Re‐emergence of a genotype VIII virulent Newcastle disease virus isolated from Chinese game fowl after 13 years

Circulation of dominant genotypes VI and VII of Newcastle disease virus (NDV) is causing significant economic losses to the poultry industry in China. However, reports of Newcastle disease (ND) outbreaks caused by genotype VIII strains of NDV are rare. In this study, a virulent genotype VIII strain of NDV, designated GXGB2011, was isolated from a vaccinated game fowl flock showing clinic signs of infection in Pinxiang county, Guangxi, China. The whole genome of the isolate was completely sequenced and was found to be comprised of 15,192 nucleotides (nt), encoding the six structural proteins in the order of 3′‐NP‐P‐M‐F‐HN‐L‐5′. The pattern of cleavage site ¹¹²RRQKR↓F¹¹⁷ in the fusion (F) protein and the intracerebral pathogenicity index (ICPI) value of 1.5 showed that the strain GXGB2011 was a velogenic NDV. The results of the challenge experiment with the 5‐week‐old SPF chickens showed that the strain was highly pathogenic with 100% morbidity and mortality of the challenged birds. Based on the detection of virus in different organs of the infected birds, the highest viral load in caecal tonsils was observed and viral levels in immune organs were higher than those in the respiratory organs. Bayesian reconstruction of complete genomes based on the sequences of 66 NDV reference strains showed that the strain belonged to the genotype VIII of NDV. Phylogenetic analysis showed that the strain was more closely related to the foreign strains gamefowl/U.S.(CA)/24225/98, 1ITTY94060 and IT‐147/94 rather than to the first domestic strains of the emergence genotype VIII in Qinghai, China during 1979–1985. In summary, the results of the study demonstrated the re‐emergence of a highly pathogenic virulent isolate of genotype VIII of NDV. These results indicate the risk that this genotype VIII of NDV may spread to commercial chickens from game fowl.     

Molecular phylogenetics of Newcastle disease viruses isolated from vaccinated flocks during outbreaks in Southern India reveals circulation of a novel sub‐genotype

Newcastle disease (ND) is an economically important, contagious poultry viral disease reported across the globe. In India, ND is endemic and episodes of ND outbreaks despite strict vaccinations are not uncommon. We isolated and characterized seven ND viruses from vaccinated commercial poultry farms during severe disease outbreaks in Tamil Nadu, in Southern India, between April 2015 and June 2016. All the seven isolates were categorized as virulent by mean death time (48–54 hr) in embryonated chicken eggs. Also, their sequences carried the virulence signature of multi‐basic amino acid residues in their fusion protein cleavage site (RRQ/RR/KRF). Phylogenetic and evolutionary distance analyses revealed circulation of a novel sub‐genotype of genotype XIII, class II ND viruses, herein proposed as sub‐genotype XIIIe. The genetic divergence between the circulating virulent strains and the vaccine strains could possibly explain the disease outbreak in the vaccinated flocks. Further, our study signifies the need to implement routine epidemiological surveillance and to revisit the current vaccination program.     

新城疫病毒治疗乳腺癌的研究进展

乳腺癌是发病率最高的女性恶性肿瘤,是不容忽视的女性健康杀手.近年来,乳腺癌的发病率持续呈上升趋势和年轻化趋势.随着乳腺癌治疗的越来越多元化、细致化,乳腺癌患者的预后得到了显著的改善.当Csatary发现新城疫病毒能够抑制晚期胃癌转移的现象后,新城疫病毒便作为一种新型的抗肿瘤生物制剂得以广泛研究.新城疫病毒治疗乳腺癌也就成为一种较好的新的辅助治疗方法.本文简要地从新城疫病毒的概况、作用机制、研究意义等方面进行阐述.     

