Hepatitis B virus infection

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Hepatitis B virus infection

Yun-Fan Liaw, Chia-Ming Chu

Since the introduction of the hepatitis B vaccine and other preventive measures, the worldwide prevalence of

hepatitis B infection has fallen. However, chronic infection remains a challenging global health problem, with more

than 350 million people chronically infected and at risk of hepatic decompensation, cirrhosis, and hepatocellular

carcinoma. An improved understanding of hepatitis B virology, immunology, and the natural course of chronic

infection, has identi? ed hepatitis B virus replication as the key driver of immune-mediated liver injury and disease

progression. The approval of potent oral antiviral agents has revolutionised hepatitis B treatment since 1998.

Conventional and pegylated interferon alfa and nucleoside and nucleotide analogues are widely authorised treatments,

and monotherapy with these drugs greatly suppresses virus replication, reduces hepatitis activity, and halts disease

progression. However, hepatitis B virus is rarely eliminated, and drug resistance is a major drawback during long

term therapy. The development of new drugs and strategies is needed to improve treatment outcomes.

Introduction

Hepatitis B virus is one of the most serious and prevalent health problems, a?ecting more than 2 billion people worldwide. Although highly e?ective vaccines against hepatitis B virus have been available since 1982, there are still more than 350 million chronic carriers, 75% of whom reside in the Asia Paci?c region. People with hepatitis B are at increased risk of developing hepatic decompensation, cirrhosis, and hepatocellular carcinoma. The estimated worldwide mortality is 0·5 to 1·2 million deaths a year.1

Substantial improvement in the understanding of hepatitis B virology and immunology during past decades, combined with the advent of highly sensitive assays, has led to new insights into the natural history of such infection. Furthermore, the approval of oral antiviral agents has revolutionised hepatitis B treatment since 1998, and enabled e? ective clinical management of the disease. Viral epidemiology

Hepatitis B virus is a double-stranded DNA virus of the hepadnaviridae family. The virus is enveloped, and contains a viral DNA genome of about 3200 bps within its core. After the virus enters a hepatocyte, the viral genome is delivered to the nucleus, and the relaxed circular DNA is converted to covalently-closed-circular DNA (cccDNA). The cccDNA serves as a template for the transcription of the viral RNA. The hepatitis B virus replication cycle includes reverse transcription of RNA intermediates to prime DNA synthesis and translation of the hepatitis B proteins, including hepatitis B surface antigen (HBsAg) and e antigen (HBeAg).2 Thus, cccDNA plays a key part in the maintenance of chronic hepatitis B infection. The virus has at least eight major genotypes (A to H), based on an intergroup divergence of more than 8% in the complete nucleotide sequence. Apart from genotypes E and G, the genotypes have sub-genotypes with a sequence di? erence of at least 4%.3

Hepatitis B virus is transmitted parenterally via apparent or inapparent percutaneous or permucosal exposure to infected blood or other body ? uids. Risk factors for infection include transfusion of unscreened blood, sexual promiscuity, sharing or re-using of syringes between injection drug users, tattooing, working or residing in a health-care setting, living in a correctional facility, renal dialysis, and long-term household or intimate non-sexual contact with an HBsAg-positive individual.1,4

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n low-prevalence areas, hepatitis B is typically a disease of young adults who acquire the infection through risky behaviour—such as unprotected sexual contact or sharing syringes with HBsAg-positive people—and through exposure to contaminated equipment used for therapeutic injections and procedures. I n high-prevalence regions, most infection occurs perinatally or during early childhood. About 90% of HBeAg-seropositive mothers (with high viral load) transmit hepatitis B virus to their o? spring, compared with 10–20% of HBeAg-seronegative carrier mothers.5 The prevalence of HBeAg is higher in Asian than in African HBsAg carrier mothers (40 vs 15%), so perinatal transmission is greater in Asians, but mainly horizontal in Africans.6,7

The prevalence of chronic hepatitis B infection is about 5% worldwide, but di?ers between regions. nfection rates are low (0·1–2·0%) in the USA and western Europe, intermediate (2·0–8·0%) in Mediterranean countries and Japan, and high (8·0–20·0%) in southeast Asia and sub-Saharan regions.1 Additionally, hepatitis B virus genotypes have a distinct

Lancet 2009; 373: 582–92

Liver Research Unit, Chang

Gung Memorial Hospital,

Chang Gung University College

of Medicine, Taipei, Taiwan

(Prof Y-F Liaw MD,

Prof C-M Chu MD)

Correspondence to:

Prof Yun-Fan Liaw, Liver

Research Unit, Chang Gung

University and Memorial

Hospital, 199 Tung Hwa North

Road, Taipei, Taiwan

livery? @https://www.wendangku.net/doc/0d17560782.html,.tw

Search strategy and selection criteria

Medline and PubMed were searched from 2000 to 2008 using

the terms “hepatitis B virus”, “HBV”, “viral hepatitis”, and

“chronic hepatitis” as search terms for hepatitis B virology,

immunology, epidemiology, and clinical liver disease status.

