QT-35 Cells Research Articles

Adaptation and characterization of Anatid herpesvirus 1 in different permissible cell lines

Duck viral enteritis is an acute, contagious infection of Anatidae family members. The disease is caused by Anatid herpesvirus 1 (AnHV-1). The infection of AnHV-1 is controlled by vaccination to the flock with chick embryo adapted attenuated vaccine in developed countries. However, its economic impact in developing countries is substantial and there is a need to understand the cell culture spectrum of the virus to produce its vaccine on a mass scale. In the present study, the permissivity of AnHV-1 for different cells was analyzed. The AnHV-1 showed enhanced replication following its serial passage in CEF, DF-1, Vero, MDCK, and QT-35 cells. The characteristic cytopathic effect (CPE) of rounding and clumping of cells were observed in CEF, DF-1, Vero, and QT-35 cell lines. The infectivity and viral replication were highest in CEF, DF-1, Vero, and QT-35 cells. In contrast, the results suggested that MDCK cells are less permissive for AnHV-1 infection with negligible CPE and reduced viral replication. Heterologous cell culture systems other than chicken embryo fibroblasts to adapted live vaccine viruses will provide a system devoid of other avian infectious agents. Moreover, it can be used for the propagation and cultivation of AnHV-1 vaccine strain for developing cell culture-based vaccines with high titer and could be an economical alternative for the existing options.

Avian Toll-like receptor 3 isoforms and evaluation of Toll-like receptor 3–mediated immune responses using knockout quail fibroblast cells

Toll-like receptor 3 (TLR3) induces host innate immune response on recognition of viral double-stranded RNA (dsRNA). Although several studies of avian TLR3 have been reported, none of these studies used a gene knockout (KO) model to directly assess its role in inducing the immune response and effect on other dsRNA receptors. In this study, we determined the coding sequence of quail TLR3, identified isoforms, and generated TLR3 KO quail fibroblast (QT-35) cells using a CRISPR/Cas9 system optimized for avian species. The TLR3-mediated immune response was studied by stimulating the wild-type (WT) and KO QT-35 cells with synthetic dsRNA or polyinosinic:polycytidylic acid [poly(I:C)] or infecting the cells with different RNA viruses such as influenza A virus, avian reovirus, and vesicular stomatitis virus. The direct poly(I:C) treatment significantly increased IFN-β and IL-8 gene expression along with the cytoplasmic dsRNA receptor, melanoma differentiation–associated gene 5 (MDA5), in WT cells, whereas no changes in all corresponding genes were observed in KO cells. We further confirmed the antiviral effects of poly(I:C)-induced TLR3-mediated immunity by demonstrating significant reduction of virus titer in poly(I:C)–treated WT cells, but not in TLR3 KO cells. On virus infection, varying levels of IFN-β, IL-8, TLR3, and MDA5 gene upregulation were observed depending on the viruses. No major differences in gene expression level were observed between WT and TLR3 KO cells, which suggests a relatively minor role of TLR3 in sensing and exerting immune response against the viruses tested in vitro. Our data show that quail TLR3 is an important endosomal dsRNA receptor responsible for regulation of type I interferon and proinflammatory cytokine, and affect the expression of MDA5, another dsRNA receptor, most likely through cytokine-mediated communication.

A flow cytometric granularity assay for the quantification of infectious virus

A flow cytometry-based assay was developed to assess the infective titer of two recombinant viruses: a recombinant herpes simplex type 2 (rHSV-2) and a recombinant canary pox (rALVAC.gfp). This method uses granularity of infected Vero and QT-35 cells, respectively, and correlates this to the infectious titer of virus samples. The percent of the cell populations with a high level of granularity could accurately be correlated to viral titers obtained through a traditional plaque assay, with R2 values greater than 0.8 using a semi-logarithmic scale. This approach offers a rapid, high-throughput method for infectious virus titration with similar accuracy to a traditional plaque assay.

C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells.

