European College of Equine Internal Medicine consensus statement on equine flaviviridae infections in Europe.

Horses and other equids can be infected with several viruses of the family Flaviviridae, belonging to the genus Flavivirus and Hepacivirus. This consensus statement focuses on viruses with known occurrence in Europe, with the objective to summarize the current literature and formulate clinically relevant evidence-based recommendations regarding clinical disease, diagnosis, treatment, and prevention. The viruses circulating in Europe include West Nile virus, tick-borne encephalitis virus, Usutu virus, Louping ill virus and the equine hepacivirus. West Nile virus and Usutu virus are mosquito-borne, while tick-borne encephalitis virus and Louping ill virus are tick-borne. The natural route of transmission for equine hepacivirus remains speculative. West Nile virus and tick-borne encephalitis virus can induce encephalitis in infected horses. In the British Isle, rare equine cases of encephalitis associated with Louping ill virus are reported. In contrast, equine hepacivirus infections are associated with mild acute hepatitis and possibly chronic hepatitis. Diagnosis of flavivirus infections is made primarily by serology, although cross-reactivity occurs. Virus neutralization testing is considered the gold standard to differentiate between flavivirus infections in horses. Hepacivirus infection is detected by serum or liver RT-PCR. No direct antiviral treatment against flavi- or hepacivirus infections in horses is currently available and thus, treatment is supportive. Three vaccines against West Nile virus are licensed in the European Union. Geographic expansion of flaviviruses pathogenic for equids should always be considered a realistic threat, and it would be beneficial if their detection was included in surveillance programs.

The First Nonmammalian Pegivirus Demonstrates Efficient In Vitro Replication and High Lymphotropism.

Members of the Pegivirus genus, family Flaviviridae, widely infect humans and other mammals, including nonhuman primates, bats, horses, pigs, and rodents, but are not associated with disease. Here, we report a new, genetically distinct pegivirus in goose (Anser cygnoides), the first identified in a nonmammalian host species. Goose pegivirus (GPgV) can be propagated in goslings, embryonated goose eggs, and primary goose embryo fibroblasts, and is thus the first pegivirus that can be efficiently cultured in vitro Experimental infection of GPgV in goslings via intravenous injection revealed robust replication and high lymphotropism. Analysis of the tissue tropism of GPgV revealed that the spleen and thymus were the organs bearing the highest viral loads. Importantly, GPgV could promote clinical manifestations of goose parvovirus infection, including reduced weight gain and 7% mortality. This finding contrasts with the lack of pathogenicity that is characteristic of previously reported pegiviruses.IMPORTANCE Members of the Pegivirus genus, family Flaviviridae, widely infect humans and other mammals, but are described as causing persistent infection and lacking pathogenicity. The efficiency of in vitro replication systems for pegivirus is poor, thus limiting investigation into viral replication steps. Because of that, the pathogenesis, cellular tropism, route of transmission, biology, and epidemiology of pegiviruses remain largely uncovered. Here, we report a phylogenetically distinct goose pegivirus (GPgV) that should be classified as a new species. GPgV proliferated in cell culture in a species- and cell-type-specific manner. Animal experiments show GPgV lymphotropism and promote goose parvovirus clinical manifestations. This study provides the first cell culture model for pegivirus, opening new possibilities for studies of pegivirus molecular biology. More importantly, our findings stand in contrast to the lack of identified pathogenicity of previously reported pegiviruses, which sheds lights on the pathobiology of pegivirus.

Prevalence of the emerging novel Alongshan virus infection in sheep and cattle in Inner Mongolia, northeastern China.

