Transmission dynamics of the recently-identified BYD virus causing duck egg-drop syndrome

Baiyangdian (BYD) virus is a recently-identified mosquito-borne flavivirus that causes severe disease in ducks, with extremely rapid transmission, up to 15% mortality within 10 days and 90% reduction in egg production on duck farms within 5 days of infection. Because of the zoonotic nature of flaviviruses, the characterization of BYD virus and its epidemiology are important public health concerns. Here, we develop a mathematical model for the transmission dynamics of this novel virus. We validate the model against BYD outbreak data collected from duck farms in Southeast China, as well as experimental data obtained from an animal facility. Based on our model, the basic reproductive number of BYD virus is high (R(0) = 21) indicating that this virus is highly transmissible, consistent with the dramatic epidemiology observed in BYDV-affected duck farms. Our results indicate that younger ducks are more vulnerable to BYD disease and that ducks infected with BYD virus reduce egg production (to about 33% on average) for about 3 days post-infection; after 3 days infected ducks are no longer able to produce eggs. Using our model, we predict that control measures which reduce contact between mosquitoes and ducks such as mosquito nets are more effective than insecticides.     

Duck egg drop syndrome virus: an emerging Tembusu-related flavivirus in China

Duck egg drop syndrome virus(DEDSV) is a newly emerging pathogenic flavivirus isolated from ducks in China.DEDSV infection mainly results in severe egg drop syndrome in domestic poultry,which leads to huge economic losses.Thus,the discovery of ways and means to combat DEDSV is urgent.Since 2010,a remarkable amount of progress concerning DEDSV research has been achieved.Here,we review current knowledge on the epidemiology,symptomatology,and pathology of DEDSV.A detailed dissection of the viral genome and polyprotein sequences,comparative analysis of viral antigenicity and the corresponding potential immunity against the virus are also summarized.Current findings indicate that DEDSV should be a distinct species from Tembusu virus.Moreover,the adaption of DEDSV in wildlife and its high homology to pathogenic flaviviruses(e.g.,West Nile virus,Japanese encephalitis virus,and dengue virus),illustrate its reemergence and potential to become a zoonotic pathogen that should not be overlooked.Detailed insight into the antigenicity and corresponding immunity against the virus is of clear significance for the development of vaccines and antiviral drugs specific for DEDSV.     

Complete Genome Sequence of Duck Tembusu Virus,Isolated from Muscovy Ducks in Southern China

We report here the complete genomic sequence of the duck Tembusu virus (DTMUV) WJ-1 strain, isolated from Muscovy ducks. This is the first complete genome sequence of DTMUV reported in southern China. Compared with the other strains (TA, GH-2, YY5, and ZJ-407) that were previously found in eastern China, WJ-1 bears a few differences in the nucleotide and amino acid sequences. We found that there are 47 mutations of amino acids encoded by the whole open reading frame (ORF) among these five strains. The whole-genome sequence of DTMUV will help in understanding the epidemiology and molecular characteristics of duck Tembusu virus in southern China.     

Complete Genome Sequence of Avian Tembusu-Related Virus Strain WR Isolated from White Kaiya Ducks in Fujian, China

Avian tembusu-related virus, which was first identified in China, is an emerging virus causing serious economic loss to the Chinese poultry industry. We report here the complete genome sequences of avian tembusu-related virus strain WR, isolated from a White Kaiya duck with disease characterized by an abrupt decrease in egg laying with ovarian hemorrhage, which will help in further understanding the molecular and evolutionary characteristics and pathogenesis of avian tembusu-related virus, the new flavivirus affecting ducks in Southern China.     

Structural,Antigenic, and Evolutionary Characterizations of the Envelope Protein of Newly Emerging Duck Tembusu Virus