The infection of chickens with avian Hepatitis E virus (avian HEV) can be asymptomatic or induces clinical signs characterized by increased mortality and decreased egg production in adult birds. Due to the lack of an efficient cell culture system for avian HEV, the interaction between virus and host cells is still barely understood. In this study, four truncated avian HEV capsid proteins (ORF2-1 – ORF2-4) with an identical 338aa deletion at the N-terminus and gradual deletions from 0, 42, 99 and 136aa at the C-terminus, respectively, were expressed and used to map the possible binding site within avian HEV capsid protein. Results from the binding assay showed that three truncated capsid proteins attached to avian LMH cells, but did not penetrate into cells. However, the shortest construct, ORF2-4, lost the capability of binding to cells suggesting that the presence of amino acids 471 to 507 of the capsid protein is crucial for the attachment. The construct ORF2-3 (aa339-507) was used to study the potential binding of avian HEV capsid protein to human and other avian species. It could be demonstrated that ORF2-3 was capable of binding to QT-35 cells from Japanese quail and human HepG2 cells but failed to bind to P815 cells. Additionally, chicken serum raised against ORF2-3 successfully blocked the binding to LMH cells. Treatment with heparin sodium salt or sodium chlorate significantly reduced binding of ORF2-3 to LMH cells. However, heparinase II treatment of LMH cells had no effect on binding of the ORF2-3 construct, suggesting a possible distinct attachment mechanism of avian as compared to human HEV. For the first time, interactions between avian HEV capsid protein and host cells were investigated demonstrating that aa471 to 507 of the capsid protein are needed to facilitate interaction with different kind of cells from different species.

Survival of turkey arthritis reovirus in poultry litter and drinking water

Turkey reoviruses (TRVs) can cause arthritis, tenosynovitis, and enteric diseases in turkeys, leading to huge economic losses. The TRVs are tentatively divided into turkey arthritis reoviruses (TARVs) and turkey enteric reoviruses (TERVs) depending on the type of disease they produce. This study was conducted to determine the survival of these viruses in autoclaved and nonautoclaved poultry litter and drinking water at room temperature (approx. 25°C). Three isolates of TARV (TARV-O'Neil, TARV-MN2, and TARV-MN4) and one each of TERV (TERV-MN1) and chicken arthritis reovirus (CARV) were used in this study. The viruses were propagated and titrated on QT-35 cells. In autoclaved dechlorinated tap water, all 5 viruses were able to survive for 9 to 13 wk. In nonautoclaved water, all 5 viruses survived for at least 2 wk. In autoclaved litter, the viruses survived for 6 to 8 wk, and in nonautoclaved litter, they survived for 6 to 8 d only. The implications of these results are discussed below.

Efficient Marek's disease virus (MDV) and herpesvirus of turkey infection of the QM7 cell line that does not contain latent MDV genome

Marek's disease virus (MDV; also known as Gallid herpesvirus 2, MDV-1) causes oncogenic disease in chickens producing clinical signs that include lymphomas, visceral tumours, nerve lesions, and immunosuppression. MDV vaccines are widely used and mostly produced using primary cells: chicken embryo fibroblast cells, duck embryo fibroblast cells, chicken embryo kidney cells or chicken kidney cells. An immortalized cell line that can be used to manufacture the virus has long been desired. In this report, we demonstrate that QM7 cells were susceptible to infection with either MDV or herpesvirus of turkey (HVT; also known as Meleagrid herpesvirus 1, MDV-3). Polymerase chain reaction analysis with primers amplifying selected MDV genes revealed that QM7 cells did not contain these sequences. However, MDV genes were detected in QT35 cells, which have been reported to harbour latent MDV virus. Transfection of naked MDV DNA initiated efficient infection of QM7 cells. In addition, QM7 cell lysate, clarified supernatant, and QM7 cell pellet infected with MDV were negative for reverse transcriptase activity, indicating absence of endogenous retrovirus. QM7 cells were also found to be free of other avian pathogens in a chick embryo inoculation test. In vivo studies of MDV growing in QM7 cells showed the virus retained its pathogenicity and virulence. In ovo experiments demonstrated that both HVT and MDV propagated in QM7 cells did not interfere with hatchability of injected eggs, and viruses could be re-isolated from hatched chicks. The results suggest that QM7 could be a good host cell line for growing both MDV and HVT.

