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No data is available regarding the pathogenesis of PCV-3 infection. The lack of virus isolation has impeded the establishment of an infection model to date. It is known that PCV-3 can be found in different tissues of domestic pig and wild boar (86, 87, 95), indicating the systemic nature of the infection. However, the point of viral entry, primary replication, organic distribution and persistence are still unsolved issues. PCV-3 has been found in feces, nasal swabs, oral fluids, and trucks transporting pigs (82, 85, 95), which allows speculating that horizontal transmission through direct contact is probably an important route. Detection of viral genome in fetuses and stillborn from farms with history of reproductive failure (21, 70, 75), as well as in semen and colostrum, points out also to vertical transmission as another likely route. Definitively, more studies are needed to ascertain the potential excretion routes of this virus.
Co-infection of PCV-3 with both PCV-2 and PRRSV has been reported (70, 78, 91, 92, 94). In fact, this was expected since both well-known pathogens are widespread in the pig population (109–111). Noteworthy, it is known that both PCV-2 and PRRSV are able to affect the immune system and, therefore, co-infections with these viruses are not unusual (112, 113). Other pathogens were also detected in PCV-3 PCR positive samples (78, 114). Very recently, PCV-3 has been found by NGS approach in pigs affected by periweaning failure-to-thrive syndrome in co-infection with PPV and Ungulate bocaparvovirus 2 (100). Since experimental and field studies demonstrated that co-infection with PPV increase the effect of PCV-2 in causing PCV-2-SD (115), at this point it cannot be ruled out that a similar effect may occur with PCV-3. Further investigations are needed to determine whether PCV-3 might act as a secondary agent up-regulating its replication once pigs are immunosuppressed or immunomodulated, or whether the frequency of co-infection is independent of the immune system affection.
In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis. Aichi virus infection has been shown to elicit an immune response. Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus (0.5% and 1.8%) was too low to find a significant association with diarrhea. In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea. Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis. Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals. Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected. However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis. Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature. This high sequence diversity has also been observed within particular outbreaks of gastroenteritis, limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified.
In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster. For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found. In addition an antibody response was observed against some but not all novel astrovirus types. Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis, suggesting that astrovirus infection may not be limited to the gastrointestinal tract.
Sun et al. analyze the phylogeny and genome compositions of 17 novel Seneca virus (SVA) isolated in China in 2017 and compare them with the genomic sequences deposited in the GenBank. SVA is a single stranded positive-sense RNA virus associated with porcine idiopathic vesicular disease (PIVD), and sudden neonatal dead reported in six countries in Asia and America. The isolated strain clustered into three distinct groups: A, B, and C, not related with the previously SVA identified in China and different from SVA identified in other countries. More effort should be directed to SVA monitoring, rapid and specific diagnosis and vaccination strategies.
Perri et al. studied the estimation of time to eliminate porcine epidemic diarrhea coronavirus (PED-CoV) in Ontario herds based in large-scale disease control program database (DCP). The analysis takes into consideration the time between the initial infection, and the confirmation of PED-CoV freedom at the minimum level of 10%. The median time to elimination varied from 23 weeks in nursery herds, to 43 weeks in farrow-to-feeder herds. Farrow-to-wean herds had the highest hazard of PED-CoV elimination. Type of herds, season and year of original diagnosis were associated with the time of negativity and reflect the complexity of the infection control practices.
Krog et al. work to determine the dynamics of infection of hepatitis E virus (HEV) by carrying out a longitudinal serological and RT-PCR fecal studies. Sows and their progeny from 2 weeks to slaughter were sampled. Antibodies were only detected in offspring born from sows with high levels of maternal antibodies (MAbs) and a few of them became shedders. All pigs seroconverted at 13–17-week-olds. By PCR 65.5% of pigs were positive at least one time during the weeks 13, 15, and 17. In 3 out of 10 slaughter pigs, HEV was detected in feces and organs. As MAbs reduced the shedder of HEV, sow's vaccination might be an option.
As a summary, this Research Topic provides a comprehensive review about the results of the combination of NGS-ISH for the diagnosis of known and unknown emerging pathogens. It's also an important discussion of potentially emerging viruses such as PCV-3, torovirus, atypical pestivirus, reemerging viruses such as PHECoV and transboundary viruses such as classical swine fever. The mechanisms used by PRRS to circumvent the host's innate immune and vaccine immune response are updated along with the development of vaccines to exosomes. The immune response against enteric coronaviruses such as PED and PDCoV and innovative vaccines for both viruses are analyzed. The role of pigs as an amplifier of the Nipah virus is reviewed, as well as the importance of vaccination to pigs for the prevention of this infection in man. The repeated transmission of human seasonal viruses to pigs has resulted in the establishment of several human-origin virus lineages globally and the failure of the current pig vaccines. A research study indicated that isolated strain Seneca virus from vesicular fluid of sows, clustered into three distinct groups, A, B, and C not related with the previously SVA identified in China, and highlight that different genotypes of SVA co-exist and spread. A study of the estimation of time to porcine epidemic diarrhea coronavirus (PED-CoV) elimination in Ontario Type of herds was carried out. The year and season of original diagnoses are associated with the time of negativity and reflect the complexity of the infection control practices. Finally, a study shows that hepatitis E virus infection is widespread in the herd, and pigs spread virus during the final stages of life, and there's a strong chance to find infected pigs at slaughter. As maternal antibodies reduce, the shedder of virus vaccinating sows might be an option.
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.
Influenza A, B and C viruses are members of the Orthomyxoviridae family that can cause influenza in humans. Influenza A viruses exist in humans, various other mammal species, and birds; migratory or domestic waterfowl are their largest reservoir. Humans are thought to be the primary hosts and reservoir of influenza B and C viruses, although both have been identified in other hosts after reverse zoonotic transmission from humans. While influenza B virus is a common seasonal human pathogen similar to influenza A virus in its clinical presentation, influenza C virus causes primarily upper respiratory tract infections in children. Clinical manifestations (cough, fever, and malaise) are typically mild, but infants are susceptible to serious lower respiratory tract infections. Influenza C viruses co-circulate with influenza A and B viruses and causes local epidemics,. Six genetic and antigenic lineages of influenza C viruses have been described, and as in influenza B viruses, are considered monsubtypic,. Co-circulation of multiple subtypes of influenza allows for rapid viral evolution through the process of antigenic shift, a property previously only shown for influenza A viruses. Thus, both influenza B and C viruses do not have pandemic potential. In contrast, the Influenza A genus includes 17 hemagglutinin and 9 neuraminidase subtypes, and reassortment among different subtypes has repeatedly generated pandemic viruses to which the human population is naïve–. It is the animal reservoirs of diverse influenza A viruses that give them the unique property within orthomyxoviruses of causing human pandemics.
