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With a possible origin from bats, the alphacoronavirus Porcine epidemic diarrhea virus (PEDV) causes significant hazards and widespread epidemics in the swine population. However, the ecology, evolution, and spread of PEDV are still unclear. Here, from 149,869 fecal and intestinal tissue samples of pigs collected in an 11-year survey, we identified PEDV as the most dominant virus in diarrheal animals. Global whole genomic and evolutionary analyses of 672 PEDV strains revealed the fast-evolving PEDV genotype 2 (G2) strains as the main epidemic viruses worldwide, which seems to correlate with the use of G2-targeting vaccines. The evolving pattern of the G2 viruses presents geographic bias as they evolve tachytely in South Korea but undergo the highest recombination in China. Therefore, we clustered six PEDV haplotypes in China, whereas South Korea held five haplotypes, including a unique haplotype G. In addition, an assessment of the spatiotemporal spread route of PEDV indicates Germany and Japan as the primary hubs for PEDV dissemination in Europe and Asia, respectively. Overall, our findings provide novel insights into the epidemiology, evolution, and transmission of PEDV, and thus may lay a foundation for the prevention and control of PEDV and other coronaviruses.

RESEARCH HIGHLIGHTSFirst report of the epidemiology of duck adenovirus 3 infection in China.Mutant DAdV-3 strains (truncated ORF67) became predominant.

With a possible origin from bats, the alphacoronavirus Porcine epidemic diarrhea virus (PEDV) causes significant hazards and widespread epidemics in the swine population. However, the ecology, evolution, and spread of PEDV are still unclear. Here, from 149,869 fecal and intestinal tissue samples of pigs collected in an 11-year survey, we identified PEDV as the most dominant virus in diarrheal animals. Global whole genomic and evolutionary analyses of 672 PEDV strains revealed the fast-evolving PEDV genotype 2 (G2) strains as the main epidemic viruses worldwide, which seems to correlate with the use of G2-targeting vaccines. The evolving pattern of the G2 viruses presents geographic bias as they evolve tachytely in South Korea but undergo the highest recombination in China. Therefore, we clustered six PEDV haplotypes in China, whereas South Korea held five haplotypes, including a unique haplotype G. In addition, an assessment of the spatiotemporal spread route of PEDV indicates Germany and Japan as the primary hubs for PEDV dissemination in Europe and Asia, respectively. Overall, our findings provide novel insights into the epidemiology, evolution, and transmission of PEDV, and thus may lay a foundation for the prevention and control of PEDV and other coronaviruses.

In 2019, flocks of Muscovy ducks presented with clinical signs typical of MDPV infection. The MDPV GD201911 strain was isolated by inoculating samples from positive birds into Muscovy duck embryos. Challenge with the isolate GD201911 caused typical MDPV disease symptoms and resulted in 25%-40% mortality, depending on the challenge dose, indicating the high pathogenicity of GD201911 for Muscovy ducks. Genome sequencing and phylogenetic analysis demonstrated that GD201911 clustered with recombinant MDPV strains, indicating that recombinant MDPV is circulating in China. Epidemiological monitoring should be performed continuously to assist with decision making for disease control.

Avian nephritis virus (ANV) infections of chicken flocks cause enteric and kidney disease, uneven growth, and runting stunting syndrome, leading to economic losses in the poultry industry. In this study, one ANV strain, designated as AH202017, was isolated from a diseased broiler flock in Anhui province, China, in 2020. Virus production in LMH cell culture was confirmed by real-time RT-PCR and immunofluorescence assay. The complete genome sequencing analysis indicated that AH202017 shares 77.5%-85.5% identity with 12 reference strains in GenBank. Phylogenetic analysis of the capsid protein revealed that AH202017 is more closely related to VIC-6a/Australia/2014 belonging to ANV genotype 2. However, the phylogenetic tree, based on the ORF1a protein and ORF1b protein, indicated that AH202017 manifests a close relationship with GXJL815/China/2017 belonging to genotype 8. In infection experiments, four infected chickens showed depression and one chicken died at 6 days post-infection, corresponding to 5% mortality. The virus was shed daily in the feces of infected chickens, and was found distributed in multiple organs. Macroscopic and microscopic lesions in the kidneys were observed. This is the first paper that describes the genomic characteristics and pathogenicity of a novel ANV strain in China.

