PCRs testing were repeated on the 50 fruit bats original samples including the Kidney, heart, lung, liver, spleen, intestine, rectal swab sample, and brain samples. Two bat’s QPCRs results were positive. One bat’s QPCRs result was positive in the lung, intestine sample (Cangyuan virus isolated) and rectal swab sample, and the Ct (Threshold Cycle) of QPCR were 19.86 ± 0.056, 19.52 ± 0.041, 19.64 ± 0.061 respectively. The Ct of another bat’s PCR were 23.07 ± 0.253, 22.53 ± 0.171 in the intestine sample and rectal swab sample, respectively.

To establish the evolutionary relationship between Cangyuan virus and other known orthoreoviruses, Homology were compared (Table 2, Table 3 and Additional file 1: Table S1, Additional file 2: Table S2 and Additional file 3: Table S3) and phylogenetic trees were constructed based on the nucleotide sequences of the L genome segments (Figure 2), the M genome segments (Figure 3) and the S genome segments (Figure 4). The Cangyuan virus L1-L3, M1-M3 segments sequence identity were 81.6% –94.2%, 83.8%–97.9%, 85.9%–97.6% ( Additional file 1: Table S1), 82.2%–94.1%, 78. 1%–95.0%, and 83.0%–93.9% (Table 2, Additional file 2: Table 2), respectively, by alignment with Pteropine orthoreovirus (PRV) species group. The phylogenetic trees for L2, L3, M1 and M2 segments demonstrated that Cangyuan virus was most closely related to Melaka and Kampar viruses, and was placed in Pteropine orthoreovirus (PRV) species group which covers all known bat-borne orthoreoviruses together with Nelson Bay orthoreovirus.

To better understand the genetic relatedness of Cangyuan virus to other known bat-borne orthoreoviruses, the published sequences for the S genome segment of bat-borne orthoreoviruses known for causing acute respiratory disease in humans were retrieved from GenBank and used to compare homology (Table 3 and Additional file 2: Table S2) and construct phylogenetic trees (Figure 4). The Cangyuan virus S1-S4 segments sequence identity were 55.3%–94.7%, 86.2%–95.5%, 86.5%–97.9%%, and 83.5%–98.2%, respectively (Table 3 and Additional file 2: Table S2). The S1 segment demonstrated a greater heterogeneity than other S segments in Pteropine orthoreovirus (PRV) species group.

Sequencing of the amplified products revealed a high homology with TAstV-2 North Carolina Q/34/1990 strain for polymerase gene and to TCoV for the 3‘UTR of turkey\UK\412\00 strain (FJ178641 and , respectively).

In order to verify the incidence of re-infection during this study, including RT-PCRs, CS, faeces and winter/summer results were compared (Table 3). For TAstV-2 search, 10% of CS and 20% of faeces were positive at winter, and 36% of CS and 20% of faeces showed an increase in the same period for TCoV, when individual RT-PCR was evaluated. In addition, the TAstV-2 was less detectable, CS 50% and faeces 72%, in the winter when multiplex RT-PCR was used (Table 3). Otherwise, the TCoV was equal detected from CS and 28% more detectable in winter for the same analysis. The results showed on Table 3, RT-PCR assayed for both virus in a single tube have 3.98 (p=0.89982) more chance to present positive results (faeces) than CS, at dry season, and faeces have 0.67 of chance to give false negative results for the same statistical analysis (p=0.67851).

In this study, viral surveillance of the sewage collected from 10 wastewater treatment plants throughout Taiwan was performed from July 2012 to December 2013. During this 18-month survey period, 300 raw sewage specimens were examined to detect the presence of viruses. The results showed that coxsackievirus type B, echoviruses, adenoviruses, and mammalian orthoreoviruses were isolated from the sewage specimens with a positive rate of 54.3%, but no poliovirus was found. Among these, enteroviruses (34.3%) and MRV (35.1%) predominated, followed by adenovirus (30.6%). Based on data provided by the WHO, at least 30% of concentrated sewage from grab samples can be expected to test positive for NPEV. Therefore, the high rate of non-polio enteroviruses in the environment can be considered proof that the environmental samples were processed and analyzed appropriately to preserve virus infectivity. This study was undertaken to supplement poliovirus surveillance in Taiwan by monitoring the possible presence of wild-type (WPV) or vaccine-derived (cVDPV) poliovirus in wastewater with a view to obtaining further evidence supporting the maintenance of Taiwan's polio-free status.

The frequencies of detection of different enteroviruses differed by geographical area and year, but overall, coxsackievirus type B strains were isolated more often than echoviruses. These findings are similar to the results of some studies on sewage surveillance in other countries [42–45]. Among coxsackievirus B strains, CVB2, CVB3, and CVB4 were the most prevalent between July 2012 and 2013 in Taiwan; however, CVB1 and CVB6 were not isolated in this study. According to a surveillance report by Taiwan's Centers of Disease Control (Taiwan CDC), among the coxsackievirus B strains, CVB3 and CVB4 were more frequent in 2012, whereas CVB2 and CVB4 predominated in 2013. This phenomenon was in agreement with the results of this study that showed the majority subtype of coxsackievirus B in 2012 was CVB3, which shifted to CVB2 and CVB4 in 2013. The results indicated that the environmental virus strains reflect the viruses circulating in the population and highlight the potential risk of viruses spreading via wastewater. In addition, the rare isolation of coxsackievirus type A in our study might be related to the lower susceptibility of the cells used to isolate the viruses or the lower resistance of the viruses in the environment and the isolation process.

Besides the enteroviruses, adenoviruses and MRV were also identified by environmental surveillance in this study. Previous studies have reported adenoviruses and MRV in contaminated surface water and wastewater [46, 47]. Adenoviruses are non-enveloped, double strand DNA virus from the family Adenoviridae and are classified into species A to G with more than 57 identified genotypes. It has been shown that adenoviruses of species B (Ad 3, 7, 11& 14), species C (Ad 1, 2 & 5), and species E (Ad 4) are associated with acute respiratory disease [48–51]; adenoviruses of species F (Ad 40 & 41) are related to acute gastroenteritis in infants and children; species D (Ad 8, 19, 37, 54) is thought to cause epidemic keratoconjunctivitis; and species B (Ad 11, 21) has been linked to hemorrhagic cystitis [52–55]. According to data on clinical adenovirus isolation in Taiwan, Ad3, Ad7, and Ad4 were found mainly during outbreaks in southern Taiwan between 1999 and 2001, and Ad3 circulated in northern Taiwan between 2004 and 2005 [56, 57]. Recently, we reported that Ad3 was the dominant strain in southern Taiwan from 2002 to 2011, and a high incidence of co-infection with Ad2 was identified.

The unexpected identification of MRV by PanEV RT-PCR may be explained by the sequence similarity between the primers for enterovirus 5′-NTR and L1 gene of MRV, as MRV was formerly classified as ECHO 10. We found MRV was positive for PanEV antibody stain, but failed to be identified by CODEHOP PCR of enteroviruses. This may be attributed to the cross-reactivity of the PanEV Blend antibody toward reoviruses, as noted by the manufacturer in the instructions provided with the kit. According to our results, a high positive rate of MRV was found in our sewage specimens. MRV belongs to the Orthorovirus genus, Spinareovirinae subfamily, Reovirus family, and is also commonly termed reovirus. MRV are non-enveloped viruses that contain 10 segmented double-stranded RNA genomes, including three large (L1-3), three medium (M1-3), and four small (S1-4) segments. It can be classified into three major serotypes: Type 1 Lang (T1L), Type 2 Jones (T2J), and Type 3 Dearing (T3D), which commonly cause asymptomatic infections or mild respiratory tract illness and enteritis in infants and children [37, 59, 60]. Other studies have reported a seropositive rate of more than 70% in 4-year-old children [27, 61]. Recently, a few novel MRV viruses were found in humans, such as, novel Type 2 MRV (MRV2TOU05), which seems to be closely related to porcine and human strains first isolated from 2 children with acute necrotizing encephalopathy in France; the mother of one patient also had influenza-like symptoms, and specific antibodies against MRV2TOU05 were detected. Another novel Type 3 MRV was isolated from a child in the United States with meningitis. The virus also showed systemic spread and was found to produce lethal encephalitis in newborn mice after peroral inoculation. Besides the novel MRV-infected pediatric cases, another novel MRV (Kampar virus) was identified from a throat swab of a 54-year-old patient with high fever, acute respiratory disease, and vomiting. Based on epidemiological tracing, there is a high probability that Kampar virus originated from fruit bats and is capable of causing human to human transmission according to the results of serological studies. In previous studies, reoviruses were commonly found in environmental water sources, and human fecal contamination has been suggested as the source of the virus [64, 65]. Our PCR sequencing data of isolated MRV showed that mammalian orthoreoviruses Types 1, 2, and 3 were present in the environment. Sequence analysis also showed that MRV1 and MRV2 persistently circulated in Taiwan and most MRV isolated in Taiwan were closely related to the reference strains isolated from patients with severe acute respiratory syndrome or meningitis. These results suggest that the reoviruses isolated from sewage may have the potential to infect humans. However, no cases of MRV infection have been reported in Taiwan to date. Since the identification of MRV requires additional molecular analysis, it may be missed by routine viral identification. In addition, this study identified MRV from L20B cells, which are not normally used in routine settings. The etiologic agent remains unknown in many cases of encephalitis (32%-75%). MRV may be a potential risk factor with public health implications. Thus, L20B cell should be tested for routine viral isolation and PCR test is needed to identify MRV when MRV is suspected in human subjects.

