PCR-sequence characterization of new adenoviruses found in reptiles and the first successful isolation of a lizard adenovirus

A consensus nested PCR was used to screen diagnostic samples from approximately 70 reptiles for the presence of adenoviruses (AdV) in the years 2006-2007. Classical virus isolation methods were also used with all samples. After adenoviruses were detected in a group of helodermatid lizards in a Danish zoo, a follow-up study was also carried out on lizards from this group (10 Mexican beaded lizards and 24 Gila monsters) over the period of a year. Adenoviruses were detected in a total of 26 lizards and snakes by PCR. The PCR amplicons from all positive animals were sequenced and the resulting polymerase gene sequences were used for phylogenetic analysis. Altogether six Agamid AdVs were amplified, with a minimal sequence variation between one another and between these and GenBank Agamid AdVs. The sequence obtained from one of the Gila monsters is identical with the GenBank Helodermatid AdV, while the sequences from the Mexican beaded lizards differ from this. In a snake collection we have detected a new AdV from an Asp viper. All of the above mentioned adenoviruses cluster in the Atadenovirus genus. However, the sequence from a new Varanid AdV detected in this study clusters outside this genus. On cell culture, viruses were isolated from three of the AdV positive helodermatid lizards (one Mexican beaded lizard and two Gila monsters) and identified as AdVs based on electron microscopy and PCR and sequencing using cell culture supernatant. This is the first report of the successful isolation of a lizard AdV.     

禽副黏病毒2型研究进展

禽副粘病毒 2型 (PMV-2 )属于副粘病毒科、腮腺炎病毒属 ,是制备人用生物制品所用SPF鸡的必检项目。该病毒呈世界性分布 ,我国鸡群中的感染也普遍存在。 PMV-2单独感染SPF鸡仅引起轻微的呼吸道症状 ,但与 IBV、MG等禽呼吸道病病原混合感染会明显加强后者的致病作用。PMV-2的 HN基因的 ORF全长为 1 743 nt,编码一个由 580个氨基酸组成的蛋白 ,F基因较为保守 ,变异不是很大。文章重点介绍了近年来关于 PMV-2病原学、流行病学、致病性、分子生物学以及诊断和防治方法的最新研究进展。     

Viruses in reptiles

ABSTRACT: The etiology of reptilian viral diseases can be attributed to a wide range of viruses occurring across different genera and families. Thirty to forty years ago, studies of viruses in reptiles focused mainly on the zoonotic potential of arboviruses in reptiles and much effort went into surveys and challenge trials of a range of reptiles with eastern and western equine encephalitis as well as Japanese encephalitis viruses. In the past decade, outbreaks of infection with West Nile virus in human populations and in farmed alligators in the USA has seen the research emphasis placed on the issue of reptiles, particularly crocodiles and alligators, being susceptible to, and reservoirs for, this serious zoonotic disease. Although there are many recognised reptilian viruses, the evidence for those being primary pathogens is relatively limited. Transmission studies establishing pathogenicity and cofactors are likewise scarce, possibly due to the relatively low commercial importance of reptiles, difficulties with the availability of animals and permits for statistically sound experiments, difficulties with housing of reptiles in an experimental setting or the inability to propagate some viruses in cell culture to sufficient titres for transmission studies. Viruses as causes of direct loss of threatened species, such as the chelonid fibropapilloma associated herpesvirus and ranaviruses in farmed and wild tortoises and turtles, have re-focused attention back to the characterisation of the viruses as well as diagnosis and pathogenesis in the host itself.1. Introduction2. Methods for working with reptilian viruses3. Reptilian viruses described by virus families3.1. Herpesviridae3.2. Iridoviridae3.2.1 Ranavirus3.2.2 Erythrocytic virus3.2.3 Iridovirus3.3. Poxviridae3.4. Adenoviridae3.5. Papillomaviridae3.6. Parvoviridae3.7. Reoviridae3.8. Retroviridae and inclusion body disease of Boid snakes3.9. Arboviruses3.9.1. Flaviviridae3.9.2. Togaviridae3.10. Caliciviridae3.11. Picornaviridae3.12. Paramyxoviridae4. Summary5. Acknowledgements6. Competing interests7. References.     