Since the first reported cases of ducks infected with a previously unknown flavivirus in eastern China in April 2010, the virus, provisionally designated Duck Tembusu Virus (DTMUV), has spread widely in domestic ducks in China and caused significant economic losses to poultry industry. In this study, we examined in detail structural, antigenic, and evolutionary properties of envelope (E) proteins of six DTMUV isolates spanning 2010–2012, each being isolated from individual farms with different geographical locations where disease outbreaks were documented. Structural analysis showed that E proteins of DTMUV and its closely related flavivirus (Japanese Encephalitis Virus) shared a conserved array of predicted functional domains and motifs. Among the six DTMUV strains, mutations were observed only at thirteen amino acid positions across three separate domains of the E protein. Interestingly, these genetic polymorphisms resulted in no detectable change in viral neutralization properties as demonstrated in a serum neutralization assay. Furthermore, phylogenetic analysis of the nucleotide sequences of the E proteins showed that viruses evolved into two distinct genotypes, termed as DTMUV.I and DTMUV.II, with II emerging as the dominant genotype. New findings described here shall give insights into the antigenicity and evolution of this new pathogen and provide guidance for further functional studies of the E protein for which no effective vaccine has yet been developed.     

Complete genome sequence of a novel flavivirus, duck tembusu virus, isolated from ducks and geese in china

Duck tembusu virus (DTMUV) is an emerging agent that causes a severe disease in ducks. We report herein the first complete genome sequences of duck tembusu virus strains YY5, ZJ-407, and GH-2, isolated from Shaoxing ducks, breeder ducks, and geese, respectively, in China. The genomes of YY5, ZJ-407, and GH-2 are all 10,990 nucleotides (nt) in length and encode a putative polyprotein of 3,426 amino acids. It is flanked by a 5' and a 3' noncoding region (NCR) of 94 and 618 nt, respectively. Knowledge of the whole sequence of DTMUV will be useful for further studies of the mechanisms of virus replication and pathogenesis.     

Propagation of vaccine strain of duck enteritis virus in a cell line of duck origin as an alternative production system to propagation in embryonated egg

Duck virus enteritis (DVE) also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with a chicken embryo-adapted live virus that is known to be poorly immunogenic and affords partial protection. Further, the risk of harboring other infectious agents in the embryo particularly the deadly and zoonotic avian influenza virus is also high. In this paper, we report propagation of a chicken embryo-adapted vaccine strain of duck enteritis virus in duck embryo fibroblast (DEF) cell line. Thirty serial passages were done in DEF cell that made the vaccine virus further attenuated which was tested in ducks. The growth behaviors of the virus in DEF cells were studied and at 30th passage level the virus titre was found to be 10^6.8 TCID₅₀/ml. Ducks were immunized with this virus and challenged after 21 days with high dose of virulent DEV. All the immunized ducks withstood challenge with no clinical symptoms in any of the ducks while all the control ducks died. DEF cell which is free from other infectious agents appears to be a good system for cultivation of duck enteritis virus vaccine strain.     

Reemerging H5N1 influenza viruses in Hong Kong in 2002 are highly pathogenic to ducks

Waterfowl are the natural reservoir of all influenza A viruses, which are usually nonpathogenic in wild aquatic birds. However, in late 2002, outbreaks of highly pathogenic H5N1 influenza virus caused deaths among wild migratory birds and resident waterfowl, including ducks, in two Hong Kong parks. In February 2003, an avian H5N1 virus closely related to one of these viruses was isolated from two humans with acute respiratory distress, one of whom died. Antigenic analysis of the new avian isolates showed a reactivity pattern different from that of H5N1 viruses isolated in 1997 and 2001. This finding suggests that significant antigenic variation has recently occurred among H5N1 viruses. We inoculated mallards with antigenically different H5N1 influenza viruses isolated between 1997 and 2003. The new 2002 avian isolates caused systemic infection in the ducks, with high virus titers and pathology in multiple organs, particularly the brain. Ducks developed acute disease, including severe neurological dysfunction and death. Virus was also isolated at high titers from the birds' drinking water and from contact birds, demonstrating efficient transmission. In contrast, H5N1 isolates from 1997 and 2001 were not consistently transmitted efficiently among ducks and did not cause significant disease. Despite a high level of genomic homology, the human isolate showed striking biological differences from its avian homologue in a duck model. This is the first reported case of lethal influenza virus infection in wild aquatic birds since 1961.     