Aside from humans, influenza C virus has been isolated only from swine in China (in 1981). Genetic analysis showed a close relation between Japanese human and Chinese swine influenza C isolates,. Serological surveys in Japan and the United Kingdom found 9.9% and 19% of swine, respectively, to have positive HI antibody titers to human influenza C viruses, suggesting that the virus is not uncommon in swine,. Swine inoculated with influenza C virus had mild respiratory disease and transmitted the virus to naive swine by direct contact. Here we characterize an orthomyxovirus isolated from a clinically ill pig and show that the virus is distantly related to human influenza C virus and readily infects and is transmissible in both ferrets and pigs. Genetic and antigenic analysis suggest that this virus represents a new subtype of influenza C virus, raising the possibility of reassortment and antigenic shift as mechanisms for influenza C virus evolution which could pose a potential threat to human health.
Another virus that we occasionally found in diarrheic post-weaned piglets, mostly associated with rotavirus, is Porcine Torovirus (PToV). The toroviruses are positive-stranded RNA, enveloped viruses, belonging to the family Coronaviridae. They have a typical morphology (discoid, kidney—and rod-shaped) and cause a serious, at times fatal, diarrheal disease in horses (Berna virus; BEV), cattle (Breda virus; BRV), gastroenteritis in humans and enteric infections in swine.
After the first detection in 1990, using ns-EM, of torovirus particles in association with RVs in the feces of piglets with diarrhea (Figure 3A), a convalescent serum was taken and its specificity was assessed by the immunoaggregation of particles molecularly characterized as PToV and by serological comparisons with both field sera and anti-PToV, as well as anti-BEV hyperimmune sera. The convalescent serum 233/90 has since then been systematically used in IAEM in suspected cases, and in this manner six more cases of PToV were identified using IAEM: one each in 1996 and 1999, and four in 2002, mostly in association with RVs and other enteric viruses (Figure 3B–E). Particles identified in these cases were further characterized by molecular methods and one sample from 2002 was defined as a new PToV strain.
Considering the low frequency of PToV in diarrheic pigs, pre-selection by IAEM or pre-screening by diagnostic RT-PCR of ToV-positive fecal specimens could allow detailed studies of the genetic diversity among ToVs.
Resende et al. provide comprehensive information about the results of the combination of NGS-ISH for the diagnosis of known and unknown emerging pathogens in tissues by NGS, and its relationship with specific lesions where it is visualized in active infection through detection of mRNA by ISH. Results on the application of PCV-2, PPV-2, Seneca virus, and Mycoplasma hyorhinis are comments.
Mora-Díaz et al. review the disease produced by porcine hemagglutinating encephalomyelitis coronavirus (PHE-CoV), a neurotropic virus affecting piglets <4 weeks old. Subjects such as: characteristic of the virus, history of the emergence of PHE-CoV, global distribution, clinical signs, pathogenesis, lesions, and diagnosis are discussed. As the infection is endemic in most swine herds, and no current vaccines are available, early exposure to old or young sows to induced maternal immunity is the only way to prevent the disease.
Klaumann et al. discuss the current knowledge on a new circovirus named porcine circovirus type 3 (PCV-3). Originally this was identified by metagenomic analyses from an outbreak of PDNS in sows associated with reproductive failure, myocarditis, and multisystemic inflammation. Thereafter it was found associated with respiratory, digestive and nervous signs in healthy pigs and wild boars. Retrospective studies detected PCV-3 as early as the 1990s. Coinfection with several virus and bacteria were reported. The authors emphasize the need of studies related to pathogenesis, the role of coinfections and their association or not with certain clinically pathological entities.
Zhou reviews classical swine fever (CSF) in China. The author discusses the epidemiology, and the geographical distribution of genotypes where 2.1, 2.1b, and 2.1c are currently dominant in China. The first one persists in an immune population by natural infection due to the high mutation rate of the enveloped glycoprotein E2. For eradication of CSF it is necessary to distinguish between the naturally infected and vaccinated animals by live attenuated marked vaccine. An experimental E2 subunit vaccine was developed in China. Besides preventive vaccination, we need culling strategies, skilled veterinarians, up-to-date diagnostic and monitoring technology, and biosecurity.
Hu et al. review the progress in the knowledge on porcine torovirus (PToV) a single-stranded RNA enteric virus found in piglets with diarrhea in North America, Africa, Asia, and Europe. The authors describe the virus morphology, the genomic structure and genotypes division, although chimeric strains with genes from porcine and bovine ToV has been identified as well as recombination with enterovirus. For epidemiological studies an indirect ELISA based in recombinant N protein expressed in baculovirus is available. Other methods included RT-PCR; qRT-PCR; and nested PCR. Prevalence of PToV is quite variable according with the country.
Montaner-Tarbes et al. analyze numerous gaps in PRSSv knowledge. Related with the biology, the scarce whole genome sequencing from different geographical origins hinder understanding the virus evolution/mutation. The function and the complex interaction of viral non-structural proteins with the target cells are reviewed. The mechanisms of the virus to avoid the innate and acquired immune response through recognition and antibody neutralization are reviewed. The new known mechanisms of dissemination mediated by cell to cell connected nanotubules and extracellular vesicles are thoroughly discussed. Later on, the development of exosomes, as a novel vaccine is analyzed.
Rajao et al. review the role of pigs in the interspecies transmission and how their susceptibility to different viruses can affect the overall epidemiology of swine influenza. The factors that have been implicated in the interspecies transmission of influenza such as receptor-binding specificity/affinity, balance between HA and NA content, host temperature and host-specific immune factors are analyzed. Surveillance of IAV in swine has shown that human viruses are transmitted to pigs more frequently than from pigs to humans. The result is the establishment of several human-origin virus lineages, antigenic diversity and failure of current swine vaccines.
McLean and Graham provide an update about Nipah virus (NiV), an RNA paramyxovirus that causes a severe neurological disease in humans. In suckling pigs, NiV infection causes high mortality and in older pigs, respiratory and neurological signs. The natural host of NiV is a fruit bat of the genus Pteropus, and pigs act as an “amplifying host”. The disease has been found in Asia in people with close contact to pigs. Recombinant NiV mutant, attenuated and subunit vaccine using several viral vectors have been studied. Currently, neither a human nor a pig vaccine has been licensed.
Gatto et al. review the information around a new RNA Pestivirus named atypical porcine pestivirus (APPV), detected in pigs with congenital tremors (CT) type AII, and splayed legs in offspring from sows by NGS technology. Viral genomes were detected in semen, preputial swabs and fluids highlighting the importance of AI in APPV epidemiology. Horizontal transmission can be made by surviving CT in healthy new-born boars, piglets and adult pigs. The virus exhibits high genetic diversity. Recently, APPV was detected in wild boars. Until the development of a vaccine, the authors recommend feedback on reproductive management in sows with CT cases.