Characterization of avian reovirus strain-specific polymorphisms

Avian reoviruses have been associated with several pathologic conditions, but correlative relationships between genotypes and specific diseases have not been demonstrated. Six avian reoviruses (883, 176, 81-5, S1133, FC, and TX) were selected for this study, and a comparative study of the pathogenic properties of the viruses in chickens, following peroral and footpad inoculation, was carried out, along with a comparison of the electrophoretic mobility of viral genomic segments and viral proteins encoded by the gene segments. The pathogenic properties of the viruses were shown to be diverse, with three distinct pathotypes being defined: Pathotype I (883) caused only a syndrome that we have termed "transient digestive system disorder" (TDSD); Pathotype II (FC, TX, and S1133) caused only "viral arthritis syndrome" (VAS), whereas Pathotype III (176 and 81-5) caused both TDSD and VAS. Likewise, the genomes of the viruses were shown to be extremely polymorphic, with a maximum of five segments co-migrating between any two strains. Considerable variation in the electrophoretic mobility of the encoded proteins also was demonstrated with pronounced variation in the molecular size of the sigma 4 protein, the purported viral attachment protein, being evident. These results show that the genomes of avian reoviruses were extremely polymorphic, preventing correlation between genotypes and pathotypes. But these studies have provided us with the genetic elements needed to characterize the gene functions involved in viral pathogenesis.     

鸭新城疫病毒分离株抗原表位和遗传演化分析

从规模化养殖场鸭群气管和泄殖腔试子分离到新城疫病毒(NDV)27株,用2株针对NDV HN单抗进行抗原表位分析,并选择4个分离株进行F基因高变区(374bp)和HN基因全长序列分析。抗原表位分析结果显示,27个鸭分离株均能与其中一株单抗C3-B7反应,而与另外一株单抗1E5反应为阴性。F基因(374bp)序列分析结果显示,4个鸭分离株均属于NDV ClassⅠ分支,分离株之间核苷酸同源性为99.2%～100%;分离株与NDV ClassⅡ毒株遗传距离为0.9%～9.9%,与NDV ClassⅠ毒株遗传距离为38.5%～41.7%。根据核苷酸序列推导的氨基酸序列表明,4个鸭NDV分离株F蛋白裂解位点氨基酸模式为:112-EROERL-117。HN基因序列分析结果显示,4个鸭NDV分离株HN基因全长1851bp,编码585个氨基酸;同源性比较发现4个鸭NDV分离株之间核苷酸同源性为99.7%～99.8%,与NDV ClassⅡ毒株核苷酸同源性为68.4%～70.5%,与NDV ClassⅠ毒株核苷酸同源性为95.8%～98.0%。本研究结果显示,鸭分离毒均属于NDV ClassⅠ弱毒,在抗原表位和基因序列上与广泛应用的NDV弱毒疫苗株(LaSota)不同,这些毒株的来源有待进一步深入研究。     

SCID mice as a tool for evaluation of heteroxenous life cycle pattern of Caryospora (Apicomplexa, Eimeriidae) species

Adult severe combined immunodeficient (SCID) mice were inoculated with oocysts of 13 different Caryospora (Protozoa, Apicomplexa) species isolated from the faeces of 10 reptilian and three raptorial bird hosts in attempt to test heteroxenous life cycle pattern. Only three reptilian isolates originated from viperid snakes, namely from Calloselasma rhodostoma, Atheris nitschei and Vipera ursinii induced lethal dermal caryosporosis in SCID mice. Neither clinical signs nor developmental stages were observed in mice infected with further nine caryosporan isolates originated from other reptilian and raptorial bird hosts. Results of this study confirmed that SCID mice represent a useful tool for evaluation of heteroxenous life cycle pattern of caryosporan coccidia and that only the Caryospora species from viperid and crotalid snakes produce dermal caryosporosis in mice     

Molecular characterization of the hemagglutinin-neuraminidase gene of porcine rubulavirus isolates associated with neurological disorders in fattening and adult pigs