Epidemiological investigation of H9 avian influenza virus,Newcastle disease virus,Tembusu virus,goose parvovirus and goose circovirus infection of geese in China

To deepen the knowledge about epidemic prevalence in the goose breeding field, a triplex PCR assay was established, and 478 samples were collected from scaled goose farms in 11 provinces in China. The results of this epidemiological investigation showed that incidence rates of H9 avian influenza and goose circovirus were the highest among five infectious diseases that were evaluated. In addition, the triplex PCR assay established remarkable sensitivity, rapidity and versatility compared to other diagnostic methods. Dual infection comprised a large proportion of the co‐infections in the field, of which the combinations of H9/Tembusu, H9/goose circovirus and goose circovirus/Tembusu co‐infected cases were more common. Epidemics were more severe in winter and spring. Additionally, significant differences in the prevalence of these infectious diseases were observed in association with different age groups. In addition, phylogenetic analysis, determined by the neighbour‐joining method, was carried out to investigate the evolution of these viruses during the study period. For the most part, virus strains isolated during the study were consistent with most goose‐origin strains isolated from the Chinese mainland over the past few years. However, mutations were observed between isolated H9 avian influenza virus strains and sequences available from GenBank, which should draw much attention.     

新城疫病毒对人肺腺癌A549细胞系的作用

目的 观察新城疫病毒对人肺腺癌A549细胞的作用.方法 应用新城疫病毒(NDV)强毒株D90对体内、体外培养人肺腺癌A549细胞进行抗癌实验,通过体外培养A549细胞观察D90对肿瘤细胞的杀伤作用,进而建立14例A549肿瘤细胞裸鼠模型,对实验后裸鼠肿瘤组织进行病理学检查,通过光镜、电镜等方法观察肿瘤细胞形态学改变从而探讨新城疫病毒D90对人肺腺癌A549细胞的杀伤作用,为临床应用奠定基础.结果 NDV强毒株D90可在体外培养条件下溶解、杀伤A549细胞.实验组与对照组比较肿瘤组织体积明显缩小(t'=4.753,P<0.01),光镜和电镜下可见肿瘤组织内血管分布减少,肿瘤细胞坏死与凋亡.结论 新城疫病毒强毒株D90能够抑制体外培养的人肺腺癌A549细胞的增殖并溶解、杀伤肿瘤细胞,能够抑制裸鼠体内肿瘤组织的生长和转移,对正常组织无影响.     

Comparative in vivo pathogenicity study of an ITA genotype isolate (G6) of infectious bursal disease virus

Recently, a new genotype of infectious bursal disease virus (IBDV), named ITA, was detected in IBD‐vaccinated Italian broilers. Genome characterization revealed ITA to be a genetically different IBDV, belonging to genogroup 6 according to a recently proposed IBDV classification. The currently available clinical data do not allow any definition of the degree of pathogenicity of the ITA‐IBDV isolates. In the present study, a pathogenicity trial was conducted by the oral inoculation of specific‐pathogen‐free (SPF) chickens. Birds were housed in poultry isolators and inoculated at 35 days of age with an ITA‐IBDV isolate (35 birds) or a strain belonging to the G1a genogroup as a comparison (35 birds). Control birds (25 birds) were contextually mock‐inoculated with sterile water. Birds were observed daily for clinical signs and at 0, 7, 14, 21 and 28 days post‐inoculation (dpi) were bled for IBDV antibody detection. At 2, 4, 7, 14, 21 and 28 dpi, five birds from each of the inoculated groups, and three from the control group, were euthanized and subjected to a post‐mortem examination; the bursa:body weight and thymus:body weight ratios were calculated. Microscopic lesions of the bursa and thymus were scored on the basis of lymphoid necrosis and/or depletion or cortex atrophy, respectively. Both viruses induced a subclinical course of disease, as neither clinical signs nor mortality were recorded during the study, even in the presence of typical IBDV gross and microscopic lesions. Bursal damage, measured by the bursa:body weight ratio, was more noticeable and precocious after ITA‐IBDV inoculation. Histopathology scores of the bursa, indicative of rapid lymphoid depletion, confirmed the aggressiveness of the ITA‐IBDV strain in this organ. This study showed that, although the ITA‐IBDV strain tested causes infection with a subclinical course, it induces severe damage to lymphoid tissues. Therefore, its circulation in birds might be a threat for the poultry industry and may jeopardize the success of the production cycle.     