Koonpaew et al. review the emergence of highly pathogenic porcine epidemic diarrhea (PED) and porcine delta coronavirus (PDCoV) as agents of watery diarrhea in suckling piglets. The authors describe aspects related to the biology pathogenesis and the host innate immune response of gastrointestinal tracts against those enteric coronaviruses. The agents evade recognition by host pattern recognition receptors (PRRs), present in resident antigen present cells (APCs) and located in gut associated lymphoid tissue (GALT), through the inhibition or blocking of interferon (IFN) induction and the signaling cascade, respectively. This knowledge will profit the development of immune modulators as well as effective vaccines.
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.
The data set supporting the results of this article is included within the article.
As reported before (Table 1) one of the viruses associated with PoRV is PCV2 (Figure 2A). PCV2 was initially isolated in 1991, was later associated with the Post-weaning Multisystemic Wasting Syndrome (PMWS), and it is still present with a high prevalence in commercial pig farms.
The small size (18–20 nm) of circovirus makes them difficult to recognize, especially when present as single dispersed particles in the background fecal material, and thus, they are more clearly observed in the intestinal contents of piglets when precipitated in aggregates by IAEM. PCV2 has an immuno-suppressive effect and, thus, it is often detected in association with other viral and bacterial agents. Thus, the use of IAEM with convalescent sera is useful for more readily identifying multiple viral infections in which PCV2 is associated with other viruses. In fact, in one study we used IAEM with anti-PCV2 convalescent sera to examine 101 animals taken from 29 industrial farms where outbreaks of PMWS were reported or lesions typical of PMWS were regularly shown at necropsy. Prior to being used in the IAEM method, the convalescent serum used was determined to be positive (titer 1:10,000) for anti-PCRV2 antibodies by testing in an in-home MAb-based competitive serological ELISA test. Forty-five samples tested positive for PCV2, and it was frequently observed in association with one or more other viruses (Table 2). Examples of the association of circovirus particles with other virions, and particularly with rotavirus and enterovirus-like virus particles, are respectively reported in Figure 2A,B.
We then compared the results obtained with IAEM and two others virological methods, immunofluorescence (IF) and PCR, in the identification of PCV2 in the organs of clinically affected pigs sampled during outbreaks of PMWS. Examinations tested different organs, such as lymph nodes and lungs using IF and PCR, and intestinal tracts using IAEM. The results confirmed the lower sensitivity of IAEM compared with PCR (concordance 44.1%) but not compared with IF (concordance 64.7%). By performing a single IAEM test, different associating particles, such as parvovirus-like, enterovirus-like, rotaviruses and PEDV, were simultaneously observed, thus more data were acquired on the interactions between PCV2 and other pathogens potentially involved in the pathogenesis of PMWS syndrome. Indeed, a higher level of PCV2 particles were detected in the caecum in comparison with the small intestine, and in several occasions only the caecum contained circovirus particles.
While previous versions of RPM have been validated and showed excellent correlation with the reference assays, more studies are needed to develop and validate novel and improved assays using this technology. More specific amplification strategy and protocols and the design of primers for the amplification of novel targets are needed to allow more widespread clinical adoption of this new tool.
Broad-range detection microarrays are used for the direct detection of microbes in complex samples and therefore require nucleic acid amplification to bring the target concentrations to detectable levels. In this study, a targeted multiplex PCR based on temperature-switch PCR (TSP) amplification technology was adopted and a commonly used multiplex PCR reagent was used, which increased assay sensitivity compared to conventional amplification strategy in the case of samples with heavy backgrounds (e.g., human and bacterial genome). The biphasic PCR parameters of the TSP allows a multiplex PCR to be performed under standardized PCR conditions without extensive optimization of each individual PCR assay. Unique buffer in the multiplex kit promotes stable and efficient primer annealing by preventing nonspecific primers from annealing to the template and increasing the local concentration of primers at the template and allows efficient extension. This improvement therefore made it easy to group the pathogens into subsets for efficient amplification without much optimization, which allowed stable performance when new primers were added into primer mix or when the primers were changed.
The combination of a targeted multiplex PCR and microarray hybridization should allow good analytical sensitivity in the case of samples with heavy backgrounds. The ability of differentiating virus types or subtypes relies on the sequences obtained from the targeted amplicons hybridization to the detector tiles. Thus, although a completely novel respiratory pathogen would not be captured by this approach, a mutant virus or an uncommon pathogen could be captured. Another benefit of association of targeted multiplex PCR and resequencing microarray is the simultaneous detection and characterization of several viruses, which allows a significant time-savings compared to conventional assays or sequencing, and could be an important element in improving emergency response capacity.
Another improvement of RPM-IVDC1 versus standard detection tools is the more comprehensive coverage of target organisms it allows. In contrast to RPM-Flu3.1, about 30% of which is dedicated to targeting all 16 HA and 9 NA alleles of avian influenza A viruses, the new resequencing array design of RPM-IVDC1 allows more coverage of respiratory viral pathogens. The two main changes of the new microarray design compared to RPM-Flu3.1 were the length of the detector tiles and the primers selected for amplification of the chosen targets. The shortening of detector tiles allows an increase in the number of probe sets to be placed on the microarray, and therefore almost all common respiratory viral pathogens could be detected by RPM-IVDC1 in a single assay. Some atypical respiratory pathogens (e.g., measles virus, rubella virus and influenza C virus) and non-respiratory virus (e.g., HHV) could be detected by RPM-IVDC1, but the probes corresponding to these are not present in RPM-Flu3.1. Furthermore, the new technology, by using one or more pairs of primers designed for amplification of conserved regions, allows rapid and efficient strain-level identification for some pathogens, which may be critical in outbreak investigations.
In addition to improvement of the amplification method and primer design, new internal controls were used in RPM-IVDC1. Two Arabidopsis thaliana genes, NAC1 and TIM, were selected as internal controls as these plant genes would be unlikely to occur naturally in clinical samples. Comparing with RPM-Flu3.1, new overlapped detector tiles for these controls were tiled on the new microarray and new primers were designed to amplify shorter detector sequences, which could improve the hybridization efficiency. The results showed the base call rates of TIM and NAC1 could reach over 90% without interference with the detection of microbial agents. As a result of new internal controls, an unqualified chip without any base called may be found and thus avoiding false-negative results.
The number of targets that needs to be validated depends on the intended purpose of the study. Control strains of febrile respiratory illness (FRI)-causing pathogens and different reference strains of influenza A virus were used to assess the performance of RPM v.1 and RPM-Flu3.1, respectively. In this study, sixteen single-virus infected reference specimens were used to evaluate the new microarray. The results showed excellent specificity of the new microarray for type- or strain-level identification of some reference samples. As the new resequencing microarray was designed to be smaller than previous versions to increase the hybridization efficiency, the lengths of the detector tiles of RPM-IVDC1 were much shorter and the numbers of the tiles were relatively limited. As a consequence, the conserved gene sequences obtained from detector tiles of some pathogens could differentiate samples at a strain level for only a few viruses (hMPV, CoV-OC43, RSVA, RSVB, PIV1, PIV3). In addition, some viruses have many serotypes, thus for these, the identification could only be achieved at species level (AdV, RV).