In this report we provide an analysis of the environmental circulating viruses in Taiwan. Our results showed that Taiwan was poliovirus vaccine strain-free in the environment two years after the oral poliovirus vaccine was replaced by the inactivated poliovirus vaccine. Although our surveillance data were negative for poliovirus, long-term monitoring is still needed to allow prompt action should WPV ever be detected. In addition, we combined cell culture and RT-PCR to assay large volumes of sewage and identify the viruses, so that infectious viruses could be detected, thereby providing meaningful data that can be applied in public health risk assessments. This is the first study to report the prevalence of MRV in sewage in Taiwan. The observations made in this investigation highlight the potential risk of MRV infection in humans. This report also suggests that continuous periodic surveillance of environmental virus is necessary to prevent the outbreak of disease or reduce casualties. Finally, since MRV was frequently identified in our environmental specimens, it is imperative that human cases with suspected MPV infection be thoroughly evaluated.

Several studies have reported the existence of AstV in goose flocks18, 19, but the prevalence and pathogenicity of AstV among domestic geese remains poorly understood due to the lack of efficient in vitro culture techniques and diagnostic assays. In this study, AAstV SD01 was successfully isolated by inoculating tissue samples into goose embryos, as confirmed by RNA detection methods similar to those for pan-AstV. We successfully cultured the virus in vitro provided a convenient method for virus propagation and laboratory diagnosis. Isolation of the AAstV from field cases and reproduction of the disease in a gosling infection model with the inoculating virus prepared in embryos fulfils Koch′s postulates.

AstV infection occurred within the first days or week of life usually resulted in a worse outcome, as the age-dependent pathogenicity of AAstV has been reported10. In this study, the mortality of young goslings caused by subcutaneous or oral inoculation indicated that AAstV SD01 was highly pathogenic. The experiment was fairly a represent of the situation in the field, where susceptible goslings are exposed to AstV soon after they are placed in contaminated houses. Apart from mortality, avian AstV infection can decrease feed intake and alter feed conversion efficiency, leading to growth repression. The decrease in body weight of infected goslings is a major concern as a 29% lighter body weight at 14 dpi has a considerable economic impact.

Histologic examination revealed the presence of a proteinaceous substance in the renal tubules, indicating that AAstV SD01 infection caused increased permeability of the kidney epithelia barrier. Degeneration and necrosis of tubular epithelial cells found in the deceased goslings provided further evidence of kidney function damage. These results could explain the development of visceral urate deposition in infected goslings. Increased epithelium permeability due to AstV infection has been reported in both human and avian species24, 25. Extra-intestinal infection with nephritis has been reported in birds infected with chicken astrovirus and avian nephritis virus16, 26. Viral RNA was found in all of the tissues sampled from the infected goslings killed on 5 dpi, indicating that the goose AstV has a wide tissue tropism and spread systemically after inoculation. Virus shedding was detected by RT-PCR and persisted in the infected goslings for about 12 days, further indicating that the virus replicated efficiently in vivo.

It is interesting that encephalitis lesions were observed in the deceased goslings (data not shown), along with the detection of AAstV SD01 RNA in the brain tissue (Fig. S2). However, no neurological symptoms were noted in either the field cases or the experimentally infected goslings. The neurologic infection of AAstV SD01 is worthy of further investigation since there are numerous reported cases of AstV-associated encephalitis and meningitis in humans and mammals9, 27. Nonetheless, based on the limited number of goslings infected in present study, it is not likely to get accurate evaluation for the virulence of the isolate.

The independence chi-square test revealed a significant relationship between RoV occurrence and the health status of turkeys (P = 0.01, φ = 0.22). The OR =6.96 indicates that the odds of disease symptoms in turkeys infected with rotaviruses are almost sevenfold higher than the odds of occurrence of these symptoms in the uninfected group. The CA plots also revealed that rotavirus infection is most strongly correlated with PEC symptoms in turkeys, and PEMS symptoms as well as healthy status correspond with turkeys uninfected with rotaviruses. The first LR model showed that only RoV infection has a significant impact on health status (diseased turkeys), with P = 0.017. The OR =5.06 indicates that the odds of disease symptoms of turkeys infected with rotaviruses are over fivefold higher than the odds of occurrence of these symptoms in the uninfected turkeys.

Many emerging infectious diseases are caused by zoonotic transmission, and the consequence is often unpredictable. Zoonoses have been well represented with the 2003 outbreak of severe acute respiratory syndrome (SARS) due to a novel coronavirus. Bats are associated with an increasing number of emerging and reemerging viruses, many of which pose major threats to public health, in part because they are mammals which roost together in large populations and can fly over vast geographical distances. Many distinct viruses have been isolated or detected (molecular) from bats including representatives from families Rhabdoviridae, Paramyxoviridae, Coronaviridae, Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, Arenaviridae, Herpesviridae, Picornaviridae, Filoviridae, Hepadnaviridae and Orthomyxoviridae.

The Reoviridae (respiratory enteric orphan viruses) comprise a large and diverse group of nonenveloped viruses containing a genome of segmented double-stranded RNA, and are taxonomically classified into 10 genera. Orthoreoviruses are divided into two subgroups, fusogenic and nonfusogenic, depending on their ability to cause syncytium formation in cell culture, and have been isolated from a broad range of mammalian, avian, and reptilian hosts. Members of the genus Orthoreovirus contain a genome with 10 segments of dsRNA; 3 large (L1-L3), 3 medium (M1-M3), and 4 small (S1 to S4).

The discovery of Melaka and Kampar viruses, two novel fusogenic reoviruses of bat origin, marked the emergence of orthoreoviruses capable of causing acute respiratory disease in humans. Subsequently, other related strains of bat-associated orthoreoviruses have also been reported, including Xi River virus from China. Wong et al. isolated and characterized 3 fusogenic orthoreoviruses from three travelers who had returned from Indonesia to Hong Kong during 2007–2010.

In the present study we isolated a novel reovirus from intestinal contents taken from one fruit bat ( Rousettus leschenaultia) in Yunnan province, China. In the absence of targeted sequencing protocols for a novel virus, we applied the VIDISCR (Virus-Discovery-cDNA RAPD) virus discovery strategy to confirm and identify a novel Melaka-like reovirus, the “Cangyuan virus”. To track virus evolution and to provide evidence of genetic reassortment PCR sequencing was conducted on each of the 10 genome segments, and phylogenetic analysis performed to determine genetic relatedness with other bat-borne fusogenic orthoreoviruses.

Pet dogs play an important role in humans’ daily lives. Recently, the emergence of new pathogens and the continuous circulation of common etiological agents in dog populations have complicated canine diseases. Among these diseases, canine infectious respiratory diseases (CIRD) and viral enteritis pose notable threats to dog health.

CIRD are complex and include canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine herpesvirus (CHV), canine reovirus, Bordetella bronchiseptica and other pathogenic agents [2–4]. Among these, CAV-2, CDV or CPIV have frequently been detected in dogs with CIRD, according to previous studies [5, 6]. Avian-origin H3N2 CIV has been detected in domestic dogs in South Korea and China since 2007 [7, 8]. H3N2 CIV is now circulating in dog populations in China, South Korea, Thailand, and even the United States [9–11]. Distinguishing these pathogens can be challenging, because dogs often show similar clinical signs of infection with these viruses, such as low-grade fever, nasal discharge and cough. These respiratory symptoms are flu-like and difficult to diagnose.