BACKGROUND: Alongshan virus (ALSV) is a novel discovered segmented flavivirus associated with human febrile illness in northeastern China. Ixodes persulcatus is considered as a candidate vector of ALSV in the endemic regions. However, the role of domesticated animals in the circulation and transmission of ALSV have not been investigated. To evaluate the prevalence of ALSV infections in domesticated animals, viral RNA and viral specific antibodies were detected in sheep and cattle in Hulunbuir of northeastern Inner Mongolia. The findings contribute to the understanding of the ecology and transmission of ALSV among different natural hosts. METHODS: A total of 480 animal serum samples were collected in Hulunbuir of northeastern China in May, 2017. Viral specific antibodies were tested by indirect enzyme-linked immunosorbent assay (ELISA) with a purified E. coli recombinant capsid protein (VP2) of ALSV (strain H3) and further detected by viral neutralization test (VNT). RNA in serum samples were extracted and detected for ALSV sequence by quantitative real-time RT-PCR. ALSV RNA positive samples were used for virus isolation. RESULTS: ALSV-specific antibodies were detected in 9.2% (22/240) of examined sheep and 4.6% (11/240) of examined cattle by ELISA, while lower serological positivity with 4.2% (10/240) for sheep and 1.7% (4/240) for cattle was confirmed by VNT. In contrast, the prevalence of ALSV RNA was much higher, ranging from 26.3% (63/240) in sheep to 27.5% (66/240) in cattle. The partial S1 (NS5-like) and S3 (NS3-like) segments of ALSVs in sheep and cattle shared high identities of more than 98% to the human and tick isolates in the studied regions. CONCLUSIONS: These results suggest that the natural infection of ALSV can be found in sheep and cattle in the endemic regions.

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus.

A simple and accurate reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the detection of porcine pegivirus (PPgV). The specific RT-LAMP primers targeting the conserved regions of NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63℃ for 60 min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 times more sensitive than that of the conventional RT-PCR and comparable to nested RT-PCR and quantitative RT-PCR (qRT-PCR). There was no cross amplification with other related RNA viruses. In the clinical evaluation, the RT-LAMP assay exhibited a similar sensitivity with nested RT-PCR and qRT-PCR. The results indicated that RT-LAMP assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of PPgV in field settings.

First description of Theiler's disease-associated virus infection and epidemiological investigation of equine pegivirus and equine hepacivirus coinfection in Brazil.

Recent advances in the study of equine pegivirus (EPgV), Theiler's disease-associated virus (TDAV) and equine hepacivirus (EqHV) highlight their importance to veterinary and human health. To gain some insight into virus distribution, possible risk factors, presence of liver damage and genetic variability of these viruses in Brazil, we performed a cross-sectional study of EPgV and TDAV infections using a simultaneous detection assay, and assessed EqHV coinfection in different horse cohorts. Of the 500 serum samples screened, TDAV, EPgV and EPgV-EqHV were present in 1.6%, 14.2% and 18.3%, respectively. EPgV-positive horses were present in four Brazilian states: Espírito Santo, Mato Grosso do Sul, Minas Gerais and Rio de Janeiro. Serum biochemical alterations were present in 40.4% of EPgV-infected horses, two of them presenting current liver injury. Chance of infection was 2.7 times higher in horses ≤5 years old (p = 0.0008) and 4.9 times higher in horses raised under intensive production systems (p = 0.0009). EPgV-EqHV coinfection was 75% less likely in horses older than 5 years comparatively to those with ≤5 years old (p = 0.047). TDAV-positive animals were detected in different horse categories without biochemical alteration. Nucleotide sequences were highly conserved among isolates from this study and previous field and commercial product isolates (≥88% identity). Tree topology revealed the formation of two clades (pp = 1) for both EPgV and TDAV NS3 partial sequences. In conclusion, the widespread presence of EPgV-RNA suggests an enzootic infection with subclinical viremia in Brazil. Horse management can influence virus spread. This first report of TDAV-infected horses outside the USA reveals the existence of subclinical viremic horses in distant geographical regions. EPgV and TDAV have similar circulating isolates worldwide. These findings contribute to global efforts to understand the epidemiology and pathogenesis of these equine viruses.

The prevalence of elevated gamma-glutamyltransferase and sorbitol dehydrogenase activity in racing Thoroughbreds and their associations with viral infection.