Reference lists of the identi? ed articles and review articles

from the past 3 years were also searched. Publications from

the past 3–5 years were selected. Earlier publications were not

excluded, however, if they were highly cited or if there were no

recent appropriate publications. Review articles covering

earlier studies on important issues were selected and cited

rather than the earlier original articles.

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geographical distribution: genotype A is prevalent in northwestern Europe and the USA; genotypes B and C in Asia; genotype D in the Mediterranean basin, Middle East, and India; genotype E in west Africa; genotype F in South and Central America; genotype G in the USA and France; and genotype H in Mexico and South America.3

Hepatitis B virus pre-core mutations occur most frequently in genotype D, followed by genotypes C and B, and are seen least frequently in genotype A.8 Accordingly, HBeAg-negative chronic hepatitis B is most common in genotype D dominant regions. The percentage of patients with chronic hepatitis B who are HBeAg negative is 80–90% in Mediterranean areas, 30–50% in southeast Asia, and less than 10% in the USA and northwestern Europe.9

Prevention

Through understanding the routes and modes of hepatitis B transmission, infection can be prevented by avoidance or interruption of transmission. Since the 1970s, serological screening of donor blood has become progressively routine, resulting in substantial reduction of transfusion-associated hepatitis B.1 Syringe-exchange programmes are run in the USA and other high-income countries, and provide free sterile syringes in exchange for used syringes, reducing transmission of blood-borne pathogens—including hepatitis B—in injection drug users. Operating at ? xed sites and on mobile van routes, the syringe-exchange programmes can make contact with otherwise hard-to-reach populations to deliver social and medical services, such as testing for hepatitis B, counselling, and vaccination.10

An e? ective hepatitis B vaccine has been available since the early 1980s, and in the early 1990s WHO recommended the addition of the hepatitis B vaccination to all national immunisation programmes. Universal vaccination programmes for newborn babies have been implemented in more than 160 countries, and international ? nancial support and a reduced cost are facilitating introduction of the vaccine into more low-income countries.11 Some high-income countries with low or very low endemicity of hepatitis B infection, such as northern European countries, apply a strategy of selective vaccination for individuals at high risk of infection, because the low burden of disease does not warrant the added cost of universal vaccination.11,12 Hepatitis B vaccination is the most e? ective preventive measure in adult populations with risk factors.1,4,10,11

Since the introduction of hepatitis B vaccination, the worldwide rates of infection have fallen. For example, the HBsAg carrier rate in Taiwanese children decreased from 10% in 1984 to less than 1% in 2004, with a 68% reduction of fulminant hepatitis in infants (0–1 year), and a 75% decline in hepatocellular carcinoma in children (aged 6–14 years).13,14 In the USA the incidence of reported acute hepatitis B fell by 81% between 1990 and 2006, from 8·5 to 1·6 cases per 100 000 population, and few cases occurred in blood recipients, dialysis patients, and health-care workers.4 However, the rate of infection among injection drug users, people with sexual risk factors, and immigrants from high-prevalence areas such as Asia has raised the burden of chronic hepatitis B infection in high-income countries, reinforcing the need to improve preventive e?orts aimed at high-risk groups.4,14 Post-exposure prophylaxis with hepatitis B immunoglobulin is e? ective and indicated for newborn infants of HBsAg-positive mothers, after percutaneous or mucosal exposure to HBsAg positive blood in health-care settings.