Canine viral enteritis is common in dogs with acute vomiting and diarrhea. Canine parvovirus (CPV) is one of the major viruses leading to acute gastroenteritis in dogs; CPV infection is characterized by fever, severe diarrhea and vomiting, with high morbidity. Puppies tend to be intolerant of CPV infection and have higher mortality than adult dogs because of myocarditis and dehydration [14, 15]. Canine coronavirus (CCoV) is characterized by high morbidity and low mortality. Dogs infected with CCoV alone are likely to have mild diarrhea, whereas the disease may be fatal when coinfection by CCoV and CPV, CDV or canine adenovirus type 1 (CAV-1) occurs [16, 17]. CAV-2 is associated with mild respiratory infection and episodic enteritis [18, 19]. Canine circovirus (CanineCV), a newly discovered mammalian circovirus, was first reported by Kapoor et al. in 2012. CanineCV has been detected in dogs with severe hemorrhagic diarrhea, and it is more common in puppies than in adults [21, 22]. Coinfection of CanineCV with other intestinal pathogens (CPV or CCoV) is closely related to the occurrence of intestinal diseases [23, 24]. Dogs with intestinal diseases are often infected with one or more viruses, and their clinical symptoms are similar [17, 25, 26], making clinical differential diagnosis difficult. To date, no multiplex PCR (mPCR) method has been developed to detect CanineCV and other enteropathogens.

An effective diagnostic tool is important for the prevention, control and treatment of CIRD and viral-enteritis-related viral diseases. Although many methods exist to detect CIRD and canine viral enteritis, most can detect only 2 or 3 pathogens, and the current lack of systematic and comprehensive detection methods makes diagnosis impractical and time consuming [4, 27, 28]. Because mPCR can simultaneously detect multiple pathogens in a timely and inexpensive manner, this technique has become increasingly popular. Therefore, in this study, two new mPCR methods were developed for the detection of canine respiratory viruses (CRV, including CAV-2, CDV, CIV and CPIV) and canine enteric viruses (CEV, including CAV-2, CanineCV, CCoV and CPV), and we indicated that the mPCR methods established here are simple and effective tools for detecting the viruses of interest.

Bacteria and parasites have been considered the primary etiological agents of gastroenteritis in commercial poultry. However, many viral infections have been associated with enteric diseases of chickens and turkeys, including coronavirus, reoviruses, rotaviruses, adenoviruses, enteroviruses, and the members of Family Astroviridae (Chicken Astrovirus-CAstV and Avian Nephritis Virus-ANV) [22–27]. Infections with the previously mentioned viruses are believed to be important in the pathogenesis of the economically important enteric disease, such as runting and stunting syndrome (RSS), which affects young chickens, mainly broiler chickens [6, 9, 28, 29] and turkeys. Recently, studies have included the enterotropic strains of infectious bronchitis virus (IBV) as a possible etiological agent of enteritis in chickens. IBV can grow in many cells of the gastrointestinal tract, and some Asian strains were described to cause lesions in the proventriculus. IBV is believed to only persist in the gastrointestinal tract of young chickens and in layers without clinical disease.

Different denominations or terms have been used to describe the enteric disease in poultry because the clinical signs are infrequent or occur independently of previous conditions, such as the presence of primary or secondary etiological agents, the immune and nutritional status of the host, and environmental conditions [3, 4, 15, 33]. According to Saif, the gastrointestinal tract (GIT) is the primary organ of the body that is exposed and a variety of injuries against it could result in inefficient utilization of nutrients during the early stages of development. Of the various signs described for enteric disease, diarrhea and lack of normal development are the most consistently reported symptoms.

The results obtained in this study demonstrate a high level of infection with one or more of the seven viruses investigated in the chickens with clinical symptoms (65.4%), as shown in Table 2. However, samples taken from chickens without symptoms (15.4%) were also positive for these viruses (Table 2), which demonstrates a similar prevalence between these two groups. This result indicates that chickens should be shedding the virus via the enteric tract without showing any clinical symptoms; therefore, these chickens are considered asymptomatic carriers or reservoir, representing a potential source of infection. Other studies showed lower levels of rotavirus infection (4.1%) in normal chickens, while higher frequencies were found in asymptomatic flocks with 30% rotavirus and 30% for CAstV from healthy flocks of turkeys. However, these studies did not survey a wide range of viruses. In an extensive survey of turkey flocks with enteric disease and healthy turkey flocks in the United States, Rotaviruses were detected slightly more frequently in healthy than in diseased flocks. Astroviruses were detected in the intestinal contents of poultry prior to the onset of clinical disease and gross pathologic changes. ANV-induced clinical disease presents as kidney lesions in young chickens but only presents as a subclinical persistent infection in mature chicken. These conditions reflect those viruses, other than reovirus, coronavirus, and chicken parvovirus, which have been identified in samples from flocks that appear healthy and may have different degrees of pathogenicity [22, 25]. In fact, there are indications that different serotypes and even strains within the same serotype can vary in their ability to produce illness and death. Moreover, several factors influence the susceptibility of chickens to enteroviruses, such as age, passive immunity level, simultaneous infection with other pathogens, and management failures, which cause stress [14, 36, 37].

Regardless of the symptoms, most of the samples (226 samples) representing 80.7% were positive for one or more of the viruses, which demonstrates the high prevalence of these viruses in Brazilian chicken flocks (Table 2). In almost half of these positive samples (115 samples or 41.1% of total), two or more viruses were detected simultaneously, which highlights the hypothesis of multicausal etiology for the enteric disease. The distribution of the seven viruses in these concomitance samples was demonstrated and is shown in Table 2.

The combinations were scattered proportionally according to the prevalence of each virus and did not present a pattern or a constant frequency. None particular combination of the viruses was observed with significant repeatability. Rarely, a single agent is the sole contributing factor to enteric disease; moreover, the presence of different combinations of viruses could result in varied disease presentations.

IBV was the most prevalent virus detected in the samples that contained a single virus (Table 2) and in all of the samples (120 samples/42.9%), as shown in Table 3. IBV is the infectious agent associated with infectious bronchitis of chickens and is responsible for outbreaks in many countries, including Brazil. Infectious bronchitis (IB) is characterized by respiratory, reproductive, and, sometimes, renal signs. However, some strains of this virus have been demonstrated to multiply in intestinal cells and were referred to as possible agents of diarrhea or may at least have some role in enteric diseases. Recently, we isolated variant strains of IBV from the intestinal contents of chickens with enteritis but without the typical respiratory, reproductive, or renal signs. In these cases, IBV was the only pathogen present, while all of the samples were negative for astrovirus, reovirus, and rotavirus. When one-day-old SPF chicks were inoculated with filtrated IBV variants, respiratory signs but not diarrheal or renal signs were observed (data not published). In addition, these samples were not screened at for other likely agents of enteritis, such as ANV or chicken parvovirus. However, the IBV strains that were detected in the intestinal contents may not play a role as direct pathogens in enteric disease.

Of the viruses investigated, ANV was the second most prevalent by absolute numbers and was detected in 83 samples (29.6%), as shown in Table 3. If IBV is not the primary causal agent, then ANV is the most prevalent pathogen causing enteric disease that was detected in this study. Originally regarded as a picornavirus, ANV was recently characterized as a new member of the Family Astroviridae in 2000 and has been detected in kidney samples from young chickens with growth deficiencies in Hungary. A recent study that was performed in the United States to detect the presence of several enteroviruses in chicken flocks demonstrated that ANV was the most prevalent virus followed by coronavirus, reovirus, CAstV, and rotavirus. The characteristic signs of avian nephritis that are caused by AVN vary from none (subclinical) to outbreaks characterized by diarrhea, growth retard, renal failure with tubulonephrosis, interstitial nephritis, uricosis, and death [37, 39].

Chicken astroviruses (CAstV) were the third and rotaviruses were the fourth most frequent agents detected in this study, with 21.1% and 17.1%, respectively (Table 3). Previously, a high frequency of CAstV was reported by Pantin-Jackwood et al., where 21 of the 34 (61.7%) analyzed samples were identified as positive for CAstV. Astroviruses cause or have been associated with acute gastroenteritis in humans, cattle, swine, sheep, cats, dogs, deer, mice, and turkeys, as well as with fatal hepatitis in ducks [26, 29, 40, 41]. However, the clinical importance of CAstV remains unclear. On the other hand, rotaviruses are frequently associated with enteric disease, but the economic significance of rotaviruses to the poultry industry has not yet been defined [10, 36, 42]. Rotaviruses were present in 46.5% of the samples and in chicken flocks from all regions of the United States that were tested during 2005 and 2006. Studies on the classification of serogroups by PAGE have indicated that group D rotaviruses are the most frequently reported group in United Kingdom flocks. Furthermore, group D rotavirus infection has recently been implicated as a contributing factor to the development of RSS in 5- to 14-day-old broilers in Germany. The A, F, and G groups have also been detected in broiler flocks [36, 43]. In Brazil, a study identified nine distinct electropherogroups using PAGE, but only three were similar to the A group profile of avian rotavirus. More recently, rotavirus was also detected in 45.3% of chicken and layer flocks in Brazil, and approximately 15% of these samples were identified as belonging to group A. In a previous study using the same RT-PCR test for the NSP4 gene, four different genotypes of rotaviruses were detected in samples from commercial turkeys, reflecting the great genetic variability of rotaviruses similar to that reported in humans and other mammals.

