hubei Province, the economic industrial heartland of China, is compounding the effects of the recent economic slowdown in China. These socio-economic pressures alone would highlight the One-Health aspect of the new Coronavirus, but there are more fundamental reasons for calling this a One Health issue.
Members of the Coronavirus family (Coronaviridae) are quintessential One Health viruses. Many coronaviruses are serious animal health threats. Dr. Oskar Seifried, a veterinarian, provided the first description of a Coronavirus in 1931; a coronavirus known as Infectious Bronchitis virus (IBV) of Chickens. Today, based on the genetic analysis, we recognise IBV as a Gammacoronavirus which produces a highly contagious disease in chickens. IBV produces not only upper respiratory tract infection, but also affects the reproductive tract, and some strains can cause nephritis. Curiously, SARS-COV-2 is also producing renal problems in severely ill patients. It is relevant from a One Health perspective to note that different serotypes and genetic types of IBV are present worldwide and that there seems to be little, if any cross-protection from one serotype to the next. Also, the veterinary community has noted that new types of IBV continue to arise as a result of recombination events in the viral genome and from mutation, making IBV challenging to identify and extremely difficult to control, even though several IBV vaccines exist. Another serious Coronoavirus is the bat enteric coronavirus HKU2, identified in China, which causes severe piglet diarrhoea and mortality, and has led to serious impacts to the livestock industry.
Based on genetic analysis, we currently divide the Coronaviruses into four genera, the Alphacoronavirus, Betacoronavirus, Deltacoronavirus, and Gammacoronavirus. The new SARS-CoV-2 falls within the Betacoronavirus genus, based on sequence identity, and is reported to have a high sequence identity to a Bat betacoronavirus.
The diversity of coronaviruses reflects the facts that this family of viruses has an RNA-dependent RNA polymerase with poor fidelity, high frequency of RNA recombination, and (for RNA viruses) have unusually large genomes. Currently, only Alphacoronavirus and Betacoronavirus have demonstrated the ability to cause human diseases. Many Alphacoronavirus produce a variety of human respiratory diseases, though most cause the symptoms of the “common cold.” The deadliest of the known coronavirus diseases in humans, Severe Acute Respiratory Syndrome (SARS), Middle East respiratory syndrome (MERS), and COVID-19 (Coronavirus Infectious disease-19) are all Betacoronavirus.
In the fall of 2002, the US National Security Agency began hearing “chatter” regarding a new serious respiratory infection in the Guangdong province of China and by the winter of 2002–2003 an alarming new disease, SARS, was making headlines worldwide. it was not until 24 March 2003, that the US CDC and Hong Kong announced that they had isolated a new Coronavirus from a SARS patient, and the virus received its name, SARS-CoV. SARS, similar to SARS-CoV-2, originated in the Republic of China with evidence that while it most likely originated in bats, entered the human population through intermediate hosts, most likely the ‘Himalayan palm civet’ (Paguma larvata) and the raccoon dog (Nyctereutes procyonoides). Thanks to the use of a massive international effort headed by the World Health Organisation (WHO), of case identification, isolation (quarantine), treatment, and contact tracing the SARS outbreak ended only a few months (July 2003), after it began and there has not been a documented SARS infection since 2004. In total, there were approximately 8096 probably cases in 29 countries and 774 deaths from the SARS outbreak.
In June 2012, the second major human coronavirus, MERS-CoV revealed itself in a sputum sample from a 60-year-old Saudi man who died of overwhelming bilateral pneumonia and renal failure; this Betacoronavirus also has a close relationship with two bat-CoVs (HKU4 and HKU5), and camelids are thought to serve as the intermediates between infected vespertilionid bats and humans. Infection of dromedary camels with MERS-CoV appears to be common on the Arabian peninsula and parts of Africa, and there is documentation not only of camel-to human transmission, but also of human-to-camel transmission. Fortunately, Human-to-Human transmission of MERS-CoV is very limited except in hospital settings. Sporadic MERS infections continue to this day.