Pathotypic and Genotypic Characterization of Two Bangladeshi Isolates of Newcastle Disease Virus of Chicken and Pigeon Origin

Two Bangladeshi isolates of Newcastle disease virus (NDV), one from a chicken and one from a pigeon, were characterized in this study. Pathogenicity of the isolates was evaluated on the basis of intracerebral pathogenicity index (ICPI). Both the isolates were found to be of velogenic pathotype having ICPI of 1.83 and 1.51 for the chicken and pigeon isolate, respectively. Genotype of the isolates was determined by phylogenetic analysis based on partial F gene sequences. A 766‐bp genome fragment spanning partial M and F gene was amplified by RT‐PCR and sequenced. The first 354 bp of the coding region of F gene and corresponding deduced amino acid sequences (residues 1–118) of these two NDV isolates were aligned with that of other NDV strains retrieved from GenBank. A phylogenetic tree constructed from the alignment showed that the chicken isolate (BD‐C162) belonged to the newly described genotype XIII and the pigeon isolate (BD‐P01) to genotype VI. Both the chicken and pigeon isolates possessed a virulent‐like fusion protein cleavage site ¹¹²RRQKRF¹¹⁷.     

Tracing the origins of genotype VIIh Newcastle disease in southern Africa

It is widely accepted that Newcastle disease is endemic in most African countries, but little attention has been afforded to establishing the sources and frequency of the introductions of exotic strains. Newcastle disease outbreaks have a high cost in Africa, particularly on rural livelihoods. Genotype VII h emerged in South‐East Asia and has since caused serious outbreaks in poultry in Malaysia, Indonesia, southern China, Vietnam and Cambodia. Genotype VII h reached the African continent in 2011, with the first outbreaks reported in Mozambique. Here, we used a combination of phylogenetic evidence, molecular dating and epidemiological reports to trace the origins and spread of subgenotype VII h Newcastle disease in southern Africa. We determined that the infection spread northwards through Mozambique, and then into the poultry of the north‐eastern provinces of Zimbabwe. From Mozambique, it also reached neighbouring Malawi and Zambia. In Zimbabwe, the disease spread southward towards South Africa and Botswana, causing outbreaks in backyard chickens in early‐to‐mid 2013. In August 2013, the disease entered South Africa's large commercial industry, and the entire country was infected within a year, likely through fomites and the movements of cull chickens. Illegal poultry trading or infected waste from ships and not wild migratory birds was the likely source of the introduction to Mozambique in 2011.     

Short time window for transmissibility of African swine fever virus from a contaminated environment

Since the introduction of African swine fever virus (ASFV ) into the Baltic states and Poland in 2014, the disease has continued to spread within these regions. In 2017, the virus spread further west and the first cases of disease were reported in the Czech Republic and Romania, in wild boar and domestic pigs, respectively. To control further spread, knowledge of different modes of transmission, including indirect transmission via a contaminated environment, is crucial. Up until now, such an indirect mode of transmission has not been demonstrated. In this study, transmission via an environment contaminated with excretions from ASFV ‐infected pigs was investigated. Following euthanasia of pigs that were infected with an isolate of ASFV from Poland (POL /2015/Podlaskie/Lindholm), healthy pigs were introduced into the pens, in which the ASFV ‐infected pigs had been housed. Introduction was performed at 1, 3, 5 or 7 days, following euthanasia of the infected pig groups. Pigs, that were introduced into the contaminated environment after 1 day, developed clinical disease within 1 week, and both ASFV DNA and infectious virus were isolated from their blood. However, pigs introduced into the contaminated pens after 3, 5 or 7 days did not develop any signs of ASFV infection and no viral DNA was detected in blood samples obtained from these pigs within the following 3 weeks. Thus, it was shown that exposure of pigs to an environment contaminated with ASFV can result in infection. However, the time window for transmissibility of ASFV seems very limited, and, within our experimental system, there appears to be a rapid decrease in the infectivity of ASFV in the environment.     