The results of RPM-IVDC1 showed excellent correlation with the reference assay for the detection of common respiratory pathogens with only one false-negative of RSVA. But since one additional occurrence of PIV2 infection, two AdVs, sixteen RVs, three FluAs and two CoV-229Es were detected by the new assay, the increased sensitivity of the assay compared with reference method is likely attributable to the use of multiple conserved primer pairs targeting specific pathogens and the high efficiency of the assay. This was particularly noticeable in the RV identification, where only 5 of the 22 RPM-identified RV infections were detected by the reference assay. The use of twelve RV type-specific primer pairs in the RPM primer mix compared to only one pair in the reference assay likely enables improved detection of RV.
The detection rate of common viruses was consistent with previous research. The most commonly detected virus in children aged under 5 years was RSV; 4 out of the 5 detected RSVs was type A. The most common etiologic agent in the adult group was RV, of which 21 out of 22 infections (95.5%) clustered within type A. In an analysis of the 68 detection-positive patients with complete clinical data, no significant seasonal predominance was observed for the total numbers of virus detected compared with previous study. Neither the obvious difference of detection rate was observed among the groups with various disease severities due to the broad detection extent of the aetiological or anetiological virus and the small sample size.
As a result of the broad range of respiratory viruses identifiable with this assay, some atypical pathogens were detected using the new microarray. One instance of measles virus, one rubella virus and one influenza type C infection were found in patients aged 13 years, 13 years and 1 month, respectively. Rubella virus is the pathogenic agent of rubella, which is a common childhood infection but could affect anyone of any age, with the possible complications of fatal pneumonia. As the causative agent of measles, the presence of the measles virus may also lead to serious problems, including measles pneumonia, which is a life-threatening complication in children. Influenza C virus, which is sometimes present in patients with a clinical diagnosis of pneumonia, could cause a variety of respiratory illnesses that cannot be clinically differentiated from those caused by other viruses. HHV is ubiquitous in humans, causing lifelong infection. After primary infection, which usually occurs during childhood, the virus remains latent in the ganglia of sensory neurons. Systemic stimuli including fever due to bacterial or viral infection can cause reaction of the virus and potentially clinical disease, resulting in active secretion in the throat. Although there are few reports of symptomatic pulmonary involvement directly attributable to HHV infection, the presence of HHV in the throat is still a highly significant and independent risk factor for the development of lower respiratory tract infections with HHV. Sequences of HHV-1 through HHV-6 were tiled on RPM-IVDC1 and 5 instances of HHV-1, 7 HHV-4 (EBV), 1 HHV-5 (CMV), 4 HHV-6 infections were detected using the RPM method, which was subsequently confirmed by PCR and sequencing. Of the 15 patients infected with HHV, 2 were co-infected with HHV-6 and HHV-4 and another one was co-infected with HHV-1 and HHV-4. HHV-6 virus, which causes roseola and fever after the primary infection, was identified in three children with ages under 5 and one aged 8. All HHV-1-positive samples and 5 (71.4%) of 7 HHV-4-positive specimens were from adult patients; the frequency of HHV in the NPAs (13%) of adult patients were lower than found in a previous study (22%), which might be due to the difference between the sample characteristics. Although it is not possible in a retrospective study, and without the concomitant detection of bacterial pathogens, to state that these atypical pathogens were causative agents, the results of this study show the capability of RPM-IVDC1 for the detection of atypical pathogens and the potential for diagnosing infectious syndromes manifesting by similar symptoms but caused by diverse etiological agents.
The accurate identification of multiple respiratory tract viral pathogens using RPM-IVDC1 in this study demonstrates that this novel assay could improve the response capacity to epidemic outbreak and be significant tool for public health. However, the false-negative detection for RSVA should be addressed prior to clinical adoption. The capability of this assay for the simultaneous detection of a broad-spectrum respiratory tract viruses should also be further validated when considering the application of this new RPM towards outbreak investigations or epidemic surveillance.
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.
In this study, C. pecorum was isolated from specimens obtained from 2
calves with diarrhea. In case 1, the bacterium was isolated from jejunum, and C.
pecorum specific omp1 genes were detected from several locations
in the intestines. Additionally, C. pecorum antigens were observed in the
jejunal villi by immunohistochemical staining. Necrotizing enterocolitis due to
Clostridium perfringens infection was also observed. Therefore, we
speculated that C. pecorum might exacerbate the disease caused by
In these 2 cases, it is possible that C. pecorum was either the primary
pathogen or an exacerbating factor causing diarrhea. On the other hand, it is thought that
C. pecorum causes asymptomatic infections. Recently, Poudel et al. reported that asymptomatic endemic C. pecorum infections
reduce growth rates in calves by up to 48%. They considered that the mechanism of growth
suppression by subclinical chlamydial infection was malabsorption of nutrients due to a
local inflammatory response to intestinal mucosal infection. Additionally, despite the
absence of clinical signs, chlamydial infection was associated with reduced serum iron
concentrations and lower hematocrit values, and infected calves were leukopenic. Mohamad and Rodolakis reported that the persistence of C. pecorum strains
in the intestines and vaginal mucus of ruminants could cause long-term sub-clinical
infection which may affect the animal’s health. This may explain the poor weight gain
observed in case 2 after recovery from diarrhea. Further studies on the pathogenesis of
C. pecorum infections are required.
Although the standard method for detecting antibodies to Chlamydiaceae
spp. in animals is still the complement fixation test, our study showed that a neutralization test was also a useful method for
diagnosis of C. pecorum infections. In addition, in the dead calf, it was
confirmed that immunohistochemistry for C. pecorum antigens was also
Genetic and antigenic analysis showed that 22–58 and 24–100 strains were more closely
related to Bo/Yokohama strain isolated from cattle with enteritis than to Bo/Maeda strain
isolated from cattle with pneumonia and Ov/IPA strain isolated from sheep with
polyarthritis. Bo/Yokohama-like C. pecorum strains might cause enteritis
more than other serotypes. However, the 2 isolates showed higher sequence identities to
Bo/Maeda and Ov/IPA strains than to 66P130 strain isolated from cattle with enteritis in
United States. Thus, sequence identities of C. pecorum omp1 gene might vary
according to their geographical background.
The isolation of similar strains from different locations in separate years suggests that
C. pecorum might be spreading among the cattle population in Yamaguchi
Prefecture. An epidemiological study of C. pecorum is being conducted
currently to clarify the seroprevalence and relationship with disease.
