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The research focused on 1,3,4-thiadiazole derivatives indicates a broad spectrum of pharmacological activities associated with good physicochemical and pharmacokinetic properties. This article presents a literature review of 2-amino-1,3,4-thiadiazole derivatives that have been evaluated for antiviral activity against several viral strains. In addition to the 2-amino-1,3,4-thiadiazole moiety, antiviral activity is also dependent on the nature of the substituents, and structure–activity studies have shown the most efficient substituents for antiviral activity in each class. Based on the literature data, the 2-amino-1,3,4-thiadiazole scaffold may be considered a possible pharmacophore group that can be incorporated into the structure of known compounds to enhance antiviral activity and contributes to the search and development of new medicines as an alternative to the treatment of viral infections.
Human cytomegalovirus (HCMV, Herpesviridae family) is a ubiquitous deoxyribonucleic acid virus that infects people of all ages. HCMV infection can be acquired through horizontal and vertical transmission. HCMV spreads from infected people through direct contact with body fluids that carry the virus, such as urine, saliva, cervicovaginal secretions, sperm and breast milk. Vertical transmission through organ transplantation, from mother to child or transmission via blood transfusion, is also possible. Blood tests indicate that 60%–90% of the adult population experienced HCMV infection at some time during their life. Although most of these infections are asymptomatic, certain patient groups such as babies that are infected before birth and children or adults with weakened immune systems due to diseases or medications (e.g., HIV-infected patients, organ transplant recipients) can develop severe illnesses that require medical treatment. HCMV is able to remain latent in several cells of the human body for a long time and can be reactivated if the person develops immune system suppression.
The first-line drugs recommended for the treatment of HCMV infection are intravenous ganciclovir or orally administered valganciclovir. Although tolerability of ganciclovir and valganciclovir is acceptable, hematological or neurological side effects can occur. Neutropenia, thrombocytopenia and anemia are the main toxic effects that limit therapy with these drugs. Serum creatinine levels may increase during ganciclovir therapy, which requires monitoring of renal function. Encephalopathy is the neurotoxic effect of ganciclovir and valganciclovir. Foscarnet is also a very effective anti-HCMV drug, and cidofovir is a broad-spectrum antiviral with good activity against HCMV. Both drugs cause a high level of nephrotoxicity that limits treatment.
Novel 2-amino-1,3,4-thiadiazole derivatives with antiviral activity against HCMV have been patented. A large number of 472 synthesized compounds were tested in an HCMV polymerase assay at a concentration of 25 μM. The degree of enzyme inhibition ranged from 20% to 100%. Among the most active compounds, four derivatives exhibited a 100% inhibition rate, 29 derivatives showed an inhibition rate of 90.1%–99.9% and 16 derivatives showed an inhibition rate of 80.3%–89.9%. Three structural series stand out among the most active 1,3,4-thiadiazole derivatives: 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamide derivatives such as 21–30, 9-octadecenamide derivatives such as 31–38 and 2-ethoxy-1-naphthamide derivatives such as 39–42 (Table 2, Table 3 and Table 4). Most of the derivatives belong to the 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamide series. At the same time, the most active compounds belong to this series, so it can be concluded that the 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamide moiety is a good scaffold for anti-HCMV activity.
Other compounds also showed good inhibitory activity. These derivatives contain a five- to six-membered saturated heterocyclic moiety, such as imidazolidinyl, tetrahydrofuryl, piperidinyl, morpholinyl, thiomorpholinyl or 5- to 10-membered aromatic or unsaturated heterocyclic moiety such as furyl, pyrrolyl, pyridyl, benzothiazolyl, etc. (e.g., derivatives 43,44).
While the synthesized derivatives have shown inhibitory activity against HCMV polymerase, their antiviral activity cannot be limited to a specific mechanism of action. These compounds may be active against cytomegalovirus by HCMV polymerase inhibition or by other mechanisms of action. In addition, during the experiments, many of these compounds also showed activity against other herpes viruses, such as varicella-zoster virus (VZV), Epstein–Barr virus (EBV), herpes simplex virus (HSV), and human herpesvirus type 8 (HHV-8). Pharmaceutical compositions containing such compounds or their pharmaceutically acceptable salts useful as antiviral agents have also been studied. Studies have been conducted for the administration of pharmaceutical preparations by parenteral, topical, oral or rectal route, depending on the purpose of their use to treat internal or external viral infections.