Efficacy and Safety of Cell-Associated Vaccines Against Marek's Disease Virus Grown in QT35 Cells or JBJ-1 Cells

The Marek's disease virus (MDV) vaccine strain CVI 988 usually is grown in primary chicken embryo fibroblasts (CEFs). We found that the strains could be grown also in the QT35 and JBJ-1 cell lines to titers in the same range as in the CEFs. Both cell lines are fibroblast-like cell lines, which can be grown in flat-bottomed tissue-culture flasks, roller bottles, and on microcarriers. For growth in QT35 cells it was necessary to adapt the virus to the cell line; for growth in JBJ-1 cells this was not necessary. We investigated the efficacy of experimental CVI 988 vaccines grown in QT35 cells and JBJ-1 cells. The efficacy studies were performed in accordance with European Pharmacopoeia (EP) monograph for live MDV disease vaccines. Groups of 1-day-old specific-pathogen-free chicks were vaccinated. Nonvaccinated control groups were included in the studies. Five to 7 days after vaccination all chickens were challenged with the very virulent MDV strain RB1B. After challenge the chickens were observed for a period of 70 days for signs of MD. The protection induced by CVI 988 grown in QT35 cells as well as JBJ-1 cells complied with the requirements of the EP that prescribe that the protection index should be at least 80%. The safety of the vaccines grown in QT35 cells and JBJ-1 cells was tested in a field study in commercial layer chickens. The vaccine virus was not safe after passaging in QT35 cells. This can be explained by the presence of fragments of the genome of MDV strains in the QT35 cell line. No signs of MD were noticed in the study in which CVI988 grown in JBJ-1 cells was tested. It is concluded that the JBJ-1 cell line is a suitable substrate for the current vaccines against MD.

Isolation and Characterization of a Turkey Arthritis Reovirus

During the spring and summer of 2011, the Minnesota Veterinary Diagnostic Laboratory at the University of Minnesota received 14 submissions of 15-to-18-week-old tom turkeys that were recumbent with wing tip bruises ("wing walkers") and uni- or bilateral swelling of the hock (tibiotarsal) joints. Gastrocnemius or digital flexor tendons were occasionally ruptured. A total of five turkey arthritis reoviruses (TARV-MN1 through TARV-MN5) were isolated in specific-pathogen-free embryonated chicken eggs and QT-35 cells. The identity of the isolates was confirmed by electron microscopy, reverse transcription-polymerase chain reaction, and gene sequence analysis. BLAST analysis on the basis of a 880 bp nucleotide sequence of the S4 gene confirmed all isolates as a reovirus. Phylogenetic analysis divided the five isolates into two subgroups: subgroup I containing TARV-MN1, -2, -3, and -5, and the other subgroup containing TARV-MN4. Isolates in subgroup I had a similarity of 97%-100% with each other, while subgroup II (TARV-MN4) had a similarity of only 89.2% with subgroup I viruses. This isolate showed 90%-93% similarity with turkey enteric reoviruses in the United States, while the other four isolates in subgroup I had 89%-97.6% similarity. These results indicate divergence within TARVs as well as from enteric viruses, which needs to be confirmed by complete genome sequence analysis. Further experimental studies are planned to determine the role of these isolates in turkey arthritis and to compare them with classical chicken reovirus.

Trichomonas gallinae, in comparison to Tetratrichomonas gallinarum, induces distinctive cytopathogenic effects in tissue cultures