In conclusion, this study showed that C. pecorum isolates similar to
Bo/Yokohama might be endemic in Yamaguchi Prefecture and cause enteric diseases in
Using the RT-PCR assay (targeting a 451 bp fragment of the S gene of PToV), 9 out of 20 farms were positive for PToV. Among the 9 farms, two farms tested positive for PToV alone, while the remaining 7 farms had mixed infection with other viruses tested; no consistent association between PoTV and these viruses was observed. For other tested enteric pathogens, PEDV, PKBV, and PRV A had high positive rates which were 55% (11 out of 20 farms), 70% (14 out of 20 farms) and 75% (15 out of 20 farms), respectively, while none of the samples were positive for PRV B and calicivirus (PSaV and PNoV). Besides, 40% (8 out of 20 farms) samples were MRV positive, and positive rates of AV and TGEV were both 25% (5 out of 20 farms). Summary of enteric pathogens present in the porcine samples obtained from diarrheic pigs was listed in Table 2.
The newly determined sequences have been deposited in the NCBI nucleotide sequence database and assigned the following accession numbers: KC340952 (farm numbers 1 and 2); KC340953 (farm number 7); KC340954 (farm numbers 8 and 11); KC340955 (farm number 10); KC340956 (farm number 13); KC340957 (farm number 14); KC340958 (farm number 18).
For 9 farms positive for PToV, the RT-PCR yielded a product of the anticipated size of 451 bp. Sequence analysis confirmed that the product was porcine torovirus specific. Those that shared the same sequence were neglected. Pairwise comparison of nucleotide sequences of the partial S gene confirmed that the strains are more closely related to the porcine torovirus. Comparison of the nucleotide (exclude the primer sequences) and deduced amino acid sequences of the fragment of the S gene of Chinese PToV strains showed that the Chinese PToV strains were highly conserved for the region, which had 90.2%–99.8% nucleotide and 93.7%–99.3 deduced amino acid identity with each other, and they formed a single lineage on the phylogenetic tree (Figure 1). The Chinese PToV strains were 90.9%–95.1% and 89.4%–96.5% identical to those porcine torovirus (AJ575372.1; GU196786.1) while only 38.3%–40.7% and 8.7%–10.8% to those of bovine torovirus. While, among the Chinese PToV strains, 6 strains clustered most closely with the the PToV Markelo/Netherlands strain, KC340955 clustered with GU196786. These results indicated that different PToV strains were circulating in China. Previous researches suggested that more than two different PToV strains could circulate simultaneously in an area; moreover individual animals could be infected by two strains during their productive life [5, 17].
In China, PToV-associated diarrhea has not been reported; even there was little information about PToV epidemiology. However, longitudinal, serological, and virological studies on PToV in piglets were carried out in Spanish and Europe and also RT-PCR method and real-time PCR were developed to detect PToV qualitatively or quantitatively in Korea [5, 6, 8, 17–19]. PToV epidemiology in China should be paid attention to. Our study first reported the existence of PToV in China, and PToV molecular epidemiology was also conducted in the study.
PToV has been detected in swine diarrhea samples and also high incidence of PToV infection in diarrhea samples was observed in our study. It was worth noting that two diarrhea samples were tested positive for PToV alone, and a previous study has reported a diarrhea sample tested positive for PToV alone when a survey for enteric pathogens in diarrheic pigs was carried out. However, the impossibility of growing the virus in culture cells has precluded the development of PToV. We could not make the conclusion if there was any necessary connection between the two, or a relationship exists between PoTV and the other enteric pathogens identified. We could not ignore the importance of PToV and diagnosis of porcine diarrhea should include PToV examination. Further studies to reveal the epidemiological status of PToV infection in China are needed to be developed. In addition to investigating the molecular epidemics using RT-PCR, further immunological method should be established to detect serological prevelence of porcine torovirus in Chinese swine herds. Future researches will focus on the epidemiology and pathogenic potential of PoTV.
To better understand the pathogenesis and epidemiology of C/OK, we performed infection studies with ferrets and swine. We first addressed the zoonotic potential of C/OK virus by conducting a pathogenesis and transmission study in the ferret model. After intranasal inoculation of ferrets, C/OK virus was first detected in nasal washes on day 3 (mean titer, 3.3 log10 TCID50/mL) (Fig. 3). C/OK virus was first detected in ferrets exposed by direct contact to inoculated ferrets on day 7, reaching a mean titer of 4.3 log10 TCID50/mL by day 10. Virus was not detected in ferrets exposed to respiratory droplets. No clinical signs of disease were observed. In the tissues of ferrets on day 5 post-inoculation (p.i.), a mean titer of 3.9 log10 TCID50/mL was observed in the nasal turbinates, but no virus was detected in the upper and lower trachea, lung, small intestine, liver, or spleen. Histopathological examination of lung tissues showed no typical influenza lesions. These results are consistent with a previous study that investigated human influenza C replication in ferret alveolar macrophage cells where viral replication with titers >104 egg infectious dose 50 from days 4 to 9 were measured with no cytopathic effects. All ferrets that were inoculated or exposed by direct contact and 1/3 of the ferrets exposed to respiratory droplets seroconverted 3 weeks after exposure as measured by HI assay (GMT = 780). To assess the pathogenicity and transmissibility of the virus in swine, we similarly challenged swine intranasally with C/OK (Fig. S1). Virus was first detected in nasal swabs on day 3 p.i. by using an rt-RT-PCR method specifically developed for C/OK virus. Virus shedding peaked at day 8 p.i. and remained detectable on day 10. Virus was detected in swine exposed by direct contact on days 7 and 9 after exposure. No clinical signs of illness were observed. Lung samples collected from inoculated swine on day 7 p.i. showed no evidence of the virus by rt-RT-PCR. Histopathological examination of lung tissues showed no typical influenza lesions. Sera collected on day 14 p.i. from donor pigs were positive for antibodies to C/OK virus in an HI assay (GMT = 30.3). All 5 pigs were positive for antibodies to C/OK. Additionally, 2 of the 5 direct contact pigs seroconverted by day 13 post exposure. These data suggest that in animals the replication kinetics is slower for C/OK virus than for influenza A viruses and infection in both swine and ferrets was limited to the upper respiratory tract. The ability of C/OK to readily transmit to contact ferrets suggests that a level of transmission potential to humans is possible. Zoonotic H5N1 and H9N2 influenza A viruses are typically unable to transmit in ferrets.
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.
168 samples of feces or intestines from piglets that died of severe diarrhea from 20 farms in southwest China were collected during the winter of 2011, when there were epidemic outbreaks of diarrhea that occurred with high morbidity and mortality, which has caused great economic losses. Of note, most of the sampled piglets were one to three weeks old, and antibiotic treatment was invalid in all sampled piglets.