Comparative pathogenicity of H5N6 subtype highly pathogenic avian influenza viruses in chicken,Pekin duck and Muscovy duck

In Japan during the 2016–2017 winter season, clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIVs) of the H5N6 subtype caused 12 outbreaks in chicken and Muscovy duck farms. These viruses have been circulating in Vietnam and China since 2014. In this study, we evaluated the susceptibility of chicken, Pekin duck (Anas platyrhynchos domesticus) and Muscovy duck (Cairina moschata) to H5N6 HPAIVs that originated in Japan, Vietnam and China. The H5N6 HPAIVs examined in this study were highly lethal to chickens compared with their pathogenicity in Pekin duck and Muscovy duck. One of five chickens infected with A/Muscovy duck/Aomori/1‐3T/2016 (MusDk/Aomori) survived despite viral shedding, although all of the chickens infected with the other viruses died. The 50% chicken lethal dose differed among the Japanese strains that shared the same gene constellation indicating that gene constellation was not a major determinant of pathogenicity in chicken. MusDk/Aomori, A/chicken/Niigata/1‐1T/2016 (Ck/Niigata) and A/duck/Hyogo/1/2016 (Dk/Hyogo) infected all Muscovy ducks inoculated; Ck/Niigata killed 50% of the ducks it infected whereas the other two did not kill any ducks. A/chicken/Japan/AnimalQuarantine‐HE144/2016 (HE144) isolated from chicken meat that originated in China was highly pathogenic to Pekin duck: all of the ducks died within 3.75 days of inoculation. This study shows that the pathogenicity of the clade 2.3.4.4 H5N6 HPAIVs differs not only between hosts but also within the same host species.     

Emergent subtype of hepatitis E virus genotype 3 in wild boar in Spain

Wild boar (Sus scrofa) is considered as the main wildlife reservoir of zoonotic hepatitis E virus (HEV) genotypes. The aim of this study was to evaluate the circulation of HEV in free‐ranging wild boar in the Doñana National Park (DNP), Spain. Blood samples were collected from 99 wild boar in the DNP during 2015. Sera were analysed in parallel using indirect ELISA and real‐time RT‐PCR. A total of 57 of the 99 tested animals (57.6%; 95%CI: 47.8%–67.3%) had anti‐HEV antibodies, indicating that this virus is widespread in wild boar in the DNP. HEV RNA was detected in one animal and phylogenetic analysis showed that the sequence isolated belonged to subtype 3r. The results suggest a potential risk of zoonotic transmission of this novel HEV‐3 subtype, which could be of public health concern. Further studies are required to assess the role of wild boar in the epidemiology of HEV‐3r and to determine the infectivity of this emergent HEV subtype in other species, including humans.     

Epidemiological investigation of the novel genotype avian hepatitis E virus and co‐infected immunosuppressive viruses in farms with hepatic rupture haemorrhage syndrome,recently emerged in China

Since 2016, hepatic rupture haemorrhage syndrome (HRHS) appeared in chickens of China and caused huge economic loss. To assess the infection status of the avian hepatitis E virus (HEV) and co‐infected viruses, including avian leukosis virus (ALV), reticuloendotheliosis virus (REV), fowl adenovirus (FAdV), and chicken infectious anaemia virus (CIAV), in farms with HRHS, 180 liver samples were collected from 24 farms in different provinces and detected by strict molecular virology methods. Results showed that the positive rates of HEV, ALV, REV, FAdV, and CIAV were 74.44%, 20.00%, 27.78%, 31.11%, and 12.22%, respectively, whereas there are also 112 samples with co‐infection, for a rate of 58%. Meanwhile, the positive rate of HEV decreased gradually with age; the lowest positive rate of ALV (5.76%) and REV (19.23%) appeared in 25–35 weeks age, during which the positive rate of CIAV was the highest (19.23%); the positive rate of HEV in layers (64.00%) was lower than that of broilers (83.33%), but the positive rates of ALV (38.46%) and CIAV (15.38%) in layers were higher than that of broilers (5.88%, 9.80%); the positive rates of HEV (75.88%) and CIAV (15.60%) in parental generation (PG) were higher than that of commodity generation (CG, 64.10%, 0.00%), whereas the positive rate of ALV showed inverse relationship (PG: 14.89%; CG: 38.46%). Additionally, phylogenetic analysis showed that all the avian HEV identified this study belong to a novel genotype, and found the close relationship between the wild strains (REV and CIAV) and corresponding isolates from contaminated vaccine. The data presented in this report will enhance the current understanding of the epidemiology characteristics in farms with HRHS in China.     