Background The circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus emergence in diverse host has been associated with severe disease. Porcine circovirus-like virus (Po-Circo-like [PCL] virus) is a CRESS DNA virus, the prevalence and pathogenicity of which are rarely studied. Methods We obtained two blood samples, four faecal samples, and two intestinal samples from a pig farm suffered from diarrheal disease in the delivery room in September 2020 and attempted to isolate and identify a causative pathogen. Subsequently, only PCL virus was positive, and qRT-PCR was designed to detect the loading titre of PCL virus. We then initiated a heightened surveillance program on the pathogenicity and epidemiology of PCL virus. Results Six PCL virus strains, with severe diarrhoea and haemorrhagic enteritis, have been found in six different pig farms in Guangdong province, China. A multiple sequence alignment of these PCL viruses and bovine circovirus-like virus/CH showed a similarity of 92.5-94.8% for the Rep protein, indicating these PCL viruses are highly homologous to Bo-Circo-like virus associated with calf diarrhoea. There were striking similarities between the PCL virus and bovine circovirus-like virus outbreaks in aetiological settings and Genomic sequence. We found that 11.2% (20/178) of diarrhoea samples and 13.3% (6/45) of pig farms were positive for PCL virus, suggesting that PCL virus may have spread widely in Pig farms. Moreover, this article underscores the risk of PCL virus spilling over and adapting to new species. Conclusions Porcine circovirus-like virus was found to be associated with porcine diarrheal disease in China.

Porcine circovirus type 3 (PCV3) has been widely detected throughout the world since it was first discovered on pig farms in 2015. PCV3 is closely associated with cardiac and multisystem inflammation, respiratory disease, congenital tremors, myocarditis, diarrhea, encephalitis and neurologic disease, and periarteritis. However, there have been few reports on the relationship between PCV3 and inflammatory pathways. The NF-κB signaling pathway plays an important role in the defense against viral infection. Here, we demonstrate that the capsid protein (Cap) of PCV3 plays a key role in the activation of NF-κB signaling in HEK-293T cells. Furthermore, PCV3 Cap promotes the mRNA expression of the pro-inflammatory cytokines IL6 and TNFα. In addition, PCV3 Cap promotes RIG-I and MDA5 mRNA expression in RIG-like receptor (RLR) signaling and MyD88 mRNA expression in Toll-like receptor (TLR) signaling but does not influence TRIF mRNA expression in TLR signaling. These results show that PCV3 Cap activates NF-κB signaling, possibly through the RLR and the TLR signaling pathways. This work illustrates that PCV3 Cap activates NF-κB signaling and thus may provide a basis for the pathogenesis of PCV3 and the innate immunity of the host.

Chicken astrovirus (CAstV) is associated with kidney disease and visceral gout, runting and stunting syndrome, and white chick hatchery disease, causing economic losses to the poultry industry worldwide. In this study, 55.6% of 36 clinical samples from Guangdong province in China were positive for CAstV, but negative for other common enteric viruses, including avian nephritis virus, infectious bronchitis virus, fowl adenovirus Group I, Newcastle disease virus, chicken parvovirus, reovirus, and rotavirus by PCRs and RT-PCRs. A CAstV strain, named GD202013, was isolated from Guangdong province in south China, and was identified by CAstV RT-PCR. A whole genome sequence analysis demonstrated that GD202013 shares 76.0 to 88.1% identity with 24 reference strains in GenBank. Phylogenetic analysis, based on whole genome and capsid protein, showed that GD202013 is more closely related to 2 US strains (GA2011/US/2011 and 4175/US/2011) belonging to subgroup Bii. Recombination analysis indicated that GD202013 is a recombinant strain formed by 3 strains: a major parent strain CkP5/US/2016, and 2 minor parent strains (GA2011/US/2011 and G059/PL/2014). In addition, the chicken embryo infection experiment demonstrated that GD202013 causes hatchability reduction, growth depression, and death of embryos. Macroscopic and microscopic lesions in the liver, kidney and small intestine were observed in the dead-in-shell embryos. This is the first report of the novel CAstV infection in China.

