Culture-adapted Strains Research Articles

Inhibition of cap-dependent gene expression induced by protein 2A of hepatitis A virus.

The viral protein 2A of hepatitis A virus (HAV) lacks the conserved 18 aa sequence found in other picornavirus proteases; hence, it is unclear whether the induction of CPE by culture-adapted HAV strains is due to 2A-mediated activity. Moreover, the cleavage sites and actual borders of HAV 2A are not known. Accordingly, a nested series of cDNA sequences encoding the segment of the HAV polyprotein (aa 760-1087) were linked to the 5'-UTR of poliovirus type 2 (Lansing strain) and inserted downstream of the gene encoding human growth hormone (GH). Following transfection of COS-1 cells, levels of GH (translation of which was entirely cap dependent) were determined in culture supernatants. Expression of HAV peptides extending from aa 764, 776 or 791 to 981 strongly inhibited cap-dependent translation of GH, whereas cap-independent expression of a reporter gene (CAT) directed by the poliovirus RNA 5'-UTR was unaffected. The inhibitory effect was absent in constructs expressing either the short peptide encompassing aa 760-836 or proteins initiated downstream of the putative cleavage site 836-837, suggesting that the boundaries of a functional HAV 2A may extend from the Gln/Ser junction 791-792 to residue 981, while peptides initiated at the Gln/Ala pair 836-837 may result from alternative cleavage. Point mutations that substituted members of the triad Ser(916), His(927) and Asp(931) abolished the inhibitory effect on cap-dependent translation, suggesting that the HAV-induced CPE may be mediated by 2A protein.

Infection of neonatal mice with sindbis virus results in a systemic inflammatory response syndrome.

Laboratory strains of viruses may contain cell culture-adaptive mutations which result in significant quantitative and qualitative alterations in pathogenesis compared to natural virus isolates. This report suggests that this is the case with Sindbis virus strain AR339. A cDNA clone comprising a consensus sequence of Sindbis virus strain AR339 has been constructed (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). This clone (pTR339) regenerates a sequence predicted to be very close to that of the original AR339 isolate by eliminating several cell culture-adaptive mutations present in individual laboratory strains of the virus (K. L. McKnight et al., J. Virol. 70:1981-1989, 1996). It thus provides a unique reagent for study of the pathogenesis of Sindbis virus strain AR339 in mice. Neonatal mouse pathogenesis of virus (TR339) generated from the pTR339 clone was compared with that of virus from a cDNA clone of the cell culture-passaged laboratory AR339 strain, TRSB, and virus from a clone of a more highly cell culture-adapted strain, HR(sp) (Toto 50). The sequence of TRSB differs from the consensus at three coding positions, while Toto 50 differs at eight codons and one nucleotide in the 5' nontranslated region. Both cell culture-adapted strains contain mutations associated with heparan sulfate (HS)-dependent attachment to cells (W.B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). TR339 caused 100% mortality with an average survival time (AST) of 1.7 +/- 0.25 days. While TRSB also caused 100% mortality, the AST was extended to 2.9 +/- 0.52 days. The more extensively cell culture-adapted virus Toto 50 caused only 30% mortality with an AST extended to 11.0 +/- 4.8 days. TRSB and TR339 induced high serum levels of alpha/beta interferon, gamma interferon, tumor necrosis factor alpha, interleukin-6, and corticosterone and induced pathology reminiscent of lipopolysaccharide-induced endotoxic shock, a type of systemic inflammatory response syndrome. However, the reduced intensity of this response in TRSB-infected mice correlated with the increased AST. Toto 50 failed to induce the shock-like cytokine cascade. In situ hybridization studies indicated that TR339 and TRSB replicated in identical tissues, but the TRSB signal was less widespread at early times postinfection. While Toto 50 also replicated in similar tissues, the extent of replication was severely restricted and mice developed lesions characteristic of encephalitis. A single mutation in TRSB at E2 position 1 (Arg) conferred HS-dependent attachment to cells and was associated with reduced cytokine induction and extended AST in vivo.

Measles virus infection in rhesus macaques: altered immune responses and comparison of the virulence of six different virus strains.