Assessment of Preparation of Samples Under the Field Conditions and a Portable Real‐Time RT‐PCR Assay for the Rapid On‐Site Detection of Newcastle Disease Virus

Newcastle disease virus (NDV), also known as virulent forms of avian paramyxovirus serotype 1 (AMPV‐1), is the causative agent of Newcastle disease affecting many species of birds and causing heavy losses to the poultry industry worldwide. Early, rapid and sensitive detection of the viruses or the viral nucleic acids is very important for disease diagnosis and control. This study aimed to evaluate sample preparation under field conditions and the application of a real‐time RT‐PCR method in the portable T‐COR4 platform for the rapid, on‐site detection of NDV on a farm. In the laboratory setting, the portable real‐time RT‐PCR assay had a similar performance compared with that obtained with a larger, stationary Rotor Gene real‐time thermocycler. In the field conditions, viral nucleic acids were manually extracted just outside of animal units with minimal equipment and real‐time RT‐PCR detection was performed with the portable thermocycler T‐COR4 placed in a nearby room. The portable assay at the farm detected viral RNA in 15 samples and reached an agreement of 83% (39/47) when the same RNA preparations were tested in the Rotor Gene thermocycler under the laboratory setting. The results demonstrated the feasibility of performing field detection but also the need to improve and further simplify sample preparation procedures.     

Shuni virus in Israel: Neurological disease and fatalities in cattle

The insect‐transmitted Shuni virus (SHUV) belongs to the Simbu serogroup of orthobunyaviruses and it is known to induce abortions, stillbirths and severe congenital malformations in ruminants and may cause neurological signs in infected horses. Here, SHUV was detected in brain samples of two Israeli cattle, which suffered from severe neurological signs that led to the deaths of the animals. During histopathological examination of the first case, a 5‐month‐old calf, small perivascular cuffs, composed mainly of neutrophils with few lymphocytes were observed in the brain stem and cerebrum. Similar infiltrates were also found to a lesser extent in the cerebellar meninges leading to the diagnosis of acute‐subacute meningoencephalitis. The histological examination of the brainstem from the second case, a 16‐month‐old heifer, revealed perivascular infiltration composed of equal numbers of macrophages and neutrophils associated with cerebral and meningeal haemorrhages. In this case encephalitis was diagnosed. Viral RNA was extracted from brain samples of both cattle that suffered from severe neurological signs and was subsequently tested by a polymerase chain reaction PCR assay specific for Simbu serogroup viruses and found positive. The presence of SHUV was subsequently confirmed by the isolation of the virus from one sample and sequence analysis of both brain samples. The comparison of the complete sequences of the coding regions of all three genome segments from both cases revealed a close relationship to Shuni viruses detected in tissue samples of aborted or malformed calves or lambs born during the last years in Israel.     

Retracted: Tilapia lake virus disease: Phylogenetic analysis reveals that two distinct clades are circulating in Israel simultaneously

Tilapia lake virus (TiLV) is an emerging viral disease that affects several tilapia species in different countries since 2014. In 2017–2018, 129 samples were collected from 14 tilapia farms in Israel. Ninety samples represented TiLV‐suspected cases (TSC), and 39 were used as control samples (CS). RT‐qPCR was performed on 89 and 39 duplicate brain and liver tissue samples from TSC samples and CS, respectively. TiLV was diagnosed in 37 (40.1%) of TSC, and two of the CS samples (5%) were also positive for TiLV. Additional validation RT‐PCR was performed on positive samples, and amplified products were sequenced. Maximum‐likelihood phylogenetic analysis of segment‐3 of 25 selected sequences revealed two distinct clades: one virtually identical to sequences from India and the second closely related to isolates from Ecuador, Thailand, Egypt and Peru, apparently imported to Israel from Thailand. Thus, our results indicate that at least two distinct clades of TiLV are circulating in Israel simultaneously. As of today, the number of TiLV sequences available in free publicly accessible databases is limited. Nevertheless, our study provides new molecular epidemiology baseline for further epidemiological studies of TiLV.     

