bubble_chart Overview

CMV infection is distributed worldwide, with humans being the only host of CMV. The infection rates vary among different countries and economic conditions. Adult CMV infection is closely related to immune function.

bubble_chart Epidemiology

CMV infection is distributed worldwide, and humans are the only host of CMV. The infection rates vary among different countries and economic conditions. Adult CMV infection is closely related to immune function. For example, those who receive immunosuppressive therapy due to organ transplantation often become ill because of latent viruses in the donated organs or transfused blood, or due to the activation of latent viruses by immunosuppression. The incidence of CMV infection is high in patients with Acquired Immune Deficiency Syndrome.

The source of the pestilence is patients and their acute carriers. The virus can be excreted in milk, saliva, and urine, persisting for several weeks to years. Humans are highly susceptible to infection, and transmission routes are diverse. It is classified as a sexually transmitted disease, often resulting from infection via semen, cervical secretions, tears, and feces (oral-genital contact, rimming). (See Table 1)

Homologous CMV infection poses a serious risk in blood transfusions and organ transplants. Multiple transfusions or a single large-volume transfusion increases the risk of primary and recurrent infections, with the risk being even higher when receiving leukocyte-containing blood. The CMV infection rate is also high after organ or bone marrow transplantation.

bubble_chart Etiology

Etiology

CMV belongs to the herpesvirus group and is the largest virus in the human herpesvirus family. It has a typical herpesvirus structure, with a maximum size composed of 162 capsomeres forming a regular icosahedron. CMV can only proliferate in human fibroblast cell cultures and cannot grow in other animal cells. Its replication is very slow, and initial isolation requires over a month to observe characteristic cellular changes: cells become rounded, swollen, with enlarged nuclei, and a large eosinophilic inclusion body surrounded by a "halo" appears around the nucleus.

CMV survives for a maximum of 2 hours in 20% ether. pH <5時，或置於56℃30分鐘，或紫外線照射5分鐘可被充分滅活。CMV的感染性對凍融或存於-20℃或-50℃均不穩定，10%的家用漂白粉可使其感染性明顯降低。

Pathogenic mechanism

CMV infection can reduce the body's immune function, particularly cellular immunity. CMV infection significantly impacts thymus development and the functions of splenic cells, mononuclear phagocytes, NK cells, and CTL cells.

1. Effects on the thymus and spleen

   In newborn guinea pigs with acute CMV infection in laboratory settings, thymus development is inhibited, and T-cell counts decrease. In adult mice infected with CMV, CMV can be detected in 88% of thymuses.

   CMV infection affects spleen function, reducing the proliferative response of splenic lymphocytes to ConA stimulation and significantly lowering IL-2 production by splenic cells.

2. Effects on immune cells

   The immunosuppression caused by CMV infection is related to viral replication within cells. CMV can replicate in mononuclear phagocytes, T cells, B cells, and some unidentified mononuclear cells, with mononuclear phagocytes being the most susceptible. Lymphocytes play a crucial regulatory and effector role in immune responses. After CMV infection, multiple immune functions of lymphocytes can be impaired.

   CMV infection often manifests as acute mononucleosis. Peripheral blood lymphocytes show weakened proliferative responses to mitogens, CMV antigens, and HSV antigens, reduced interferon induction, and a decline in the CD4/CD8 ratio from 1.7±0.7 to 0.2±0.2, along with decreased T-cell activity. These changes can persist for a considerable time; even 10 months after infection, most patients' T-cell subset ratios have not fully normalized.

   The immunosuppressive effects of CMV infection are primarily due to functional abnormalities in infected large mononuclear cells and CD8 cells. Mononuclear phagocytes play a pivotal role in anti-CMV immunity, not only by directly phagocytizing and killing the virus but also by processing and presenting antigens, secreting cytokines, and regulating and amplifying immune responses. After CMV infection, mononuclear phagocyte function is impaired: CMV-infected macrophages exhibit reduced phagocytic capacity, decreased intracellular oxygen free radical production, altered expression of Fc and complement receptors, diminished antigen-presenting function, and reduced IL-1 production and responsiveness to IL-1 and IL-2. Moses et al. detected decreased IL-1 activity using thymocyte proliferation assays, and reduced IL-1 production can lead to an imbalance in the TH/TS cell ratio.

   NK cells have an antagonistic effect on the spread of CMV. NK cells actively participate in the entire process of anti-CMV infection, but high NK activity is not necessarily a protective response; rather, it may serve as evidence of active infection. Although NK cells cannot prevent the occurrence of primary CMV infection, once infection is established, NK cells can emerge early in the course of CMV infection, playing a role in limiting its spread and localizing the infection. NK cells and CTL cells are important effector cells against CMV. In the early stages of CMV replication, before the production of sexually transmitted disease viral particles, they can lyse infected cells, preventing the virus from spreading between cells late abortion. In mouse models, during the first 3-5 days of viral activity, the antiviral effect is mediated by NK cells, and their activity can be enhanced by IFN. Between days 6-21, cytotoxic activity of CTL cells is observed in the spleen and peripheral blood. The level of NK and CTL cell activity determines the host's susceptibility to CMV infection and the ease of recovery. However, during CMV infection, the activity of NK and CTL cells is also severely affected. Additionally, specific cellular immunity plays a role in preventing CMV reinfection. A study examining T-cell responses in 20 kidney transplant recipients with CMV infection found that 14 exhibited CMV cytotoxic responses, while the 6 who lacked such responses experienced severe clinical outcomes. Thus, the presence of specific T cells helps prevent CMV reinfection.

3. Antibodies have the effect of reducing the virulence of CMV infection.

   After the body is infected with CMV, various antibodies can appear. Although specific antibodies, including neutralizing antibodies, are present in breast milk, cervical secretions, and saliva, CMV can still be detected, indicating that antibodies cannot prevent viral spread. Antibodies passively acquired by the fetus from the mother cannot block infections transmitted intrauterinely, through the birth canal, or via breast milk. Studies have shown that injecting 0.2 ml of high-titer anti-CMV globulin into the peritoneal cavity or veins of mice before a lethal CMV challenge can fully protect the animals from death. One month later, when challenged again with CMV, all animals survived, demonstrating that antibodies can reduce the virulence of CMV.

After initial infection, CMV persists indefinitely in host cells in a latent state. It may involve multiple tissues and organs, and autopsies suggest that the lungs, liver, pancreas, salivary glands, central nervous system, and intestines may also be sites of viral latency. The severity of congenital infection is related to the lack of ability to produce precipitating antibodies and the T-cell response to CMV. After CMV infection in children and adults, activated T lymphocytes with a cytotoxic suppressor phenotype appear in the peripheral blood. If the host's T-cell function is impaired, latent viruses may reactivate and cause various syndromes. Chronic stimulation following tissue transplantation provides conditions for CMV reactivation and disease induction. Certain potent immunosuppressants targeting T cells, such as anti-thymocyte globulin, are associated with a high incidence of clinical CMV syndromes. Additionally, CMV can functionally act as a cofactor, reactivating latent HIV infection.

bubble_chart Clinical Manifestations

The natural history of CMV infection is complex. After primary infection, viral shedding often persists for weeks, months, or even years before the infection becomes latent. Recurrent infections with renewed viral shedding are common. Even many years after primary infection, latent virus may reactivate, and reinfection with different antigenically distinct strains can occur. The clinical manifestations of CMV infection are related to the individual's immune function and age. As shown in Table 2, the symptoms and signs arising from vertical transmission, horizontal transmission, or iatrogenic infection are diverse.

Table: Clinical Course and Types of CMV Infection (Baerlocher et al.)

1. Neonatal infection (congenital)

a. Severe type: jaundice, anemia, hepatosplenomegaly, thrombocytopenic purpura, central nervous system involvement, pneumonia, myocarditis)

b. Mild type: asphyxia, cardiomegaly, hyperbilirubinemia

2. Asymptomatic period after birth, reinfection with CMV during infancy (congenital/acquired?)

a. Systemic type

b. Respiratory type (whooping cough-like) c. Hepatosplenomegaly

d. Gastrointestinal type

e. Renal type?

3. Acquired type

a. Influenza-like type

b. Mononucleosis-like type

c. Respiratory type

d. Gastrointestinal type

e. Hepatitis

f. Due to special medical immunosuppression

g. Asymptomatic type?

4.

2. Consequences of item 3, namely mental
3. and motor developmental delay

   Regarding acquired CMV infection, it is commonly observed clinically as post-transfusion mononucleosis. In cases of immune dysfunction, vasculitis

4. retinitis
5. pneumonia, and gastrointestinal infections may occur. Additionally, most patients are complicated by Guillain-Barré syndrome.

bubble_chart Diagnosis

Diagnosis of CMV infection cannot be made based solely on clinical manifestations. Isolation of the virus from clinical specimens, along with a fourfold or greater increase in antibody levels or sustained elevation of antibody titers, will aid in diagnosis.

1. Virus Isolation

   The best specimens for virus isolation include saliva, urine, genital secretions, breast milk, and leukocytes. These are inoculated into human fibroblast cells for propagation and isolation. Cytopathic effects (CPE) may appear within one day or several weeks. After fixation and HE staining, giant cells can be observed, featuring intranuclear inclusion bodies, perinuclear halos, and eosinophilic cytoplasmic inclusion bodies, resembling "owl's eye." Monoclonal or polyclonal antibody fluorescence staining can also be used for detection.

2. Serum Antibody Detection

   Commonly used methods include complement fixation (CF), indirect immunofluorescence (IIF), enzyme immunoassay (EIA), indirect hemagglutination (IHA), and radioimmunoassay (RIA) to detect CMV-IgG and IgM antibodies. When a single serum sample confirms past CMV infection, additional serum samples should be collected immediately and at intervals of 2, 4, and 8 weeks. The combination of virus isolation can diagnose primary infection.

3. DNA Probe

   Widely used for CMV detection, the 32P-labeled probe is the most sensitive. For certain specimens, hybridization methods may be more sensitive than virus isolation.

4. Polymerase Chain Reaction (PCR)

   (1) Specimen Collection and Processing

   Specimens include patient blood, urine, and glandular tissue. Buffy coats prepared from whole blood can be stored at -80°C; urine specimens can be stored in liquid nitrogen. Repeated freeze-thaw cycles should be avoided.

Urine specimens are centrifuged at 2500 rpm for 10 minutes to remove cellular debris, and the supernatant is collected. Since urine contains PCR inhibitors, pretreatment with polyethylene glycol (PEG6000) is required: mix 50μl of urine supernatant with 50μl of 20% PEG6000 and 25μl of 2mol/L NaCl, then incubate in an ice bath for 6 hours. Centrifuge at 15,000 rpm for 30 minutes to collect the precipitate. Centrifuge again at 6400 rpm for 3 minutes; remove as much supernatant as possible and resuspend the precipitate in distilled water. This suspension can be directly used for PCR amplification, which involves heating at 100°C for 10 minutes followed by rapid cooling in an ice bath.

(2) Template DNA Preparation

1. Template DNA Preparation from Blood Specimens

   Method A: Add NaCl to serum to a final concentration of 150mmol/L; take 10μl of this serum, heat at 70°C for 45 seconds, and use directly for PCR amplification.

Method B: Preparation from buffy coats (BCP): (1) Wash BCP twice with PBS and collect the precipitate. (2) Add 500μl of 6mol/L guanidine hydrochloride and homogenize. (3) Add 30μl of 10mmol/L EDTA, 10mmol/L NaCl, 30μl of 20% Sarcosyl, and 5μl of proteinase K (10mg/ml), mix well, and incubate at 60°C for 1 hour. (4) Add 1130μl of ethanol and precipitate at -20°C for 1–2 hours. (5) Centrifuge to collect DNA precipitate. (6) Wash twice with 70% ethanol, then resuspend the precipitate in a small volume of 10mmol/L Tris-HCl, 1mmol/L EDTA. Store at 4°C for later use.

2. Preparation of Template DNA from Frozen Tissue Specimens: (1) Cut a 5–10 μm frozen tissue section using a cryostat and place it in a 1.5 ml plastic tube. (2) Add 10% buffered formalin. (3) After 10 minutes, centrifuge for 1–2 minutes, gently decant the supernatant, and wash the precipitate twice with ethanol. (4) Dry at room temperature for 10–60 minutes. (5) Add extraction buffer (100 mmol/L Tris-HCl, 4 mmol/L EDTA, pH 8.0, 400 μg/ml proteinase K) to just submerge the precipitate (approximately 50–100 μl); crush the precipitate. Formalin-fixed or paraffin-embedded tissues can also be processed similarly after dewaxing and drying. (6) Incubate at 37°C overnight. (7) Place in boiling water for 7 minutes to inactivate proteinase K. (8) Centrifuge to collect the supernatant, and take 1–10 μl for PCR amplification.

3. Preparation of Template DNA from Urine Samples: (1) Mix 100μl of urine supernatant with 100μl of 6mol/L guanidine isothiocyanate, 7μl of 2mol/L NaCl, and 20μl of glass powder suspension (DNA PREP, Asahi Glass Co, Tokyo). (2) After standing at room temperature for 10 minutes, centrifuge at 6400r/min for 2 minutes and collect the precipitate. (3) Wash the precipitate once with 50% ethanol, 10mmol/L Tris-HCl (pH 7.4), and 50mmol/L NaCl, then centrifuge at 6400r/min for 1 minute. (4) Repeat the washing step twice and collect the precipitate. (5) Add 50μl of distilled water and incubate at 55°C for 15 minutes. (6) Centrifuge at 15000r/min for 2 minutes and collect the supernatant (containing HCMV DNA) for PCR amplification. Heat this suspension at 100°C for 10 minutes and rapidly cool it in an ice bath.

(3) Primers and Probes

The design of primers and probes is based on the published sequences of the promoter region and the first four exons of the CMV immediate-early protein gene, the advanced-stage antigen gp64 gene, and the phosphoprotein pp71 gene. Commonly used primers and probes are listed in Table 3.

(4) PCR Amplification Steps

1. 10× Reaction Buffer: 100mmol/L Tris-HCl (pH 8.4), 500mmol/L KCl, 25mmol/L MgCl2, 2mg/ml gelatin.

   Taq DNA Polymerase: 5U/μl.

   10× dNTP: 2.0mmol/L each of the four dNTPs.

   Primers: 100pmol/L.

2. Conventional PCR Amplification

   10× Reaction Mixture (50μl): Reaction Buffer 5μl

   10× dNTP 5μl

   Template DNA 5μl

   Taq DNA Polymerase 0.2μl (IU)

   Primers 0.5μl each

   Distilled Water 3.8μl

   Add 1–2 drops of mineral oil.

   Heat the reaction mixture at 94°C for 5 minutes; then perform 35–40 cycles of 95°C for 30s, 55°C for 40s, and 72°C for 60s.

3. Nested PCR Amplification: (1) The reaction mixture composition is the same as (2), except the primers are IE2a and IE4b (covering the entire exon 3, 721bp), each at 100pmol. Perform 20 cycles of 94°C for 60s, 52°C for 150s, and 72°C for 480s. (2) Take 2μl of the above amplification product, add 100pmol each of primers IE3a and IE3b, and supplement with other reagents. Then perform 20 cycles of 94°C for 60s, 52°C for 150s, and 72°C for 180s.

(5) Product Identification

Analysis of amplification products can be performed using solid-phase (filter membrane) hybridization and liquid-phase hybridization assays.

1. Solid-Phase Hybridization:

   (1) The prehybridization solution consists of 3×SSPE, 5×Denhardt, 0.5% SDS, and 25% formamide. The filter membrane containing the amplified products is prehybridized at 42°C for 30–60 min. (2) Add the labeled probe (10 cpm/μg, 2 ng/ml) and hybridize for 30–60 min. (3) Wash the filter membrane three times at room temperature with 0.2×SSPE and 0.1% SDS, 5 min each time; wash once at 60°C, 10 min each time; then wash once more at room temperature, 5 min each time. (4) Autoradiography.

2. Liquid-phase hybridization and electrophoresis analysis:

   (1) Mix 1/10 volume of the amplification product with 0.5–1.0 pmol of end-labeled probe. (2) Add NaCl to a final concentration of 150 mmol/L, sodium phosphate to 10 mmol/L, and EDTA solution to 1 mmol/L, adjusting the total volume to 20 μl. (3) Incubate at 95°C for 10 min, then at 56°C for 60 min. (4) Centrifuge for 10 s, add sample buffer, and perform electrophoresis on an 8% polyacrylamide gel. (5) After electrophoresis, stain with ethidium bromide, expose to X-ray film, and perform autoradiography.

HCMV DNA is very large, and its nucleotide sequence has not yet been fully elucidated. Although its DNA exhibits polymorphism in restriction enzyme fragment lengths, the discreteness of its genome remains unclear. Using a single primer pair for PCR amplification can identify 90% of wild-type HCMV isolates, while employing two or more primer pairs targeting different HCMV sequences can detect nearly all viral strains. Therefore, depending on the situation, two or more primer pairs located in different regions of the genome can be used for PCR detection.

During the preparation of HCMV template DNA, small DNA fragments are sometimes generated, which can lead to a certain level of background products during PCR, affecting result interpretation. In such cases, nested PCR can be employed. The basic principle involves two-step amplification of the target DNA: first, a pair of primers is used to amplify a long DNA fragment including the target DNA; then, a small amount of the amplified product is taken, and a second amplification is performed using primers specific only to the target DNA. By controlling the number of amplification cycles in each step, false positives caused by small DNA fragments can be prevented. This method also avoids false-negative results.

PCR detection of HCMV specimens is highly sensitive. From the supernatant of HCMV-infected tissue cultures, DNA sequences equivalent to a few dozen viral particles or 1–5 PFU can be detected. Southern hybridization analysis of amplification products using non-radioactive oligonucleotide probes can detect HCMV DNA sequences at levels as low as 1 pg in urine samples. With this system, as few as one viral genome in 4×10^4 cells can be detected, which is 2×10^3 times more sensitive than dot blot hybridization.

PCR technology has clinical value for detecting HCMV infections because viral DNA in body fluids appears earlier than clinical symptoms or serological evidence of infection, serving as an early indicator of HCMV infection. Since HCMV can be transmitted through intraplacental infection, birth canal infection, and other routes, and infected newborns have a high mortality rate, early diagnosis using PCR and timely treatment are crucial for eugenics and prenatal care. This method can also determine whether donors in organ or tissue transplants are HCMV-positive, as HCMV is associated with many severe conditions. Furthermore, PCR provides stable detection indicators and allows semi-quantitative analysis, making it a useful tool for evaluating the efficacy of various antiviral drugs.

bubble_chart Treatment Measures

If primary cytomegalovirus infection is detected in the early stage of {|###|}pregnancy{|###|}, the {|###|}pregnancy{|###|} should be terminated as soon as possible. For those infected in the middle or {|###|}advanced stage{|###|} of {|###|}pregnancy{|###|}, further examinations should be conducted to check for fetal abnormalities, and corresponding treatment measures should be taken. For those with clinical symptoms or congenital cytomegalovirus disease, antiviral drugs such as cytarabine, vidarabine, and interferon can be used, but the efficacy still requires further observation.

For the treatment of cytomegalovirus infection, various antiviral agents such as GCV, anti-cytomegalovirus immunoglobulin preparations, interferon, and transfer factor can be used. However, these drugs do not address the root cause, and the virus often rebounds latently after discontinuation. Given that this virus may be one of the {|###|}disease causes{|###|} of {|###|}Acquired Immune Deficiency Syndrome{|###|}, scholars worldwide are dedicated to research on controlling its infection. Recently, American researchers have developed two live vaccines, which have shown promising results in initial trials. One is derived from the AD169 strain, and the other is made from the TOWn strain. After parenteral administration, both have demonstrated significant efficacy against cytomegalovirus, with elevated CMV antibodies leading to enhanced immune function.

bubble_chart Prevention

(1) Engage in conscious physical fitness exercises to enhance the body's immune function and disease resistance, especially for women of childbearing age, to reduce the severe harm of cytomegalovirus to the fetus. (2) Pregnant women or patients with chronic wasting diseases and weakened immunity should be protected and kept away from sources of pestilence. (3) Pay attention to environmental hygiene and food hygiene. (4) Those with cytomegalovirus-positive breast milk should not breastfeed. (5) Immunoprophylaxis is still under research and exploration.