I mmunoglobulin is further indicated for prophylaxis after exposure to body ? uids (sexual exposure) and for the prevention of hepatitis B recurrence after liver transplantation.11

Pathophysiology

Hepatitis B virus is not cytopathogenic. In acute infection, clinical hepatitis B becomes apparent after an incubation period of 45–180 days. The elimination of hepatitis B virus by non-cytopathic mechanisms begins several weeks before the disease onset. Hepatitis B virus DNA clearance is mediated largely (up to 90%) by antiviral cytokines that are produced by cells of the innate and adaptive immune responses—including tumour necrosis factor α, interferon alfa, or interferon beta.15–17 After viral DNA declines, a cytolytic immune response with hepatocyte apoptosis and necrosis ensues, coincident with the onset of clinical hepatitis and a rise in serum alanine aminotransferase (ALT). The recognition of infected hepatocytes by virus-speci? c CD8 cytotoxic T cells, via class

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human lymphocyte antigen (HLA-

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)-presenting hepatitis B virus peptides, is presumed to be the main mechanism causing both liver damage and virus control. Cytotoxic T cells further recruit various antigen-non-speci? c in? ammatory cells into the liver by secreting cytokines, initiating a cascade of immunological events leading to necro i n? ammation.18–20

A vigorous, multispeci?c CD4 and CD8 response is associated with viral clearance.21n individuals with chronic hepatitis

B infection, the hepatitis B virus-speci? c CD4 and CD8 response is insu? cient;21 and can cause a persistent in? ammatory response that is ine? ective for hepatitis B virus clearance.22

Timely conventional liver biochemical tests are essential for diagnosis of hepatitis—including measurement of ALT for the degree of hepatocellular damages, bilirubin for conjugation and excretion function, and albumin values and prothrombin time for liver synthesis function. Liver biopsy is important for the grading of necroin?ammation and staging of ? brosis. Ultrasonography and other imagining methods are non-invasive ways to detect cirrhosis and hepatocellular carcinoma23—the judicious use of these methods and assays is crucial to di? erentiate the nature and severity of

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disease. Testing for hepatitis B virus markers is mandatory for the detection and diagnosis of hepatitis B. Serological markers of hepatitis B are HBsAg and its antibody (antiHBs), HBeAg and its antibody (antiHBe), and immunoglobulins G and M antibody to hepatitis B virus core antigen (I gG antiHBc and I gM antiHBc). HBsAg seropositivity indicates infection, and HBeAg is a surrogate marker of viral replication with high hepatitis B virus DNA. HBsAg assays should be done in those at risk for hepatitis B infection, people with ALT elevation or evidence of liver disease, and anyone about to receive immunosuppressive treatment or chemotherapy.

The ?rst serological marker to appear in acute hepatitis B infection is HBsAg, usually 1–6 weeks before the manifestation of clinical symptoms. I gM antiHBc appears 1–2 weeks after HBsAg, and persists for up to 6 months after HBsAg is cleared.23 Previously undiagnosed chronic HBsAg carriers with acute exacerbation of hepatitis B or superinfection with other hepatitis virus(es) presenting as clinical acute hepatitis, are seronegative for IgM antiHBc.24 Serum IgM antiHBc assay is, therefore, mandatory for the serodiagnosis of acute hepatitis B. HBeAg and hepatitis B virus DNA are present early during acute infection. Both markers usually disappear when serum ALT peaks, or soon thereafter, and before HBsAg seroclearance, which occurs within 1–2 months, and are followed by the appearance of antiHBs several weeks later (? gure 1). Previous infection is diagnosed by the detection of antiHBc and antiHBs.23

Presence of HBeAg for more than 10 weeks indicates a high likelihood of transition to persistent infection. Persistence of serum HBsAg for more than 6 months implies progression to chronic infection. People with HBeAg-positive chronic infection usually have high levels of hepatitis B virus DNA, whereas serum concentrations are lower in patients with HBeAg-negative infection. Serial testing showing a hepatitis B virus DNA concentration of less than 2×103 IU/mL and normal ALT values is needed to verify an inactive carrier state.25,26 Serum hepatitis B virus DNA assay is a direct measure of viral load. I t is particularly useful for assessment of risk of disease progression and candidacy for antiviral therapy, monitoring treatment response, and to distinguish active hepatitis B from the inactive carrier state with other causes of high ALT. PCR-based assays, with high sensitivity and a wide dynamic range (101–10? I U/mL), are the mainstay for measurement of hepatitis B viral load. WHO has established an international standard for hepatitis B virus quanti? cation units, in which 1 I U is equal to about ? ve genome equivalents.27 Assays for hepatitis B virus genotypes and mutations are available and becoming increasingly important in the clinical ? eld.