Parvoviruses are known to cause gastrointestinal disease in mammalian species and have been implicated as a cause of malabsorption syndrome in chickens and enteritis in turkeys [9, 44]. However, the role of chicken parvoviruses in disease has not yet been determined. Previous studies using electron microscopy have identified parvovirus-like particles in the samples of chickens with enteric disease, but it was not possible to confirm the presence of parvoviruses until the development of new molecular tools such as PCR. In this survey, 12.1% of the chickens were positive for chicken parvovirus, which is slightly more than those which were positive for reovirus and adenovirus and indicates that chicken parvovirus should be considered as an important etiological agent of enteric disease in chicken. In a nationwide survey in the United States, a high prevalence of chicken parvovirus (77%) was detected in 54 chicken samples.

Fowl Adenovirus subgroup I had a frequency 9.6% positive samples among the viruses investigated in this study (Table 3). The subgroup I and II adenoviruses are considered widely distributed in poultry and are commonly found in enteric samples [10, 47]. Although the association between adenovirus and disease is well established for subgroup II (turkey hemorrhagic enteritis and related viruses and subgroup III (egg drop syndrome)), the role of most subgroup I avian adenoviruses as pathogens is not well defined.

Avian reoviruses are frequently detected in the intestinal tracts of poultry with enteric disease and are widely distributed in poultry. Avian reoviruses were identified in this study of lowest frequency with only 7.9% (Table 3). Avian reoviruses were identified in 62.8% of the chicken flocks tested from all regions of the United States during 2005 and 2006. Avian reoviruses have been isolated from a variety of tissues in chickens that are affected by assorted disease conditions including viral arthritis/tenosynovitis, respiratory disease, immunosuppression, and enteric disease or malabsorption syndrome. Moreover, avian reoviruses have also been demonstrated to cause a synergistic effect and enhance the pathogenicity of other agents, such as chicken anemia virus, Escherichia coli, and infectious bursal disease virus. However, the severity of the effects depended on the strain of reovirus. A clear relationship is frequently reported between arthritis/tenosynovitis and reovirus infection. However, the role of avian reoviruses in enteric disease remains unclear, when we consider other primary pathogens associated with enteric problems in chickens [49, 50].

The association between viruses and age showed that the viruses could be detected at different stages in the broilers, breeders, and pullet/layer hens; for example, virus was detected from the first week of age to the last week, especially in broilers. An important finding was the detection of CAstV in one-day-old breeder chicks, which may constitute an indicator of vertical transmission. According to McNulty et al., in a longitudinal study carried out in young poultry flocks, the avian rotavirus is normally detected after two weeks of age because of the modulation of the passive maternal immunity. However, Tamehiro et al. reported samples that were positive for rotavirus in one-week-old broiler chickens. Furthermore, Pantin-Jackwood et al. demonstrated the presence of rotavirus in samples collected from poults before placement, in addition to the presence of astrovirus throughout their lifetime.

In conclusion, the primary viruses detected in this study were the IBV, ANV, CAstV, and rotavirus, followed by chicken parvovirus, adenovirus, and reovirus. However, the astroviruses (ANV and CAstV) should be regarded as the most important pathogens because coronavirus (IBV), although present in a higher percentage of samples, has not been demonstrated to cause pathogenicity in the enteric tract in our previous experiments (data not published). Detection of virus was high among chickens with and without clinical signs of disease, which demonstrates the high prevalence of these viruses in asymptomatic carriers and indicates that these carries may represent a potential source of infection. Moreover, the presence of CAstV in one-day-old breeder chicks that was detected in this study may suggest a vertical transmission. In addition, the injuries that these viruses cause can interfere with the absorption capacity of the intestinal tract during the first weeks of life of these birds, which enhances the negative impact on productivity for the rest of the production cycle. This is the first study to demonstrate the presence of these viruses in association with enteric disease in Brazil. Experimental challenges should be conducted to establish the role of these viruses and to analyze their impact on commercial chicken productivity.

The relationship between co-infection with two or three viruses and the health status of turkeys was identified in χ2 statistics (P = 0.046, φ = 0.26). The health condition of turkeys deteriorated with an increasing number of viruses; the OR =2.98 indicates that the odds of PEC symptoms in turkeys infected with one virus are almost threefold higher (and above fourfold higher for PEMS symptoms; OR =4.13) than the odds of occurrence of these symptoms in the uninfected turkeys. Additionally, the odds of PEMS symptoms (in relation to PEC symptoms) in turkeys infected with two viruses are about 1.27-fold higher than the odds of occurrence of these symptoms in birds infected with one virus and they are about 1.39-fold higher in turkeys infected with one virus than in the uninfected turkeys. Similarly, the CA diagrams also indicated that healthy turkeys had no virus infection, and infections with two or three different viruses most commonly resulted in disease symptoms. The second logistic regression model also indicated that the odds of disease symptoms in turkeys infected with one virus are more than 3.5-fold higher than in turkeys free from these infections (OR =3.61, P = 0.01) and that younger age of turkeys is a factor stimulating illness. The OR =2.75 (P = 0.04) indicated that the odds of PEC or PEMS symptoms in turkeys up to 4 weeks of life are more than 2.5-fold higher than in turkeys 5 to 12 weeks of age.

Epidemiological investigation by the Centre for Health Protection of Hong Kong showed that five other members of staff at the NTNAMC were hospitalized for respiratory tract infection, with onset of symptoms from November 6–24, 2012. The details of the five other patients have been reported previously.

Astroviruses (AstVs) are non-enveloped, positive-sense, single-stranded RNA viruses belonging to the Astroviridae family. Currently, two genera: namely Mamastrovirus and Avastrovirus are distinguished within this family. The genus Mamastrovirus includes astrovirus species isolated from humans and a number of mammals. Isolates originated from avian species, such as turkey, chickens, ducks, and other birds are classified into the genus Avastrovirus1, 2. AstVs have been detected in humans and a variety of animal species, including non-human primates, other mammals and avian species3–5. Their genomes are 6.8–7.9 kb in length, consisting of a 5′-untranslated region (UTR), three open reading frames (ORFs), a 3′-UTR and a poly (A) tail6. The high degree of genetic diversity among AstVs and their recombination potential signify their capacity to cause a broad spectrum of diseases in multiple host species3, 7, 8. Human classical AstVs are a frequent cause of acute gastroenteritis in young children and the elderly, occasionally with encephalitis8.

In poultry, AstV infections have been found to be associated with multiple diseases, such as poult enteritis mortality syndrome, runting-stunting syndrome of broilers, white chick syndrome, kidney and visceral gout in broilers and fatal hepatitis of ducklings, leading to substantial economic losses9–16. Increasing evidence indicates that there is a high degree of cross species transmission of AstVs between domestic birds, and even the potential to infect humans17. By comparison, fewer AstV infection cases have been described in domestic goose flocks. Bidin et al.18 reported the detection of avian nephritis virus infection in Croatian goose flocks and provided evidence that this AstV was associated with stunting and pre-hatching mortality of goose embryos. Studies to detect AstV genomes from the clinical samples of geese suggested that these viruses might distribute widely among goose flocks, as seen in other poultry flocks19, 20. In February 2017, an outbreak of disease was reported in a goose farm in Weifang, Shandong Province, China. Affected flocks (containing 2000–3000 goslings) experienced continuous mortality rates ranging from 20 to 30% during the first 2 weeks of the outbreak despite antibiotic and supportive treatment. We conducted a systematic investigation to identify the causative agent of this disease and report here the isolation and characterization of a genetically distinct avian AstV. The pathogenicity of this virus was evaluated by experimental infection of goslings.

To the best of our knowledge, this is the first report of a co-infection of psittacine birds with avian adenovirus and C. psittaci associated with an outbreak of human psittacosis. In this study, we have identified a novel adenovirus that was most closely related to Duck adenovirus A of the Atadenovirus genus in the epidemiologically linked Mealy Parrots. In contrast, this novel adenovirus was not identified in any of the healthy parrots and other detained animals without C. psittaci infection. Psittacine adenovirus HKU1 antigen was detected in lung and liver tissue cells using immunostaining, which indicated active viral replication instead of latent infection. A positive correlation between adenovirus viral loads with C. psittaci bacterial loads was observed in lung specimens, which suggested a possible synergistic interaction between adenovirus and C. psittaci in disease pathogenesis.