BACKGROUND: In racehorses, serum gamma-glutamyltransferase (GGT) activity is positively correlated with cumulative days in training and, when ≥100 IU/L, has been associated with poor performance. The prevalence of increased GGT activity in North American Thoroughbreds and its aetiopathogenesis are unknown. Four emerging viruses, pegivirus E (PgV E; equine pegivirus), hepacivirus A (HcV A; equine hepacivirus), pegivirus D (PgV D; Theiler's disease virus), and equine parvovirus-hepatitis (EqPV-H) have been identified in horses with clinical and subclinical hepatopathy. Available prevalence data indicate these viruses may commonly infect racehorses and contribute to increased liver enzyme activity in this population. OBJECTIVES: To investigate the association between viral infection and increased liver enzyme activity in racing Thoroughbreds. STUDY DESIGN: Cross-sectional study. METHODS: Prerace blood samples were collected from 802 Thoroughbreds and tested for GGT and sorbitol dehydrogenase (SDH) activity, and the presence of PgV E, HcV A, PgV D and EqPV-H nucleic acid. RESULTS: Increased SDH and/or GGT were detected in 56.2% of the 802 serum samples. The infection prevalence and relative risk (RR) of having concurrently increased liver enzyme activity were: PgV E = 18.2% (RR = 0.820, 95% CI = 0.662-0.978, P = 0.03), HcV A = 2.5% (RR = 1.132, 95% CI = 0.719-1.466, P = 0.6), PgV D = 0.5% (RR = 0.875, 95% CI = 0.165-1.598, P>0.9), EqPV-H = 2.9% (RR = 0.916, 95% CI = 0.564-1.266, P = 0.7). MAIN LIMITATIONS: Longitudinal samples were not tested. CONCLUSIONS: While viral infection was common among Thoroughbreds in this study, infection did not explain the high prevalence of increased liver enzyme activity. In fact, PgV E infection was associated with a reduced risk of having increased liver enzyme activity, indicating PgV E is unlikely to be a cause of hepatitis in horses. Importantly, like GGT, increased SDH activity was highly prevalent in this study, and provides additional evidence that hepatocellular injury was occurring in these horses.

Semi-quantitative duplex RT-PCR reveals the low occurrence of Porcine Pegivirus and Atypical Porcine Pestivirus in diagnostic samples from the United States.

Porcine Pegivirus (PPgV) and Atypical Porcine Pestivirus (APPV) are two recently identified porcine viruses. In this study, the identification of two viruses by metagenomic sequencing, and a duplex semi-quantitative RT-PCR was developed to detect these pathogens simultaneously. The PPgV strain Minnesota-1/2016 had a 95.5%-96.3% nucleotide identity and clustered with the recently identified US PPgV strains, which is a distant clade from the German PPgV strains. The APPV strain Minnesota-1/2016 shared an 87.3%-92.0% nucleotide identity with the other global APPV strains identity but only shared an 82.8%-83.0% nucleotide identity with clade II consisting of strain identified in China. Detection of both PPgV and APPV was 9.0% of the diagnostic cases. Co-infection of PPgV and APPV was identified in 7.5% of the diagnostic cases. The occurrence and genetic characterization of PPgV and APPV further enhance our knowledge regarding these new pathogens in the United States.

Genomic characterization of a novel pegivirus species from free-ranging bottlenose dolphins (Tursiops truncatus) in the Indian River Lagoon, Florida.

We report the discovery of the first cetacean pegivirus (family Flaviviridae) using a next-generation sequencing approach. One of two infected bottlenose dolphins had elevated activities of liver enzymes, which may suggest hepatocellular injury. Further research is needed to determine the epidemiology and pathogenicity of dolphin pegivirus.

Virus discovery reveals frequent infection by diverse novel members of the Flaviviridae in wild lemurs.

Lemurs are highly endangered mammals inhabiting the forests of Madagascar. In this study, we performed virus discovery on serum samples collected from 84 wild lemurs and identified viral sequence fragments from 4 novel viruses within the family Flaviviridae, including members of the genera Hepacivirus and Pegivirus. The sifaka hepacivirus (SifHV, two genotypes) and pegivirus (SifPgV, two genotypes) were discovered in the diademed sifaka (Propithecus diadema), while other pegiviral fragments were detected in samples from the indri (Indri indri, IndPgV) and the weasel sportive lemur (Lepilemur mustelinus, LepPgV). Although data are preliminary, each viral species appeared host species-specific and frequent infection was detected (18 of 84 individuals were positive for at least one virus). The complete coding sequence and partial 5' and 3' untranslated regions (UTRs) were obtained for SifHV and its genomic organization was consistent with that of other hepaciviruses, with one unique polyprotein and highly structured UTRs. Phylogenetic analyses showed the SifHV belonged to a clade that includes several viral species identified in rodents from Asia and North America, while SifPgV and IndPgV were more closely related to pegiviral species A and C, that include viruses found in humans as well as New- and Old-World monkeys. Our results support the current proposed model of virus-host co-divergence with frequent occurrence of cross-species transmission for these genera and highlight how the discovery of more members of the Flaviviridae can help clarify the ecology and evolutionary history of these viruses. Furthermore, this knowledge is important for conservation and captive management of lemurs.

Genomic sequencing and characterization of Theiler's disease-associated virus identified in commercial equine sera in China.

