Rabbit model of rotavirus infection.

A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines.     

Possible role of infectious simian foamy virus DNA in persistent infection.

Simian foamy virus type 7 (SFV-7) can persist in peripheral blood leukocytes of SFV-7-infected rabbits. Rabbit peripheral blood leukoyctes cultured in vitro did not support the growth of SFV-7, but low titer of virus could be detected after 4 to 5 days incubation. The DNA derived from SFV-7 infected primary rabbit kidney cells is infectious. The presence of SFV-7 DNA provirus in infected rabbit kidney cells during replication may contribute one of the mechanisms of persistance.     

Growth of Murine Cytomegalovirus in Various Cell Lines

Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV.     

Isolation and Characterization of a Herpes-like Virus from New Zealand Albino Rabbit Kidney Cell Cultures: a Probable Reisolation of Virus III of Rivers

An agent which possesses the physical, chemical, cytopathic, histological, and electron microscopic attributes of a herpes group virus was isolated from an uninoculated batch of primary rabbit kidney cell cultures. Preliminary evidence indicates that antibodies against the agent are found in some sera of other "normal" New Zealand albino rabbits. In cell cultures, the virus grew best and almost exclusively in cells of rabbit origin. On the basis of these facts, the name herpesvirus cuniculi (HC) is suggested for the isolate. A batch of anti-herpesvirus bovis antiserum prepared in rabbits was found to be "contaminated" with unsuspected neutralizing antibodies against HC. Caution is mandatory when using rabbits, rabbit tissues, or rabbit sera for work with any herpes group virus unless precautions are taken to rule out unsuspected infection with or antibodies against HC. This agent may well represent a reisolation of virus III, a rabbit herpes virus, described by Rivers in 1923; the isolation of this virus has not been reported since 1940. It is important to reemphasize the existence of this agent in an animal which is commonly used for laboratory investigation of herpes group viruses.     

Herpesvirus sylvilagus in cottontail rabbits: attempted laboratory transmission by two insect species.

The vector potential of the rabbit flea (Cediopsylla simplex) and a mosquito (Aedes triseriatus) was investigated for Herpesvirus sylvilagus transmission among cottontail rabbits (Sylvilagus floridanus). Twelve groups of 12-50 fleas were fed on three viremic cottontails for 2-21 days before transfer to 12 susceptible rabbits. Standard interrupted feeding trials employed five groups of 6-12 mosquitoes, two viremic donor cottontails anf five healthy recipients. No evidence of virus was detected from recipients' blood nor did they develop specific antibody. Virus acquisition and persistence in the insects was evaluated by attempting to recover the virus from 19 pools of mosquitoes engorged on viremic blood and 36 pools of engorged fleas or those living on viremic hosts for 1-21 days. Results were negative.     

Rabbit as a Novel Animal Model for Hepatitis E Virus Infection and Vaccine Evaluation

Background

The identification of hepatitis E virus (HEV) from rabbits motivated us to assess the possibility of using rabbits as a non-human primate animal model for HEV infection and vaccine evaluation.

Methodology/Principal Findings

First, 75 rabbits were inoculated with seven strains of genotypes 1, 3, 4, and rabbit HEV, to determine the appropriate strain, administrative route and viral dosage. Second, 15 rabbits were randomly divided into three groups and vaccinated with 0 µg (placebo), 10 µg and 20 µg of HEV candidate vaccine, HEV p179, respectively. After three doses of the vaccination, the rabbits were challenged with 3.3×10⁵ genome equivalents of genotype 4 HEV strain H4-NJ703. The strain of genotype 1 HEV was not found to be infectious for rabbits. However, approximately 80% of the animals were infected by two rabbit HEV strains. All rabbits inoculated with a genotype 3 strain were seroconverted but did not show viremia or fecal viral shedding. Although two genotype 4 strains, H4-NJ153 and H4-NJ112, only resulted in part of rabbits infected, another strain of genotype 4, H4-NJ703, had an infection rate of 100% (five out of five) when administrated intravenously. However, only two out of fifteen rabbits showed virus excretion and seroconversion when inoculated orally with H4-NJ703 of three different dosages. In the vaccine evaluation study, rabbits vaccinated with 20 µg of the HEV p179 produced anti-HEV with titers of 1∶10⁴–1∶10⁵ and were completely protected from infection. Rabbits vaccinated with 10 µg produced anti-HEV with titers of 1∶10³–1∶10⁴ and were protected from hepatitis, but two out of the five rabbits showed virus shedding.

