Pathogenesis of a bovine enterovirus-1 isolate in experimentally infected calves

The pathogenesis and virulence of Bovine enterovirus-1 (BEV-1) in cattle is largely unknown. Reports concerning its virulence suggest that there might be an association between BEV-1 infections and a range of diseases in cattle that vary from respiratory to enteric to reproductive disease and infertility. In the current study, the pathogenesis associated with acute infection of BEV-1 in calves experimentally inoculated with the Oklahoma isolate of BEV-1 was described. Although interpretation of the study was limited by lack of an effective control group, results suggest that an association between inoculation of BEV-1, virus localization, and the potential development of lesions in the brain and heart probably exists. In the experiment, BEV-1 virus localized to the terminal ileum, ileocecal and cecocolonic junctions, spiral colon, and ileocecal lymph nodes; BEV-1 virus was detected in the cytoplasm of enterocytes, lamina propria macrophages, endothelium, neurons of the submucosal and myenteric plexi, and lymphocytes of the submucosal lymphoid tissue. Although no clinical signs were noted following acute infection, BEV-1 was localized in the cerebellar white matter of a calf with encephalitis and in the heart of another calf with coronary arteritis. The current study suggests that the BEV-1 isolate is infectious to young calves and that BEV-1 potentially can have a similar pathogenesis to that observed in natural or experimental enterovirus infections in other species.     

Serological survey of bovine enterovirus type 1 in different mammalian species in Turkey

The bovine enterovirus type 1 (BEV-1) infection has a wide range of host spectrum including humans. In this study, seroprevalence of BEV-1 was investigated in eight mammalian species. Blood serum samples were collected from 244 humans, 1520 cattle, 272 horse, 126 dog, 281 sheep, 477 goat, 18 camel (Camelus dromedarius) and 82 gazelle (Gazella subgutturosa subgutturosa) in different regions of Turkey. Microneutralization tests showed that gazelle and camel did not have any seropositivities, but seropositivities were detected in humans (30.3%), cattle (64.8%), horse (12.8%), dog (3.2%), sheep (32.8%) and goat (27.6%).     

用荧光定量RT-PCR方法检测猪瘟病毒

为了建立能特异检测不同基因型猪瘟病毒(Classical swine fever virus,CSFV),同时又能区分其他瘟病毒的基因检测方法,本实验针对CSFV基因组5′端非编码区设计并合成了简并引物和TaqMan探针,在优化反应条件的基础上,成功地建立了特异检测CSFV的荧光定量RT-PCR检测方法。再以已知滴度的CSFV石门株血毒总RNA反转录产物建立标准品,该标准品可以用于定量临床样品中的CSFV滴度,所建立的荧光定量PCR方法可以灵敏地检测出10~(-0.82)个TCID_(50)病毒含量。最后用建立的方法对108份临床样品进行检测并同时进行病毒分离,荧光定量PCR方法检测出73份阳性样品且与病毒分离的符合率为100%,而常规RT-PCR只检测出54份阳性样品,表明本荧光定量RT-PCR法在检测猪瘟病料上具有潜在的应用价值。     

Investigation of Marek’s disease virus from chickens in central Ethiopia

Marek’s disease (MD) is a lymphoproliferative and neuropathic disease of domestic chickens and less commonly, turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpesvirus. In Ethiopia, MD is believed to be introduced with importation of exotic and crossbred to improve the poultry production and has been reported to be a potential threat to the poultry sector both in backyard and commercial farming systems. This study was aimed at isolation and molecular analysis of MD virus isolates circulating in chicken population in the central part of Ethiopia where commercial farms are populated. From September 2013 to January 2014, clinical and post-mortem examination were conducted on diseased chickens suspected of MD virus infection. Representative spleen and feather follicle samples were collected following sterile procedure, and infectious virus isolation was performed using primary chicken fibroblast cell culture. Cell culture inoculated with suspension of pathological samples developed characteristic MD virus cytopathic effect of rounding of the cells and small plaques. Further analysis of the virus was conducted by conventional PCR amplifying the ICP4 gene fragment from eleven tissue samples using MD virus specific primers. PCR products were further sequenced and analyzed. Nucleotide sequence similarity search of the local isolates resulted a high degree of sequence similarity with Gallid Herpes virus type 2 strain (Marek’s disease virus type 1, JN034558). To our knowledge, the present study is the first report conducted on virus isolation and molecular characterization of MD virus isolates circulated in Ethiopia. Eleven ICP4-like gene fragment (318 bp) sequences generated in the present study were uploaded in the public database (KU842366–76). Further research on virus isolation, genetic characterization, and infection dynamics is recommended targeting chickens of all age groups reared in different agro-ecological zones under different production system.