In birds, many adenoviral infections are subclinical, but some infections can lead to severe disease,. Previous studies have suggested that avian adenoviruses may cause immunosuppression in birds. Aviadenovirus can lead to immunosuppressive disease, such as the hydropericardium syndrome. Fowl adenovirus and chicken anemia virus co-infection causes much more severe disease than either virus alone. Avian adenoviruses have been shown to directly infect lymphocytes and dendritic cells in the spleen and causes depletion of lymphocytes,. Fowl adenovirus 1 has been shown to affect antibody response of chicks to Brucella abortus

[38]. We speculate that our novel adenovirus may have caused immunosuppression among the infected parrots, and therefore a larger number of Mealy Parrots were infected by C. psittaci with a higher bacterial load, leading to a higher chance of zoonotic transmission to humans. Other studies have also proposed that reovirus and avian pneumovirus infection may cause immunosuppression leading to avian chlamydiosis,. Experimental infection showed that avian pneumovirus could exacerbate acute C. psittaci infection in turkeys. Alternatively, in a report demonstrating adenovirus-C. psittaci co-infection in a parakeet using electron microscopy and antigen detection without molecular confirmation, Desmidt M et al proposed that C. psittaci could cause immunosuppression, which can lead to reactivation of latent adenovirus infection. However, no further studies have been performed to verify this hypothesis.

In psittacine birds, adenovirus infection manifests as depression, anorexia, diarrhea and cloacal hemorrhage. Gross examination may show hepatomegaly, splenomegaly, dilatation of the duodenum and proventriculus, swollen kidneys, and edema, congestion, and hemorrhage of the lungs. Histological changes may include necrosis in the liver and the spleen. A case of inclusion body hepatitis has been described. The pathological features in our infected parrots were typical of C. psittaci infection, but some of these are actually indistinguishable from adenovirus infection as one Mealy Parrot was also positive for the Psittacine adenovirus HKU1 on immunofluorescence staining.

Current ICTV guidance on adenovirus taxonomy specifies species designation by phylogenetic distance, host range, DNA hybridization, nucleotide composition, cross-neutralization and gene organization at the right end of the viral genome. With the exception of nucleotide composition and cross-neutralization, for which no data are available, our data and analysis of the Psittacine adenovirus HKU1 are consistent with it being a novel member of the genus Atadenovirus. Notably, the genome of the Psittacine adenovirus HKU1 has a higher G+C content than other Atadenovirus genomes, which might be viewed as a feature against its designation as a novel Atadenovirus. Indeed, the high A+T content of the first few identified Atadenoviruses was considered characteristic among adenoviruses and was the basis for naming the genus. Nonetheless, the snake adenovirus 1, a member of the Atadenovirus, has a genomic G+C content of 50.2%, which is lower than but comparable to the G+C content of 53.5% for the Psittacine adenovirus HKU1. Hence, we consider that the high genomic G+C content of the present Psittacine adenovirus HKU1 is not a major contraindication to its inclusion in the genus Atadenovirus.

Besides the genomic G+C content, there are several unusual genomic features in the Psittacine adenovirus HKU1. Firstly, a second viral protein, fiber-2, is present in our novel adenovirus, but not in any other Atadenovirus. The fiber-2 protein has also been found in the Aviadenovirus, like the fowl adenovirus. Since fiber proteins are responsible for binding to cellular receptor, it has been postulated that the presence of different fiber proteins may determine the tropism of the adenovirus. It remains to be determined whether the fiber-2 protein is important for the virus to infect psittacine hosts. Another interesting feature is the predicted CCU start codon of the IVa2 protein. The annotation is made on the basis of sequence conservation among other adenoviral IVa2 proteins, and the lack of an in-frame ATG in its vicinity. The function of the non-canonical CCU start codon may be augmented by the presence of an upstream Kozak sequence ACCACC. The use of CCU as the start codon is rare in other organisms, but has been reported in psittacine viruses. The other notable genomic feature in the Psittacine adenovirus HKU1 is the intron between the two exons of 33K, which usually overlaps with coding sequences in other adenoviruses, but not in the present case.

Virus and bacteria often act synergistically in causing diseases in humans or animals. C. psittaci-avian pneumovirus co-infection has been associated with an outbreak in turkeys, C. psittaci-fowlpox virus co-infection with an outbreak in hens and C. psittaci-reovirus with an outbreak in budgerigars. Infectious bursal disease virus and chicken anaemia virus can cause immunosuppression, leading to secondary bacterial infection such as bacterial chondronecrosis with osteomyelitis. However, it is unclear whether co-infection in birds can increase the risk of transmission of avian pathogens to humans. Our investigation suggested that the novel Psittacine adenovirus may have been associated with immunosuppression among infected birds, leading to higher C. psittaci bacterial loads in the lungs of psittacine birds, and hence increasing the risk of infection in exposed humans.

At present, the emergence of new pathogens and the continuous circulation of common etiological agents in dogs have made canine diseases more complex and difficult to diagnose. Dog infectious diseases mainly include respiratory and intestinal viral diseases, including CRV (CAV-2, CDV, CIV and CPIV) and CEV (CAV-2, CanineCV, CCoV and CPV). However, the traditional methods of virus identification and isolation are time consuming, causing delays in treatment initiation. A few methods for detecting virus-induced respiratory or enteric disease have been developed [4, 27, 28, 34], but no previous study had developed a systematic way to detect both CRV and CEV in dogs. Here, we developed two mPCR methods for detection of the most frequently coinfected viruses; these methods could be performed to diagnose dogs according to their clinical symptoms.

Primer design is the first and most important step in the process of establishing a detection method, and the following conditions must be satisfied: primers were designed to bind to conserved sequence regions, to have similar annealing temperatures, and to lack dimers or hairpin structures. In these novel mPCR methods, the primer combination produced amplicons that were easy to distinguish from each other, the primer annealing temperatures were similar, and degenerate bases were required only infrequently. The specificity, sensitivity and reproducibility tests all showed good results.

The mPCR methods were tested on 20 NS and 20 AS samples collected from dogs with symptoms of respiratory disease or enteric disease. The ratio of positive samples to total samples was 80% (16/20) for CRV detection and 85% (17/20) for CEV detection. Because the sample number was insufficient, these results were not statistically significant. However, CPV and CDV clearly remain two of the more serious and epidemic diseases in dogs in worldwide at present [35–38]. Epidemiological monitoring of CPV is particularly important because CPV evolves at a rapid rate, similar to that of Porcine Circovirus 3 [39, 40]. Because a small number of dogs were negative for the viruses tested by the CRV or CEV detection assays, although they suffered respiratory illness or intestinal problems, we suggest that some viruses with low prevalence and pathogenic bacteria may also cause disease in dogs [2, 41]. A variety of pathogenic bacteria are often present along with viruses in canine infections [42, 43], and thus, it is essential to expand the coverage of mPCR detection in the future. For example, CIRD also include CHV-1, canine reovirus, and Bordetella bronchiseptica and so on. At the same time, other pathogens causing serious zoonotic diseases, such as pseudorabies virus, should also be monitored in future [44, 45].

In this study, the detection of CanineCV was added to an mPCR method for the first time, because coinfection of this pathogen with other pathogens is common. Though the pathogenic mechanism of CanineCV is unclear, epidemiological testing is important for future research. CanineCV was not detected from the AS clinical samples; perhaps the limited source of these clinical samples was responsible for this result. We didn’t get a lot of clinical samples because it was not easy to get disease samples. CAV-2 mostly replicates in the lower respiratory tract and was detected in the NS samples; however, the CAV-2 primer pair used in this study was probably able to amplify the CAV-1 DNA virus despite the optimization performed. Notably, the live vaccine strains used may have an unavoidable impact on disease detection using the methods developed in this study. Additionally, discriminating between wild-type infections and vaccines is important, and therefore, a trend exists toward later development of broad-spectrum and accurate mPCR detection methods. Sometimes, cross contamination may lead to experimental failure. It is worth noting that PCR pretreatment and post-treatmen performed in different isolation zones can effectively avoid pollution. Besides, regular air spray cleaning will also play a role.