Detection of influenza D virus in bovine respiratory disease samples,UK

Influenza D is a newly described virus of cattle, pigs and small ruminants first detected in North America during 2011. Cattle have been shown to be the main viral reservoir and mounting evidence indicates that infection with influenza D may contribute to the development of bovine respiratory disease. The virus has been detected across the United States, Europe and Asia. To date, influenza D has not been reported in the UK. During the winter and spring of 2017/2018, we performed molecular testing of cattle submitted for post‐mortem examination where respiratory disease signs were present. We detected influenza D virus in 8.7% of cases, often as the sole viral agent and always in conjunction with bacterial co‐infection with one or more agents. Viral RNA was present in both the upper and lower respiratory tract and pathological changes in lung tissues were observed alongside signs of concurrent bacterial infections. Sequencing of one UK isolate revealed that it is similar to viruses from the Republic of Ireland and Italy.     

Attenuated and non‐haemadsorbing (non‐HAD) genotype II African swine fever virus (ASFV) isolated in Europe,Latvia 2017

A non‐haemadsorbing (non‐HAD) ASF virus (ASFV) genotype II, namely Lv17/WB/Rie1, was isolated from a hunted wild boar in Latvia in 2017. Domestic pigs experimentally infected with the non‐HAD ASFV developed a nonspecific or subclinical form of the disease. Two months later, these animals were fully protected when exposed to other domestic pigs infected with a related virulent HAD genotype II ASFV.     

Genetic characterization of African swine fever virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype

African swine fever virus (ASFV ) is one of the most threatening infectious diseases of pigs. There are not sufficient data to indicate the importance of the sylvatic cycle in the spread and maintenance of the disease locally and potentially, globally. To assess the capacity to maintain ASF in the environment, we investigated the presence of soft tickreservoirs of ASFV in Gorongosa National Park (GNP ) and its surrounding villages. A total of 1,658 soft ticks were recovered from warthog burrows and pig pens at the wildlife/livestock interface of the GNP and viral DNA was confirmed by nested PCR in 19% of Ornithodoros porcinus porcinus and 15% of O. p. domesticus . However, isolation of ASFV was only achieved in approximately 50% of the PCR ‐positive samples with nineteen haemadsorbing virus isolates recovered. These were genotyped using a combination of partial sequencing of the B646L gene (p72 ) and analysis of the central variable region (CVR ) of the B602L gene. Eleven isolates were classified as belonging to genotype II and homologous to contemporary isolates from southern Africa, the Indian Ocean and eastern Europe. Three isolates grouped within genotype V and were similar to previous isolates from Mozambique and Malawi. The remaining five isolates constituted a new, previously unidentified genotype, designated genotype XXIV . This work confirms for the first time that the virus currently circulating in eastern Europe is likely to have a wildlife origin, and that the large diversity of ASFV maintained in wildlife areas can act as a permanent sources of different strains for the domestic pig value chain in Mozambique and beyond its boundaries. Their genetic similarity to ASFV strains currently spreading across Europe justifies the need to continue studying the sylvatic cycle in this African country and other parts of southern Africa in order to identify potential hot spots of ASF emergence and target surveillance and control efforts.     

Revisiting the genetic diversity of classical swine fever virus: A proposal for new genotyping and subgenotyping schemes of classification

Classical swine fever (CSF ) is a highly contagious febrile viral disease caused by CSF virus (CSFV ), and it is considered one of the most important infectious diseases that affect domestic pigs and wild boar. Previous molecular epidemiology studies have revealed that the diversity of CSFV comprises three main genotypes and different subgenotypes defined using a reliable cut‐off to accurately classify CSFV at genotype and subgenotype levels. However, a growing number of CSFV both complete genome and full E2 gene sequences have been submitted to GenBank (more than 500 sequences are currently available, revised on December 1, 2017). Therefore, the aim of this study was to revisit the taxonomy of CSFV at genotype and subgenotype levels, to unify nomenclature and to provide an update to the classification of CSFV . We propose here a new genotyping scheme with five well‐defined CSFV genotypes (CSFV Genotypes 1–5) and 14 subgenotypes (seven for each of the CSFV Genotype 1 and CSFV Genotype 2). The findings showed in this study are relevant for molecular epidemiology approaches and will help to better understand the genetic diversity and spreading of CSFV at a global scale. The update in the classification of CSFV will allow the scientific community to establish more accurately the links among different outbreaks of the disease.