Background Porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global pig industry. Currently available PEDV vaccine strains have limited protective effects against PEDV variant strains. Methods In this study, the highly virulent epidemic virus strain CT was serially passaged in Vero cells for up to 120 generations (P120). Characterization of the different passages revealed that compared with P10 and P64, P120 had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. Results Pathogenicity experiments using P120 in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal PEDV antigen distribution; the piglet survival rate in the P120 group was 100%. Furthermore, whole-genome sequencing identified 13 amino acid changes in P120, which might be responsible for the attenuated virulence of P120. Conclusions Thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines.

Porcine circovirus 3 (PCV3) is a novel circovirus that is associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multi-systemic inflammation. The type I Interferon (IFN) signaling pathway is an important innate immune signaling pathway for defense against viral infection. Many mammalian viruses inhibit host innate immune signaling through diverse strategies. Here, we found that the PCV3 capsid protein (Cap) significantly inhibited IFN-β-stimulated response element (ISRE) promoter activity, suggesting that Cap suppresses IFN signaling. However, Cap did not affect expression and phosphorylation levels of STAT1 and STAT2 and did not interrupt the heterodimerization of pSTAT1 and pSTAT2. Although Cap interacted with KPAN1, it did not block the interaction between KPNA1 and pSTAT1 or the nuclear translocation of pSTAT1 and pSTAT2. Interestingly, we found that Cap inhibited the activation of ISRE promoter induced by IRF9-S2C. Mechanistically, Cap interacted with the transactivation domain of STAT2, a key protein in type I IFN signaling. In addition, we found that Cap bound to ISRE and prevented ISRE binding of IRF9-S2C. This work is the first to describe the mechanism of inhibition of IFN signaling by PCV3 Cap.

Summary Porcine circovirus type 3 (PCV3) is a novel circovirus that was associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multisystemic inflammation. Recently, a PCV3 strain was identified from pyretic and pneumonic piglets in Guangdong province, China. This virus strain was sequenced and designated PCV3-China/GD2016. The complete genome of PCV3-China/GD2016 is 2,000 bp in length and shared 99.1% and 99.1% nucleotide identities with PCV3/29160 and PCV3/2164, respectively. [Corrections added after initial online publication on 13 March 2017: The numbers ‘98.5%’ and ‘97.4%’ has been changed to ‘99.1%’ and ‘99.1%’ in the previous sentence.] Phylogenetic analysis based on the complete genome showed that PCV3-China/GD2016 clustered with the emerging PCV3 and separated with other virus in genus Circovirus. The results of this study suggest that PCV3 has existed within the pigs of China. It is urgent to investigate the pathogenicity and epidemiology of this novel circovirus China.

Duck Tembusu virus (DTMUV) is a new pathogen that produces an acute and potent disease in ducks which has caused serious economic losses in China. In this study, a virulent strain of DTMUV, designated as ZJSBL01, was attenuated by serial passages in BHK-21 cells supplied with 5-Fluorouracil (5-FU) for 50 passages to induce mutation and attenuation. Growth kinetics of different passages of ZJSBL01 strain in BHK-21 cells show that these viruses have similar replication characteristics. The virus was highly attenuated after 40 passages in BHK-21 cells supplied with 5-FU, based on mortality, morbidity, and viral load in inoculated Sheldrake ducklings. In addition, all of the ducklings immunized with ZJSBL01-P40, the virus obtained at passage 40 of ZJSBL01, showed seroconversion on day 14 post inoculation. Moreover, P40 did not cause clinical symptom for layding ducks. Immunization with ZJSBL01-P40 could provide effective protection against the virulent parental ZJSBL01 strain. Seventeen amino acid substitutions were observed in the polyprotein of ZJSBL01-P40 compared with parental ZJSBL01. These results indicate that ZJSBL01-P40 may be a live vaccine candidate for prevention of DTMUV-disease.