Most of our existing knowledge about ToV is based on the study of BToV and EToV, or the members of the Coronaviridae. The lack of an adaptive culture system and infection model to grow the virus hampers the study of viral characteristics and development of diagnostic tools. On the other hand, as PToV has not caused great economic losses, people do not pay attention to it, resulting in a lack of research on treatment and prevention.
The limited studies of sequence diversity may impede development of accurate diagnostic assays and vaccine production. Especially, a lack of study of the variability of the S gene also limits our understanding of the serology of PToV. So far, though many test methods have been established, there are no commercially available diagnostic kits. The expression of structural proteins by various means is important in order to screen for antibodies against PToV, and monoclonal antibodies are needed for further research on important topics like the mechanism of pathogenesis. In particular, the sequence of structural genes, as well as their processing and modification, may affect host specificity of the virus.
Intertype recombination events that have occurred in Europe (31) and Japan (32), among other places, remind us not to underestimate the danger posed by PToV from the possibility of cross-species infection. The mechanism of pathogenesis of PToV is still unclear, and its role during co-infections with other swine enteric pathogens such as PRV A, PAstV, PEDV, TGEV, PKV and Salmonella spp. is unknown (11, 30, 46). Considering the prevalence of asymptomatic PToV infections, more research is needed to explore whether it may aggravate the diseases caused by other swine pathogens.
The worldwide distribution of PToV has been proven, with a high infection rate in pigs. However, due to limitations of diagnostic assays and asymptomatic infections (4, 13), there are not many reports on epidemiology. In 1998, Kroneman et al. performed a neutralization assay using EToV to detect cross-reacting antibodies and found that 81.4% (96/118) of the pig serum samples collected from farms in the Netherlands contained EToV-neutralizing antibodies (4). A qRT-PCR method was applied to detect PToV in rectal swabs collected from piglets at a farm in northeastern Spain in 2010, with a positive detection rate of 39.6% (19/48) (9). A longitudinal serological and virological study of PToV in Spain detected serum antibody levels by N protein ELISA, and fecal shedding by qRT-PCR based on the N gene. Seroprevalence in one hundred and twenty piglets at 1, 3, 7, 11, and 15 weeks-of-age was 92, 58, 91, 100, and 100% positive, respectively, and the corresponding 30 sows were all seropositive, reflecting the process of maternal antibody decline and subsequent immune response. As for fecal shedding in a 36-piglet subpopulation, 92% (33/36) of piglets had detectable PToV RNA at some age (39). Another epidemiological study in Spanish farms was done in 2012, with serum samples collected from 100 farms tested by N protein ELISA, revealing a total seroprevalence of 95.7% (2550/2664) and prevalence at different ages ranging from 59.4 to 99.6%. The lowest seroprevalence was detected in 3-week-old piglets (98/165) (45).
Shin et al. examined the prevalence of PToV in Korea in 2007, revealing 6.4% (19/295) of diarrheic pig samples were positive by RT-PCR (11). Among samples from diarrheic pigs collected in Korea during 2004–2005 and 2007, 36% (31/86) were positive by SYBR Green qRT-PCR (42). RT-PCR targeting the S gene was used to test stool and intestinal samples of diarrheic piglets from 20 farms in southwest China collected in the winter of 2011, with 45% (9/20) farms positive for PToV. In addition, 7 of those 9 farms had mixed infection with other swine viruses including PEDV, PKV, porcine rotavirus group A (PRV-A), transmissible gastroenteritis virus (TGEV), PAstV and mammalian orthoreovirus (MRV) (12). In Sichuan Province in the southwest of China, 872 fecal samples collected from diarrheic swine in 2011–2013 were tested by RT-PCR based on the conserved region of the S gene. An overall positive rate of 37.96% (331/872) was found, with positive co-infection with PEDV, TGEV or PRV-A in 4.1% (36/872) of these samples. Among the different ages tested, piglets at 1–3 weeks-of-age had the highest infection rate of 42.47% (295/697) (30).
Diarrhoea is the most common infectious cause of neonatal death in pigs. Prevention of this condition can be achieved by immunization of the sow with the aim of ensuring transfer of immunoglobulin A in colostrum and milk. Indeed, outbreaks of infectious diarrhoea in neonates are associated to the lack of specific immunity in the sow.
Classically, infectious neonatal diarrhoea has been seen as a problem caused by single aetiological agents. However, with the development of molecular techniques, a growing number of evidences indicated that many agents can be found in the faeces of affected animals [19, 21]. The problem now is to distinguish which of those agents primary cause diarrhoea, which others act as secondary or associated agents and which of them are part of the enteric microbiome without involvement in the disease.
In this work, we examined the RNA virome present in faecal samples from 47 cases of diarrhoea plus four non-diarrhoeic negative controls in which specific bacteriological agents were excluded. The approach taken using NGS did not include any previous enrichment or PCR step; hence, the results obtained represented not only the RNA viruses present in a sample analysed but also their relative abundances. This may contribute to our understanding of the role of each virus in a given case.
In the samples examined, the predominance of Rotaviruses reads reinforce the notion of these viruses as primary agents of neonatal diarrhoea; although, occasionally, KobuV and SAV may have a role in this process. The examination of the relative proportions of viral reads versus the number of reads showed that for any given case always one virus predominated, representing more than 45% of the mammalian viral reads obtained. This value can be tentatively proposed as a cut-off for the assignment of an etiological agent. In contrast, for all the other viruses examined, the relative abundance of their genomes would be more consistent with a subclinical infection. Whether this could be the consequence of a limited virulence, because of some level of passive immunity, or other causes, cannot be addressed in the present study.
Since the NGS filtering method used in the present study could only detect known agents, additional analyses to screen for potential viral motifs were performed. The de novo assembly and virus detection analyses were not able to identify any contig that could be related to the presence of an unknown RNA virus.
Interestingly, in the non-diarrhoeic samples analysed the number of mammalian reads was very scarce, but a higher number of plant and fungi viruses was reported. This is somewhat surprising, since the examined animal were suckling piglets (less than 1 week of age) that do not eat feedstuff. It is difficult to explain the origin of those viruses, since samples were taken from the rectum and thus, environmental contamination is little likely.