Measles remains a major cause of childhood mortality, with questions about virus virulence and pathogenesis still requiring answers. Rhesus macaques were infected with 5 different culture-adapted strains of measles virus, including 2 from patients with progressive vaccine-induced disease, and a sixth nonculture-adapted strain, Bilthoven. All caused infection detectable by reverse transcriptase-polymerase chain reaction and induction of antibody. Chicago-1 and Bilthoven induced viremias detectable by leukocyte cocultivation. Bilthoven induced Koplik's spots, conjunctivitis, and rash. Lymphopenia and depressed interleukin (IL)-2 production were followed by monocytosis and eosinophilia. All monkeys, including 41 involved in a primate facility outbreak, showed suppressed responses to phytohemagglutinin. As the rash resolved production of IL-2, IL-1beta, tumor necrosis factor-alpha, IL-6, and IL-5 mRNA increased. Monkeys are useful for studies of measles immunopathogenesis, but virus strains must be carefully chosen. Increased virulence of vaccine strains isolated from immunocompromised infants with fatal infections was not evident.

Isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses

Abstract Objective To isolate bovine coronaviruses from the respiratory tracts of feedlot cattle and compare antigenic and biological properties of these strains with bovine enteric coronaviruses. Animals 5- to 8-month-old mixed-breed cattle at 4 feedlots. Procedure Samples were obtained from the nasal passages for testing. The 13 samples with the highest magnitude of positive values for bovine coronavirus (BCV) were cultured. Ten strains of bovine respiratory coronavirus (BRCV) were adapted successfully to serial passage. After observation of cytopathic effects (CPE) and confirmation of BRCV by immune electron microscopy and immunofluorescence testing, cell culture-adapted strains were cloned by limiting dilution. These isolates then were compared with a panel of bovine enteric coronaviruses (BECV), using hemagglutination (HA), receptor-destroying enzyme activity (RDE), hemagglutination inhibition (HI), and virus neutralization (VN) assays. Antigenic relatedness values then were calculated. Results The BRCV were detected in 105 of 488 (21.5%) of the cattle tested. Of 13 strains tested, 10 were isolated in cell culture. Six of the BRCV strains were similar to 2 strains obtained from neonatal calves with diarrhea and 2 strains from adult cattle with winter dysentery. The other 4 BRCV isolates had high RDE activity against mouse erythrocytes but differed from other strains of BECV. Nine of 10 BRCV isolates had properties similar to the 2 BECV subtypes. Conclusions and Clinical Relevance The BRCV can be isolated from nasal passages of cattle entering feedlots. Most BRCV were similar to BECV strains, although a few had unique properties. Vaccines developed to protect against enteric strains also may protect against respiratory tract strains. (Am J Vet Res 1999;60:1227–1233)

Endothelial cell infection in vivo by equine infectious anaemia virus.

Equine infectious anaemia virus (EIAV) infection of horses is characterized clinically by recurrent episodes of fever, thrombocytopenia and anaemia. In vivo, the only site of virus replication that has been previously demonstrated for EIAV is the tissue macrophage. In this study, in situ hybridization for EIAV was combined with immunohistochemistry for cell-type-specific markers to identify infected endothelial cells. EIAV-infected endothelial cells and macrophages were detected in horses infected with either virulent wild-type or with weakly virulent tissue culture-adapted strains of EIAV. The role of endothelial cell infection in the pathogenesis of EIAV remains undefined, but could contribute to the development of thrombocytopenia. However, endothelial cell infection does not appear to be a determinant of virulence for EIAV.

The furin protease cleavage recognition sequence of Sindbis virus PE2 can mediate virion attachment to cell surface heparan sulfate.