     

Studies on pathogenesis following single and double infection with viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) were bath challenged with viral hemorrhagic septicemia (VHS) virus or infectious hematopoietic necrosis (IHN) virus or with both viruses simultaneously. The viral distribution and development of histologic lesions were examined using immunohistochemistry, while virus titer in kidney was determined by viral titration in cell culture. Single infections with VHS virus and IHN virus showed similar distributions of virus in internal organs. The early identification of virus in gill epithelium, 1 and 2 days postinfection (PI) for VHS virus and IHN virus, respectively, indicates that this organ is the point of entry for both viruses. The detection of VHS virus at 1 day PI and 3 days PI for IHN virus is indicative of kidney and spleen being the target organs for these viruses. A simultaneous infection of VHS virus and IHN virus resulted in both viruses establishing an infection. Further double infection did not result in a statistically significant lower titer of both viruses in kidney but a more restricted distribution of IHN virus in internal organs compared with the single infected group. The most striking finding is that, for IHN virus, virus was not detected in the brain in situ in the double-infected group. This study provides support for the conclusion that simultaneous infection with two piscine rhabdoviruses in a susceptible host results in some degree of interaction at the cell level, leading to a reduced systemic distribution of IHN virus.     

新发林麝环状类病毒的鉴定和分析

利用病毒宏基因组学(viral metagenomics)方法,本研究从95份林麝粪便样品中检测到1株新型圆环病毒样(Circovirus-like)病毒,经过拼接获得其全基因组序列,该毒株命名为ls-cyc.其基因组全长约3552 nt,GC含量39.00％,含有2个开放阅读框(ORF),即ORF1和ORF2.其中O...     

Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts.

Bovine herpesvirus 1 (BHV1) and caprine herpesvirus 1 (CapHV1) are useful models to study virus-host interactions, as well as pathogenicity and latency, when comparing the outcome of infection in the natural and the foreign hosts. Molecular seroepidemiological analyses revealed that cross-reacting antibodies were mainly induced by glycoprotein gI (gB analogue), by the major capsid protein and by nonstructural proteins, whereas the most virus-specific antibodies were elicited by glycoproteins gIII and gIV. These glycoproteins, especially gIII (gC analogue), might therefore play an important role in the virus-host-interactions. As a basis for further studies, we re-evaluated observations concerning experimental infections with BHV1 and CapHV1 in the natural and the foreign hosts. All parameters indicated that both viruses were able to infect either host, but that the pathogenicity was restricted to the natural host. Latent virus could be reactivated exclusively from cows infected with BHV1. It was possible neither to reactivate BHV1 from goats, nor to reactivate CapHV1 from either species. The experiments indicated that the outcome of infection in the natural and the foreign host is dependent on host and viral factors, whereby gIII is only one important virus component involved. Further investigations in the host and host cell range of BHV1 and CapHV1 will help to clarify the role of factors responsible for virus-host-interactions.     

Isolation and characterization of an adventitious avian leukosis virus isolated from commercial Marek's disease vaccines