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.
The main objective is to prevent economic losses caused by viral immunosuppressive infection. Strategy control is based on different approaches including, good management, application of biosecurity standards, immunization of birds, and genetic selection.
The good management has an important role in optimizing turkey’s performances and in maintaining bird health and welfare. Delivering constantly a good quality air, a high quality water and feed is essential and required at all stages of growth. Control of mycotoxins, as an immunosuppressive agent, must be continually performed. Litter management is a key of the pathogens control.
Application of strict biosecurity measures is fundamental to prevent exposition to immunosuppressive viruses. Increasing bird’s resistance is a complement but essential tool through good vaccination, as the best tool inducing specific protection. Vaccines must be administrated properly for all animals in the same flock, in order to achieve uniform immune response. However, vaccines are not available for the main viral diseases in turkeys. The control of other immunosuppressive agents (bacteria, mycotoxins, parasites, and stress) must be considered to prevent vaccination failures.
Currently, turkeys can be vaccinated against few immunosuppressive viral diseases, such as HE, ND, aMPV, and Reoviruses. Live vaccines used against HE are effective in preventing disease outbreaks. However, they are immunosuppressive, predisposing young animals to opportunistic infections and vaccination failures.
Control of ND is based on different types of vaccines. Lives vaccines, worldwide used in poultry industry, can stimulate local protection. Inactivated vaccines required for a long lasting immunity. Currently, vectored vaccines, using glycoprotein F and administered in ovo or to 1-day old poults, are effective and safety
Vaccination against aMPV is performed to prevent lesions in the upper respiratory tract and the decrease in egg production. Live vaccines are used in young poults, while inactivated vaccines are reserved to adults.
The development of vaccination against Reoviruses interest chickens. In general, strain vaccine protected against viral arthritis with partial protection against RSS.
Although TCoV was identified as the causative agent of Bluecomb disease of turkey poults over 50 years ago (28), vaccines are not available to control the disease. Recent assays of vaccination using protein spike are performed with promoted results (Chen et al., 2018).
The MD is well controlled by vaccination in chickens. Despite the appearance of natural disease in commercial turkeys flocks, vaccination is not performed. However, possible immunization of poults at the hatchery with Rispens strain is suggested (Blake-Dyke and Baigent, 2013).
Cytokines may be used as therapeutic agents for viral diseases and for vaccines adjuvants (Wigley and Kaiser, 2003). IFN-α induces increase of antibody titer in turkeys immunized by NDV DNA vaccine (Rautenschlein et al., 2000a). While, addition of IFN-γ to NDV DNA vaccine, is accompanied by more rapid humoral response and increased protection to NDV challenge in turkeys and chickens vaccinated in ovo (Rautenschlein et al., 1999; Cardenas-Garcia et al., 2016).
Given the limitations associated with vaccination, several assays of genetic selection have been performed. Genetic resistance to MD is well documented, with a special focus on major histocompatibility complex. Notable correlation is demonstrated between resistance of chickens to MD and B21 allele, which is accompanied by reduction of infected T-cells. Mapping genes suggested the presence of a resistance gene in the natural killer region within chromosome 1 (Bumstead, 1998), whereas birds with a B-19 haplotype suffer 100% mortality (Briles et al., 1977). Certain types of artificial selection, as higher growth rates, influence negatively immune competence in turkeys (Husby et al., 2011).
Recently, insertion of transgenes that target AIV into the genomes of chickens allowed to limit virus spread, but this approach is incapable de prevent emergence of disease (Looi et al., 2018).
Genetic selection birds for optimal immune response to used vaccines may complete optimizing natural resistance to viral diseases. Selection for enhanced innate immunity is possible because of the existence of toll-like receptors in chickens and turkeys. Possible interactions between adjuvants and immunogenetics may lead to develop novel vaccines.
Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software). Student’s t-test, one-way ANOVA and two-way ANOVA were used as appropriate. P values < 0.05 were considered to be significant.
DNA vaccination provided a new and valuable approach to the development of poultry vaccines and offered advantages in flexibility of design, speed, simplicity of production, and the ability to elicit both cellular and humoral immune responses. DNA vaccines against influenza in poultry have been in development since 1993, and recently, the USDA conditionally approved the first DNA vaccine against H5N1 for chickens. DNA vaccines are amendable for stockpiling to control future influenza H5N1 outbreaks. The pandemic AIV strains have undergone antigenic shift or drift, which allows them to avoid immunity elicited by the poultry influenza vaccines. Recent AIV vaccine development studies have indicated the need for additional systemic vaccine challenge studies against highly pathogenic AIV. Moreover, full protection has been demonstrated against poultry diseases, such as DTMUV, IBD, and ND. DNA vaccines also suffer from several pitfalls where in vivo efficacy and stability are still problems. Additionally, a single DNA vaccination in poultry is often insufficient to induce robust humoral and cell-mediated immunity as well as confer full protection. Therefore, booster immunization is often required. Both biological and physical carriers, with their appropriate antigens and adjuvants, offer the possibility to overcome the disadvantages of DNA vaccines. Although DNA vaccines carrying different antigens have been delivered by different types of carriers and adjuvants, very few have been evaluated by challenges with the pathogens in question. Thus, additional in vivo field trials should be carried out to identify the efficiency and safety of the currently available carriers, antigens, and adjuvants to combat infectious diseases of veterinary pathogens.
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.
Commensal bacteria are essential for shaping intestinal immune responses; however, the beneficial role of commensal bacteria is not restricted to the intestinal mucosa. Recent studies suggest a link between the intestinal microbiota and antiviral immunity at non-intestinal mucosal surfaces, such as those in the lung. We previously demonstrated that antibiotic-treated mice show impaired adaptive immune responses to influenza virus infection when compared with water-fed mice. Mice treated with antibiotics showed reduced expression of pro-IL-1β, pro-IL-18, and NLRP3 mRNA in the lung, resulting in reduced secretion of mature IL-1β after intranasal infection with influenza virus (Fig. 2). As a result of impaired inflammasome activation in the lungs of antibiotic-treated mice, the number of DCs migrating from the lung to the mLNs was lower than that in water-fed mice after influenza virus infection. Importantly, the administration of a TLR ligand, lipopolysaccharide, was able to restore both the migration of respiratory DCs to mLNs and the virus-specific T cell responses in antibiotic-treated mice. Two follow-up studies identified the molecular mechanism that links the intestinal microbiota to antiviral immunity. The induced expression of antiviral defense genes by peritoneal macrophages derived from antibiotic-treated mice, or by splenic DCs derived from germ-free mice, was diminished after stimulation with influenza virus or poly(I:C). This suggests that the microbiota primes APCs by providing tonic type I IFN signals that induce efficient viral recognition and the generation of antiviral adaptive immune responses. Although the main reason of antibiotic treatment to patients infected with influenza virus is to protect the patients from the secondary bacterial infection that causes sever pneumonia, these studies suggest that the administration of antibiotics to patients infected with influenza virus may have negative effects. Although it is still unclear whether it is the species of bacteria or the overall composition of the microbiota that is important for antiviral immunity, the use of gnotobiotic animals and genomic sequencing analysis of the microbiota will highlight new strategies for the development of effective influenza vaccines.
The enteric viral infections, mainly rotavirus, are a global cause for concern. Among the four serogroups of rotaviruses identified in poultry, the RVD has gained more importance due to its involvement in runting and stunting syndrome. With the advent of molecular techniques, the diversity of RVD strains is beginning to be explored. However, still, the exact prevalence and annual losses associated with RVD in poultry industry are unknown. There are many gaps that need to be filled and warrant attention, such as:
▪Studies on host–pathogen interactions, whether they are alike other enteric viruses or not.▪As RVD is found in both symptomatic and asymptomatic birds, factors responsible for its virulence and pathogenicity are to be studied.▪To date, very few sequences are available, and only for some of the genes of RVD strains. Once enough sequence data are available, a nucleotide sequence-based classification system can be established for RVD, as was achieved for RVAs.▪Only one complete genome sequence is available so far, despite the widespread distribution of RVD in chickens.▪The function of additional ORF (ORF-2) encoded by the 10th segment of RVD is still not defined.▪The development of sensitive and specific diagnostic tests, including the improvement of available ones, is of prime importance.▪The development of specific treatment by means of antivirals.