"Blue eye disease" is a viral infection of swine endemic in Mexico, which produces fatal encephalitis accompanied by respiratory signs and corneal opacity in suckling piglets. An atypical blue eye disease outbreak presented high rates of neurological signs in fattening and adult pigs from 2000 to 2003. In order to identify the basis of increased neurovirulence, the hemagglutinin-neuraminidase (HN) gene of several porcine rubulavirus isolates were sequenced and compared with that of La Piedad Michoacan virus and other isolates that did not produce neurological disorders in weaned pigs. Nine amino acid mutations distinguished the high neurovirulent PAC6-PAC9 viruses, whereas five mutations characterized the low neurovirulent PAC2 and PAC3 viruses. HN protein three-dimensional models showed that the main conformation and functional domains were preserved, although substitutions A223T and A291D occurred in PAC2 and PAC3 viruses, as well as A511K and E514K presented in PAC6-PAC9 viruses considerably modified the properties of the HN protein surface. The increased positive charge of the HN protein of PAC6-PAC9 viruses seems to be associated with their increased neurovirulence.     

Amebiasis in four ball pythons, Python reginus.

Between September 13th and November 18th in 1999, four ball pythons, Python reginus kept in the same display, showed anorexia and died one after another. At necropsy, all four snakes had severe hemorrhagic colitis. Microscopically, all snakes had severe necrotizing hemorrhagic colitis, in association with ameba-like protozoa. Some of the protozoa had macrophage-like morphology and others formed protozoal cysts with thickened walls. These protozoa were distributed throughout the wall in the large intestine. Based on the pathological findings, these snakes were infested with a member of Entamoeba sp., presumably with infection by Entamoeba invadens, the most prevalent type of reptilian amoebae.     

Virological and molecular characterization of a mammalian orthoreovirus type 3 strain isolated from a dog in Italy

A mammalian orthoreovirus (MRV) strain was isolated from a pup with fatal diarrhea, which had a concurrent infection by canine parvovirus type 2. The reovirus isolate showed an atypical hemagglutination pattern and a retarded electrophoretic mobility of the S1 segment, which is characteristic of MRV type 3 (MRV-3). Assignment of the isolated virus to MRV-3 was confirmed by type-specific RT-PCR assays, targeting the S1 gene, and by subsequent sequence analysis of the PCR product. By phylogeny based on the S1 gene of several MRVs, the isolate fell into lineage E, along with the murine strain T3C9/61 and the bovine strains T3C18/61 and T3C31/59. Conversely, L1 sequences were found to segregate regardless of the viral type. A total of 110 fecal samples, 56 nasal and 31 ocular swabs from dogs with diarrhea or nasal/ocular discharge were tested by a nested-PCR assay specific for reoviruses, and no sample was found to contain MRV RNA, a finding that is apparently in contrast with the seroprevalence (25.77%) observed in dogs.     

Isolation of avian paramyxoviruses from sentinel ducks and turkeys in Minnesota

In a 4-year study (1980-1983) involving the use of sentinel ducks that intermingled with wild ducks, a total of 98 paramyxovirus (PMV) isolates (84 Newcastle disease virus, 14 PMV-6) were obtained from 3652 sentinel duck cloacal samples (2.7% isolation rate) collected between June and mid-November each year. PMV infection of sentinel ducks appeared to be seasonal, with the onset of infection occurring between mid-July and mid-August. PMV was not isolated from sentinel turkeys that co-mingled with sentinel ducks during the last 2 years of the study. However, there was serological evidence that the sentinel turkeys were infected with PMV. These findings indicate that wild waterfowl are a natural reservoir of PMV and suggest that interspecies transmission of certain PMV serotypes may occur between waterfowl and turkeys.     

Resistance of chickens to challenge with the virulent Herts 33 strain of Newcastle disease virus induced by prior infection with serologically distinct avian paramyxoviruses.

Results indicate that some degree of protection from challenge by Newcastle disease virus (NDV)/Herts 33 was conferred on chickens by prior infection with PMV/turkey/Wisconsin/68, PMV/turkey/Ontario/6661/68, PMV/Netherlands/449/75 and PMV/parakeet/England/39/78 viruses, all of which are serologically related but distinguishable from NDV. Except for one bird which survived challenge three weeks after infection with Robin/Hiddensee/19/75, no protection was seen in chickens infected with other unrelated avian paramyxoviruses. In contrast to infection with NDV-B1, birds protected by infection with avian paramyxoviruses showed large increases in NDV haemagglutination inhibition (HI) titres after challenge. In these birds considerable increases in the homologous HI titres were also seen after challenge.     