On December 31 of 2019, China revealed that there was a growing number of cases of a mysterious pneumonia in Wuhan City, Hubei Province. On January 7th 2002, Chinese authorities announced the detection of a novel human betacoronavirus, provisionally named 2019-nCoV by the WHO (and later renamed, SARS-CoV-2) as the agent responsible for the pneumonia outbreak in Wuhan. By 10 January a virus genome was released, and published and multiple other centres soon provided additional laboratory details on the new betacoronavirus. The on-going outbreak of SARS-CoV-2 has already caused far more infections than SARS or MERS and in a far shorter time, most likely because a significant percent of patients do not become seriously ill in a time-frame that would rapidly lead to their detection. Based on the current numbers for COVID-19 (death rate of 2–3 per 100) the new coronavirus appears less deadly than SARS (mortality ~ 1 in 10) or MERS (mortality ~ 3/10), but more deadly than seasonal flu (mortality ~ 0.5 to 1 per 1000).
While SARS-CoV-2 is spreading at alarming rates in China, there has not yet been sustained human-to-human transmission outside of China. Neither SARS-CoV nor MERS-CoV were characterised by sustained person to person transmission, but were characterised by large clusters with superspreading events. It is uncertain whether COVID-19 will vanish, similar to SARS, or become an established disease that follows seasonal patterns. It is still possible to contain this outbreak using vigorous early case detection, early isolation of suspected and confirmed cases, treatment of cases, contact tracing and social detention measures in China where there is risk of transmission is high. With each day, there is more data suggesting that the outbreak could break out as a pandemic and the fear is that sustained-human-to-human transmission in a low-to- middle-income country could lead to massive numbers of patients with acute respiratory disease and death. Multiple countries have experienced cases of COVID-19, many with severe illness including a few deaths outside of China, and nearly all countries are experiencing the social and economic costs of this new outbreak. However, so long as current public health measures are sustained, the general population outside of China are unlikely to be exposed to this virus at the current time. The goal is to block any potential chains of transmission, however, with so many unknowns, we cannot assure that these measures might not prevent the eventual establishment of a pandemic- in other words an ongoing, widespread transmission of the virus outside of China.
Given the sudden rapid superspreading event currently at play in China, it is imperative to learn as much as possible about this new coronavirus and to compare it to what we know about other human coronaviruses. One important area of research is determining the cell receptor for the new coronavirus. The previously recognised coronaviruses enter host cells by attaching their Spike (S) protein to a cellular receptor. Most Alphacoronaviruses use aminopeptidase-N (APN), but one Alphacoronavirus, NL6, uses Angiotensin-converting enzyme 2 (ACE2) as its receptor for entry into host cells. The SARS-CoV also uses the ACE2 receptor as its entry method into cells, this ACE2 is commonly present on cells of the respiratory and enteric tract. The MERS-CoV uses the host cell receptor dipeptidyl peptidase 4 (DPPR or CD26) a cell receptor that is similarly abundant in respiratory and enteric tracts. Recent evidence suggests SARS-CoV-2 uses ACE2 as its entry point; data that should speed the development of an effective drug and eventually a safe and effective vaccine.
One Health approaches attempt to strategize the coordinated efforts of multiple overlapping disciplines, including environmental surveillance and environmental health. Primary components of the approach lie in animal health and environmental aspects. At the time of writing, the host from which the SARS-CoV-2 entered the human population is unknown although the suspicion is that food markets are likely sources for the original spillover. While the search for the natural host highly implicates bats, search for the intermediary host, if any, is ongoing with the suggestions of the pangolin as a host far from certain. While it is premature to implicate any one particular urban source or natural host, the ensuing search will give insight into pathogens with potential to cross over into human transmission. This approach of environmental surveillance forms part of the PREDICT strategy for detecting viruses with potential for spillover into human.
The order Nidovirales encompasses a diverse group of viruses that includes significant veterinary and human pathogens (1–6). These viruses cause a variety of diseases that range from mild enteric infection to severe respiratory disease or hemorrhagic fever (7, 8). Examples of disease-causing nidoviruses include the severe acute respiratory syndrome (SARS) coronavirus, a number of other coronaviruses that cause typically mild respiratory disease in humans, and agriculturally important animal pathogens, such as equine arteritis virus, porcine reproductive and respiratory syndrome virus, and yellow head virus. Nidoviruses are characterized by their overall genome architecture, distinct pattern of gene expression, and presence of a conserved set of functional domains in their nonstructural polyproteins. The nidoviruses cluster into five major groups, which have been taxonomically categorized into four families: Arteriviridae, Roniviridae, Mesoniviridae, and Coronaviridae. Viruses in the Coronaviridae family (subfamilies Torovirinae and Coronavirinae) have the largest known RNA genomes, an attribute thought possible because of a virally encoded proofreading exonuclease (ExoN) that increases replication fidelity (9–12). Although nidoviruses are known to infect mammals, birds, fish, and crustaceans, no nonavian reptile nidovirus has been previously described.