Natural history

The spectrum of acute hepatitis B infection ranges from asymptomatic infection to self-limited hepatitis, to fulminant hepatitis and it depends on various viral and host factors. Symptomatic hepatitis is rare in neonates (less than 1%) and occurs in about 10% of children 1–5 years old.1,22 Fulminant hepatitis is very rare in paediatric patients, with most reported cases being in infants born to HBeAg-negative, HBsAg-carrier mothers.28 One proposed explanation is that the absence of HBeAg in maternal blood fails to induce immunological tolerance,29 thus allowing vigorous immune clearance of hepatitis B virus from the infant liver. A third of acute infections in adults are symptomatic,22 and fulminant hepatitis occurs in less than 1% of cases, with a mortality of about 70%. Fulminant hepatitis B is related to an enhanced immune response with rapid viral clearance, which means serum HBsAg and hepatitis B virus DNA might be undetectable at the time of clinical presentation, and the diagnosis is made only by the presence of serum IgM antiHBc.2

Resolution of hepatitis B with HBsAg seroclearance occurs in more than 95% of adult patients. However, small amounts of hepatitis B virus DNA can still be detected by PCR in serum and peripheral mononuclear cells years after recovery from hepatitis, indicating a state of occult infection.30 Hepatitis B can be transmitted via organ transplantation, and hepatitis B reactivation might occur under immunosuppressive treatment—or by chemotherapy in such cases.31 The risk of chronicity is correlated closely with the patient’s age at the time of infection. I nfection persists in about 90% of infants infected at birth, 20–30% of children infected between the ages of 1 and 5 years, 6% of infection in children

Figure 1: Serological and clinical changes after acute hepatitis B virus (HBV) infection

Shaded bars indicate the duration of seropositivity in self-limited acute hepatitis B infection. Pointed bars indicate that HBV-DNA and HbeAg can become seronegative during chronic infection. Only IgG antiHBc is detectable after resolution of acute hepatitis or during chronic infection. Y axis is schematic concentration of ALT and antiHBs.

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aged 5–15 years and only 1–5% of patients infected as adults.1

One possible explanation for the high chronicity in infants is that the fetus develops tolerance to the virus in utero after the transplacental passage of viral proteins.32The natural course of chronic hepatitis B infection consists of distinct phases resulting from the interaction between the virus, hepatocytes and host immune response. Typically, chronic infection acquired perinatally or during infancy has three phases: immune tolerant, immune clearance, and inactive residual.33 Hepatitis B re-activation can occur in some patients with inactive disease and trigger immune-mediated liver injury.34,35 Such patients, therefore, enter a variant phase of immune clearance (? gure 2).23,36 Adult-acquired chronic infection has a similar clinical course, except that there is no obvious initial immune tolerant phase.37The mechanisms of immune tolerance during the ? rst phase are not well understood. Work in mice suggests that a transplacental transfer of maternal HBeAg could induce a speci? c unresponsiveness of helper T cells to HBeAg, and result in an ine? ective cytotoxic T-cell response to nucleocapsid antigens in neonates.32 Patients in the immune-tolerant phase are usually young, asymptomatic, and HBeAg seropositive, with high viral loads (more than 2×10? to 2×10? IU/mL) but normal serum ALT levels and near-normal liver histology. There is usually no, or only minimum, disease progression while serum ALT concentrations remain normal.38The mechanisms triggering loss of immune tolerance are mostly unknown, but a high viral load seems necessary to maintain the tolerant state.39 The immune-clearance phase is associated with falling

serum hepatitis B virus DNA concentrations, but their causal relation remains unclear. Other ? ndings that

characterise the transition from the immune tolerant to the immune clearance phase include a shift of

antigen (HBcAg) from nucleus to cytoplasm,40 and a

gradual accumulation of pre-core or core-promoter mutations.41,42

During the immune-clearance phase, hepatitis activity and intermittent acute ALT increase can ? are to over ? ve times the upper limit of normal, usually without

apparent symptoms. This activity of ? ares can be complicated by hepatic decompensation. The rises in