Commercial Marek's disease (MD) vaccines produced by two manufacturers were tested for possible contamination with avian leukosis virus (ALV). Samples of MD vaccines manufactured by two companies (A and B) were received from a breeder company; samples were also received directly from vaccine company B. Using virus isolation tests, samples initially tested positive for subgroup E (endogenous) ALV. However, upon repassage, the vaccines also tested positive for exogenous ALV. The isolated exogenous ALV proved to be a subgroup A virus, as determined by flow cytometry using polyclonal chicken antibodies specific for various subgroups of ALV, and by DNA sequencing of the envelope glygoprotein (gp85). The exogenous ALV isolated from MD vaccines was inoculated in chickens from ADOL lines 15I(5) x 7(1) and 0 to determine its pathogenicity and compare it with that of Rous-associated-virus-1 (RAV-1), the prototype strain of ALV-A. Each chicken from each line was inoculated with approximately 10,000 infectious units of RAV-1 or the ALV-A isolated from vaccines termed B-39 virus at 7th day of embryonation. At hatch, and at 4, 8, and 16 wk of age, chickens were tested for viremia and cloacal shedding; chickens were also observed for ALV-induced tumors within 16 wk of age. Viremia and cloacal shedding results suggest that chickens from both lines were susceptible to infection with either virus. Within 16 wk of age, the proportion of ALV tumors induced by strain B-39 in line 0 and line 15I5 x 7(1) chickens was 0% and 12%, respectively, compared with 62% and 67% in chickens inoculated with RAV-1. The data indicate that commercial MD vaccines produced by two manufacturers were contaminated with endogenous subgroup E and an exogenous subgroup A ALV. Further, data from biological characterization suggest that the ALV-A isolated from commercial MD vaccines is of low oncogenicity, compared with that of RAV-1. GenBank accession numbers: The gp85 gene sequences of ALV isolated from commercial Marek's disease vaccines have been deposited in GenBank and assigned the following accession numbers: A46 subgroup A, DQ412726 ; B53 subgroup A, DQ412727; A46 subgroup E, DQ412728; B53 subgroup E, DQ412729.     

Genetic characterization of maedi-visna virus (MVV) detected in Finland

The aim of the study was to characterize the small-ruminant lentiviruses (SRLVs) detected in Finland by defining their phylogenetic relationships and by studying the evolution of the virus based on a well-known epidemiology. The study material comprised lung tissue samples of 20 sheep from 5 different farms, a cell-cultured virus from one of the original sheep lung samples, and a blood sample of a goat. The sheep were identified as positive during seroepidemiologic screenings in 1994-1996 and the goat in 2001. Initial classification of a 251 nucleotide sequence within gag gene amplified from the uncultured samples as well as from the cell-cultured virus showed that the SRLVs were genetically close and that they were more closely related to the prototype ovine maedi-visna viruses (MVVs) than to the caprine arthritis-encephalitis virus (CAEV). The lentivirus detected from the goat aligned within the cluster of the Finnish ovine viruses, demonstrating a natural sheep-to-goat transmission. Further phylogenetic analysis of the proviral gag, pol and env sequences confirmed the initial classification and showed that they constituted a new subtype within the diverse MVV group. The sequence analyses also showed that the virus had remained genetically relatively stable, in spite of the time given for virus evolution, an estimated 20 years, and in spite of the virus crossing the host species barrier.     

The requirement of environmental acidification for Ibaraki virus infection to host cells

The effect of environmental acidification on Ibaraki virus (IBAV) infection was tested using endosomal inhibitory chemicals and low pH treatment. Treatment of target cells with endosomal inhibitors significantly decreased the progeny virus production. IBAV outer capsid proteins, VP5 and VP2, were removed from virion when purified IBAV was exposed to low pH environment. Further experiment showed that the exposure to low pH buffer facilitated IBAV infection when the cellular endosomal pathway was impaired by bafilomycin A1. Results obtained in this study suggest that acidic environment is essential to initiate IBAV infection.     

Serological investigations of red foxes (Vulpes vulpes L.) for determination of the spread of tick-borne encephalitis in Northrhine-Westphalia

Serum samples from 786 red foxes shot between January 1995 and August 1996 in the southern half of Northrhine-Westphalia, located in western Germany, were tested for the presence of antibodies against tick-borne encephalitis (TBE) virus using the Immunozym FSME IgG All Species-ELISA (Immuno, Heidelberg, Germany) as a screening test: 759 sera were negative, 23 (2.9%) were borderline, and four (0.5%) were positive. Nine of the 27 ELISA reactive sera were confirmed by the TBE Western-Blot (Immuno, Heidelberg, Germany). Furthermore these 27 sera were tested for neutralizing antibodies by means of a plaque reduction neutralization test (PRNT) against TBE and West Nile viruses. Only one single serum was found to have a neutralization titre (+1:800 PRNT80) against TBE virus. All other 26 sera were negative for neutralizing antibodies against TBE or West Nile virus. Since the titre of the single serum is low, it can be interpreted that if TBE virus is present, its prevalence is extremely low. Northrhine-Westphalia is not classified as a TBE-endemic area. Further calculated serological testing of game and virological investigation of collected ticks in the affected area seem to be meaningful and necessary.     