Our results agree with a number of studies that reported different Rotavirus species as agents of neonatal diarrhoea (reviewed in). One interesting observation from the present study is that coinfections or RVA-RVC and RVB-RVC was very common, suggesting the need for simultaneous testing all three agents in cases of neonatal diarrhoea. Regarding KobuV, our results also agree with an increased prevalence of this agent observed in cases of diarrhoea in suckling piglets worldwide: Brazil, Korea and Vietnam; despite several studies have observed non-significant differences in KobuV infection between diarrheic and healthy piglets in several European countries [12, 16, 31]. As mentioned above, two different patterns were observed in cases where KobuV was present: one with high proportion of KobuV reads and high number of viral reads, and others where these two circumstances were not fulfilled. The first pattern could correspond to clinical cases caused by KobuV, while the second could correspond to secondary or concomitant infections by this agent. A similar pattern would apply to SAV. Regarding the other agents, since they were always in combination and their reads were not preeminent, its role must be seen as secondary at best. For instance, no differences in Astrovirus prevalence between diarrheic and non-diarrheic piglets were reported [32, 33], nor for Enterovirus prevalence between healthy and diarrheic pigs. Interestingly, PEDV was seldom found in our samples and when found only traces could be detected. Prior to this, a higher frequency for this pathogen could be expected. However, while PEDV has caused recently severe epidemics in America, in Europe the incidence seems to be much lower. Of note is the total absence of TGEV, a virus which was widespread in Europe, but has consistently declined, or even disappeared, after the apparition and fast spread worldwide in the nineties of Porcine Respiratory Coronavirus.
In any case, it is somewhat surprising to detect that many different viruses (up to seven in a single sample) in so young animals, even though the pattern was already observed in diarrhoeic piglets. While it is easy to understand that the introduction of a new enteric virus in the farm will result in its rapid spread and the eventual development of an epidemic, it is more difficult to understand how so many viruses can be present in the maternities without generating a herd immunity resulting in colostral and lactogenic protection of suckling piglets. One possibility is that, most often, maternal immunity could be enough to limit the replication of those agents but not to completely prevent the infection; namely only provided partial immunity. In other cases, on farms reporting disease even in sows, RVA was present. This is compatible with its assumed role as primary agent. We have recently described the introduction of a new RVA strain that rapidly spread across pig farms in Spain and that was also present in the samples of this study as well.
It is worth noting that the phylogenetic analyses for all the examined viruses indicated the existence of local clusters except for RVA (discussed above). This, together with the depth of the corresponding branches in the trees, suggested a long evolutionary history on a local basis. Certainly, we cannot discard the existence of other clusters for any of the reported viruses. However, the detection of several new genotypes for RVB and RVC plus the relative low nucleotide identity with other available sequences in GenBank reinforces the idea of local evolution at least for these two viruses. Moreover, with the results obtained we propose to define for RVB one new VP4 (P) and two new VP7 (G27 and G28) genotypes; as well as genotypes G14 and G15 for RVC. The geographical pattern observed in the RVB phylogenetic trees agrees with the notion that RVB genotypes may be specific for host species and region. Regarding KobuV, no differences in the clustering pattern were observed between those strains coming from cases in which KobuV was predominant or not. This suggests that phylogenetic clustering probably is not predictive of virulence for this virus and most probably, other causes (i.e. immunity) are more relevant with regards to the clinical expression of the disease. Similarly, for SAV no differences were seen, and all isolates clustered together within genogroup III, the most commonly reported porcine SAV.
Advances in the enteric microbiology research have improved the understanding of etiology of infectious gastroenteritis, as well as the involvement and transmission modes of enteric pathogens. This has enabled the design of specific control strategies limiting the losses due to consequent severe infections. Although, bacteria and viruses are both responsible for gastroenteritis, the latter have had more impact on public health (Gunn et al., 2015). As of date, non-bacterial acute gastroenteritis, and respiratory infections are the leading causes of global deaths in both humans (mainly children) and animals (Dominguez et al., 2009; Bok and Green, 2012; Dhama et al., 2015). Since the identification of the first enteric virus, Norovirus (Caliciviridae), in 1972 using electron microscopy, a range of viruses, such as Rotavirus (Reoviridae), Picobirnavirus (Picobirnaviridae), Astrovirus (Astroviridae), enteric Adenovirus (Adenoviridae), Sapovirus (Calciviridae), Torovirus (Coronaviridae), Parechovirus, Bocavirus, and Aichivirus (Picornaviridae), and many more, have been found to be associated with gastroenteritis infections (Cheng et al., 2008; Dhama et al., 2009, 2014; Malik et al., 2011, 2014; Ahmed et al., 2014; Yip et al., 2014; Sidoti et al., 2015; Kattoor et al., 2017; Delmas et al., 2018; Kattoor et al., 2019). The enteric viruses known as of now along with their respective advanced diagnostic methods are tallied in Table 1. Although acute viral gastroenteritis is more common in immune-compromised and young individuals (Krones and Högenauer, 2012), it is also seen in the aged individuals, which may be due to changes in physiology and the waning of immunity with time (Estes and Kapikian, 2007).
New viruses are emerging at a faster pace, apparently as a feature of their rapidly changing genetic makeup due to the accumulation of point mutations, reassortments or recombinations (Malik et al., 2016; Kattoor et al., 2017). For an example, a new porcine coronavirus, has emerged through recombination between the transmissible gastroenteritis virus and a porcine epidemic diarrhea virus (Boniotti et al., 2016). Enteric viral diseases are diagnosed by identifying the causative viral agents in feces/body fluids or viral antigens and/or antibodies in the serum of patients. Conventional methods to achieve this, however, are either inefficient, cumbersome or time consuming, because of the pace of change of the virome. There were not much significant approaches available in the past, and in the recent time various techniques have come up offering a modern field for advances in bio-techniques for the easy, quick and reliable diagnosis and discovery of new viruses. In clinical laboratories, polymerase chain reaction (PCR)-based assays are considered as gold-standard for the detection of viruses, but when it comes to multiple detections of similar types of viruses simultaneously, variations in the properties of viral nucleic acids make the amplification difficult (Fout et al., 2003; Fong and Lipp, 2005). Among different techniques used to explore new viruses, such as conventional and next-generation sequencing, metagenomics has been a promising approach to study the unrevealed viral genomes since more than a decade (Garza and Dutilh, 2015; Martinez-Hernandez et al., 2017). This allows researchers to study the genetic material directly from pooled samples and bypass the need for culturing the virus in vitro as well. Virome capture sequencing is another approach for vertebrate viruses, in which several million probes covering the genomes of several viral taxonomies are used to enrich virus targets (Briese et al., 2015). A new metagenomic sequencing method, ViroCap, based on the target nucleic acid capture and enrichment detects viral sequences with up to 58% variation from the references used to select capture probes (Wylie et al., 2015).
Nevertheless, several diagnostic methods have been developed over the last two decades, seeing the constant evolution of viruses, newer, sensitive, efficient, and rapid diagnostics are still warranted for the effective diagnosis (Liu et al., 2007; Saminathan et al., 2016). This paper systematically describes and discusses the features, advantages and limitations primarily of advanced diagnostic tools devised for the sensitive and quick detection of enteric viruses worldwide (Figure 1).