Conclusions/Significance

Rabbits may be served as an alternative to the non-human primate models for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected.     

Detection and transmission of infectious hematopoietic necrosis virus in rainbow trout.

Detection and transmission of Infectious Hematopoietic Necrosis Virus in rainbow trout (Salmo gairdneri) was studied at a commercial trout hatchery. Transmission of virus was demonstrated via water, feed and contaminated eggs. If eggs from carrier females were incubated several weeks in virus-free water, the resulting fry did not become infected. However, if fry subsequently became infected they were lifetime carriers. Infectious virus was readily detectable in most tissues of moribund fish; in carriers it was detected in sex products of spawning fish, and in samples from the intestine of post-spawning fish, but not in samples from blood, feces, kidney, or liver. The carrier rate was not significantly different between sexes. It was concluded that adult carriers are the reservoir of infection and that transmission occurs primarily when carriers shed virus and expose susceptable fish or eggs.     

NASBA快速检测禽流感H5亚型病毒

采用建立的依赖核酸序列的扩增(Nucleicacidsequencebasedamplification,NASBA)对禽流感病毒3株H5亚型、1株H1、H3、H6亚型、3株禽流感H9亚型、5株不同宿主来源的新城疫病毒、鸭肝炎病毒、鸭瘟病毒、SPF鸡胚尿囊液及禽流感(H9)疫苗、新城疫疫苗、传染性法氏囊病疫苗、传染性支气管炎疫苗进行检测,结果NASBA(H5试剂)仅检测到禽流感病毒H5亚型,表明方法的特异性强。采用已知禽流感病毒A/Chicken/HK/1000/97(H5N1)的鸡胚尿囊液(ELD5010-7.5/mL),经10倍连续稀释,将经典的鸡胚病原分离法和NASBA进行比较,二种方法的灵敏度相当。用A/Chicken/HK/1000/97(H5N1)病毒人工感染SPF鸡、商品鸡,采用NASBA和病原分离法同时对人工感染鸡的粪拭子、血液进行了动态检测;采集感染死亡鸡的组织脏器,共检测了101个组织脏器,两种方法的符合率为90%(87/97)。     

Bovine herpesvirus 5 glycoprotein E is important for neuroinvasiveness and neurovirulence in the olfactory pathway of the rabbit

Glycoprotein E (gE) is important for full virulence potential of the alphaherpesviruses in both natural and laboratory hosts. The gE sequence of the neurovirulent bovine herpesvirus 5 (BHV-5) was determined and compared with that of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gE open reading frames showed that they had 72% identity and 77% similarity. To determine the role of gE in the differential neuropathogenesis of BHV-1 and BHV-5, we have constructed BHV-1 and BHV-5 recombinants: gE-deleted BHV-5 (BHV-5gEDelta), BHV-5 expressing BHV-1 gE (BHV-5gE1), and BHV-1 expressing BHV-5 gE (BHV-1gE5). Neurovirulence properties of these recombinant viruses were analyzed using a rabbit seizure model (S. I. Chowdhury et al., J. Comp. Pathol. 117:295-310, 1997) that distinguished wild-type BHV-1 and -5 based on their differential neuropathogenesis. Intranasal inoculation of BHV-5 gEDelta and BHV-5gE1 produced significantly reduced neurological signs that affected only 10% of the infected rabbits. The recombinant BHV-1gE5 did not invade the central nervous system (CNS). Virus isolation and immunohistochemistry data suggest that these recombinants replicate and spread significantly less efficiently in the brain than BHV-5 gE revertant or wild-type BHV-5, which produced severe neurological signs in 70 to 80% rabbits. Taken together, the results of neurological signs, brain lesions, virus isolation, and immunohistochemistry indicate that BHV-5 gE is important for efficient neural spread and neurovirulence within the CNS and could not be replaced by BHV-1 gE. However, BHV-5 gE is not required for initial viral entry into olfactory pathway.     

Experimental Absidia corymbifera infection in rabbits: Clinicopathological studies

Absidiosis was produced experimentally in rabbits by intravenous inoculation of 1.4×10⁵ spores of Absidia corymbifera. Infected rabbits exhibited a rise in body temperature, anorexia, dullness, listlessness, diarrhoea, occasional blindness, convulsions and death in some cases. Mortality occurred mainly between 6 to 9 days post infection (DPI) and overall mortality was 50 per cent during the three week observation period. No significant difference was observed in erythrocytic indices viz., Hb, PCV, TEC in control and infected rabbits. However, erythrocyte sedimentation rate was considerably increased in the infected rabbits. A state of leucocytosis was observed in the infected rabbits, which was due to increase in the relative percentage of neutrophils and decrease in lymphocytes. There was a significant increase in blood urea nitrogen concentrations of infected rabbits from 3 to 14 DPI as compared to controls, but serum creatinine values were not significantly altered at any stage of infection. The cause of death was attributed to kidney failure and uraemia in infected rabbits. The rabbit was found to be a suitable model for the study of absidiosis.     

Transfection of RNA from Organ Samples of Infected Animals Represents a Highly Sensitive Method for Virus Detection and Recovery of Classical Swine Fever Virus

Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.     

Study on potato virus M (PVM) occurrence in potato fields in Iran

57 native potato tuber samples collected from different potato growing region of Iran, planted on single rows in Karaj College experimental station. Plant samples of each single row plus 9.25 Fresh foliage samples collected from fields under new introduced cultivars were tested for potato virus (PVM) infection during growing season. Also 78 weeds and field crops belonging to Solonacae and Leguminosae from or neighboring to potato field were tested. Results indicated that PVM was not found on any plant other than potatoes. PVM was detected on 16 samples of 57 old vars, Virus was not seen in any samples collected from fields under new varieties. Results show that PVM is limiting in this crop. PVM detecting is difficult using assay hosts. Best test plants were French bean var Red kidney, Showing pinpoint necrotic LL, also Datura metel and Nicotiana debneyi are useful for virus detection showing chlorotic local lesion. Also microprecipition and gel diffusion test can be used for virus detection but Elisa was the best method. PVM infected plant showed 11-19.5 percent yield decrease in 3 cultivars tested.     

兔出血症病毒遗传变异分析及RT—PCR检测方法的建立

目的获得兔出血症病毒（浙江分离株）近20年间遗传变异概况,建立兔出血症病毒的RT—PCR检测方法。方法对1989～2006年间的7株RHDV浙江分离株的衣壳蛋白基因（VP60）进行了克隆与测序,并与国内、外RHDV的基因组序列进行了遗传比对和分析;根据兔出血症病毒株的VP60设计引物,对20只人工感染兔的实质脏器、全血和350只实验兔全血样本进行了检测。结果7株RHDV的VP60基因均由1740个核苷酸组成,编码580个氨基酸。它们与参考序列之间的氨基酸同源性为94.0%～99.8%。系统发生树分析结果显示,RHDV可划分为2个大的基因群,浙江（中国）的RHDV分离株主要集中于C亚群。RT—PCR检测方法表明20只实验兔全部扩出预期条带,而350只实验兔全血检测中出现13只RHDV可疑样本。经血凝抑制试验检测,13例PCR阳性样品中有10个HAI阳性,3个阴性。检测敏感度达100%,特异性为99.12%,经统计检验,kappa=0.865,表明PCR检出RHDV的结果与HAI高度一致。结论RHDV基因群之间的遗传距离有逐渐加大的趋势。我们建立的RT—PCR法可用于RHDV浙江分离株的检测,用RT—PCR检测全血中RHDV方法的建立为活体检测RHDV打下了基础。     

Postexposure immunoprophylaxis against B virus (Herpesvirus simiae) infection.

Local infiltration of antiserum into sites inoculated with B virus protected rabbits from an otherwise fatal encephalomyelitis. Treatment was effective when delayed for six hours but not after 24 hours. Homologous rabbit antisera were more effective than heterologous monkey antisera, and protection was unrelated to neutralisation titres. Protection apparently depended not on neutralisation of inoculated virus but on destruction of infected cells before they produced progeny virus. Normal human immunoglobulin able to neutralise B virus did not protect. Intravenously administered antibody was effective only if large doses were given. The findings suggest that persons bitten or scratched by monkeys latently infected with B virus may be treated successfully by immunoprophylaxis with specific antibody. Stocks of human or of more readily available simian antisera should be held in laboratories where such animals are used.     

An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation.