ALT and ? ares are attributable to the host’s immune

response against hepatitis B virus with resultant apoptosis and necrosis, thus, higher ALT concentrations

usually indicate a more vigorous immune response against hepatitis B virus and more extensive hepatocyte damage.43 The occurrence of hepatitis ? are varies in patient cohorts, but has reached 25% of patients per year during the ? rst 3–5 years of follow-up in hospital-based studies 43–45 The overall occurrence of hepatic failure in patients was estimated to be 0·5%.24 Hepatitis ? ares can lead to cirrhosis but will eventually

be followed by HBeAg seroconversion to antiHBe. The estimated yearly proportion of spontaneous HBeAg seroconversion is 2–15%, depending on factors such as age, ALT concentrations, and hepatitis B virus genotype.41–46 In Asia, HBeAg seroconversion occurs at a mean age of 30–35 years, with most cases (90%) occurring before age 40.45–48 Patients infected with genotype B seroconvert earlier and more frequently than do those with genotype C.3,47,49–51 In native Alaskans with chronic hepatitis B infection, the median age of HBeAg seroclearance is younger than 20 years in patients with genotypes A, B, D, and F, but over 40 years in patients with genotype C.52HBeAg seroconversion is usually followed by clinical remission and a life-long inactive state with an excellent outcome, although a few patients may develop hepatocellular carcinoma.34,35,53–56 Spontaneous HBsAg seroclearance can occur, reported at a rate of less than 1% per year in early studies.53–57 A study with 1965 patients showed that HBsAg seroclearance occurred at a rate of 1·2% per year, and rose to 1·8% per year in individuals over age 50; to a 45% cumulative incidence after 25 years of follow-up.58 Patients with virus genotype A and B infection have a higher likelihood of HBsAg seroclearance than do patients with other genotypes.59,60 HBsAg seroclearance usually confers an excellent long-term outcome if there is no pre-existing cirrhosis or viral superinfection.61,62 However, a small amount of hepatitis B virus DNA may persist in a state of occult infection.31Figure 2: Natural course of chronic hepatitis B virus (HBV) infection acquired perinatally and during infancy The reactivion phase is similar in every aspect to the immune-clearance-phase, except for HBeAg status.

Adult-acquired infection usually presents in the immune-clearance or reactivation phase (inset). The events during

the immune-clearance and reactivation phases could lead to cirrhosis and hepatocellular carcinoma (HCC) (adapted from Liaw 21

with permission). HBeAg=hepatitis B e antigen; antiHBe=hepatitis B e antigen antibody; pre C=pre core; BCP=basal core promoter; ALT=serum alanine aminotransferase.

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After HBeAg seroconversion, 1–4% patients have HBeAg seropositive hepatitis again (HBeAg reversion), whereas a greater proportion of patients develop HBeAg-negative chronic hepatitis B because of reactivation of the hepatitis B virus with pre-core or core promoter mutations that abolish or downregulate HBeAg production.34,35,42,63 Results from Taiwanese studies showed relapse in 2–3% of patients per year,34,45 with a cumulative rate of 20–25% after 15 years of follow-up.64 The proportion of relapse was very low in patients who HBeAg-seroconverted before age 30,55,64 but was raised in men, patients with genotype C, those who HBeAg-seroconverted after age 40,65 and in patients with serum hepatitis B virus DNA concentrations greater than 10 000 copies per mL.66 These age-related ? ndings show that early HBeAg seroconversion, or a short HBeAg-positive phase, are associated with an improved chance of sustained remission.48

Cirrhosis or hepatocellular carcinoma, or both, can develop during the natural course of chronic hepatitis B infection. Results of large population-based studies with mostly (85%) HBeAg-negative, HBsAg-positive people older than 30 years at recruitment have shown that the risk of cirrhosis, hepatocellular carcinoma, and mortality increases proportionally with increasing viral DNA concentrations, starting with at least 1×104 copies per mL.67–69 The study ? ndings suggest hepatitis B virus replication, with subsequent immune-mediated liver injuries, is the main driver of disease progression70 (? gure 3). Further risk factors for the development of cirrhosis include: male sex; increasing age;67–69,71,72 HBeAg positivity;72 virus genotype C (vs B);47,49,73–76 HBeAg reversion or virus reactivation;34,47persistent seropositivity for HBeAg77 or viral DNA;78 persistent raised ALT;79 viral superinfections;80 as well as the severity (hepatic decompensation), extent (bridging hepatic necrosis), and frequency of hepatitis ? are, and the duration of hepatic lobular alterations.71

At least a third of patients with cirrhosis are seropositive

for HBeAg or hepatitis B virus DNA at presentation,81 and disease progression can continue after cirrhosis development.82 The 5-year probability of hepatic decompensation is 15–20%, and is four-fold higher in patients with active viral replication than in patients without.83 The yearly rate of hepatocellular carcinoma occurrence is 3–6%.81,82 The estimated 5-year survival rate

of patients with compensated cirrhosis is 80–85% and 30–50% in patients with decompensated cirrhosis.81 Hepatocellular carcinoma mostly develops in patients with cirrhosis, therefore, hepatocellular carcinoma and cirrhosis share the same risk factors, with a raised risk in patients with a family history of hepatocellular carcinoma.84 Viral factors also contribute to hepatocellular carcinoma development, including hepatitis B virus DNA level, genotypes, and naturally occurring mutations such as hepatitis B virus pre-S and basal core promoter A1762T/