In conclusion, these newly established mPCR methods provide an efficient, sensitive, specific and low-cost testing tool for the detection of CRV (CAV-2, CDV, CIV and CPIV) and CEV (CAV-2, CanineCV, CCoV and CPV). The use of Taq Master Mix makes the detection process more convenient and reduces the chance of contamination during the process of sample addition; PCRs can be initiated by simply adding enzyme, ddH2O, premixed primers, and template, and thus, this method is superior to other mPCR detection methods. Here, detection of CanineCV was added to mPCR for the first time, making this method suitable for the further study of coinfection by CanineCV and other pathogens. This study provides a novel tool for systematic clinical diagnosis and laboratory epidemiological surveillance of CRV and CEV among dogs.

The World Health Organization (WHO) Global Polio Eradication Initiative (GPEI) was established in 1988 and successfully prevented wild-type poliovirus (WPV) transmission in the Americas, the Western Pacific (WPR), and Europe (EUR) [1–3]. The Southeast Asia Region (SEAR), home to a quarter of the world's population, was also certified polio-free in March 2014. WHO certified Taiwan, along with the entire WPR, as polio-free in 2000 and Taiwan changed its immunization strategy from oral (OPV) to inactivated polio vaccine (IPV) in 2010. To date, WPV remains endemic in Afghanistan, Nigeria, and Pakistan. Numerous outbreaks in heretofore polio-free regions have been reported recently in China (2011), Somalia (2013), Ethiopia (2013), and Kenya (2013) caused by importation [5–7]. Besides WPV, cases of circulating vaccine-derived poliovirus (cVDPV) causing acute flaccid paralysis (AFP) have risen since 2000, and have been identified in eight countries in 2013 and in two countries in May 2014.

Normally, acute flaccid paralysis (AFP) surveillance is the gold standard for poliovirus surveillance in eradication initiatives; under certain circumstances, environment surveillance is also employed to monitor the circulation of poliovirus in populations in order to better understand its evolution and transmission [9–13]. For instance, although certified as polio-free in 2002, Israel isolated WPV in routine environmental sewage samples in early February 2013, and immediate steps were taken to implement national supplementary immunization with OPV to prevent its spread. Recently, the WHO included environmental poliovirus surveillance in a new strategic plan as part of its global eradication initiative to supplement AFP surveillance. In Taiwan, AFP surveillance has long been established for poliovirus surveillance of the population, but environmental surveillance is not routinely performed.

Besides poliovirus in populations, enteroviruses, adenoviruses, reoviruses, and noroviruses are often found in environmental raw sewage [16–19]. These groups of viruses can cause a broad range of asymptomatic to severe gasterointestinal or respiratory infections, or even more acute conditions such as meningitis and paralysis, thus constituting a considerable public health problem in the community. Among these fecal-oral viral pathogens, reovirus is usually the most abundant virus detected in environmental water [22, 23]. Mammalian orthoreovirus (MRV), which belongs to the family Reoviridae and the genus Orthoreovirus, are a common class of enteric viruses capable of infecting a broad range of mammalian species, including humans. Previous studies have indicated that reoviruses have a high endemic infection rate in humans and seroconversion was found in more than 70% of 4-year-old children. Although reovirus infection in humans usually induces mild respiratory or gastrointestinal symptoms, there are reports of human reovirus-associated neurological disease [26, 27]. Several studies also described the isolation of reovirus strains directly from cerebrospinal fluid (CSF) or neural tissues obtained from patients with meningitis or encephalitis [28–31]. In addition, immunocompromised, pediatric, and elderly populations may become susceptible to severe bacterial respiratory disease due to an initial reovirus infection [32, 33].

In response to the international threat of WPV importation and the changes to the national vaccination policy, we adopted the WHO guidelines for environmental surveillance of circulation in Taiwan. Two-phase Dextran 40/Polyethylene glycol (PEG) separation and cell culture were performed to monitor environmental viral circulation. We successfully isolated enteroviruses, adenoviruses, and mammalian orthoreoviruses, but no poliovirus was detected in sewage collected islandwide. Our results showed a high incidence of MRV, which may cause human disease, and thus further research is warranted.

Immunosuppression is a common condition in intensive breeding, where stress factors are diverse and constantly present. The pressure supported by the immune system of birds can have several origins: environmental, management, nutritional, infectious, and parasitic. Transient or permanent immunosuppression induces considerable economic losses in terms of performance, secondary infections, mortality, vaccination failures, condemnation in slaughterhouse, and poor animal welfare conditions.

Viruses-induced immunosuppression in turkeys is a major cause of decrease in profitability. Despite the knowledge of many features of virus’s effects on immune system of birds, several molecular and immunological aspects are still unclear. The interaction between immunosuppressive viruses and other stressors is not yet well explained. On the other hand, some immune mechanisms, particularly related to cellular mediated immune response, due to viral infection are insufficiently explored and clarified.

Diagnosis and evaluation of immunosuppression due to viral diseases are based on field and laboratory criteria. The role of avian veterinarians is fundamental in term of early detection of immunosuppression. They will be challenged with emergent viruses and immunosuppressive in particular, in turkey industry.

Preventing of immunosuppression needs an integrate approach, where the research development and the field observations play an important role in the refining of turkey industry strategies for a better and efficient controlling programs. The maintaining of appropriate management, environmental, and nutritional conditions is essential to minimize stressors. Application of strict biosecurity and vaccination programs of breeders and their progenitor, against immunosuppressive and other major diseases are currently practical and feasible measures to prevent introduction and propagation of pathogens and enhance the quality of life for animals. In addition, development of new controlling methods, bases on novel generation of vaccines, administration of cytokines and genetic resistance, is still being tested despite the promoter results relative to increase in disease resistance of birds.

To investigate whether the GD isolatewould be the causative agent for the ongoing goslings gout in China, 5-day-old goslings were infected with GD. All the infected goslings showed clinical signs with depression and started to excrete white feces at day 3 post infection, and grew slower than the non-infected control goslings as described in Fig. 4a. At day 7 post infection, three inoculated goslings were killed and the tissues were collected. At necropsy, the typical gout pathological changes were found, including kidney with swelling and white substance-filled ureters. The histopathological assay also demonstrated the similar pathological signs to that of naturally infected goslings, including necrosis and abscission of renal tubular epithelial cells, presence of protein cast in renal tubules (Fig. 4c). Moreover, the virus could be efficiently isolated from the liver, spleen and kidney of the infected goslings at day 7 post infection, but not from the non-infected control goslings as described in Fig. 4b. Although all the infected goslings had clinical signs and histopathological changes associated with gout, all these infected goslings were survival during 2 weeks observation post infection. In addition, the neutralizing antibody titer against GD from the infected goslings could reach 1:6400 at day 14 post infection. All these data demonstrated that the GD isolate could efficiently infect the goslings and cause diseases associated with gosling gout.

Bats (order Chiroptera) are natural reservoirs for zoonotic viruses that cause some of the deadliest diseases in humans, including filoviruses (such as Ebola and Marburg viruses), lyssaviruses, severe acute respiratory syndrome (SARS)-related coronaviruses and henipaviruses (e.g. Hendra and Nipah viruses) [1–3]. Despite being hosts to such an array of pathogens, bats generally show mild or no clinical symptoms to their presence, a phenomenon that is largely a mystery and a potential biomedical treasure trove that could offer new insights into the treatment and control of such pathogens in humans and affected animals. The lack of illness does not mean that bat cells are not infected by such viruses. Bat cells are susceptible to infections with paramyxoviruses and filoviruses, and show varying degree of permissiveness to virus replication, which is a pre-requisite for the hosts to acquire carrier status. Bat lung epithelial cells (TB1-Lu) of Tadarida brasiliensis display resistance to reovirus infection; infected cells show no cytopathic effects and rapid decline in virus production; however, low virus release is maintained for at least 2 months. Murine encephalomyocarditis virus, in contrast, causes severe cytopathic damage in TB1 Lu cells, and Ebola virus shows persistent infection in such cells.

Recently, two novel influenza viruses, H17N10 and H18N11, were identified in bats by deep sequencing analyses (although live viruses have not been directly isolated) which have understandably caused much speculation about their zoonotic potential. These viruses are, however, highly divergent from conventional mammalian and avian influenza A viruses. Chimeric virus housing the six core genes from bat H17N10 virus replicated well in human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Furthermore, the chimeric bat virus failed to reassort with conventional influenza viruses in MDCK cells. Bat viral ribonucleopolymerase (vRNP) complex subunits (PB1, PB1 and PA) were not functionally interchangeable with corresponding human virus-derived vRNP subunits suggesting there is limited reassortment potential between bat and human influenza viruses. However, vRNP from bat H17N10 virus is able to drive with high efficiency the non-coding region of human H1N1 virus (A/WSN/1933) in vRNP minigenome reporter assays, highlighting the possibility of viable reassortment between bat and human influenza viruses. Although the issue of functional reassortment between native bat and conventional influenza A viruses has not been fully resolved, its likelihood is presently considered low.