Molecular characterization of Brazilian isolates of orf virus

Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modified methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed field gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was confirmed by these hybridizations.     

通过病毒分离鉴定和基因检测首次发现虎流感

应用F81猫肾传代细胞，从高热、拒食和间有神经症状的死亡率病科中分离获得1株病毒，经形态学、理化学、生物学和血清学系统鉴定，证明为流感病毒。采用流感病毒核蛋白基因引物，对分离病毒及该虎病科进行RT－PCR扩增和序列分析，结果从分离病毒和虎病科中均扩增出与理论值大小相符的464bp基因片段；其序列与A、B、C3型流感病毒相比较，同源性分别为84．9％、35．0％、24．8％。由此说明，所分离病毒为A型流感病毒，命名为/蘧／哈尔滨（中国）／01／2002。用此分离病毒静脉接种于3月龄家猫3号，均出现与病虎相似的临床症状，其中1号耐过，2只死亡。死亡猫剖检变化与死虎相似，主要是呈肺炎病变，并可从病科中回收到所接种病毒。从此分离病毒为HA抗原，进行虎与猫血清的HI抗体检测，结果康复虎血清比其病初血清、康复猫血清比其接种前血清HI抗体均增高4倍以上，表明该病毒具有致病性，是引起该虎与试验猫发病甚至死亡的病原。     

马立克病病毒编码的miR-M4-5p调控宿主靶基因的筛选及鉴定

血清1型马立克病病毒（MDV-1）感染引起的鸡马立克病（MD）一直以来被认为是研究病毒诱导肿瘤发生机制的理想模型。此前研究发现，MDV-1编码的miR-M4-5p是宿主癌基因miR-155的病毒同源物，从基因组中敲除miR-M4-5p可显著降低MDV-1的致病性和致瘤性，表明miR-M4-5p可能是MDV-1诱导肿瘤发生的重要调控因子。为进一步揭示miR-M4-5p的调控机制，以CEF细胞总RNA反转录产物cDNA为模板，利用hybrid-PCR技术构建了miR-M4-5p的候选靶基因文库。通过基因克隆、PCR鉴定及序列比对分析，获得128个候选基因序列，有73个基因的3'-UTR存在miR-M4-5p的潜在结合靶点，其中23个3'-UTR结合靶点与miR-M4-5p完全互补配对。通过双荧光素酶报告试验和qRT-PCR分析，对miR-M4-5p与3'-UTR的体内外相互作用以及候选靶基因的表达水平进行分析和验证，最终鉴定DPT、TMEM230和DCLK1为miR-M4-5p调控的宿主靶基因。本研究为后续进一步阐明miR-M4-5p在MD肿瘤发生中的调控机制奠定了重要基础。     

Diagnosis of mixed infections with myxovirus influenzae A equi 2 and herpes virus equi 1 among Danish stud horses

Examination of nasopharyngeal secretion and organ material from clinical cases of respiratory diseases in horses, using inoculation of embryonated hen eggs and rabbit and horse kidney cell cultures, resulted in the isolation of influenza virus and herpes virus. In 2 cases, both viruses were present in the same specimen.On the basis of the physio-chemical, cytological and serological criteria, the viruses were found to be identical with influenza virus type A equi 2 and herpes virus equi type 1.The methods for serological diagnosis and characterization of the influenza and herpes viruses are discussed. Detailed serological examinations of blood specimens from the clinical cases confirmed the aetiological significance of the isolates and the presence of mixed infections.Further characterization of a strain of influenza virus was carried out by inoculation experiments on horses. These included reproduction of clinical influenza, reisolation of the strain in casu and a study of the antibody formation.     