In the present study the interaction of three genetically different clonal cultures of Trichomonas gallinae and Tetratrichomonas gallinarum with a permanent chicken liver (LMH) and a permanent quail fibroblast (QT35) cell culture was studied. Proliferation of T. gallinae cells was associated with a disintegration of the cell monolayer. The initial lesions on the LMH monolayer consisted of a progressive accumulation of the flagellate, forming clumps attached to the monolayer. A prolonged incubation time was characterized by appearance of holes in the cell monolayer with accumulation of trichomonads at their periphery. According to the severeness of the monolayer disruption differences among three tested T. gallinae clones were noticed. Furthermore, filtrates obtained either from axenic cultures of T. gallinae or from infected cell cultures produced a cytopathogenic effect similar to the protozoal cells, on both types of cell cultures. However, the destructive effect of the flagellates and their cell-free filtrates was much more pronounced on the LMH monolayer in comparison with the QT35 cells. Furthermore, freshly seeded LMH and QT35 cells suspended in cell-free filtrates of T. gallinae were unable to form a confluent monolayer. In comparison to T. gallinae, clonal cultures of T. gallinarum or their cell-free filtrates produced no effect on both types of monolayers. Interestingly, the cell-free filtrates obtained from both trichomonad species had an effect on the viability of both cell cultures. However, the cytotoxic effect of T. gallinarum filtrates was less severe than that recorded by T. gallinae. Consequently, for the first time a destruction of specified monolayers induced by T. gallinae-free filtrates could be demonstrated.

Pathogenicity of a Quail (Coturnix coturnix japonica)-Derived Marek's Disease Virus Rescued from the QT35 Cell Line

The QT35 cell line was established in 1977 from methylcholanthrene-induced tumors in Japanese quail. It was later shown that at least some of the QT35 cell lines were latently infected with Marek's disease (MD) virus (MDV). An MDV-like herpesvirus, named quail MDV (QMDV), was isolated from QT35 cells in 2000 by Yamaguchi et al. To determine the pathogenicity of QMDV, we inoculated 10-day-old specific-pathogen-free chickens with QMDV JM (virulent), RB-1B (very virulent), or 584A (very virulent plus). In addition, we inoculated 5-day-old Japanese quail with QMDV, JM, or RB-1B. QMDV is pathogenic in chickens with a tumor incidence comparable to JM. QMDV also caused MD in three out of 18 infected Japanese quail. In conclusion, QMDV is a virulent MDV, and its presence in QT35 cells has implications for the use of QT35 cells for vaccine production.

Permissibility of different cell types for the growth of avian metapneumovirus

Vero cells are commonly used for the growth of avian metapneumovirus subtype C (aMPV-C). This study was conducted to evaluate 17 different cell types for the growth of a Minnesota strain of aMPV-C. The virus was inoculated into these cell types and virus growth was monitored by the development of cytopathic effects (cpe) and immunofluorescence. Virus growth was obtained in 6 of 17 cell types tested with the highest virus titers observed in BGM and DF-1 cells. The flow cytometric analysis of cells at 72 h post inoculation found the highest number of infected cells in BGM cells followed by QT-35 cells. At 48 h post inoculation, DF-1 and BGM cells showed the highest number of infected cells. These results suggest that BGM, QT-35, and DF-1 cells can be used for high titer propagation of aMPV-C.