Isolation of the enteric viruses in cell culture system from fecal samples is the most conventional way of confirmatory diagnosis. Although the cultivation of viruses in cell culture is time and labor intensive, taking from days to weeks before the virus is adapted to cell culture; it is still the ideal and gold standard method for the virus detection worldwide. Many new cell lines have been developed for easy propagation of enteric viruses and are given in Table 2.
New methods for the easy cultivation, better preservation and isolation of viruses have been developed; for example, cryopreserved cell culture, virus isolation in co-cultured cells and virus identification in transgenic cell lines. Inclusion of genetic elements in the transgenic cell lines has helped in the rapid and accurate detection of some viruses. For culturing gastrointestinal viruses in the laboratory, induced intestine-like human intestinal organoids (iHIOs) with differentiation of human embryonic or induced pluripotent stem cell lines have been used successfully for culturing rotavirus (McCracken et al., 2011; Spence et al., 2011). This also increases the potential for successful isolation and propagation of other enteric viruses, although there is still a need to develop better isolation or cultivation methods for enteric viruses. For other enteric viruses viz. Calicivirus, except for murine Norovirus and porcine Sapoviruses (Cowden strain), establishment of an efficient human Norovirus and Sapovirus cultivation system is still lacking (Greenberg et al., 1982; Sidoti et al., 2015). Jones et al. (2015) were the first to report the in vitro cultivation of GII.4-Sydney human Norovirus strain in B cell line (BJAB cell line) and achieved the modest level of viral output, ranging from 0.5 to 3.5 logs. Four days were found optimum for infection and analysis assays. Recent attempts to grow human Noroviruses have been established in human induced pluripotent stem cells derived intestinal epithelial cells (iPSC–derived IECs) (Sato et al., 2019).
Here, we detected a high prevalence of bovine astrovirus by RT-PCR from rectal swabs
collected from young calves of cattle and water buffalo with diarrhea. However, more studies
are needed to determine whether the persistent diarrhea observed in these calves was mainly
associated with the high prevalence of astroviruses, as previous studies reported that
bovine astroviruses were not directly associated with severe diarrhea in calves under
natural conditions [4, 24]. However, other reports [14, 26] indicated that bovine astrovirus may evolve to severe
diarrhea in co-infections with other gastrointestinal viruses, as in the case of BAstV
co-infection with BRV or BToV (Breda virus).
Nonetheless, the epidemiology of bovine astrovirus remains unclear, especially considering
the limited number of studies of cattle and water buffalo. While excretion of BAstV may
occur in up to 60–100% of calves on farms, only 5
(2.4%) of 209 rectal swabs collected from asymptomatic adult cattle were positive for BAstV
. In the present study, sampling at different
time points demonstrated a high prevalence of up to 56.52% among calves with diarrhea.
Moreover, in our complementary study, astrovirus was detected alongside other
gastrointestinal viruses, including BEV, BCoV, BRV and BVDV, in 87.5% of cases.
Surprisingly, 12.5% of these cases were positive for bovine astrovirus, but yet negative for
other tested gastrointestinal viruses. In contrast, titers of BRV and BToV, which have been
previously reported as principal gastrointestinal co-infecting viruses with bovine
astrovirus, were minimal or undetectable in this study. From these results, it is clear that
astrovirus may be directly associated with diarrhea or possibly linked to other factors,
such as poor hygienic conditions or associated with other non-viral pathogens, such as
bacteria or parasites, which are known causative agents of diarrhea in calves. Similar
results were reported in Korea, where co-infection of bovine astrovirus with
gastrointestinal viruses, other than BRV and BToV, was associated with clinical symptoms of
diarrhea in 20- and 14-day-old calves.
Our phylogenetic analysis showed that all isolates in this study were closely related to
BAstV-B76-2/HK, BAstV-B18/HK, CcAstV-1/DNK/2010 and CcAstV-2/DNK/2010, but highly divergent
from a BAstV NeuroS1 isolate previously associated with neurologic disease in cattle in the
U.S.A.. Nonetheless, our results support that
proposal that BAstV and CcAstV may be different
strains of the same virus, and water buffalo may be a new host of the BAstV variant of this
virus. Moreover, most sequences derived from one farm belonged to one subgroup, although
sequence analysis clearly demonstrated origins from different strains. In addition, no
significant genetic differences were observed between the bovine and water buffalo
astrovirus strains investigated in this study (Table
4). These results further support previous evidence that one farm could serve as
a reservoir of different bovine astrovirus strains, suggesting possible outbreaks of novel
astroviruses due to mutation or recombination events. Notably, considering the history of
astroviruses, recombination events have been described in cattle, swine, humans and poultry
22], and co-infection of two different astroviruses
in one head of cattle has been previously reported as well. Consequently, it is important to screen for and control astrovirus infection
in order to prevent eventual outbreaks of highly pathogenic astroviruses resulting in
mutation or recombination events on farms. Furthermore, the astrovirus isolates from cattle
and water buffalo isolated in this study displayed relatively close relationships,
indicating that these animals were infected with diverse astroviruses, which probably
evolved from the same viral ancestor. Consequently, given these close relationships,
evidence exists of possible cross-infection between the two hosts; therefore, control
measures against bovine astrovirus should also be taken into consideration when screening of
viruses in water buffalo populations.
Appreciation of the genetic diversity and evolution of astroviruses among wild and domestic
animal populations is important to fully understand this challenging gastrointestinal virus
. However, the organization of the BAstV genome
has not yet been fully described. In this study, a sequence motif upstream of ORF2 was
predicted to be the signal of the putative promoter for subgenomic RNA (sgRNA) synthesis in
bovine astrovirus as well as that for the ORF1b/ORF2 overlap sequence and the most 3′-end of
ORF2. It has been suggested that in astroviruses [13,
23], a conserved sequence acts as a putative
promoter for sgRNA synthesis upstream of ORF2. In human astroviruses, the ORF1b/ORF2 overlap
is 8 nt in length, while in duck astroviruses, the ORF1b/ORF2 junctions are not overlapped
[6, 13, 23]. In contrast, the overlap in the ORF1b/ ORF2 junction
of bovine astroviruses may be longer (56 nt) than that of the other known astroviruses.
Moreover, compared with a previous report, the
3′-end of ORF2 of bovine astroviruses was highly conserved, although our results suggested
further studies and analysis of more sequences from several novel bovine astroviruses to
fully characterize ORF2 of bovine astrovirus.
In summary, this study is of interest at both the epidemiological and genetic levels. These
results will certainly contribute to the understanding of the evolution and pathology of
bovine astrovirus in cattle and water buffalo, as not only did we provide useful reference
material for further studies, but also isolated bovine astrovirus in water buffalo for the
first time. The present study is thus far among the largest epidemiological investigations
of bovine astrovirus conducted at the farm level in the dairy industry in China.