Cell culture-adapted Sindbis virus strains attach to heparan sulfate (HS) receptors during infection of cultured cells (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). At least three E2 glycoprotein mutations (E2 Arg 1, E2 Lys 70, and E2 Arg 114) can independently confer HS attachment in the background of the consensus sequence Sindbis virus (TR339). In the studies reported here, we have investigated the mechanism by which the E2 Arg 1 mutation confers HS-dependent binding. Substitution of Arg for Ser at E2 1 resulted in a significant reduction in the efficiency of PE2 cleavage, yielding virus particles containing a mixture of PE2 and mature E2. Presence of PE2 was associated with an increase in HS-dependent attachment to cells and efficient attachment to heparin-agarose beads, presumably because the furin recognition site for PE2 cleavage also represents a candidate HS binding sequence. A comparison of mutants with partially or completely inhibited PE2 cleavage demonstrated that efficiency of cell binding was correlated with the amount of PE2 in virus particles. Viruses rendered cleavage defective due to deletions of portions or all of the furin cleavage sequence attached very poorly to cells, indicating that an intact furin cleavage sequence was specifically required for PE2-mediated attachment to cells. In contrast, a virus containing a partial deletion was capable of efficient binding to heparin-agarose beads, suggesting different requirements for heparin bead and cell surface HS binding. Furthermore, virus produced in C6/36 mosquito cells, which cleave PE2 more efficiently than BHK cells, exhibited a reduction in cell attachment efficiency correlated with reduced content of PE2 in particles. Taken together, these results strongly argue that the XBXBBX (B, basic; X, hydrophobic) furin protease recognition sequence of PE2 can mediate the binding of PE2-containing Sindbis viruses to HS. This sequence is very similar to an XBBXBX heparin-HS interaction consensus sequence. The attachment of furin protease cleavage sequences to HS may have relevance to other viruses whose attachment proteins are cleaved during maturation at positively charged recognition sequences.

A recombinant measles vaccine virus expressing wild-type glycoproteins: consequences for viral spread and cell tropism.

Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism.

Comparative Evaluation of Cell Culture-Adapted and Chicken Embryo-Adapted Fowl Pox Vaccine Strains

Two types of vaccines, chicken embryo adapted (VacCE) and cell culture adapted (VacCC), were tested for their efficacy to elicite the immune response in birds vaccinated at 2 and 8 wk of age. The cell-mediated immune response studied by blastogenesis assay showed that birds vaccinated at the second week of age by both VacCE and VacCC vaccines had significant increase in T-lymphocyte count at 21 days postvaccination (PV) and 7 days postchallenge (PC), whereas in birds vaccinated at 8 wk of age, a significant increase was seen at 21 days PV and 7 days PC with the VacCC vaccine. The rise in passive hemagglutination titers was observed up to 21 days PV and 7 days PC in birds vaccinated at 2 wk of age. However, only the birds vaccinated with VacCC at 8 wk of age showed rise in titers at days 21 PV and 7 PC. Birds were challenged 90 days PV by scarification on the thigh region, and the birds vaccinated with VacCC showed 90% and 70% protection when vaccinated at 2 and 8 wk, respectively. The birds vaccinated with VacCE showed only 60% and 20% protection at the corresponding levels, respectively.

Experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by RT-PCR.

SummaryA reverse transcriptase PCR (RT-PCR) targeting a 407 bp fragment of the nucleocapsid gene of bovine coronavirus (BCV) was developed for detection of BCV RNA in feces of experimentally inoculated cattle. The sensitivity and specificity of the RT-PCR were confirmed using tissue culture-adapted BCV strains and feces of 2 calves inoculated with BCV. Ten nonpregant, BCV seropositive, adult dairy cows were inoculated with winter dysentery (WD) (n = 8) or calf diarrhea (CD) (n = 2) strains of BCV intranasally and orally (n = 2) or through a surgically-placed duodenal catheter (n = 8) with and without dexamethasone treatment or feeding ice water. The 6 cows inoculated with BCV intranasally and through a duodenal catheter (2 of 2 cows given CD BCV and 4 of 6 cows given WD BCV) developed mild diarrhea, and BCV was detected in diarrheal feces by RT-PCR, ELISA or immune electron microscopy. These results suggest that CD and WD strains of BCV can cause diarrhea in adult cows in conjunction with host or environmental factors and that RT-PCR might be useful to diagnose BCV infections in calves and adult cows.

The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity.

Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5 - carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

Continuous in vitro culture of Plasmodium falciparum using microaerophilic gas generators and portable incubator

AnaeroPack Malaria Culture System (SUGIYAMA-GEN Co., Ltd.) using AnaeroPack.plas (5% O2, 5% CO2) and AnaeroPack.CO2 (15% O2, 6% CO2) was evaluated by comparing with the standard laboratory in vitro continuous culture technique. Two culture-adapted strains of Plasmodium falciparum, SGE-1 (chloroquine sensitive strain) and K1 (chloroquine resistant strain), were continuously cultured for 26 days in vitro under the 3 systems. The parasite proliferation curves under the different set systems were paralleled in both strains, which demonstrate that this AnaeroPack Malaria Culture System is useful for the culture-adapted strains of P. falciparum. Although further test using isolates from falciparum malaria patients should be carried out, the AnaeroPack Malaria Culture System seems promising for the culture in the field studies.

The role of the chemokine receptor CXCR4 in infection with feline immunodeficiency virus.

Infection with feline immunodeficiency virus (FIV) leads to the development of a disease state similar to AIDS in man. Recent studies have identified the chemokine receptor CXCR4 as the major receptor for cell culture-adapted strains of FIV, suggesting that FIV and human immunodeficiency virus (HIV) share a common mechanism of infection involving an interaction between the virus and a member of the seven transmembrane domain superfamily of molecules. This article reviews the evidence for the involvement of chemokine receptors in FIV infection and contrasts these findings with similar studies on the primate lentiviruses HIV and SIV (simian immunodeficiency virus).

The microculture tetrazolium assay (MTA): another colorimetric method of testingPlasmodium falciparumchemosensitivity

Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

Epizootics of bovine ephemeral fever on dairy farms in Saudi Arabia

In 1990 and 1996, field veterinarians suspected the clinical occurrence of bovine ephemeral fever among dairy and conventional cattle in different regions of Saudi Arabia. The disease has a seasonal occurrence; it begins in early summer (May) and ends in late autumn (November). The mortality rate is low: 0.3% to 0.6%. The morbidity rate ranged from 5% to 61% within the different age groups of one affected herd in the 1996 outbreaks and from 3.4% to 19% among four affected herds in the 1990 outbreaks. A sudden sharp drop in milk production occurred in lactating animals, some of which had become dry by the end of the outbreaks. Trials to isolate the causative virus in cell culture and in baby mice were unsuccessful. Serum neutralisation tests, which used a cell culture-adapted vaccine strain of bovine ephemeral fever virus as an antigen, revealed the presence of specific antibodies with significantly increased titres in the convalescent sera of affected animals. In addition, the testing of paired sera from non-affected heifers and from both dry and milking cows, performed twice, with an interval of 21 days, revealed the presence of neutralising antibodies. In the 1990 outbreaks, comparative serological studies indicated a high percentage (67.5%; 27/40) of seropositive animals in herds in which bovine ephemeral fever had been previously suspected. No antibodies were detected in animals of herds which had no recorded clinical history of bovine ephemeral fever. Following serological confirmation of the prevalence of bovine ephemeral fever in Saudi Arabia, some dairy farms started using a live imported vaccine to control the disease. This study discusses the epizootiological findings in regard to bovine ephemeral fever, as well as its economic impact on four affected dairy farms in 1990. In addition, the authors evaluate the efficacy of immunoprophylaxis in another dairy herd during the same outbreaks.

Rotavirus Disease, but Not Infection and Development of Intestinal Histopathological Lesions, Is Age Restricted in Rabbits

The rabbit model of rotavirus infection has proved to be useful for assessing active immunity and protection after infection or vaccination with virus or virus-like particles. One limitation of the rabbit model is that after experimental infection of rabbits, clinical diarrhea is not routinely induced. Lack of diarrhea in the rabbit model has been proposed to be due to the fluid absorptive capability of the cecum or attenuation of virus strains through tissue culture adaptation. To test whether a wild-type lapine rotavirus strain BAP (BAPwt) isolated from diarrheic rabbits would cause disease on passage in rabbits, 1-, 2-, 10-, and 16-week-old rabbits were orally inoculated with BAPwt, its tissue culture-adapted counterpart strain (BAP-2), tissue culture-adapted lapine strain ALA, or PBS. Lapine rotavirus infection in 1-week-old, but not ≥2-week-old, rabbits resulted in the development of disease characterized by soft, wet, yellow-to-brownish-green partially formed-to-liquid stools observed only at the time of virus antigen shedding. The level and duration of virus shedding after infection were prolonged in 1-week-old rabbits compared with rabbits ≥2 weeks of age. Although diarrhea was not observed beyond the first 2 weeks of life, histopathological changes, including villus shortening and fusion, increased vacuolation of epithelial cells, and mononuclear infiltration of the lamina propria, were observed throughout the small intestine between 12 and 120 h after ALA infection in 1-week-old, 1- to 2-month-old, and 11-month-old rabbits. In 11-month-old rabbits, onset of intestinal damage appeared to be slightly delayed, was less severe, and was not observed in the duodenum. There were no differences in the immune responses to rotavirus infection in rabbits of different age groups (1 week to 5 years of age). All lapine rotavirus-inoculated rabbits seroconverted and were protected from virus challenge at 28 days postinoculation. Like in mice, rotavirus disease is age restricted in rabbits.