Duck Tembusu virus (DTMUV) has spread to the major duck-farming region in China, causing acute egg-production drop in Chinese duck population. In this study, we characterized a DTMUV strain (named GD2014) isolated from an egg-production drop duck farm in Guangdong province, South China. The virus was pathogenic to Muscovy duck embryos and caused severe egg production drop for laying Muscovy ducks. The genome sequence of GD2014 shared 97-99% homologies with other waterfowl-origin Tembusu viruses, and shared 89% identities with MM1775 strain isolated from mosquito. Phylogenetic analysis of entire open reading frame (ORF), E gene and NS5 gene indicated that GD2014 belonged to Ntaya group. These results have implications for understanding the orgin, emergence and pathogenicity of DTMUV as well as for the development of vaccines and diagnostics based on epidemiological data.

Recently, a novel atypical porcine pestivirus (APPV) in pig was reported. In this study, two APPV strains, APPV-China/GZ01/2016 (GZ01) and APPV-China/GD-SD/2016 (GD-SD), were identified in two newborn piglet herds with congenital tremor from China. The open reading frame of the two strains shared an 83.5% nucleotide identity. Phylogenetically, the APPV strains were placed into two groups: GZ01 belonged to group I and GD-SD belonged to group II. A high viral load was detected in the cerebellum (quantification cycles ＜ 26). Further studies should be carried out to thoroughly elucidate the development of congenital tremors caused by APPV.

Duck Tembusu virus (DTMUV) is an infectious pathogen that can cause epidemics in egg-laying ducks. Here, we isolated and characterized a DTMUV, designated GDLH01, thought to be responsible for the noticeable egg drop in Muscovy duck flocks in South China since 2011. The genome sequence of GDLH01 shared 97-99% homology with other avian-origin Tembusu viruses, and 99.5% homology with the mosquito-borne strain SDMS recently reported in China. Phylogenetic analysis based on the nucleotide sequence of the entire open reading frame confirmed that the isolate was of avian origin and closely related to a mosquito-borne strain. Our findings characterize a novel Tembusu virus circulating in Muscovy ducks in South China and emphasize the importance of reinforcing biosecurity measures and developing vaccines to prevent the spread of this viral pathogen.