The use of antibiotics in reptiles: a review

The rationale of antibiotic therapy in reptiles, the significance of reptilian bacterial isolates and their antibiotic sensitivity are discussed. Individual antibiotic studies in snakes and terrapins are reviewed and the extrapolation of dose rates from endotherms (mammals) to ectotherms (reptiles) is shown to be unreliable.     

Prevalence of reoviruses in commercial chickens

The prevalence of reoviruses in commercial chickens with the runting/stunting syndrome, tenosynovitis, and normal chickens was investigated. Reoviruses were isolated from 3-week-old chickens affected with the runting/stunting syndrome and from older chickens with tenosynovitis; viruses were isolated from tissues with and without lesions. Reoviruses were also frequently isolated from rectal contents of normal 3-week-old chickens, and there was serological evidence of previous reovirus infection in all flocks of adult meat breeder chickens examined. The widespread occurrence of reoviruses in both normal and diseased chickens indicates that the isolation of reoviruses from tissues of chickens with lesions does not necessarily imply any aetiological relationship of reovirus with disease, even in the absence of other known causes. However, the occurrence of reovirus in normal chickens does not preclude an aetiological relationship with disease and further investigation of strain variation and possible virulence factors in avian reoviruses is required.     

A comparison of the pathogenicity of four avian reoviruses in chickens

Four avian reoviruses were orally inoculated into 1-day-old chickens to determine pathogenicity, virus persistence in the intestinal tract, and effects on body weight gains. Avian reoviruses Reo-25 and W3-492 belonged to two separate serotypes, and viruses TC 897 and W3-410 were antigenically related to W3-492. Isolate W3-492, which was highly pathogenic, was very rarely recovered from cloacal swabs collected 2 weeks postinoculation, but inoculated chickens gained significantly less weight (P less than or equal to 0.001) than uninoculated controls during the 5-week test study. Isolate Reo-25 persisted the longest in the intestinal tract, and isolates TC 897 and W3-410, of intermediate persistence, had no significant effect on body weights. There was no apparent correlation between serotype and pathotype of avian reoviruses.     

新城疫病毒山东分离株ShD-5-06 F和HN基因序列分析

从山东济南某非典型新城疫发病鸡群中分离到一株新城疫病毒株（ShD-5—06）,研究其生物学特性表明,该病毒具有新城疫强毒株的一些特征。从该分离株扩增出其F和HN基因,并与标准株进行同源性比较,为探讨NDV是否发生变异提供理论依据。本试验通过RT—PCR法特异性地扩增出F和HN基因全基因序列,并对其与已经发表的序列进行核苷酸序列测定和分析。结果表明,ShD-5—06株的F和HN基因开放性阅读框架（ORF）为1662bp和1716bp,分别编码489个和571个氨基酸。与国外发表的部分新城疫病毒强毒株和弱毒株之间相应序列进行比较,F基因核苷酸序列的同源性在84．1％～88．7％之间,氨基酸同源性在88．1％～93．3％之间;HN基因核苷酸序列的同源性在82．19,5～87．4％之间,氨基酸同源性在88．6％～90．9%之间;F蛋白裂解位点区（112～117）氨基酸组成与强毒株一致,说明NDV山东分离株（ShD-5—06）为新城疫强毒株。     

Pathogenicity evaluation of different Newcastle disease virus chimeras in 4-week-old chickens

The aim of this study was to evaluate the disease-inducing ability of four chimeric Newcastle disease viruses (NDV) by clinicopathological assessment. The infectious clones were previously generated by insertion of hemagglutinin–neuraminidase (HN) and/or fusion (F) genes from virulent strains (Turkey North Dakota and California 02) into a mesogenic strain (Anhinga) backbone. Groups of 4-week-old chickens were inoculated via eye drop instillation, clinical signs were monitored daily, and necropsies with collection of tissues were performed at 2, 5, 10, and 14 days post infection. Tissue sections were evaluated for histopathology and immunohistochemistry for NDV nucleoprotein. All viruses replicated successfully in the natural host, although viral recovery, seroconversion, and extent of immunohistochemical staining were greatest from birds infected with those viruses containing both F and HN genes from the same virulent virus. There was minimal to no increase in clinicopathologic disease due to infection with the chimeras compared to the recombinant backbone. However, all birds developed histological evidence of encephalitis. The results suggest that the inherent virulence of Turkey North Dakota and California 2002 strains is due to more than the simple presence of their F and HN genes.     