Sequence Analysis of the VP2 Hypervariable Region of Nine Infectious Bursal Disease Virus Isolates from Mainland China

The VP2 hypervariable region of infectious bursal disease virus (IBDV) from nine Mainland Chinese strains was amplified by reverse transcriptase/nested polymerase chain reaction and cloned into pGEM-T vector. The nine isolates, which were from the center (HN3), the north (Bj-1, B2/28, HD96), the east (JS-18 and AH-2), the northeast (D11-2, C4-2), and the west (Ts) of China, were sequenced and compared with each other and with six reference IBDV sequences. Clustering analysis separated the nine isolate into two groups. The six virulent isolates, propagated in bursae, formed the first group. They revealed only one to three amino acid changes from the very virulent (vv) European and Japanese isolates, suggesting that they might have the same origin as European and Japanese vvIBDV strains. On the basis of their distinct geographic origins, extensive dissemination of vvIBDV in China was indicated. (The other three chicken embryo fibroblast cell cultured isolates with mild pathogenicity were placed in the second group.) Their sequences correlated closely with those of the culture-adapted strains (Cu-1 (4) and Cj-801). None of the nine isolates showed very close sequence relationship with the antigenic variant strains from the USA. Although antigenic variants have been reported in China, the reverse transcriptase/polymerase chain reaction-restriction endonuclease analyses of the nine viruses tested herein were not similar to any U.S.A. variant strains on the basis of computer software analysis. Our results and conclusions agree with a previous molecular study of IBDV isolates from the south of China.

Utilization of chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus to study the molecular basis of virulence.

Chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus (HAV) were constructed to evaluate the effect of the 2C gene of AGM-27 on HAV replication in cell culture and virulence in tamarins (Saguinus mystax) and chimpanzees (Pan troglodytes). Kinetic studies and radioimmunofocus assays demonstrated that replacement of the 2C gene of HAV/7, a cell culture-adapted strain of HM-175, with that of AGM-27 drastically reduced the ability of the virus to replicate in cultured cells. Intragenic chimeras containing AGM-27 sequences in either the 5' or 3' half of the 2C gene replicated in cell culture at an intermediate level. Whereas HAV/7 is attenuated for tamarins, a chimera containing the simian virus 2C gene in the HAV/7 background was virulent in tamarins, demonstrating that the simian virus 2C gene alone can confer the phenotype of virulence to an otherwise attenuated virus. Clusters of AGM-27-specific residues near both ends of the 2C protein were required for virulence since a chimera containing AGM-27 sequences in the carboxy-terminal half of 2C was partially attenuated for tamarins while one containing AGM-27 sequences only in the amino-terminal half of 2C was even more attenuated. Chimeras containing either the entire or only the 3' half of the simian virus 2C gene in the HAV/7 background were attenuated for chimpanzees.