Theiler's disease-associated virus (TDAV) could be the aetiological agent of Theiler's disease. Horses experimentally inoculated with equine plasma containing TDAV develop acute and chronic infections with viraemia. Since its first identification in 2013, TDAV has not been detected in equines in the epidemiological studies that have been conducted. Until now, only one genome sequence of TDAV (HorseA1_serum) had been obtained. In this study, we sequenced the genome of four TDAV strains (A/China, F/China, H/USA and I/USA) in commercial equine sera used for cell culture propagation in China using three rounds of RT-PCR. The PCR primers were designed based on the HorseA1_serum genome sequence. All four TDAV strains had a polyprotein gene that was 9567 nt long, the same nucleotide length as the polyprotein gene of HorseA1_serum. Sequence analysis demonstrated the genetic diversity of TDAV. The nucleotide similarity of the polyprotein genes of the TDAV strains ranged between 90.3 and 93.6 %, with a high amino acid similarity that ranged from 98.2 to 98.8 %. Phylogenetic analysis using the polyprotein gene showed that A/China, F/China, H/USA and I/USA were clustered together with HorseA1_serum in the genus Pegivirus D. This study enriches our knowledge of the genetic diversity of TDAV.

Hepacivirus A Infection in Horses Defines Distinct Envelope Hypervariable Regions and Elucidates Potential Roles of Viral Strain and Adaptive Immune Status in Determining Envelope Diversity and Infection Outcome.

Hepacivirus A (also known as nonprimate hepacivirus and equine hepacivirus) is a hepatotropic virus that can cause both transient and persistent infections in horses. The evolution of intrahost viral populations (quasispecies) has not been studied in detail for hepacivirus A, and its roles in immune evasion and persistence are unknown. To address these knowledge gaps, we first evaluated the envelope gene (E1 and E2) diversity of two different hepacivirus A strains (WSU and CU) in longitudinal blood samples from experimentally infected adult horses, juvenile horses (foals), and foals with severe combined immunodeficiency (SCID). Persistent infection with the WSU strain was associated with significantly greater quasispecies diversity than that observed in horses who spontaneously cleared infection (P = 0.0002) or in SCID foals (P < 0.0001). In contrast, the CU strain was able to persist despite significantly lower (P < 0.0001) and relatively static envelope diversity. These findings indicate that envelope diversity is a poor predictor of hepacivirus A infection outcomes and could be dependent on strain-specific factors. Next, entropy analysis was performed on all E1/E2 genes entered into GenBank. This analysis defined three novel hypervariable regions (HVRs) in E2, at residues 391 to 402 (HVR1), 450 to 461 (HVR2), and 550 to 562 (HVR3). For the experimentally infected horses, entropy analysis focusing on the HVRs demonstrated that these regions were under increased selective pressure during persistent infection. Increased diversity in the HVRs was also temporally associated with seroconversion in some horses, suggesting that these regions may be targets of neutralizing antibody and may play a role in immune evasion.IMPORTANCE Hepacivirus C (hepatitis C virus) is estimated to infect 150 million people worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. In contrast, its closest relative, hepacivirus A, causes relatively mild disease in horses and is frequently cleared. The relationship between quasispecies evolution and infection outcome has not been explored for hepacivirus A. To address this knowledge gap, we examined envelope gene diversity in horses with resolving and persistent infections. Interestingly, two strain-specific patterns of quasispecies diversity emerged. Persistence of the WSU strain was associated with increased quasispecies diversity and the accumulation of amino acid changes within three novel hypervariable regions following seroconversion. These findings provided evidence that envelope gene mutation is influenced by adaptive immune pressure and may contribute to hepacivirus persistence. However, the CU strain persisted despite relative evolutionary stasis, suggesting that some hepacivirus strains may use alternative mechanisms to persist in the host.

Identification and genetic characterization of equine Pegivirus in China.