The herpes simplex virus type 1 (HSV-1) ICP34.5 gene is a neurovirulence gene in mice. In addition, some ICP34.5 mutants have been reported to have a reduced efficiency of induced reactivation as measured by in vitro explantation of latently infected mouse ganglia. However, since spontaneous reactivation is almost nonexistent in mice, nothing has been reported on the effect of ICP34.5 mutants on spontaneous reactivation in vivo. To examine this, we have deleted both copies of the ICP34.5 neurovirulence gene from a strain of HSV-1 (McKrae) that has a high spontaneous reactivation rate in rabbits and used this mutant to infect rabbit eyes. All rabbits infected with the ICP34.5 mutant virus (d34.5) survived, even at challenge doses greater than 4 x 10(7) PFU per eye. In contrast, a 200-fold-lower challenge dose of 2 x 10(5) PFU per eye was lethal for approximately 50% of rabbits infected with either the wild-type McKrae parental virus or a rescued ICP34.5 mutant in which both copies of the ICP34.5 gene were restored. In mice, the 50% lethal dose of the ICP34.5 mutant was over 10(6) PFU, compared with a value of less than 10 PFU for the rescued virus. The ICP34.5 mutant was restricted for replication in rabbit and mouse eyes and mouse trigeminal ganglia in vivo. The spontaneous reactivation rate in rabbits for the mutant was 1.4% as determined by culturing tear films for the presence of reactivated virus. This was more than 10-fold lower than the spontaneous reactivation rate determined for the rescued virus (19.6%) and was highly significant (P < 0.0001, Fisher exact test). Southern analysis confirmed that the reactivated virus retained both copies of the ICP34.5 deletion. Thus, this report demonstrates that (i) the ICP34.5 gene, known to be a neurovirulence gene in mice, is also important for virulence in rabbits and (ii) in vivo spontaneous reactivation of HSV-1 in the rabbit ocular model, although reduced, can occur in the absence of the ICP34.5 gene.     

Experimental Infection of Rabbits with Rabbit and Genotypes 1 and 4 Hepatitis E Viruses

Background

A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77–79% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection.

Methods

Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination.

Findings

Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose (genome equivalents), with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation.

Conclusions

These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis.     

Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats

Ashe, Warren K. (National Institute of Dental Research, Bethesda, Md.), Henry W. Scherp, and Robert J. Fitzgerald. Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats. J. Bacteriol. 90:1719-1729. 1965.-A serially transmissible cytopathic agent was isolated from histologically normal submaxillary glands, but not from various other tissues or specimens, from 74 of 97 gnotobiotic and conventional rats. Triturates of the glands or subsequent culture supernatant fluids induced specific cytopathic effects (CPE) in monolayer cultures of primary rabbit kidney cells (14 passages), a line of human skin cells (8 passages), and HeLa cells (17 passages). Transfer of supernatant fluids containing infected cells enhanced transmissibility. Neutralization of the CPE was demonstrated with sera of gnotobiotic and conventional rats and with homologous rabbit antiserum. A cold hemagglutinin specific for rabbit erythrocytes is associated with, but separable from, the infectious particle. Cultures of the agent induced no discernible effect on inoculation by various routes into suckling, weanling, or adult conventional mice, rats, and hamsters. Weanling and adult rabbits were also unaffected. Cultures for bacteria in gland extracts and infected cell supernatant fluids were uniformly negative. Negative cultures on PPLO media and negative arginine deiminase tests indicated that this agent is not a Mycoplasma. The data indicate that it is a virus whose biological and physical properties distinguish it from the known cytomegaloviruses. Because it has been found so far only in rat submaxillary glands, this agent is designated provisionally as RSMG virus.     

Herpesvirus sylvilagus in cottontail rabbits: antibody prevalence and flea burden relationships

A serologic survey of a wild cottontail rabbit (Sylvilagus floridanus) population in southern Wisconsin was conducted from November-March, 1971-72 and November-April, 1972-73, to determine prevalence of antibody against Herpesvirus sylvilagus. Flea burdens on each live-trapped cottontail were quantified by species. All but six of the 5029 fleas collected were Cediopsylla simplex. No correlation was found between flea infestation and viral antibody. Of 101 cottontail rabbits trapped, only six had specific antibody as determined by plaque neutralization in rabbit kidney cell culture. Three of the six developed antibody between January and March of the trapping season. Blood samples from 46 captured rabbits were negative for virus.