Development of a Blocking ELISA for Detection of Serum Neutralizing Antibodies against Newly Emerged Duck Tembusu Virus

Background

Since April 2010, domesticated ducks in China have been suffering from an emerging infectious disease characterized by retarded growth, high fever, loss of appetite, decline in egg production, and death. The causative agent was identified as a duck Tembusu virus (DTMUV), a member of the Ntaya virus (NTAV) group within the genus Flavivirus, family Flaviviridae. DTMUV is highly contagious and spreads rapidly in many species of ducks. More than 10 million shelducks have been infected and approximately 1 million died in 2010. The disease remains a constant threat to the duck industry; however, it is not known whether DTMUV can infect humans or other mammalians, despite the fact that the virus has spread widely in southeast China, one of the most densely populated areas in the world. The lack of reliable methods to detect the serum antibodies against DTMUV has limited our ability to conduct epidemiological investigations in various natural hosts and to evaluate the efficiency of vaccines to DTMUV.

Methodology/Principal Findings

A neutralizing monoclonal antibody (mAb) 1F5 binding specifically to the E protein was developed. Based on the mAb, a blocking enzyme-linked immunosorbent assay (ELISA) was developed for the detection of neutralizing antibodies against DTMUV. The average value of percent inhibition (PI) of 350 duck serum samples obtained from DTMUV-free farms was 1.0% ±5.8% (mean ± SD). The selected cut-off PI values for negative and positive sera were 12.6% (mean +2SD) and 18.4% (mean +3SD), respectively. When compared with a serum neutralizing antibody test (SNT) using chicken embryonated eggs, the rate of coincidence was 70.6% between the blocking ELISA and SNT, based on the titration of 20 duck DTMUV-positive serum samples.

Conclusions/Significance

The blocking ELISA based on a neutralizing mAb allowed rapid, sensitive, and specific detection of neutralization-related antibodies against DTMUV.     

Possible waterborne transmission and maintenance of influenza viruses in domestic ducks.

Two duck farms in Hong Kong were examined monthly for 1 year for the occurrence and persistence of influenza viruses within the duck communities. The predominant virus in one community was H3N2, a virus antigenically related to the pandemic Hong Kong strain. This virus was isolated monthly throughout the year from feces or pond water or both, indicating a cycle of waterborne transmission. Viruses of the same antigenic combination were isolated 1 and 2 years after the last sampling occasion, implying persistence in the community. Infection was asymptomatic. Maintenance of virus appeared to be dependent upon the continual introduction of ducklings susceptible to infection onto virus-contaminated water; the feces of ducks 70 to 80 days old were generally free of detectable virus despite the exposure of the ducks to virus in pond water. In the second community, in which ducklings were not introduced after the initial sampling, the prevailing viruses, H7N1 and H7N2, also present asymptomatically, ceased to be detected once the ducks were 70 to 80 days old. The normal practice of raising ducks of different ages on the same farm, wherein the water supplies are shared, as typified by the first community, appears to be instrumental in maintaining a large reservoir of influenza viruses in the duck population of southern China.     

Isolation and characterization of a hepatitis B virus endemic in herons.

A new hepadnavirus (designated heron hepatitis B virus [HHBV]) has been isolated; this virus is endemic in grey herons (Ardea cinerea) in Germany and closely related to duck hepatitis B virus (DHBV) by morphology of viral particles and size of the genome and of the major viral envelope and core proteins. Despite its striking similarities to DHBV, HHBV cannot be transmitted to ducks by infection or by transfection with cloned viral DNA. After the viral genome was cloned and sequenced, a comparative sequence analysis revealed an identical genome organization of HHBV and DHBV (pre-C/C-, pre-S/S-, and pol-ORFs). An open reading frame, designated X in mammalian hepadnaviruses, is not present in DHBV. DHBV and HHBV differ by 21.6% base exchanges, and thus they are less closely related than the two known rodent hepatitis B viruses (16.4%). The nucleocapsid protein and the 17-kilodalton envelope protein sequences of DHBV and HHBV are well conserved. In contrast, the pre-S part of the 34-kilodalton envelope protein which is believed to mediate virus attachment to the cell is highly divergent (less than 50% homology). The availability of two closely related avian hepadnaviruses will now allow us to test recombinant viruses in vivo and in vitro for host specificity-determining sequences.     

Characterization of a highly pathogenic H5N1 avian influenza A virus isolated from duck meat

Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.     