Construction and evaluation of an attenuated vaccine for foot-and-mouth disease: difficulty adapting the leader proteinase-deleted strategy to the serotype O1 virus

Over the last few years we have utilized a system to genetically engineer foot-and-mouth disease virus (FMDV) to produce live-attenuated vaccine candidates. These candidates have been generated in the genetic background of a tissue culture-adapted strain of serotype A12 virus. Based on this A12 system, we created a virus lacking the sequence encoding the leader (L) proteinase ( Piccone et al., 1995), and demonstrated that this leaderless virus, A12-LLV2 was avirulent in bovine and swine, and could be used as an attenuated vaccine ( Mason et al. 1997, Chinsangaram et al. 1998). The current study shows that a similar leader-deleted chimeric virus containing the genome of the type A12 virus with a substituted type O1 capsid coding region from a bovine-virulent virus can be constructed, and that the virus has low, but detectable virulence in swine. A second chimera specifying a tissue culture-adapted type O1 capsid lacking the RGD cell binding site, was avirulent in swine, but was not sufficiently immunogenic to provide protection from challenge. These results are described with respect to mechanisms of attenuation and antigen formation in live-attenuated virus-inoculated animals.

Molecular Characterization of Seven Chinese Isolates of Infectious Bursal Disease Virus: Classical, Very Virulent, and Variant Strains

Seven infectious bursal disease virus (IBDV) strains isolated from China have been characterized in this study, including a classical strain CJ801, an attenuated strain GZ911, a variant strain GZ902, and four very virulent strains G9201, G9302, F9502, and HK46. With the use of reverse transcription-polymerase chain reaction, the full-length VP2 genes were amplified and the hypervariable regions were sequenced. Protein sequences of the hypervariable region (a.a. 143-382) of the field isolates confirmed their identities. CJ801 has the highest identity to the classical strains STC and 52/70. GZ902 has the highest identity to the American variant strains A, E, and GLS, and they share unique amino acid residue at positions 249K and 254S, which are not present in standard serotype 1 strains. Attenuated strain GZ911, like other cell culture-adapted strains, has substitutions at positions 279(D to N) and 284 (A to T) as well as in the serine-rich heptapeptide region. Hence, these substitutions may take an important role in the reduced virulence of these strains. The four very virulent strains have the highest identity to the European very virulent strain UK661 and Japanese strain OKYM. These strains share unique amino acid residues at positions 222A, 256I, and 294I, which are not present in other less virulent strains. The very virulent strains isolated in Guangdong (G9201, G9303) and Fujian (F9502) Provinces have one to five amino acid substitutions at the two hydrophilic domains of VP2 comparing with UK661 and OKYM, indicating that new very virulent strains are evolving. Phylogenetic analysis suggests that Chinese very virulent IBDVs and European very virulent strains are derived from similar origin.

An Equine Herpesvirus Type 1 Recombinant with a Deletion in the gE and gI Genes Is Avirulent in Young Horses

The cell culture-adapted KyA strain of equine herpesvirus type 1 (EHV-1) has been found to be attenuated in young horses (Matsumuraet al.,1996,Vet. Microbiol.48, 353–365). The KyA strain lacks at least six genes in its genome, including those encoding glycoproteins gE and gI. To elucidate whether EHV-1 glycoproteins gE and gI play a role in viral virulence, we have constructed an EHV-1 recombinant that has the genes encoding both gE and gI deleted from its genome and its revertant. Growth properties of the deletion mutant virusin vitrowere compared with those of the parent and the revertant viruses. Plaque size of the mutant virus in fetal horse kidney (FHK) cells was significantly smaller than those of the parent and the revertant viruses. In one-step growth experiments, however, the yields of infectious virus from FHK cells infected with the deletion mutant, the parent, or the revertant virus were approximately the same. The results suggested that gE and/or gI of EHV-1 promoted cell-to-cell spread of the virus, but that these glycoproteins were not involved in the process of virus maturation and release or in virus attachment and penetration. Subsequently, the virulence of mutant and revertant viruses was examined in young horses. No clinical signs were observed in six horses, including three colostrum-deprived foals inoculated intranasally with the deletion mutant virus, whereas three colostrum-deprived foals inoculated intranasally with the revertant virus manifested clinical signs typical for EHV-1 respiratory infection (i.e., pyrexia, nasal discharge, and swelling of submandibular lymph nodes). The results obtained fromin vivostudies revealed that the EHV-1 mutant defective in both gE and gI genes was avirulent in young horses, suggesting that gE and/or gI of the EHV-1 have an important role in EHV-1 virulence. However, the EHV-1 mutant defective in both gE and gI genes induced only a partial protectivity in inoculated foals from manifestation of respiratory symptoms after challenge infection.