Ball pythons (Python regius) have become one of the most popular types of reptiles sold and kept as pets (13). Native to West Africa, these snakes make popular pets because of their relatively modest size (≤1.5 m), docile behavior, and ease of care. Selective captive breeding has resulted in a tremendous variety of colors and patterns (morphs), many of which command high prices. Since the late 1990s, veterinarians have been aware of respiratory tract disease as a common syndrome affecting ball pythons. This syndrome is often characterized by pharyngitis, sinusitis, stomatitis, tracheitis, and a proliferative interstitial pneumonia. The clinical and epidemiological characteristics suggested an infectious etiology.
In this study, we investigated the pathology and etiology of this disease. We obtained case samples from 7 collections around the United States, performed necropsies, and collected multiple tissues for light microscopy and samples of lung for transmission electron microscopy (TEM). Although TEM of the lung suggested a viral etiology, traditional molecular diagnostic methods did not identify an agent. Metagenomic sequencing was used to identify and assemble the genome of a novel virus in the order Nidovirales. Here we describe clinical and pathological manifestations of this disease, ultrastructural findings, tissue tropism, disease association, and subgenomic RNA expression and analyze the genome of this virus in the context of related viruses.
Viruses belonging to the Coronaviridae family have a single stranded positive sense RNA genome of 26–31 kb. Members of this family include both human pathogens, such as severe acute respiratory syndrome virus (SARS-CoV)1, and animal pathogens, such as porcine epidemic diarrhea virus2. Currently, the International Committee on the Taxonomy of Viruses (ICTV) recognizes four genera in the Coronaviridae family: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. While the reservoirs of the Alphacoronavirus and Betacoronavirus genera are believed to be bats, the Gammacoronavirus and Deltacoronavirus genera have been shown to spread primarily through birds3. The first three species of the Deltacoronavirus genus were discovered in 20094 and recent work has vastly expanded the Deltacoronavirus genus, adding seven additional species3.
By contrast relatively few species within the Gammacoronavirus genus have been identified. There are currently two recognized species in the Gammacoronavirus genus: avian coronavirus (ACoV) and beluga whale coronavirus SW1 (SW1). ACoVs infect multiple avian hosts and include several important poultry pathogens, such as infectious bronchitis virus (IBV) and turkey coronavirus (TCoV)5. IBV was first described in the United States6 but has since been described around the globe7. Turkey Coronavirus is the cause of acute enteritis in domestic turkeys8. The second species in the Gammacornavirus genus SW1 was first discovered in beluga whales9 but has since been detected in other cetaceans, such as Indo-Pacific bottlenose dolphins10. Despite IBV being the first discovered coronavirus and the impact it has on the poultry industry11, the number of identified species within the Gammacoronavirus genus remains small in comparison to the other coronavirus genera. Coronaviruses from several other avian hosts for which partial sequences are available suggest relatedness to IBV and TCoV. These viruses, which include goose coronavirus (GCoV), were tentatively classified as part of the ACoV species. An approximately 3 kb region, including the nucleocapsid gene and several accessory genes, of GCoV were previously sequenced from a greylag goose in Norway12.
Here we present the full genome of Canada goose coronavirus (CGCoV) sequenced directly from the cloacal swab of a Canada goose, which expired in a mass die-off in a remote region near the arctic in Nunavut, Canada. Our analyses demonstrate that it should be classified as a novel species in the Gammacoronavirus genus.
Infectious bronchitis (IB) is a serious and highly contagious disease of chickens, accompanied by decreased egg production and poor egg quality in laying flocks. Avian infectious bronchitis virus (IBV) was first reported in the USA, replicating in the respiratory tract and some epithelial cells of gut, kidney, and oviduct. IBV commonly predisposed the birds to secondary infection with some bacterium, such as Escherichia coli and Mycoplasma gallisepticum, resulting in complicated disease process and increased mortality. The clinical disease and production problems frequently cause catastrophic economic losses to the poultry industry all over the world. IBV belongs to the genus Coronaviridae, family Coronaviridae, order Nidovirales, and possesses a single stranded positive-sense RNA genome encoding four structure proteins, phosphorylated nucleocapsid (N) protein, small envelope protein (E), integral membrane glycoprotein (M), and spike glycoprotein (S). The S glycoprotein on the outside of the virus contains epitopes associated with serotype differences, and is cleaved post-translationally by cellular proteases into the S1 and S2 subunits. The globular S1 subunit forms the tip of a spike, extending outward, plays a role in attachment and entry into the host cell, which has relation to induce virus neutralizing antibody and hemagglutination inhibition antibody, whereas the S2 subunit anchors the S1 moiety to the viral membrane. Coding for the heavily glycosylated spike glycoprotein, the error-prone nature of RNA polymerase made the S1 gene could easily generate nucleotide insertions, deletions, point mutations, and RNA recombination under vaccine pressure, to bring about new variation strain and change of tissue tropism. It is documented that only a few amino acid differences amongst S proteins are sufficient to have a detrimental impact on cross-protection. Antigenically different serotypes and newly emerged variants of field chicken flocks lead to vaccine breaks.
Recently, more than 20 serotypes within IBV have been identified worldwide. The complex epidemiology characterize of IB raised the control difficulty. In China, since IBV strains were first isolated and identified in 1982, various live-attenuated and inactivated vaccines derived from Massachusetts (Mass) serotype strains have been widely and extensively used in chicken farms to reduce the adverse effect of the IBV. However, the disease continues to emerge and cause serious production problems, even occurred in routinely vaccinated layer and breeder flocks in China, and the situation gets worse as time progressed.
It was documented that nephropathogenic type IB has become more and more prevalent in China. The unprecedented economic losses caused by the nephropathogenic IB suggested that selecting the appropriate vaccine strain against the IB outbreaks is of great importance. However, the integrated natures of novel circulating IBV strains in mainland China were not well-learned.
The previous study by other researchers has been revealed that the variation in S1 sequences was closely confirmed relative to the emergence of novel strains, and S1 gene sequence was a good predictor of challenge of immunity in chickens. This study was conducted to identify the IBV strains that have escaped immune defenses conferred by vaccination in China. The genetic characterization of recent IBV field isolates in China was performed by sequencing the whole S1 genes, sequence alignment and phylogenetic analysis compared with other reference strains.
Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded, positive-sense RNA virus in the genus Alphacoronavirinae of the family Coronaviridae. PED, which is caused by PEDV, is characterized by severe diarrhea, dehydration, and high mortality rates in the affected swine. PEDV variants belonging to genogroup 2 (G2) have been emerging in China in a large-scale outbreak characterized by approximately 80–100 % morbidity rates and high mortality rates among suckling piglets since late 2010. Highly virulent PEDV strains, phylogenetically related to the G2 Chinese PEDV variants (shared ≥99.5 % nt identity), suddenly emerged in the United States in May 2013 and rapidly spread throughout the country, causing severe economic losses [3, 4]. The second PEDV variant in the US, designated S INDEL PEDV, with insertions and deletions in the N terminal region (S1 subunit) of the spike (S) protein same as the G1 PEDV, was identified subsequently. The S INDEL strains caused reportedly milder disease in the field, indicating that the S gene contains major virulent determinants. The other large S-deletion PEDV strains were also identified, including one Korean field strain with a 204-aa deletion, and a US cell-adapted strain with a 197-aa deletion. Whether these S-deletion PEDV strains have distinct pathogenic characteristics, have not been described.
In July of 2013, fecal samples from diarrheal sows, collected in a pig farm in Jiangsu province of east China, were submitted to our lab for routine laboratory diagnostics. PEDV positivity was confirmed by reverse transcription PCR (RT-PCR) according to the method reported previously. One of the PEDV-positive samples, from a sow with very mild clinical sign, was used to inoculate into Vero cells to isolate the virus, as described previously. This PEDV strain, designated FL2013 strain, was successfully passaged and propagated, as characterized by typical PEDV-induced cytopathic effects (CPE), such as cell fusion and syncytia formation (data not shown), which was similar to what we observed for the CHGD-01 strains. The other field PEDV samples collected from pigs with severe clinical sign could not adapt to Vero cells. The complete genomic cDNA of the FL2013 strain was further determined using the 3rd cell culture passaging virus by amplification of twelve regions covering the PEDV genome as described previously. The sequences were assembled and analyzed using DNASTAR program and MEGA6.0 program.