Single-cycle green fluorescent protein (GFP) reporter virus (human A/WSN/33) was variably able to infect all eleven bat cell lines, derived from seven bat species. Similar number of infected cells were found among all seven bat cell lines by immunocytochemical detection of viral nucleoprotein (NP). Human virus-derived vRNP complex was shown to perform better than avian virus-derived vRNP complex in the same A/WSN/33 viral backbone at progeny virus release, based mostly on the use of TB1-Lu bat cells, which appear inherently resistant to influenza virus infection. Although there is limited potential for reassortment between human and bat influenza viruses, Pteropus alecto kidney cells were able to produce reassorted progeny from human H1N1 (A/WSN/1933) and highly pathogenic avian influenza (HPAI) H5N1 (A/Vietnam/1203/04) viruses. Collectively, these findings appear to indicate that bat cells are susceptible to infection with conventional mammalian and avian influenza viruses. However, we are unclear about the relative permissiveness of bat respiratory epithelial cells to conventional influenza viruses in the production of viable progeny. Although bats are not known to act as hosts for human and avian influenza viruses, the potential epidemiological significance of avian influenza virus infection in bats was highlighted by the recent discovery that around 30 out of 100 free ranging Eidolon helvum (fruit bats) in Ghana were serologically positive for avian H9 virus.

We report here on the relative susceptibility of lung epithelial cells from three diverse bat species, T. brasiliensis (a medium insectivorous bat), E. helvum, (a large fruit bat) and C. perspicillata (a small mainly fruit, and insect eating bat), to avian and human influenza A viruses. We found that all three species of bat cells were more resistant than control Mardin-Darby canine kidney (MDCK) cells, in terms of reduced progeny virus production and higher cell viability, which appeared not to depend on JAK/STAT signalling. Although the three species of bat cells showed variation in resistance to infection, they were relatively more permissive to avian than human influenza viruses which could be important in the ecology of avian influenza viruses.

A throat swab was taken from the patient at the time of his first clinical examination and transported in viral transport medium (VTM) to the National Public Health Laboratory for virus isolation. The sample was treated with antibiotics (C. penicillin 100,000 I.U./ml and streptomycin 100 µg/ml) for an hour before being inoculated in duplicate (100 µl and 200 µl, respectively) into freshly confluent monolayers of MDCK (ATCC, CCL-34), Vero (ATCC, CCL-81) and Hep-2 (ATCC, CCL-23) cells cultured in a 24-well tissue culture plate. The plate was incubated at 37°C in 5% CO2 and examined daily for the presence of CPE in cultured cells. Supernatant from cultures with visible syncytial cytopathic effect (CPE) after 3 days was taken for further analysis by serial passage in different cell lines available in the laboratory.

The investigation conducted in this study was approved by the ethics committee of the Malaysian National Public Health Laboratory. All patients (subjects) in this manuscript have given written informed consent (as outlined in the PLoS consent form) to publication of their case details. No identification of the subjects is to be revealed in any publication.

It has been reported that avastrovirus infections can cause enteritis, diarrhea, hepatitis of ducklings, nephritis of chickens, and decrease of egg-hatching rate of different fowls7,10–17. Based on the amino acid sequences of the ORF2-encoded capsid protein, these AAstVs can be divided into three species: Avastroviruses 1, 2, and 3 (The International Committee on Taxonomy of Viruses, ICTV, 2016). In this study, a novel goose astroviruses GD was identified and isolated from goslings with gout symptom. The infection study further confirmed that the GD belonging to Avastroviruses 3 could be the causative agent of the ongoing gosling gout disease in China. Although CAstVs could induce gout disease in chickens7, CAstVs have only about 35.1–35.8% homology with GD on amino acid sequence of capsid, and there is no recombinant event happened between them, which indicate that the GD is an under recognized novel astrovirus. In comparison with the goose astrovirus recently identified in geese with enteritis in China8, the novel goose astrovirus showed only about 60% identity in nucleic acid. This highlights at least two species of goose astrovirus associated with different diseases are circulating in goslings flocks in China.

The infection study with GD could reproduce the clinical signs and histopathogenesis associated with the gout disease, but all the infected goslings were survival during 2 weeks observation post infection. The high morbility and low mortality for the infection study with GD might be related with the infection route and dose, and the age. Although the pathogens including fowl adenovirus, goose hemorrhagic polyomavirus, goose reovirus, goose coronavirus, goose parvovirus, and duck Tembusu virus were not able to be detected in the diseased goslings, we could not exclude the potential co-infections with other pathogens, which may exacerbate the clinical symptoms of gout. In addition, whether the high protein level in the feed promotes the occurrence of gout also need to be further investigated. The high-titer neutralizing antibody to GD from the survival geese highlighted the novel goose astrovirus could efficiently induce enough adapt immunity in goslings. However, whether this adapt immunity can protect goslings completely from the novel goose astrovirus need to be further studied.

The mechanism for the gout disease induced by either CAstV reported by Bulbule et al. or GD identified in this study need to be further elucidated. Moser et al. reported that astroviruses could increase the permeability of the epithelial cells18. The increased permeability of the kidney epithelial cells induced by the astroviruses might contribute to the gout disease7. In addition, due to lacking arginase in poultry, ammonia cannot be processed into urea instead of purine, hypoxanthine, and xanthine, then oxidized to uric acid, forming sodium urate and calcium urate, excreted through kidney finally. If the rate of urate formation is greater than the excretory capacity of the urinary organs, gout can be caused with the urate deposits on visceral surfaces19. Therefore, factors that cause renal and urinary tract injury or urine concentration and urinary excretion disorder can promote the formation of urate deposits. In this study, the novel astrovirus GD could cause kidney damage, which maybe the main cause of gosling gout in China.

In summary, this is the first demonstration of a gout-disease associated novel goose astrovirus efficiently isolated by LMH cells. The specific sequences of the astroviruses detected in samples from the five different provinces demonstrated the widespread of the novel goose astroviruses in China. However most astrovirusesare difficult to grow in cell culture20, the novel goose astrovirus GD could be isolated efficiently in LMH cells in this study, which provides an efficient proliferation system for the avastrovirus in vitro. This efficient system might provide a good tool for developing inactivated vaccine and investigating the pathogenicity mechanism of goose astrovirus in the future. The full genomic RNA of the novel goose astrovirus was sequenced and publicated in Genbank,which pave ways for further developing vaccines and diagnostic methodsin controlling the gout disease in goslings. During the preparation of the revised version for this manuscript, other groups in China recently also reported the novel goose astrovirus21–23. Different from those recently reported by other groups, we efficiently isolate such novel goose astrovirus in vitro using the cell culture system (LMH cells) and reproduce the associated gout disease by inoculating the cell cultured virus GD. Of course, the originator, host range, variation, molecular pathogenesis, and potential zoonotic infection of the novel goose astrovirus need to be further studied.

Acute respiratory infections are a major global health problem responsible for about 3.9 million deaths worldwide each year. These infections are of the top five causes of mortality worldwide and the leading cause of mortality among children under five years of age in many developing countries. Acute respiratory infections are most often caused by viruses. Over 200 viral serotypes are associated with human respiratory diseases including Influenza A and Influenza B virus, respiratory syncytial virus (RSV), parainfluenza virus (PIV), human adenovirus (HAdV), human coronavirus (HCoV), human rhinovirus (HRV), human metapneumovirus (HMPV) and human bocavirus (HBoV). In addition, two human polyomaviruses (HPyV), KIPyV and WUPyV, have been detected in patients with respiratory infections. These infections affect all age groups, but nearly all severe episodes occur in children under five years, the elderly and immunocompromised individuals (e.g., HIV-infected patients). In adults, viral respiratory infections are the cause of 30%–50% of pneumonia cases, 80% of asthma complications and 20%–60% of chronic obstructive pulmonary disease exacerbations. Consequently, common viral respiratory infections cause a greater economic burden than many other clinical conditions in terms of medical expenses and productivity losses. The World Health Organization has supported the monitoring of acute respiratory diseases worldwide since 1977.