To the Editor: Avian influenza A viruses generally do not cause disease in aquatic birds, the natural reservoir of these viruses (1). Influenza A(H5N6) was first isolated from mallards by Garcia et al. in 1975 (2). Influenza viruses continue to evolve and reassort to generate novel, highly pathogenic viruses. Novel H5 highly pathogenic avian influenza virus subtypes, such as H5N2, H5N5, and H5N8, have been reported (3,4). Highly pathogenic influenza A viruses are endemic to many countries (http://www.oie.int/en/animal-health-in-the-world/update-on-avian-influenza/2015/), cause tremendous economic losses to the poultry industry, and represent a serious threat to public health. In March 2014, an influenza A(H5N6) outbreak caused the death of 457 birds in Laos (http://www.oie.int/wahis_2/public%5C..%5Ctemp%5Creports/en_imm_0000015052_20140507_182757.pdf). During the same month, a flock of ducks in Guangdong Province in southern China exhibited typical respiratory signs of influenza A virus infection. This flock also had 70% decreased egg production and a slightly increased mortality rate. Throat swab specimens were taken from the symptomatic and dead ducks, and the samples were used to inoculate chicken embryos for virus isolation. Hemagglutination (HA) and neuraminidase (NA) inhibition assays were performed to identify the subtype of the isolated virus, which was designated A/duck/Guangdong/GD01/2014 (H5N6) (GD01/2014). The complete RNA genome was amplified by reverse transcription PCR and cloned into the pMD-19T vector for sequencing (5). The complete genome sequence of the GD01/2014 virus was submitted to GenBank (accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"KJ754142-KJ754149","start_term":"KJ754142","end_term":"KJ754149","start_term_id":"639126630","end_term_id":"639126646"}}KJ754142-KJ754149). Multiple-sequence alignments showed that the HA gene of GD01/2014 shared 97.5% nt identity with A/wild duck/Shandong/628/2011 (H5N1) and NA genes shared 96.6% and 98.3% nt identity with A/swine/Guangdong/K6/2010 (H6N6) and A/duck/Shantou/1984/2007 (H6N6), respectively. All internal genes shared high levels of nucleotide identity (97.6%–98.5%) with A/wild duck/Fujian/2/2011(H5N1). The whole genes of GD01/2014 and the H5N6 viruses in Laos (LAO/2014) shared 98.2%–99.7% nt identity, indicating the same genotype. Phylogenetic analysis of the HA gene revealed that the isolated virus belonged to clade 2.3.4.6 (Technical Appendix Figure) (6). The NA gene of GD01/2014 was clustered with some H6N6 viruses circulating in China (Technical Appendix Figure, panel B). The 6 internal genes of GD01/2014 were closely related with A/wild duck/Fujian/2/2011(H5N1) and A/wild duck/Fujian/1/2011(H5N1) (Technical Appendix Figure, panels C–H). Phylogenetic analysis showed that all 8 genes of GD01/2014 and LAO/2014 were closely related although genetically distant from the earlier isolated H5N6 viruses (Technical Appendix Figure). These findings suggest that GD01/2014 and LAO/2014 are reassortants of wild duck H5N1 and H6N6 viruses, both of which have pathogenic and potential pandemic capacity in southern China. A previous report that H5N1 and H6N6 co-infected a duck suggests that GD01/2014 might be generated from the co-infection of H5N1 and H6N6 in the same host (7). The intravenous pathogenicity index of GD01/2014 was 3.0, which indicates that the isolate is highly pathogenic for chickens. GD01/2014 had multiple basic amino acids (LRERRRKR/GLF) at the cleavage site between HA1 and HA2; this characteristic is typical of highly virulent influenza viruses (8). The HA protein contained E190, R220, G225, Q226, and G228 (H3 numbering) residues at the receptor-binding pockets, indicating that the virus preferentially binds to the sialic acid-2,3-NeuAcGal of the avian-like receptor (9). The HA protein has 7 potential N-glycosylation sites (PGSs); the HA1 protein has 5 PGSs; the HA2 protein has 2 PGSs. The NA protein of GD01/2014 and LAO/2014 had a deletion of 11 aa residues at positions 59–69 (N6 numbering) in the NA stalk region. Moreover, a deletion of 5 aa residues from positions 80–84 in the nonstructural 1 protein was found in GD01/2014 and LAO/2014. The position 627 and 701 of the polymerase basic 2 protein were E and D, respectively, characteristics of the avian influenza virus (10). In summary, in 2014, outbreaks of H5N6 virus occurred in China, Laos, and Vietnam and caused the deaths of infected humans in Sichuan province, China (http://www.oie.int/en/animal-health-in-the-world/update-on-avian-influenza/2014/; http://www.wpro.who.int/china/mediacentre/releases/2014/20140507/en/). We characterized the novel reassortant H5N6 virus in China and found that it was the same genome type as and was highly homologous with the H5N6 virus in Laos. The findings in this study are also supported by the previous genetic characterization of these viruses by Wong et al. (11). However, the adaptation, host range, and virulence of this reassortant H5N6 virus are still unclear and should be further investigated. Furthermore, the potential for infection, outbreaks, and pandemic in other poultry and mammals should be carefully monitored. Technical Appendix. Phylogenetic relationship of genes of A/duck/Guangdong/GD01/2014(H5N6), a novel virus isolated from ducks in Guangdong Province in southern China during March 2014, compared with reference strains from GenBank. Click here to view.(1.1M, pdf)

As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSR↓GLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSR↓GLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks.