Persistent rat cytomegalovirus (RCMV) infection of the salivary glands contributes to the anti-RCMV humoral immune response

The salivary glands are the major sites of persistent replication of rat cytomegalovirus (RCMV). At several months post infection (pi), infectious RCMV is usually still produced in the salivary glands but not in any other organ or tissue of the rat. To investigate whether the persistence of RCMV in the salivary glands is crucial to the pathogenesis of viral infection, we monitored the progression of RCMV-induced disease in rats from which the salivary glands had been surgically removed (desalivated) as well as in sham-operated rats, both after a lethal and sublethal challenge with RCMV. Desalivation did not have a significant effect on either RCMV-induced morbidity or mortality. As expected, at 1 year pi, relatively high levels of infectious virus were detected in the salivary glands of sham-operated rats, whereas neither infectious virus nor RCMV DNA could be detected in liver, spleen and lungs of these animals. Infectious virus and viral DNA were also undetectable in organs from desalivated animals at 1 year pi. Surprisingly, a difference was found between desalivated and sham-operated rats in the titers of anti-RCMV IgG antibodies, which were significantly higher in sham-operated rats than in desalivated animals at 183, 295 and 365 days pi. This finding indicates that the persistence of RCMV in the salivary glands may contribute significantly to the anti-RCMV humoral immunity of infected rats.     

Rat cytomegalovirus replication in the salivary glands is exclusively confined to striated duct cells

The salivary gland is the preferred organ for cytomegalovirus (CMV) replication and viral persistence. In order to identify the nature of infected cells and to study viral replication in more detail, several experiments were conducted. Using the rat CMV (RCMV) model, acutely infected young adult rats (6 weeks of age) and new-born rats (3 days of age) were infected, and submandibular, parotid and sublingual glands were harvested at different time points after infection. For identification of the nature of infected cells, immunohistochemistry, in situ hybridisation and electron microscopic techniques were used. In young adult animals, the submandibular gland was the preferred organ for RCMV replication. The parotid and sublingual glands contained fewer viruses than the submandibular gland. In contrast, in new-born rats, the main site of RCMV replication was the sublingual gland, while the submandibular and parotid glands contained low amounts of virus. No virus could be detected in the parotid glands. In all glands of RCMV-infected animals, the infection was exclusively confined to striated duct cells. Infection resulted in a cellular inflammatory response which was mostly located in the interlobular duct region, whereas only few inflammatory cells were found in the neighbourhood of infected striated duct cells. This phenomenon may contribute to the long persistence of the virus in this organ. Received: 25 May 2000 / Accepted: 26 May 2000     

A rat cytomegalovirus strain with a disruption of the r144 MHC class I-like gene is attenuated in the acute phase of infection in neonatal rats

Summary.  We previously generated an RCMV strain in which the r144 gene, encoding a major histocompatibility complex class I homolog, had been deleted (RCMVΔr144). To investigate the role of r144 during acute infection of neonatal rats, we infected three days-old neonatal rats with either RCMVΔr144 or wild type (wt) RCMV and the presence of infectious virus as well as viral DNA in various organs was determined at either 3, 5 or 21 days p.i.. In addition, we as-sessed both type and number of inflammatory cells in these organs. Interestingly, a significantly lower concentration of infectious virus as well as viral DNA was found in spleens of RCMVΔr144-infected rats than in those of wt RCMV-infected animals at 3 days p.i.. At the same time point, a significantly lower amount of infiltrating NK cells and monocytes/macrophages was seen in the spleens of RCMVΔr144-infected rats than in spleens of rats infected with wt RCMV. At 21 days p.i., RCMVΔr144-infected rats were found to have lower virus titers in the salivary glands than wt RCMV-infected animals. Significant differences between RCMVΔr144- and wt RCMV-infected rats were detected neither at other time points nor at other sites. We conclude that after infection of neonatal rats, the replication of RCMVΔr144 is severely restricted compared to wt RCMV. Received May 4, 2001 Accepted July 4, 2001     

The r131 Gene of Rat Cytomegalovirus Encodes a Proinflammatory CC Chemokine Homolog which is Essential for the Production of Infectious Virus in the Salivary Glands

Rat cytomegalovirus (RCMV) possesses two adjacent genes, r131 and r129, which have the potential to encode CC chemokine homologs. Interestingly, the amino acid sequences encoded by both genes show similarity to the sequence of the murine CMV (MCMV) MCK-2 protein, which is encoded by the m131/129 gene. In order to study the significance of the r131 gene in the pathogenesis of RCMV infection, we generated two different virus strains in which the r131 open reading frame is disrupted. Replication of these null mutant strains, designated RCMVdeltar131a and RCMVdeltar131b, was evaluated in vitro and in vivo. Both strains were found to replicate with a similar efficiency as wild-type (WT) RCMV in vitro. However, in contrast to WT virus, neither RCMVdeltar131a nor RCMVdeltar131b established a high-titer infection in the salivary glands of immunocompromised rats. Furthermore, in a local, rat footpad infection model, both recombinant viruses induced a significantly lower amount of paw swelling than did WT RCMV. Also, a higher number of infiltrating macrophages was observed in paws infected with WT RCMV than in those infected with the recombinants. Taken together, these results suggest that r131 (i) promotes inflammation at initial sites of inoculation and, subsequently, efficient virus dissemination to or infection of the salivary glands and (ii) might be involved in the persistence of virus infection, at least in the spleen. In addition, our data indicate that r131 represents the functional homolog of the MCMV m131/129 gene.     

An animal model for therapeutic intervention studies of CMV infection in the immunocompromised host

Summary An experimental rat model to study acute cytomegalovirus infections is described. Eight-week old male Brown Norway rats, immunosuppressed by total body irradiation, were infected with rat cytomegalovirus (RCMV). The effects of infection were determined by survival rates and the presence of virus or viral components in different organs was assayed by plaque test, immunoperoxidase staining, dot-blot DNA hyridization and in situ DNA hybridization. At days 10-post infection nearly 90% of the animals had died. Spleen, liver and bone marrow were heavily infected. Interstitial pneumonia was observed. Pathological findings strongly resembled the full scale of lesions in human CMV infections. Anti-RCMV hyperimmune serum was effective against mortality from RCMV infection and viral spread to lungs and liver was prevented. This model is appropriate for studies on the pathogenesis and antiviral therapy of CMV infections in the immunocompromised host.     

Infection with rat cytomegalovirus (CMV) in the immunocompromised host is associated with the appearance of a T cell population with reduced CD8 and T cell receptor (TCR) expression

Infection with human cytomegalovirus (HCMV) mostly results in a chronic subclinical infection; the immune system is unable to eliminate the virus and is apparently in equilibrium with the persistent virus. In the immunosuppressed host this equilibrium is disturbed, resulting in clinical infection. Rat cytomegalovirus (RCMV) infection in its host can be used as a model for HCMV infection. Using flow cytometry we examined the effect of acute RCMV infection on the composition of leucocyte subsets in the peripheral blood of both immunocompetent and immunosuppressed (5 Gy total body irradiation) Lewis rats. Special attention was paid to the natural killer (NK) cells and the CD8⁺ T cells known to be involved in the control of viral infections. Furthermore, we determined the presence of leucocyte subsets in the internal organs by immunohistochemistry. In immunocompetent rats, infection caused a small increase in NK cells and a large increase in CD8⁺ T cells. In contrast, infection of immunosuppressed rats caused a marked increase in NK cells and a small increase in CD8⁺ T cells, consisting of T cells with reduced expression of both CD8 and TCR. This phenomenon is characteristic of anergic CD8⁺ T cells, possibly explaining the ability of the virus to escape elimination by the immune system. The increase of NK cells in the peripheral blood of immunosuppressed, RCMV-infected rats could also be detected in kidney, liver, lung and pancreas, but not in salivary gland. This could explain the long persistence of infectious virus in the salivary gland.     

Detection of Epstein-Barr virus antigens and DNA in major and minor salivary glands using immunocytochemistry and polymerase chain reaction: possible relationship with Sjogren's syndrome.

This study has investigated the presence of Epstein-Barr virus (EBV) in parotid (n = 12), submandibular (n = 15), and minor salivary glands (n = 25) using immunohistochemical methods for detection of EBV-encoded antigens and the polymerase chain reaction (PCR) for detection of viral DNA. Major salivary glands were from patients without connective tissue disease. Labial glands were from patients with primary Sjogren's syndrome (n = 10), rheumatoid arthritis (n = 8), or from normal individuals (n = 7). None of the glands exhibited specific reactivity for lytic (EA-D, EA-R, VCA) or latent (EBNA-2, LMP) viral antigens. Antibodies to EA-D, when used at 20-50 times their optimal concentration, gave lumenal staining of ducts and acini of all the specimens tested (n = 14), irrespective of the presence (n = 8) or absence (n = 6) of EBV-DNA by PCR. Ductal immunoreactivity for the EBV/C3d (CR2, CD21) receptor was found in 40 per cent of specimens. PCR detected EBV-DNA in 64 per cent submandibular, 46 per cent parotid, and 80 per cent of minor glands. There were no significant differences in the detection of EBV-DNA between specimen/patient groups. Only type A EBV was detected by strain typing PCR. These results indicate that EBV (type A), undetected immunocytochemically, is commonly present at low copy numbers within salivary glands irrespective of a clinical diagnosis of Sjogren's syndrome.     

Biology of Mouse Thymic Virus, a Herpesvirus of Mice, and the Antigenic Relationship to Mouse Cytomegalovirus

Mouse thymic virus (TA) is a herpesvirus which produces extensive necrosis of the thymus of newborn mice 7 to 14 days after infection. Infectious virus can be recovered from the thymus for only 10 days after infection, with highest titers occurring between days 5 and 7. In mice 5 days old or less, TA infects thymus cells and produces massive necrosis. TA also infects the salivary glands and persists as a chronic infection. Newborn mice infected with TA have no detectable humoral immune response. Infected adult mice respond, and humoral antibody is detected 7 days after infection. Titers are maintained for months thereafter. Regardless of the age of the mice inoculated with TA, persistent infection was established in the salivary glands, but no evidence for thymus involvement was observed when adults were infected. TA does not cross-react serologically by immunofluorescent, complement fixation, or virus neutralization tests with mouse cytomegalovirus; however, interestingly, the epidemiology of the two herpesviruses are similar. Both mouse cytomegalovirus and TA were isolated from the same animals in populations of laboratory and wild mice. Evidence of infection with mouse cytomegalovirus and TA were most apparent by virus isolations, since humoral antibody responses are rarely observed. All strains of mice tested were susceptible to TA infection. However, in some strains maximum necrosis occurred at 7 days, compared with 10 to 14 days for other strains. The difference in age susceptibility and the target tissue of thymus in newborn mice suggests that TA is a model herpesvirus for studying the effects of viral infections on humoral and cell-mediated immunological functions.     

A double antibody sandwich ELISA for rapid diagnosis of virus infection and to measure the humoral response against infectious bursal disease on clinical material.

A double antibody sandwich ELISA (DAS-ELISA) was developed and employed for simultaneous direct detection of infectious bursal disease virus (IBDV) from bursal samples and to measure the humoral response, using the same basic immunoreagents. The purified and non-purified antigen, capture antibody and chicken hyperimmune sera were prepared, and standardized for this purpose. The DAS-ELISA was applied to both 80 bursal suspensions and 224 corresponding serum samples from vaccinated and non-vaccinated commercial flocks. Bursae samples were collected at 2 weeks of age, and submitted to histological examination, virus isolation in specific pathogen-free chickens embryos, and the DAS-ELISA technique. Serum titres obtained in indirect ELISA and serum neutralization test were compared with those in DAS-ELISA. The agreement was 80% between DAS-ELISA, and the conventional techniques, with high sensitivity (87%) and specificity (90%).     

Improved detection and quantification of mouse cytomegalovirus by real-time PCR

During latent cytomegalovirus (CMV) infection, viral presence cannot be detected by plaque assay. Therefore, we assessed the applicability of real-time PCR for temporal determination of virus dissemination in two different mouse strains. Eight-week-old BALB/c and C57BL/6J mice were infected with mouse CMV (MCMV) and sacrificed at 1, 2, 4, 6, 14 and 28 days post infection. Real-time PCR was used to determine MCMV copy number in the heart, bone marrow cells, aorta and blood. In lung, liver, salivary gland and spleen the presence of MCMV was determined both by plaque assay and real-time PCR. In analogy with the plaque assay, the real-time PCR technique revealed higher numbers of MCMV genomic copies in all organs obtained from BALB/c mice when compared with C57BL/6J mice, demonstrating the applicability of the technique. A significant correlation was observed between both assays when a positive test result was seen with both assays. Nonetheless, lower viral infectivity titers were found compared to real-time PCR data. Thus, the real-time PCR technique is more sensitive in detecting the presence of MCMV and is therefore well suited for (dose-response) intervention studies aimed at studying virus eradication.     

Use of enzyme amplification in an ELISA to increase sensitivity of detection of barley yellow dwarf virus in oats and in individual vector aphids

A new technique of alkaline phosphatase amplification in an ELISA (amplified ELISA) was used to increase the sensitivity of detection of barley yellow dwarf virus (BYDV) from oat plant sap and in individual vector aphids. Amplified ELISA differs from conventional direct double antibody sandwich ELISA (DAS-ELISA) in the enzyme substrate reaction. The bound enzyme-labelled antibody catalyzes the conversion of NADP to NAD which is then used in a secondary enzyme-mediated cyclic reaction producing a red-coloured end product. Amplified ELISA was compared with DAS-ELISA for the detection of BYDV and each assay was done with both monoclonal and polyclonal antibody reagents. Both types of antibodies detected BYDV from oat sap and amplified ELISA increased the sensitivity of detection sufficiently to allow a diagnostic test to be completed in less than 2 h using microtitre plates precoated with antibodies. However, in the amplified ELISA using polyclonal antibodies the absorbance values obtained with the healthy oat sap samples were much greater than those obtained in the DAS-ELISA, or with the monoclonal antibodies, and were too large to be acceptable for reliable diagnostic tests. Both monoclonal and polyclonal antibodies were used successfully to detect BYDV in individual virus-carrying Rhopalosiphum padi by amplified ELISA and there was little nonspecific background reaction in the control samples with either of the antibodies.     

Multiple organ involvement during experimental cytomegalovirus infection is associated with disseminated vascular pathology

Since much of the pathogenesis of cytomegalovirus (CMV) disease is still unknown and vascular involvement may be of importance, a rat model was used to study the nature and course of CMV-induced vascular pathology. In this model, local CMV infection was established by subcutaneous inoculation of rat-specific CMV (RCMV) in the sole of the foot. Signs of endothelial activation, including leucocyte adhesion, preceded detectable RCMV infection of these cells. Ultimately, vasculitis and thrombotic occlusion were accompanied by diffuse tissue inflammation and necrosis. Generalized RCMV infection was induced in rats by intraperitoneal administration of the virus, which resulted in multiple organ pathology, including haemorrhages, inflammation, and gastrointestinal ulceration. RCMV-encoded antigens were found especially in mononuclear inflammatory cells in the organs and peripheral blood. In addition, multiple haemorrhages and disturbed haematological parameters indicated diffuse intravascular coagulopathy. In conclusion, this study provides evidence for extensive vascular involvement and haematological consequences during disseminated CMV infection. The nature and chronology of RCMV-induced pathological vascular events were demonstrated, indicating the importance of endothelial damage. These data and further study may lead to a better understanding of the pathogenesis of CMV multiple-organ disease. © 1998 John Wiley & Sons, Ltd.     

Restriction of viral dissemination from the midgut determines incompetence of small brown planthopper as a vector of Southern rice black-streaked dwarf virus

Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, is transmitted by the white-backed planthopper in a persistent-propagative manner. In this study, we found that another planthopper species, the small brown planthopper (SBPH), could acquire SRBSDV but not transmit it. To identify the transmission barrier for SRBSDV in SBPHs, sequential infection by SRBSDV in the organs of SBPHs was studied with immunofluorescence for viral antigens. SRBSDV initially entered the epithelial cells of the midgut, then viroplasms, the sites for viral replication, formed in the midgut of viruliferous SBPHs. Furthermore, SRBSDV spread within the midgut, but failed to disseminate from the midgut into the hemocoel or into the salivary glands. All these results indicated that the inability of SBPH to transmit SRBSDV could be due to the restriction of viral dissemination from the midgut of SBPH, which led to the failure of viral spread to the salivary glands for virus transmission.     

Quantitative measurement of infectious murine cytomegalovirus genomes in real-time PCR

Acute virus replication in murine (M) CMV infected C57BL/6 (Cmv1(r)) mice is severely limited by Ly49H+ NK cells, but not in MCMV infected BALB/c or BXD8 (Cmv1(s)) mice that lack Ly49H+ NK cells. Interestingly, other NK cell receptors may also play a role in MCMV immunity, as CMV encoded gp40 protein can diminish expression of protein ligands recognized by the NK cell receptor NKG2D. To determine whether other additional gene products might influence MCMV immunity, we designed an efficient, sensitive and reliable method for screening resistance or susceptibility phenotypes in mice. Although multiple methods are frequently used to detect and quantify infectious MCMV in mouse tissue samples collected during acute viral infection, these are not readily adaptable to high-throughput screening strategies. Hence, we utilized real-time PCR for detection and quantitative measurement of infectious MCMV genomes present in various tissues of infected mice. MCMV genomic sequence was accurately and reproducibly detected over the range 10(2)-10(8) molecules in mouse genomic DNA samples using this methodology. Importantly, it was found that quantitative real-time PCR and viral plaque assay measurements of MCMV in tissues collected from infected mice, including resistant and susceptible strains, were directly correlated. Moreover, quantitative real-time PCR results obtained during a 3-week time-course study of virus replication in spleens, livers and salivary glands of infected mice demonstrated sensitive, accurate and reproducible detection and measurement of infectious MCMV.     

Simultaneous quantification of two plant viruses by double-label time-resolved immunofluorometric assay

For simultaneous and sensitive detection of two antigens in one sample, monoclonal antibody (MAb) to potato virus M (PVM) was labelled with a lanthanide Eu3+ and MAb to potato virus X (PVX) with another lanthanide Sm3+. A mixture of the labelled MAbs was used as a tracer. After performing the immunoreactions, the fluorescence of the dissociated lanthanides was measured at different wave-lengths in a time-resolved fluorometer to quantificate the PVX and PVM amount in a sample. Double-label time-resolved fluoroimmunoassay (TR-FIA) detected 1 ng/ml of each virus and was therefore more sensitive for simultaneous detection of PVX and PVM than reported for a single virus detection with double antibody sandwich ELISA (DAS-ELISA).     

Comparison of a Transplant Multiplex Viral Panel on the ICEPlex System with Real-Time PCR for Detection of Cytomegalovirus,Epstein-Barr Virus,and BK Virus in Clinical Specimens

We compared a multiplex viral transplant panel on the ICEPlex system to real-time PCR for the detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus (BKV). The sensitivities of the ICEPlex were 83.3%, 95.5%, and 65.5% for the detection of CMV, EBV, and BKV, respectively. Interestingly, the multiplex assay detected dual infections in 16/280 (5.7%) samples tested.     

Histopathologic and ultrastructural studies of disseminated cytomegalovirus infection in strain 2 guinea pigs

Inbred strain 2 guinea pigs developed severe disseminated disease during acute experimental guinea pig cytomegalovirus (GPCMV) infection. A high mortality rate (100%) resulted, with most animals dying between 10 and 14 days after high dose (7.5 X 10(5) TCID50) virus inoculation. Infectious virus was recovered from many tissues, including spleen, lungs, liver, pancreas, heart, adrenals, kidneys, and salivary glands. The rate of GPCMV isolation from these tissues ranged from 50 to 100%. Gross lesions were observed in the spleen, liver, and lungs. On histologic examination, lesions were also seen in many other organs, including heart, pancreas, kidneys, adrenals, brain, intestines, and salivary glands. Intranuclear viral inclusions were present in many cell types of various organs. Under electron microscopic examination, cells with viral inclusions were easily found in the spleen, and liver, but less readily in the lungs, kidneys, salivary glands, and other organs. Most of the intranuclear inclusions consisted of electron-dense fibrils (10 nm diameter), viral nucleocapsids (100 nm), and tubular structures (60 nm diameter). Dense bodies and enveloped dense virions containing single or multiple capsids were present in the cytoplasm of many infected cells. The morphologic developments of GPCMV in these visceral tissues of strain 2 guinea pigs resembled those seen in GPCMV-infected cultured guinea pig cells but differed from those observed in the infected salivary gland duct cells. Strain 2 guinea pigs are a useful animal model for studying disseminated infection in CMV-associated human diseases.     

Direct detection of human cytomegalovirus in urine specimens from renal transplant patients following polymerase chain reaction amplification

A polymerase chain reaction (PCR) assay was used to amplify human cytomegalovirus (HCMV) directly from urine specimens taken from renal transplant patients. In serial urine samples from patients who had at least one specimen positive for HCMV; the PCR assay consistently detected the presence of HCMV DNA sequences, whereas virus detection by other tests such as enzyme-linked immunosorbent assay (ELISA), nonradioactive DNA hybridization assay, and virus isolation were variable. Of 37 specimens positive by PCR, 36 were positive by either ELISA, hybridization assay, or virus isolation. Infectious virus was detected in 13 of the 37 PCR-positive urines. HCMV DNA was detected by PCR in all samples that were positive for HCMV by either hybridization assay or virus isolation. The viral genome copy number was determined by PCR assay for several urine samples that were positive by virus isolation but negative for HCMV by ELISA or hybridization assay. Viral genome copy number estimates indicated the presence of HCMV at very low levels in these urines verifying the fidelity of the virus isolation procedures. The consistency of the PCR assay makes it an ideal method for detection of infection and monitoring antiviral drug therapy in patients infected with HCMV.