The Influenza virus belongs to the Orthomyxoviridae family and causes respiratory infections in about 20% of the global population every year. The 1918 flu pandemic was caused by Influenza A subtype H1N1 and killed 50 million people around the world. The Asian Influenza caused by Influenza A subtype H2N2 occurred in 1957 and the Hong Kong Influenza caused by Influenza A subtype H3N2 took place in 1968 and made far fewer victims than the 1918 Spanish flu. About 70 people died in Asia in 2004–2005 due to the H5N1 strain of avian flu. The 2009 flu pandemic (swine flu) was the second pandemic involving a strain of Influenza A virus. It was classified as Influenza A H1N1 2009 and the genetic material originated from three different species: human, avian and swine. The chemotherapy or prophylaxis of Influenza infections comprises agents blocking the Influenza A virus M2 proton-selective ion channel (amantadine, rimantadine) and neuraminidase inhibitors (zanamivir, oseltamivir, laninamivir, peramivir). Both classes can induce virus resistance and therefore there is an urgent need to develop new antiviral agents with novel mechanisms of action. An alternative concept has recently emerged and it is based on the idea of designing new molecules targeting host cell factors that are hijacked by the virus during its replication. Host-targeting antivirals are an alternative strategy for addressing host structures involved in the virus life cycle. This type of inhibitors could exhibit a significantly greater barrier for selecting drug-resistant viruses and, in addition, display broad-spectrum antiviral activity when interacting with a cellular target common to several viruses. The host factor-directed antiviral therapy is recently studied. This is increasingly recognized as a relevant approach to combat viral resistance and provides broad-spectrum antiviral agents.

Many studies are currently being developed to find new Influenza inhibitors. Tatar et al. synthesized 2-phenylamino-1,3,4-thiadiazole derivatives 45–48.

The antiviral activity against some respiratory viruses such as Influenza A H1N1, Influenza A H3N2, Influenza B, Parainfluenza-3, RSV, Reovirus-1and Feline Coronavirus was investigated and the results are summarized in Table 5. No activity was observed at the highest concentration tested or at subtoxic concentration against Influenza B and RSV.

The in vitro antiviral assay showed N-{3-(methylthio)-1-[5-(phenylamino)-1,3,4-thiadiazol-2-yl]propyl}benzamide 45 as an Influenza A H3N2 virus subtype inhibitor. With an EC50 value of 31.4 μM, the derivative 45 was the most potent among the tested compounds and moderate active compared to standard drug oseltamivir, but a promising scaffold for future developments. Derivatives 47 and 48 exhibited activity against Parainfluenza-3 and Reovirus-1 and probably the thiourea moiety favors antiviral activity on these strains (Figure 5).

The session on hepatitis virus infection and liver cancer provided a research update on: the molecular virology and immunology of HBV; host factors related to hepatitis virus infection; the genetic landscape of virus-associated hepatocellular carcinoma (HCC); integrated genomics to identify drivers of human liver cancers; chemical-viral interaction between aflatoxin and HBV in induction of HCC; and antibody therapeutics targeting glypican-3 (GPC3) for the treatment of liver cancer. Through an extensive clinical network of patients in China, randomized clinical trials are being conducted to study new treatment strategies for chronic HBV infection involving nucleotide analog reverse transcriptase inhibitors and peginterferon. An ongoing effort to prevent mother–child transmission of HBV through a short-term antiviral therapy with tenofovir disoproxil fumarate during late pregnancy, reported a significantly lower HBV transmission rate compared to the control group. This session also included a discussion of host factors related to hepatitis virus infection using a genome-wide association study (GWAS). A genetic analysis using GWAS, identified host factors for various human multifactorial diseases, as well as interferon (IFN) lambda for drug response, and HLA II genes for susceptibility to chronic HBV infection (CHB). Frequencies of HLA-DP risk alleles are high in Asian populations, whereas frequencies of HLA-DP protective alleles are high in European populations. These findings could explain the high incidence of CHB in Asian countries and suggest that host genetic factors are important to viral infections. Another talk outlined the genomic and epigenomic associations in HBV-related liver cancer using data obtained from whole genome bisulfate sequencing and whole genome sequencing. Clonal HBV integrations preferentially occurred in inactive chromatin regions; massive rearrangements were detected in the integrated HBV genome, and a negative correlation exists between HBV rearrangement number and total somatic mutation number. These observations could be useful for understanding the progression of HBV-related liver cancer. It was noted that liver cancer, including HCC and cholangiocarcinoma, is the second leading cause of cancer death (about 9.1% of total cancer deaths) with a significant burden in low- and middle-income countries in Asia and sub-Saharan Africa. Etiological factors associated with HCC include infection with HBV and exposure to high levels of aflatoxin B1 (AFB1) in the diet. HCV-associated HCC is becoming the most rapidly rising solid tumor in the United States and Japan. The development of highly effective drugs that cure HCV infection is a major advance that, hopefully, will diminish the role of HCV in liver cancer. Ongoing clinical investigations are defining the utility of GPC3, a cell surface proteoglycan differentially expressed in HCC, and other promising antibody therapeutics to treat liver cancer.

Pyridone 6 (Merck), a JAK inhibitor, was applied at 5 μM to cells for 20 h prior to infection at 37 °C. Cells were rinsed twice with PBS and then infected with USSR H1N1 virus at 1.0 MOI. DMSO treated cells were infected as controls. After 2 h infection, cells were rinsed three times with PBS and fresh infection medium was added with corresponding inhibitor and incubated for a further 22 h before virus titration on MDCK cells using spun supernatants from infected cells.

Effective DNA vaccine delivery is required to induce a strong and long-lasting immune response that can produce high and sustained levels of antigen production at targeted sites. Delivery routes of DNA vaccines can be generally grouped into those that are mucosal or systemic. Relative proportions of different administration routes of inoculation in poultry were calculated from the data summarized in Table 1 and presented in Figure 1B. The most extensively used routes for the delivery of poultry DNA vaccines include IM (55%), oral (23%), in ovo (IO) (11%), eye drop (ED) (4%) and intranasal (IN) (3%) (Figure 1B). Although some new delivery methods and routes are under development or being tested in poultry, conventional IM injection is still considered the dominant DNA vaccine delivery route. The majority of poultry DNA vaccines (approximately 55%) were applied as naked DNA through IM injection into the leg, chest or thigh muscles of poultry, and some promising results have been obtained. Full protection against a highly virulent H5N1 AIV infection was elicited in quails by IM immunization of a DNA vaccine encoding the H5 gene. Ideally, DNA vaccine delivery should not be invasive. However, most of the parenteral routes commonly used were needle-based deliveries and thus might cause complications in vaccinated chickens. Compared with the parenteral routes, oral administration in poultry is faster and much easier to administer for mass application without requiring highly trained manpower and no risk of needle-stick injury or cross-contamination. Oral immunization is able to induce mucosal immune responses and was performed as the second most popular route, with approximately 23% of poultry vaccinations. IO, which is specific to poultry, is the third most popular route of vaccination, at approximately 11% (Figure 1B).

Encapsulation of naked DNA with a carrier has been proposed as a solution to improve the controlled release of antigens that could increase the efficacy of DNA vaccines. Regardless of live, attenuated, killed or DNA vaccines, noninvasive vaccinations, including IN and oral delivery, could reduce stress, pain and cost of vaccinations and increase the safety of vaccination in large flocks of birds.

Furthermore, successful IN and oral delivery tend to raise better mucosal immunity than the other routes against poultry respiratory viruses, such as infectious bronchitis virus (IBV), NDV, and AIV. Thus, the design of carriers should help improve the efficacy and stability of DNA vaccines for IN or oral delivery. The carrier must be able to resist degradation and attack by the immune system and have sufficient safety profiles to become a successful delivery system.

Many other viruses have been considered as immunosuppressive agent in turkey. Respiratory viruses have usually negative effect on immune system, such as Newcastle disease virus (NDV) avian influenza viruses (AIV), and avian Metapneumoviruses (aMPV).

Newcastle disease (ND) is a worldwide disease causing severe economic losses. NDV can damage lymphoid tissues and decrease macrophage secretion and their phagocytosis role. Necrosis of lymphocytes and apoptosis of peripheral blood lymphocytes and mononuclear cells have been also reported (Cheville and Beard, 1972; Agoha et al., 1992; Lam, 1996).

aMPV is the causal agent of turkey rhinotracheitis (TRT). Replication of this virus in epithelial cells the upper respiratory tract can impair the mucociliary functions and increase deeper bacterial infections, with E. coli and Ornithobacterium rhinotracheale (Majo et al., 1997; Jirjis et al., 2004). Being an immunosuppressive pathogen, aMPV is able to reduce reactions to phytohaemagglutinin and immune responses to sheep red cells in poults. Infected animals showed lower thymus weight (Timms et al., 1986). Furthermore, it has been shown that aMPV can interfere with HEV vaccines and subsequently, reduce immune response in turkeys (Chary et al., 2002).