The FL2013 PEDV genomic sequence had the size of 28,044 nt excluding the polyadenosine tail (GenBank accession no. KP765609). Twenty available PEDV sequences with complete genomes were used for multiple alignment and phylogenetic analysis (Table 1). The phylogenetic trees based upon either the complete genome (Fig. 1a) or S gene (Fig. 1b) showed that the FL2013 strain belongs to the genogroup G2b as proposed by Huang et al.. The S gene harbors two significant insertions at aa 58 to 61 (QGVN) and 142 (N), and a deletion of two aa (DI) between aa positions 167 and 168 at the N-terminus in comparison with the prototype CV777 strain (Fig. 2), which is the unique sequence signature of the virulent PEDV strains in the genogroup G2 [2–4]. However, the extreme C-terminus of the FL2013 S gene has a unique 21-nt deletion, leading to a 7-aa deletion (FEKVHVQ) in comparison with the other G2 PEDV sequences (Fig. 2). Interestingly, this unique deletion was also found in a G1 Korean strain SM98 (GenBank accession no. GU937797; Fig. 2). The complete S gene was sequenced in the original fecal sample, the 5th, 10th and 20th cell culture passages, respectively, and no sequence alterations were found, indicating that the 7-aa deletion was naturally present in the PEDV field strain and was stable in cell culture passaging. In addition, we did not find significant mutations on the other structural genes, non-structural genes and untranslated regions (UTR) potentially associated with attenuation.
Since the FL2013 strain was isolated from a sow with very mild clinical sign, we were interested to determine its virulence by experimental infection in newborn piglets in comparison with the virulent G2 strain. Thirty-six 3-day-old piglets, negative for PEDV RNA were assigned into three groups with 12 in each. Piglets in each group were housed with their mothers (PEDV RNA and antibody negative) with no artificial supply of colostrum and milk. Group A serves as negative control, whereas piglets in groups B and C were challenged orally with the FL2013 strain and the virulent CHGD-01 strain at 1.0 × 105 50 % tissue culture infectious doses (TCID50)/3 mL, respectively. Two piglets from each group were euthanized and necropsied at 3 days post-inoculation (dpi) for histopathologic examinations and immunohistochemistry staining according to Stevenson et al.. The remaining 10 piglets in each group were examined monitored for clinical signs and recorded to evaluate survival rate for 25 dpi. The result showed that Group-B piglets, together with the negative control group-A piglets did not show clinical sign, and all 10 remaining piglets in each group survived during the observation period (Fig. 3). Group-C piglets had PED signs characterized by acute vomiting and watery diarrhea, and immunohistochemistry staining showed abundant viral antigen in the severely atrophic villi (Fig. 3). In contrast, less PEDV positive staining was found in the intestinal villi of group-B samples, and the villi was only slightly damaged (Fig. 3). 10 of 10 remaining piglets in group C died within 7 dpi. The comparative pathology study indicated that the FL2013 strain had reduced virulence to newborn piglets.
In summary, we identified an apparently attenuated G2 PEDV field strain with 7-aa deletion at the 3′-end of the S gene. Genetic characterizations of various PEDV field strains are of importance for demonstration of PEDV outbreaks and development of efficacy vaccines. Findings from present study reveals that variant PEDV strains circulating in swine-producing areas in China, and provides new molecular epidemiological data for a better understanding of this disease. It is highly possible that the sequence deletions (especially the 7-aa deletion), and other unknown mutations found in the variant strain FL2013 might have contributed to the reduced severity of the clinical disease in the piglets. Whether the changes in the genomic sequence, insertions and deletion, could collectively alter the efficiency of viral replication and RNA synthesis in the FL2013 strain leading to reduced pathogenicity will be investigated in further. More studies will be conducted to test this hypothesis. In addition, the FL2013 isolate may serve as a potential vaccine candidate that could protect the Chinese piglets from the infection caused by the virulent strain of PEDV.
The median probability (PContBatch) that a batch of blood collected at the abattoir abroad is contaminated with PEDV, due to the slaughtering of at least one infected pig, was 6.0 % (90 % Prediction Interval: 0.3 %; 22.6 %).