In 2013, two new viruses, equine pegivirus (EPgV) and Theiler's disease-associated virus (TDAV), both belonging to the genus Pegivirus within the family Flaviviridae, were identified. To investigate the geographical distribution and genetic diversity of these two viruses in China, we screened EPgV and TDAV infection in imported race horses and Chinese work horses by using reverse-transcription polymerase chain reaction (RT-PCR). EPgV was detected in 10.8 % (8/74) of the total horses tested, with a prevalence of 5.8 and 22.7 % in the race horses and work horses, respectively. No TDAV infection was found. A near full-length genome sequence of EPgV was obtained that showed an identity of 89.5-90.6 % at the nucleotide level and 98.1-98.3 % at the amino acid level with an American strain, C0035, and another Chinese strain, LW/216, respectively. Phylogenetic analysis showed two different clusters of the sequences from the race horses and work horses, indicating a difference in virus origin. Our results demonstrated a higher positive rate of EPgV in the Chinese work horses than in the imported race horses, a moderate genetic diversity of EPgV strains worldwide and possibly no liver pathogenesis for EPgV infection.

New virus of the family Flaviviridae detected in lumpfish (Cyclopterus lumpus).

In this study, we determined the complete coding sequence of a putative new member of the family Flaviviridae, named "Cyclopterus lumpus virus" (CLuV), which is associated with a serious disease in lumpfish (Cyclopterus lumpus). The virus was present in all tissues tested, but pathology was primarily observed in the liver and kidneys. CLuV shows low but distinct similarity to the unassigned Tamana bat virus (TABV). Unlike other known members of the family Flaviviridae, translation of the entire CLuV polyprotein is dependent on a - 1 ribosomal frameshift in the NS2A region.

Molecular characterization of a genetically divergent equine pegivirus strain identified in China.

Equine pegivirus (EPgV) is a newly discovered equine virus, which is taxonomically classified in the Pegivirus genus of the Flaviviridae family. Until now, only the complete genome sequence of the first reported EPgV strain, from the USA (strain name: C0035) is available on online databases. Considering this, horse serum samples were collected from horses in China and screened for EPgV RNA by RT-PCR. One EPgV strain, LW/2016, was obtained and its near-complete genome sequence was acquired by standard PCR. Further analysis of its nucleotide sequence indicates LW/2016 is genetically divergent from C0035, with a nucleotide identity of 89.02%. These two viruses clustered into two independent branches following phylogenetic analysis of on the NS3 and NS5B genes. To our knowledge, this is the first report of genetic divergence in the EPgV genome.

A Review of Flaviviruses that Have No Known Arthropod Vector.

Most viruses in the genus Flavivirus are horizontally transmitted between hematophagous arthropods and vertebrate hosts, but some are maintained in arthropod- or vertebrate-restricted transmission cycles. Flaviviruses maintained by vertebrate-only transmission are commonly referred to as no known vector (NKV) flaviviruses. Fourteen species and two subtypes of NKV flaviviruses are recognized by the International Committee on Taxonomy of Viruses (ICTV), and Tamana bat virus potentially belongs to this group. NKV flaviviruses have been isolated in nature almost exclusively from bats and rodents; exceptions are the two isolates of Dakar bat virus recovered from febrile humans and the recent isolations of Sokoluk virus from field-collected ticks, which raises questions as to whether it should remain classified as an NKV flavivirus. There is evidence to suggest that two other NKV flaviviruses, Entebbe bat virus and Yokose virus, may also infect arthropods in nature. The best characterized bat- and rodent-associated NKV flaviviruses are Rio Bravo and Modoc viruses, respectively, but both have received limited research attention compared to many of their arthropod-infecting counterparts. Herein, we provide a comprehensive review of NKV flaviviruses, placing a particular emphasis on their classification, host range, geographic distribution, replication kinetics, pathogenesis, transmissibility and molecular biology.

ICTV Virus Taxonomy Profile: Flaviviridae.

The Flaviviridae is a family of small enveloped viruses with RNA genomes of 9000-13 000 bases. Most infect mammals and birds. Many flaviviruses are host-specific and pathogenic, such as hepatitis C virus in the genus Hepacivirus. The majority of known members in the genus Flavivirus are arthropod borne, and many are important human and veterinary pathogens (e.g. yellow fever virus, dengue virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the Flaviviridae, which is available at www.ictv.global/report/flaviviridae.

Antibody Responses to Zika Virus Infections in Environments of Flavivirus Endemicity.