Once the basic information is available about RVD, its prevention should acquire the attention of researchers by means of developing vaccines to prevent its spread and to save one of the fastest-growing sectors, the poultry industry.
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.
To investigate the possible infection route, three groups of 1-day-old goslings were kept at separate isolator. Birds were inoculated orally (n = 6) or intranasally (n = 6) at 5-day old with the astrovirus isolate at the dose as experiment 1. Five goslings were kept as uninfected control. Mortality were checked daily. Cloacal swabs were collected for virus shedding detection as described in experiment 2.
The infected cells were fixed with 4% paraformaldehyde (Sangon Biotech) for 10 min, followed by rinsing with PBS. 0.2% v/v triton X-100 (Sigma) diluted in PBS was applied to each well for 10 min. After washing three times with sterile PBS, the cells were overlaid with PBS containing 5% w/v bovine serum albumin (Sangon Biotech) and incubated for 30 min at 37° C. The mouse antiserum anti-capsid P2 was used as the primary antibody with a 1:200 dilution in PBS. After 45min incubation at 37 °C, the cell monolayers were rehydrated by rinsing three times with PBS. Coverslips were stained using goat anti-mouse IgG conjugated with FITC (KPL) and incubated for a further 45 min at 37° C. The stained cells were rinsed with PBS, then dyed with Hoechst33342 (Solarbio) and rinsed again. The stained cells were then examined under a confocal microscope.
Each plasmid was used as a template to evaluate the sensitivity of the mPCRs. For CRV, the minimum detection limits for pMD-CAV-2, pMD-CDV, pMD-CIV and pMD-CPIV were 1×103 (Fig 3A), 1×103 (Fig 3B), 1×104 (Fig 3C) and 1×104 (Fig 3D) viral DNA copies, respectively. For CEV, the minimum detection limits for pMD-CAV-2, pMD-CanineCV, pMD-CCoV and pMD-CPV were 1×104 (Fig 4A), 1×104 (Fig 4B), 1×103 (Fig 4C) and 1×103 (Fig 4D) viral DNA copies, respectively. The sensitivity test results revealed that the minimum simultaneous detection limit for mPCR of CRV was 1 × 104 viral copies (Fig 3E), and the limit for CEV (Fig 4E) was also 1×104 viral copies.
To date, it seems difficult to eliminate RVs from commercial flocks, as rotaviruses are ubiquitous as well as quite resistant to environmental conditions. The titre of AvRVs was found to decrease after treatment at 56 °C for 30 min, but the viral infectivity is difficult to inactivate completely. Further, it was recognized that AvRVs were resistant to chloroform and were stable at pH 3. In general, sodium hypochlorite is an effective virucidal disinfectant, but for highly resistant AvRVs, glutaraldehyde had stronger activity, which is also true for enveloped viruses. The spread of AvRVs can be prevented by maintaining good sanitation and hygiene by means of thorough cleaning and disinfection to reduce the environmental contamination between flocks. Although RV disease is vaccine preventable, effective and safe vaccines are available only for human RVAs. As it is difficult to grow atypical AvRVs in cell culture, and also due to high antigenic variations among them, no vaccines are available so far for the prevention of AvRVs.
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 evaluate the specificity of the mPCRs, we performed specificity assays on CRV and CEV with CRV- and CEV-specific primers, respectively. Similar procedures were used to detect possible cross-reaction of CRV and CEV primers with RNA/DNA extracted from MDCK cells or from other pathogens (RABV, E. coli and Salmonella enterica). The nucleic acid extraction products of the MDCK cells, E. coli and Salmonella enterica were used directly as PCR templates. In contrast, the viral RNA extraction products of RABV required RT prior to use as templates. Both the individual plasmid and premixed plasmids were tested separately in this assay. The empty pMD-18T vector was used as a negative control.