Growth of vaccine strains of avian pneumovirus in different cell lines

The isolation of avian pneumovirus (APV) (avian metapneumovirus) is usually performed in embryonated chicken eggs or chicken embryo fibroblast cell cultures followed by adaptation in continuous cell lines such as Vero cells. This study was conducted to find a suitable cell line that could be used to propagate vaccine strains of APV to high titre. For this purpose, we compared the growth of two Vero cell-adapted vaccine strains of APV (P63 and ca-APV) in seven different cell types with their growth in Vero cells. The cell types used were BGM-70, MA-104, QT-35, BHK-21, McCoy and DF-1 cells and primary turkey embryo fibroblast cells. When compared with growth in Vero cells, both viruses yielded higher titres in BGM-70 cells, while P63 also produced higher titres in MA-104 cells. In another experiment, the two viruses were grown and titrated in Vero cells under various cell culture conditions, such as age of cells, seeding concentration, and time of harvest. None of these cell culture variables were found to affect virus titres. L'isolement de pneumovirus aviaires (AVP; métapneumovirus aviaire) est généralement réalisé sur œufs embryonnés de poulet ou sur cultures cellulaires de fibroblastes d'embryon de poulet suivi par une adaptation sur lignées cellulaires continues telles les cellules Vero. Cette étude a été conduite pour trouver une lignée cellulaire favorable qui pourrait être utilisée à la propagation des souches vaccinales d'AVP avec un titre élevé. Dans ce but nous avons comparé la croissance, de deux souches vaccinales d'AVP (P63 et ca-APV), sur sept types cellulaires différents à celle sur cellules Vero. Les types cellulaires utilisés ont été des cellules BGM-70, MA-104, QT-35, BHK-21, McCoy et DF-1 ainsi que des cellules primaires de fibroblastes d'embryon de dinde. En se référant à la croissance des deux virus sur cellules Vero, les titres les plus élevés ont été obtenus sur les cellules BGM-70, et avec le virus P63 les titres ont également été supérieurs sur les cellules MA-104. Dans un autre essai, les deux virus ont été cultivés et titrés sur cellules Vero avec différentes conditions de culture, tels l’âge des cellules, la concentration cellulaire et le temps de récolte. Aucune de ces conditions de culture n'a eu d'effet sur les titres en virus. Die Isolierung von aviärem Pneumovirus (AVP, aviäres Metapneumovirus) wird üblicherweise in embryonierten Hühnereiern oder Hühnerembryofibroblastenzellkulturen mit nachfolgender Adaptation an permanenten Zelllinien wie Verozellen durchgeführt. Diese Studie wurde unternommen, um eine geeignete Zelllinie zu finden, die für die Anzüchtung von APV-Vakzinestämmen mit hohen Titern verwendet werden könnte. Aus diesem Grund verglichen wir das Wachstum von zwei Verozell-adaptierten APV-Vakzinestämmen (P63 und ca-APV) in sieben verschiedenen Zelltypen mit ihrer Vermehrungsrate in Verozellen. Die verwendeten Zelltypen waren BGM-70-, MA-104-, QT-35-, BHK-21-, McCoy- und DF-1 Zellen sowie primäre Putenembryofibroblastenzellen. Im Vergleich zur Vermehrung in Verozellen erreichten beide Viren in BGM-70-Zellen höhere Titer, während P 63 sich auch in MA-104-Zellen mit höheren Titern anzüchten ließ. In einem anderen Experiment wurden die beiden Viren in Verozellen unter verschiedenen Wachtumskonditionen wie Zellalter, Einsaatkonzentration und Erntezeitpunkt angezüchtet und titriert. Keine dieser Zellkulturvariablen hatten einen Einfluss auf die Virustiter. El aislamiento de pneumovirus aviar (APV; avian metapneumovirus) se lleva a cabo habitualmente en huevos embrionados de pollo o cultivos celulares de fibroblastos de embrión de pollo seguido por adaptación en líneas celulares continuas como las células Vero. Este estudio fue realizado con el objetivo de encontrar una línea celular que pudiera usarse para propagar y obtener títulos elevados de cepas vacunales de APV. Para este propósito, se comparó el crecimiento de dos cepas vacunales de APV adaptadas a cultivo celular (P63 y ca-APV) en siete diferentes tipos de células con su crecimiento en células Vero. Se utilizaron diferentes líneas celulares: BGM-70, MA-104, QT-35, BHK-21, McCoy y células DF-1, así como cultivos celulares primarios de fibroblastos de embrión de pavo. Cuando se compararon con el crecimiento en células Vero, ambos virus produjeron también títulos altos en las células MA-104. En otro experimento, los dos virus crecieron y se titularon en células Vero bajo varias condiciones de cultivo celular, como la edad de la células, concentración de siembra y tiempo de cultivo. Ninguna de esas variables de cultivo afectó el título vírico.

Use of a Japanese quail fibrosarcoma cell line (QT-35) in serologic assays to determine the antigenic relationship of avian metapneumoviruses.