Characterization of Avian Paramyxoviruses Isolated from Feral Ducks in Northern Japan: The Presence of Three Distinct Viruses in Nature

Seventy-eight strains of avian paramyxoviruses (PMV) were isolated from cloacal and/or tracheal swabs taken from 1,342 feral ducks, comprised of spot-bill ducks, mallards, pintails, teals, falcated teals, wigeons and buffie-heads, in Wakuya-cho, Miyagi Prefecture, Japan, between 1976 and 1979. Five and a half percent of the ducks were positive for virus. Serological and structural characterization indicated that three different avian paramyxoviruses arc prevalent in the Japanese feral duck population. The first group of PMV was Newcastle disease virus (NDV), and in vivo pathogenecity tests in embryonated chicken eggs and 1-day-old chicks revealed that all the NDV strains isolated were avirulent. The second and most prevalent strain was closely related to PMV-4, duck/Hong Kong/D3/75 strain. The viruses of the third group were recovered only from pintails. They cross-reacted antigenically with PMV-3 when antisera to the PMV-3 reference strains, turkey/Wisconsin/68 and parakeet/Netherlands/449/75, were employed. However, no cross-reaction was observed when antiserum to pintail/ Wakuya/20/78, the prototype of this group, was used. The viruses of the third group also differed in viral polypeptide profile from the reference strains of PMV-3.     

Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity

Five new hepadnaviruses were cloned from exotic ducks and geese, including the Chiloe wigeon, mandarin duck, puna teal, Orinoco sheldgoose, and ashy-headed sheldgoose. Sequence comparisons revealed that all but the mandarin duck viruses were closely related to existing isolates of duck hepatitis B virus (DHBV), while mandarin duck virus clones were closely related to Ross goose hepatitis B virus. Nonetheless, the S protein, core protein, and functional domains of the Pol protein were highly conserved in all of the new isolates. The Chiloe wigeon and puna teal hepatitis B viruses, the two new isolates most closely related to DHBV, also lacked an AUG start codon at the beginning of their X open reading frame (ORF). But as previously reported for the heron, Ross goose, and stork hepatitis B viruses, an AUG codon was found near the beginning of the X ORF of the mandarin duck, Orinoco, and ashy-headed sheldgoose viruses. In all of the new isolates, the X ORF ended with a stop codon at the same position. All of the cloned viruses replicated when transfected into the LMH line of chicken hepatoma cells. Significant differences between the new isolates and between these and previously reported isolates were detected in the pre-S domain of the viral envelope protein, which is believed to determine viral host range. Despite this, all of the new isolates were infectious for primary cultures of Pekin duck hepatocytes, and infectivity in young Pekin ducks was demonstrated for all but the ashy-headed sheldgoose isolate.     

Immunological and genomic analyses of two serotypes of avian paramyxovirus isolated from wild ducks in Japan

A total of 11 avian paramyxoviruses isolated from migrating feral ducks in Niigata, Japan, were characterized by serological and genomic analyses. Hemagglutination inhibition and immuno-double-diffusion tests with antisera specific for the isolated hemagglutinin-neuraminidase polypeptides of reference strains indicated that, of these, eight isolates possessed hemagglutinin-neuraminidase antigen closely related to that of duck/Hong Kong/D3/75, and the remaining three isolates possessed antigen closely related to that of duck/Hong Kong/199/77. RNA analysis of the eight isolates identified serologically as duck/Hong Kong/D3/75 by oligonucleotide mapping revealed that these isolates were genetically very similar to each other but different from the reference strain and isolates reported previously. The oligonucleotide maps of duck/Hong Kong/199/77-like isolates appeared to be very similar to each other, suggesting the same origin, but not to the duck/Hong Kong/199/77 virus.     

Development and Evaluation of a DAS-ELISA for Rapid Detection of Tembusu Virus Using Monoclonal Antibodies against the Envelope Protein

Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin.     

Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to H5N1

Aquatic birds are the natural reservoir for most subtypes of influenza A, and a source of novel viruses with the potential to cause human pandemics, fatal zoonotic disease or devastating epizootics in poultry. It is well recognised that waterfowl typically show few clinical signs following influenza A infection, in contrast, terrestrial poultry such as chickens may develop severe disease with rapid death following infection with highly pathogenic avian influenza. This study examined the cellular response to influenza infection in primary cells derived from resistant (duck) and susceptible (chicken) avian hosts. Paradoxically, we observed that duck cells underwent rapid cell death following infection with low pathogenic avian H2N3, classical swine H1N1 and 'classical' highly pathogenic H5N1 viruses. Dying cells showed morphological features of apoptosis, increased DNA fragmentation and activation of caspase 3/7. Following infection of chicken cells, cell death occurred less rapidly, accompanied by reduced DNA fragmentation and caspase activation. Duck cells produced similar levels of viral RNA but less infectious virus, in comparison with chicken cells. Such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 fatal to ducks. The induction of rapid death in duck cells may be part of a mechanism of host resistance to influenza A, with the loss of this response leading to increased susceptibility to emergent strains of H5N1. These studies provide novel insights that should help resolve the long-standing enigma of host-pathogen relationships for highly pathogenic and zoonotic avian influenza.     

1株含H9N2内部基因的H5N1亚型基因重配禽流感病毒的全基因测序及遗传进化分析

在对华东地区家养水禽中流感病毒的带毒状况的流行病学监测过程中,从表观健康家鸭体内分离到一株H5N1亚型禽流感病毒A/duck/Shandong/009/2008(简称Dk/SD/009/08)。为了解该毒株的基因组构成,对该分离株进行全基因测序。测序结果显示:该毒株HA裂解位点处的氨基酸序列为PLRERRRK-R/GL,符合高致病性禽流感病毒的分子特征,且参照H5N1国际统一命名准则,Dk/SD/009/08的HA基因属于2.3.4进化支。BLAST结果显示,HA、NA、NP及NS基因均与H5N1亚型病毒的核苷酸一致性最高,而RNA聚合酶基因(PB2、PB1、PA)及M基因则与H9N2亚型病毒的亲缘关系最近,故推测该分离株可能是一株天然重组病毒;遗传进化分析进一步表明,流行于华南地区鹌鹑中的G1-like H9N2亚型病毒可能为该分离株提供部分的内部基因。     

Factors influencing the levels of fatty acid synthase complex activity in fowl

There is a notable discrepancy between the FAS (fatty acid synthase) activity of four types of fowl (egg chicken, meat chicken, egg duck, and meat duck) with distinctively different body fat levels. There is a 14.8 fold difference per unit body weight between the maximum and minimum FAS activities. The three major factors affecting this discrepancy are liver weight per unit body weight, which is 2.3 times greater in meat ducks than in egg chickens, the amount of FAS protein per gram of liver, which is 1.85 times greater in meat ducks than in egg chickens, and the FAS specific activity in meat ducks, which is 3.5 times greater in meat ducks than in egg chickens. Within the same species of egg chickens, the abdomen fat per kg of body weight at 470 days after egg production is 66 times greater than 90 days before egg production and the liver FAS activity is increased 9.6 fold. The 9.6 fold FAS activity increase resulted from an increase in the specific activity, since the liver weight per kilogram of body weight remained constant at approx. 20 grams and the FAS weight per gram of liver also remained constant at approx. 4.5 mg. This shows that the control of the basic FAS activity level which is closely related to the level of body fat does not mainly arise from genetic control. For the same kind of fowl, the control of the basic FAS activity level occurs after gene expression. It is suggested that control may be imposed in the folding phase when new peptides give rise to functional proteins.     

Zoonotic Echinostome Infections in Free-Grazing Ducks in Thailand

Free-grazing ducks play a major role in the rural economy of Eastern Asia in the form of egg and meat production. In Thailand, the geographical location, tropical climate conditions and wetland areas of the country are suitable for their husbandry. These environmental factors also favor growth, multiplication, development, survival, and spread of duck parasites. In this study, a total of 90 free-grazing ducks from northern, central, and northeastern regions of Thailand were examined for intestinal helminth parasites, with special emphasis on zoonotic echinostomes. Of these, 51 (56.7%) were infected by one or more species of zoonotic echinostomes, Echinostoma revolutum, Echinoparyphium recurvatum, and Hypoderaeum conoideum. Echinostomes found were identified using morphological criteria when possible. ITS2 sequences were used to identify juvenile and incomplete worms. The prevalence of infection was relatively high in each region, namely, north, central, and northeast region was 63.2%, 54.5%, and 55.3%, respectively. The intensity of infection ranged up to 49 worms/infected duck. Free-grazing ducks clearly play an important role in the life cycle maintenance, spread, and transmission of these medically important echinostomes in Thailand.