鹅细小病毒HN株的分离鉴定及全基因组序列分析

为研究鹅细小病毒(GPV)基因遗传变异特征,采集海南某养鹅场疑似鹅细小病毒感染的病料,将其处理后接种番鸭胚成功分离到一株病毒,经PCR鉴定为鹅细小病毒,命名为HN株,并获得了其全基因组序列,将该序列与GenBank数据库中登录的16条鹅和番鸭细小病毒基因序列进行了比对分析。结果显示,该株病毒基因组全长为5 106bp,由ITR、NS、VP构成,其中ITR为444bp,NS1为1 844bp,VP1为2 199bp;HN株与SHFX1201株的NS1基因和VP1同源性最高,分别达到99.8%和99.7%,与番鸭细小病毒株FM的NS1基因同源性最低,为82.7%;与90-0215株VP1同源性最低,为80.1%。HN株的遗传进化树可以看出,GPV可以分成明显的2个基因亚群,HN株与鹅细小病毒匈牙利株(B)、欧洲疫苗株(VG32/1)和台湾株(82-0321V、82-0321、06-0329)均处在第I亚群,且与安徽分离株Y株以及SHFX1201株同源性最接近,番鸭源匈牙利株FM单独处于第Ⅱ亚群。本研究丰富了GPV的数据资料,为研究GPV分类地位以及遗传进化关系提供了依据,同时也为研究GPV流行趋势和疫苗的开发奠定了基础。     

Isolation of viruses from outbreaks of suspected tenosynovitis (viral arthritis) in chickens

Specimens from the legs of chickens from 84 outbreaks of suspected tenosynovitis were examined for the presence of viruses by culture in chick embryo lung or liver cell monolayers. All samples were from broilers or broiler breeders, ranging in age from dead-in-shell embryos to 36 weeks old. Twenty-five outbreaks (29.8 per cent) yielded viruses of which 12 were reoviruses alone, 12 adenoviruses alone, and one, a mixture of both types of virus. Rupture of the gastrocnemius tendon was seen in 12 outbreaks and viruses were isolated from six of these: three were reoviruses and three were adenoviruses. Approximately half the affected flocks from which specimens were received were in the six to 14 week age range. With one exception, all the reovirus isolations were made from chickens of 11 weeks or under, while adenovirus isolations showed more scatter with regard to age.     

Enteric viruses in diarrheic turkey poults

Thirty-three intestinal samples from 10-to-21-day-old diarrheic turkey poults were examined for the presence of enteric viruses by electron microscopy. Samples originated from 32 flocks in six commercial operations located in six states. Mortality in these flocks ranged from 3 to 15%, and birds from recovered flocks varied greatly in size. Rotavirus-like agents (RVLA) were the most common viruses associated with diarrhea outbreaks in the flocks examined, occurring in five out of six operations. Other viruses detected either singly or in combination, in order of prevalence, were astroviruses, reoviruses, rotaviruses, enteroviruses, and adenoviruses. With the exception of RVLA and rotaviruses, the other viruses were identified solely on the basis of morphology. Salmonellae were isolated from only one of the intestinal samples. By electron microscopy, RVLA were morphologically indistinguishable from rotaviruses, occurring as both 55-nm single-shelled and 70-nm double-shelled particles. However, immune electron microscopy was useful for antigenic differentiation of these two viruses. Turkey rotaviruses reacted with antisera to porcine and bovine rotaviruses, whereas turkey RVLA did not. Neither turkey rotaviruses nor RVLA reacted with antisera to porcine para-rotavirus or an antigenically distinct bovine rotavirus (bovine rotavirus-like agent). Similarly, convalescent anti-turkey RVLA serum (from recovered specific-pathogen-free poults) reacted with homologous virus but did not react with mammalian or avian rotaviruses or reoviruses. Further, RVLA were found to possess RNA electrophoretic migration patterns unlike those of conventional rotaviruses or reoviruses. This trait was used as an additional means of differentiating these viruses.