The ability of a Japanese quail fibrosarcoma cell line (QT-35) to support the replication of avian metapneumoviruses belonging to the 3 subgroups A (14/1 virus), B (Colorado virus), and C (Hungary virus) enabled the development of assays for the detection and evaluation of virus-specific antibodies. On the basis of the results of enzyme-linked immunosorbent assay (ELISA), plaque reduction neutralization assay (PRNA), immunofluorescent assay (IFA), and Western blot analysis, some degree of antigenic cross-reactivity was observed between prototype viruses belonging to each of the 3 subgroups A, B, and C. The antigen produced in QT-35 cells was found to be superior with respect to its reactivity with virus-specific antibodies, as determined when used in ELISA and IFA. Standardization of both the input virus and the virus-specific antibodies in PRNA enabled a more detailed analysis of the antigenic relationship between these viruses. Specifically, it was observed that 14/1 virus shared more neutralizing regions with Hungary and Colorado viruses than did either of these viruses with 14/1 virus. In addition, Hungary virus shared comparatively fewer neutralizing epitopes with the Colorado virus than did 14/1 virus. Western blot analysis of the reactivity patterns of virus antigen, produced in QT-35 cells, with subgroup-specific antibodies identified a cross-reactive protein migrating at approximately 18 kD. These assays and the information from the Western blot will enable further analysis of avian metapneumovirus isolates to determine antigenic relationships.

Plaque assay for avian metapneumovirus using a Japanese quail fibrosarcoma cell line (QT-35)

A plaque assay for detecting, isolating and titrating avian pneumoviruses using a Japanese quail fibrosarcoma cell line (QT-35) is described. Plaques are produced after application of either an agarose or carboxymethyl-cellulose (CMC) overlay onto cell monolayers infected with representative avian metapneumoviruses which belong to subgroup A, B or C. Virus plaques can be easily visualized by light microscopy or after staining. The parameters affecting plaque appearance include: cell seeding concentration, virus strain, overlay composition and incubation time following infection. Optimal conditions for plaquing involve seeding QT-35 cells at 40 000 cells per cm 2 when using a 1.5% CMC overlay or 100 000 cells per cm 2 when using a 1.0 or 0.8% agarose overlay. In both cases, cell monolayers are infected with virus 24 h post-seeding and clearly visible plaques develop in 6 days. Due to the robust nature of these cells, the incubation time can be extended to a maximum of 13 days after infection in order to produce larger plaques.

Detection of avian pneumovirus in wild Canada (Branta canadensis) and blue-winged teal (Anas discors) geese.

Choanal cleft swab samples from 770 wild Canada geese (Branta canadensis) and 358 blue-winged teal (Anas discors), captured for relocation or banding, were examined for the presence of avian pneumovirus (APV) RNA by reverse transcription (RT)-polymerase chain reaction (PCR) and for virus isolation. The swab samples were pooled into groups of 5 or 10. Sixty eight of 102 (66.7%) pooled goose samples were RT-PCR positive for APV RNA. Thirteen of 52 (25.0%) pooled blue-winged teal samples were RT-PCR positive for APV RNA. APV RNA-positive samples were inoculated onto chick embryo fibroblasts (CEF) and QT-35 cells. Infectious APV was isolated from five Canada goose pooled samples in CEF and from one Canada goose pool in QT-35 cells but not from blue-winged teal.

Evaluation of a Japanese quail fibrosarcoma cell line (QT-35) for use in the propagation and detection of metapneumovirus

A Japanese quail fibrosarcoma cell line (QT-35) was evaluated and compared to Vero cells for its utility in metapneumovirus propagation, titration and serological detection by indirect immunofluorescence staining. Cell characteristics such as growth kinetics at different passage levels and seeding density in 96-well plates using various media formulations were studied in order to determine suitable assay parameters. Specifically, QT-35 cells supported the replication of a subgroup A metapneumovirus, strain 14/1, when maintained in DMEM containing a high level of glucose (4500 mg/l) and 2% gamma-irradiated fetal bovine serum (γ-FBS). There appeared to be a decreased ability of metapneumovirus produced in chicken embryo fibroblast (CEF) cells to replicate to high titers in QT-35 cells, however, this apparent restriction was overcome after the second passage resulting in high titered stock. Metapneumovirus produced in Vero cells and propagated in QT-35 cells produced high titered stock after the first passage. Viral titers determined in Vero and QT-35 cells were comparable, when the latter cell line was used at passage levels ≤20 and seeded between 5.0×10 4 and 1.0×10 5 cells/0.33 cm 2 in hgDMEM containing 10% γ-FBS, with a reduction to 2% γ-FBS when the virus was applied to the cell monolayers 24 h post-seeding. After infection with metapneumovirus, QT-35 cells exhibited syncytia, similar to those in metapneumovirus-infected Vero cells, which were readily detected by indirect immunofluorescent (IF) staining.

Canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells.

Canine parvovirus (CPV) enters and infects cells by a dynamin-dependent, clathrin-mediated endocytic pathway, and viral capsids colocalize with transferrin in perinuclear vesicles of cells shortly after entry (J. S. L. Parker and C. R. Parrish, J. Virol. 74:1919-1930, 2000). Here we report that CPV and feline panleukopenia virus (FPV), a closely related parvovirus, bind to the human and feline transferrin receptors (TfRs) and use these receptors to enter and infect cells. Capsids did not detectably bind or enter quail QT35 cells or a Chinese hamster ovary (CHO) cell-derived cell line that lacks any TfR (TRVb cells). However, capsids bound and were endocytosed into QT35 cells and CHO-derived TRVb-1 cells that expressed the human TfR. TRVb-1 cells or TRVb cells transiently expressing the feline TfR were susceptible to infection by CPV and FPV, but the parental TRVb cells were not. We screened a panel of feline-mouse hybrid cells for susceptibility to FPV infection and found that only those cells that possessed feline chromosome C2 were susceptible. The feline TfR gene (TRFC) also mapped to feline chromosome C2. These data indicate that cell susceptibility for these viruses is determined by the TfR.

Transactivation of latent Marek's disease herpesvirus genes in QT35, a quail fibroblast cell line, by herpesvirus of turkeys.

The QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95-103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16, meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisense meq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies.

Isolation of Avian Pneumovirus from an Outbreak of Respiratory Illness in Minnesota Turkeys

Antibodies to avian pneumovirus (APV) were first detected in Minnesota turkeys in 1997. Virus isolation was attempted on 32 samples (28 tracheal swabs, 4 pools of trachea and turbinates) that were positive for APV by reverse transcriptase polymerase chain reaction (RT-PCR). The cell cultures used were chicken embryo fibroblast (CEF), Vero cells, and QT-35 cells. Five virus isolates were obtained from these samples, and the identity of the isolates was confirmed by RT-PCR. Four isolates were obtained by inoculation of CEF cells, and 1 isolate was obtained in QT-35 cells after 3-7 blind passages in cell cultures. Vero cells did not yield any isolate on primary isolation; however, all 5 isolates could be adapted to grow in Vero cells following primary isolation in CEF or QT-35 cells. This is the first report of isolation of APV in Minnesota and also the first report of primary isolation of APV in QT-35 cells.

A Rapid Virus Neutralization Assay for Newcastle Disease Virus with the Swine Testicular Continuous Cell Line

Five continuous cell lines, swine testicular (ST), human rectal tumor (HRT 18), fetal rhesus monkey kidney (MA104), bovine turbinate (BT), and quail tracheal (QT35), were evaluated and compared with chicken embryo fibroblasts (CEFs) for their ability to propagate B1 or Texas GB strains of Newcastle disease virus (NDV). The NDV Texas GB strain replicated in all the continuous cell lines used in this study. Only the ST and QT35 cells produced a cytopathic effect (CPE) similar to that produced in CEFs. However, the ST cell line remained attached while displaying CPE, whereas infected QT35 cells detached, as did the CEFs. The B1 strain of NDV replicated in ST cells, MA104 cells, and CEFs but with less CPE as compared with the Texas GB strain. Pretreatment with trypsin did not enhance CPE with either NDV strain at the level tested. Sera evaluated for neutralizing antibody titers to NDV were significantly higher in titer when the ST cell line was used and compared with CEFs. A high correlation was found between the microscopic examination and the tetrazolium dye (MTT) microassay methods for determining the viral neutralization endpoint, thus suggesting the ST cell line and MTT microassay could be used as an alternative to CEFs and microscopic examination for evaluating neutralizing antibodies titers to NDV.