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).
The viral diseases panel was chaired by Drs. Richard Kuhn (Purdue University, U.S.A.) and Jiro Arikawa (Hokkaido University Graduate School of Medicine, Japan), with secretariat Dr. Eun Chung Park (NIAID, U.S.A.). The 1.5-day panel meeting focused on the following session topics: 1) hemorrhagic viruses including Ebola, Lassa, and Severe fever with thrombocytopenia syndrome virus (SFTSV), a highly pathogenic and recently emerged virus in Asian countries including Japan, Korea, and China; 2) enteric viruses including norovirus, rotavirus, poliovirus, and EV71, a virus causing hand and foot and mouth disease in Southeast Asian countries; 3) arboviruses including dengue, SFTSV, and chikungunya viruses; and 4) rabies virus and included aspects such as epidemiology, pathogenesis, structural biology, animal models, vaccines and antiviral developments, and human challenge models for dengue virus. There were significant presentations by junior/early-stage career scientists and discussions during the meeting were robust due to active participation and engaging discussions. Notable presentations from early-stage investigators selected from abstract submissions included a talk on a clinical trial of an investigational vaccine, called AGS-v, against mosquito saliva peptide. If proven effective, this vaccine can protect against mosquito-borne viral infections. Another presentation was on a broad-spectrum antiviral peptide that is shown to be active against Zika virus given post infection in an animal model. The viral diseases panel will reconvene in 2020.
The hepatitis panel, “New Approaches to Hepatitis B Virus (HBV) Therapy” was chaired by Drs. Takaji Wakita (National Institute of Infectious Disease, Japan), Christopher Walker (Nationwide Children’s Hospital, U.S.A.), and Rajen Koshy (NIAID, U.S.A.; secretariat). The keynote lecture by Dr. Raymond Chung (Massachusetts General Hospital, U.S.A.) kicked off the two-day meeting with a discussion on the current understanding of a cure for chronic hepatitis B and the challenges for the complete elimination of HBV because of the stable and self-perpetuating replication intermediate of HBV DNA. The panel meeting included four sessions: 1) Chronic hepatitis B in Asia—scope of the problem and exploring new treatments; 2) markers of a functional cure for chronic hepatitis B; 3) preclinical animal models for testing HBV curative therapies; and 4) regulation of HBV replication and new targets for HBV therapy. In addition to the panelist presentations, posters were presented by early-stage investigators. The hepatitis panel meeting featured exceptional presentations, collaborative discussions, successful outcomes, and will be reconvened in 2020.
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).
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.
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.
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.
According to the viral genome sequence, a codon-optimized gene of capsid P2 was synthesized and cloned into pET-30a (Novagen). The recombinant capsid P2 protein was expressed in E.coli BLR (DE3) cells (Novagen) under the inducing of isopropyl-β-d-thiogalactopyranoside (Sangon Biotech) at concentrate of 1mmol/L and purified using Ni-NTA Agarose (Qiagen). After verified using convalescent goose sera by Westernblot, 200 μg purified recombinant protein was inoculated to 8-week old Balb/c mouse in abdomen at an interval of 10 days. 10 days after the 3rd immunization, the mouse blood was collected to prepare antisera.
The MDCC-MSB1 cells with positive PCR results were harvested by centrifugation, washed with PBS (pH = 7.4), and lysed with lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, and 1× complete cocktail protease inhibitor. Whole lysates were boiled for 10 min in the presence of 5× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. After centrifugation at 12,000 × g for 2 min, equivalent sample amounts were separated by 12% SDS-PAGE and transferred to pure nitrocellulose blotting membranes (PALL, 66485, USA). After blocking with 5% skim milk, the membranes were incubated with primary antibody at 37°C for 1.5 h, followed by the IRDye 800CW secondary antibody for 1 h at 37°C. Proteins were visualised using the Odyssey system (Li-Cor, USA).