Zika virus (ZIKV) infections occur in areas where dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and other viruses of the genus Flavivirus cocirculate. The envelope (E) proteins of these closely related flaviviruses induce specific long-term immunity, yet subsequent infections are associated with cross-reactive antibody responses that may enhance disease susceptibility and severity. To gain a better understanding of ZIKV infections against a background of similar viral diseases, we examined serological immune responses to ZIKV, WNV, DENV, and YFV infections of humans and nonhuman primates (NHPs). Using printed microarrays, we detected very specific antibody responses to primary infections with probes of recombinant E proteins from 15 species and lineages of flaviviruses pathogenic to humans, while high cross-reactivity between ZIKV and DENV was observed with 11 printed native viruses. Notably, antibodies from human primary ZIKV or secondary DENV infections that occurred in areas where flavivirus is endemic broadly recognized E proteins from many flaviviruses, especially DENV, indicating a strong influence of infection history on immune responses. A predictive algorithm was used to tentatively identify previous encounters with specific flaviviruses based on serum antibody interactions with the multispecies panel of E proteins. These results illustrate the potential impact of exposure to related viruses on the outcome of ZIKV infection and offer considerations for development of vaccines and diagnostics.

Proposed update to the taxonomy of the genera Hepacivirus and Pegivirus within the Flaviviridae family.

Proposals are described for the assignment of recently reported viruses, infecting rodents, bats and other mammalian species, to new species within the Hepacivirus and Pegivirus genera (family Flaviviridae). Assignments into 14 Hepacivirus species (Hepacivirus A-N) and 11 Pegivirus species (Pegivirus A-K) are based on phylogenetic relationships and sequence distances between conserved regions extracted from complete coding sequences for members of each proposed taxon. We propose that the species Hepatitis C virus is renamed Hepacivirus C in order to acknowledge its unique historical position and so as to minimize confusion. Despite the newly documented genetic diversity of hepaciviruses and pegiviruses, members of these genera remain phylogenetically distinct, and differ in hepatotropism and the possession of a basic core protein; pegiviruses in general lack these features. However, other characteristics that were originally used to support their division into separate genera are no longer definitive; there is overlap between the two genera in the type of internal ribosomal entry site and the presence of miR-122 sites in the 5' UTR, the predicted number of N-linked glycosylation sites in the envelope E1 and E2 proteins, the presence of poly U tracts in the 3' UTR and the propensity of viruses to establish a persistent infection. While all classified hepaciviruses and pegiviruses have mammalian hosts, the recent description of a hepaci-/pegi-like virus from a shark and the likely existence of further homologues in other non-mammalian species indicate that further species or genera remain to be defined in the future.

The effect of Tembusu virus infection in different week-old Cherry Valley breeding ducks.

To study the effect of Tembusu virus (TMUV) infection on Cherry Valley Breeding ducks of different ages, 350 five-week-old ducks were divided into 14 groups. Ducks in seven experimental group were respectively infected with 1.265×10(5) mean embryo lethal dose (ELD50) of TMUV-AHQY strain (in 4.2mL) by intravenous route. Ducks in control groups were inoculated with Phosphate-buffered Saline (PBS) in the same way. Clinical symptoms, gross and microscopic lesions, viral loads and serum antibodies were detected and recorded for 20days after infection. Some ducks infected at 7 and 21 week s of age showed severe clinical symptoms including depression and inappetence, and no obvious clinical symptoms were seen in other week-old infected ducks. Severe gross lesions including hepatomegaly, meningeal congestion, myocardial hemorrhage, intestinal, myocardial and pulmonary edema were observed in ducks infected at 7, 18 and 21 weeks of age. No or mild gross lesions were observed in ducks infected at 14 and 16 weeks of age. The main microscopic lesions including hyperaemia, degeneration and necrosis of different cells and inflammatory cellular infiltration mainly consisting of mononuclear cells or lymphocytes were observed in ducks infected at 7 and 21 week of age. But relatively intact structures and rare lymphocytic infiltration were presented in ducks infected at 14 and 16 weeks of age. Viral antigen was more frequently observed in organ slices collected from 7 week-old infected ducks and few positive staining was found in 14 and 16 week-old infected ducks. Less viral loads in different tissues and swabs were detected by a quantitative real-time PCR assay. The level of viral loads in the tissues of ducks infected at 14 and 16 weeks of age was very lower than that of ducks infected at 7 and 21 weeks of age. Meanwhile, less viral copy numbers were detected in swab samples collected from 14 and 16 week-old infected ducks. Ducks infected at 14-week-old developed significantly higher serum neutralizing antibody titers than those infected at other week of age. These results indicated that the effect of TMUV infection on Cherry Valley ducks is partly related to weeks of age. 7-10 week-old and 18-21 week-old ducks were more susceptible to TMUV infection, but 14-16 week-old ducks were more resistant to this disease.