Infectious dose‐dependent accumulation of live highly pathogenic avian influenza H5N1 virus in chicken skeletal muscle—implications for public health

Highly pathogenic avian influenza viruses (HPAIV) of H5N1 subtype are a major global threat to poultry and public health. Export of poultry products, such as chicken and duck meat, is a known source for the cross‐boundary spread of HPAI H5N1 viruses. Humans get infected with HPAI H5N1 viruses either by close contact with infected poultry or through consumption of fresh/undercooked poultry meat. Skeletal muscle is the largest soft tissue in chicken that has been shown to contain virus during systemic HPAIV infection and supports productive virus infection. However, the time between infection of a chicken with H5N1 virus and presence of virus in muscle tissue is not yet known. Further, it is also not clear whether chicken infected with low doses of H5N1 virus that cause non‐fatal subclinical infections continue to accumulate virus in skeletal muscle. We investigated the amount and duration of virus detection in skeletal muscle of chicken experimentally infected with different doses (10², 10³ and 10⁴ EID₅₀) of a HPAI H5N1 virus. Influenza viral antigen could be detected as early as 6 hr after infection and live virus was recovered from 48 hr after infection. Notably, chicken infected with lower levels of HPAI H5N1 virus (i.e., 10² EID₅₀) did not die acutely, but continued to accumulate high levels of H5N1 virus in skeletal muscle until 6 days post‐infection. Our data suggest that there is a potential risk of human exposure to H5N1 virus through meat from clinically healthy chicken infected with a low dose of virus. Our results highlight the need to implement rigorous monitoring systems to screen poultry meat from H5N1 endemic countries to limit the global spread of H5N1 viruses.     

Characterization of Env antigenicity of feline foamy virus (FeFV) using FeFV-infected cat sera and a monoclonal antibody

To characterize neutralizing antigenicity in relation to env genotypes of feline foamy virus (FeFV), serological analyses were performed using FeFV-infected cat sera and several field isolates including two env genotypes (F17- and FUV-types). Since three cats from which FeFV were isolated were found to have undetectable titers of virus neutralization (VN) antibodies, even to the homologous virus, VN antibodies were further examined with complement supplementation as an enhancement factor. With the presence of complement, the VN titers of FeFV-infected cat sera increased drastically. Although most of serum samples neutralized strains of either env genotype, sera sampled from two cats neutralized all the strains examined at similar titers, suggesting that superinfection with both env genotypes of FeFV might have occurred in the two cats. Further, we produced a monoclonal antibody (mAb) specifically neutralizing FeFV strains of FUV-type. The mAb was shown to have higher affinity to an epitope on Env of FUV-type than that of F17-type by immunoprecipitation assay. This study supplies basic information important for studies on FeFV vector development as well as on the relationship between the virus and the host immune response.     

Serological Investigations of Red Foxes (Vulpes vulpes L.) for Determination of the Spread of Tick‐borne Encephalitis in Northrhine‐Westphalia

Serum samples from 786 red foxes shot between January 1995 and August 1996 in the southern half of Northrhine‐Westphalia, located in western Germany, were tested for the presence of antibodies against tick‐borne encephalitis (TBE) virus using the Immunozym FSME IgG All Species‐ELISA^® (Immuno, Heidelberg, Germany) as a screening test: 759 sera were negative, 23 (2.9 %) were borderline, and four (0.5 %) were positive. Nine of the 27 ELISA reactive sera were confirmed by the TBE Western‐Blot (Immuno, Heidelberg, Germany). Furthermore these 27 sera were tested for neutralizing antibodies by means of a plaque reduction neutralization test (PRNT) against TBE and West Nile viruses. Only one single serum was found to have a neutralization titre (+ 1 : 800 PRNT₈₀) against TBE virus. All other 26 sera were negative for neutralizing antibodies against TBE or West Nile virus. Since the titre of the single serum is low, it can be interpreted that if TBE virus is present, its prevalence is extremely low. Northrhine‐Westphalia is not classified as a TBE‐endemic area. Further calculated serological testing of game and virological investigation of collected ticks in the affected area seem to be meaningful and necessary.