G1764A double mutations.68,85–89 Other con t ributing factors are habitual alcohol consumption, cigarette smoking, and

a? atoxin exposure.90

Management

Acute hepatitis B in adults is sel?imiting in more than 95% of cases, therefore, antiviral therapy is indicated only for patients with protracted severe acute hepatitis or fulminant hepatitis B.25 Management of patients with chronic hepatitis B infection should include thorough patient assessment and counselling. Although patients are usually asymptomatic, they can be anxious and attribute a wide range of negative psychological, social, and physical symptoms to their condition.91 Counselling should include dietary and lifestyle advice, including guidance to increase physical activity and control alcohol use. Further, health-care providers and patients should discuss the behaviours that lead to superinfection, prevention of such infection and preventive measures against transmission of hepatitis B to intimate contacts. The importance of the long term hepatocellular carcinoma surveillance, with ultrasonography supplemented with α-fetoprotein assay, should be emphasised to patients older than 40 years, with advanced

?brosis or cirrhosis, and with a family history of hepatocellular carcinoma.25,36,81

Hepatitis B virus replication is key to liver injury and disease progression,70 and, therefore, the main aims of treatment are to suppress the virus to achieve HBeAg seroconversion or undetectable viral-DNA levels, or both; stop or reduce hepatic necroin? ammation; and prevent the development of hepatic decompensation. Long term goals are to reduce cirrhosis and hepatocellular carcinoma development, and ultimately extend survival. Viraemic

Figure 3: Hepatitis B virus (HBV) replication and the outcomes of chronic Hepatitis B infection

Note that Hepatitis B e antigen (HBeAg) seroconversion is followed by remission in most people but that HBeAg-negative (–) hepatitis (HBV-DNA ≥ 2x103–2x10? IU/mL) may develop. Patients who remain HBeAg seropositive or develop HBeAg-negative hepatitis have a high occurrence of cirrhosis (~4 and ~3% per year). Most hepatocellular carcinoma (HCC) develops in patients who have cirrhosis.

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patients with an ALT concentration of twice the upper limit of normal or more, or substantial liver disease, are candidates for drug therapy. Of note, ALT ? are may precede spontaneous HBeAg seroconversion.43 Obser-vation for 3 months before considering drug therapy is acceptable.25,36 Liver biopsy is recommended to assess the necroin?ammation grade and ?brosis stage, because signi? cant ? brosis could have developed in patients with normal ALT, except during the immunotolerant phase.92 Before drug therapy is initiated, the e? cacy, advantages and disadvantages, and cost of available therapies should be discussed with the patient.36

Approved and widely used agents are conventional interferon alfa and pegylated interferon-alfa-2a; the nucleoside analogues lamivudine, entecavir, and telbivudine; and the nucleotide analogue adefovir dipivoxil. Tenofovir, another nucleotide analogue approved for the treatment of HIV infection, was more e?ective than adefovir in hepatitis B,93 it has been approved in European countries and USA, and will probably be approved worldwide. Drug therapy should be selected according to the patient’s condition, the drug’s mechanism, rapidity of action, potency, convenience of administration, adverse e? ect pro?le, and cost (see table).

Interferon-based therapy

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nterferon alfa and pegylated interferon have immunomodulating activity. Theoretically, interferons are the ideal treatment for patients with chronic hepatitis B, but the response rate after 4–6 months of interferon alfa is only 30–40% in HBeAg-positive patients, with a risk di?erence of 23–25% against untreated controls.94 48 weeks of pegylated interferon therapy yields a sustained HBeAg seroconversion rate of 32% when assessed 24 weeks after completion of therapy.95–97 Patients with genotype A (vs D) or B (vs C) infection tend to have a better response to interferon or pegylated interferon.3,95–97 HBeAg seroconversion response

is sustained in more than 80% of people, and can be followed by HBsAg loss,77,98,99 which is the desired end point.

Patients with HBeAg-negative chronic hepatitis B respond to interferon alfa therapy but often relapse after treatment completion.78,100 The combined response to pegylated interferon therapy (normal ALT+hepatitis B virus DNA concentration less than 2×10? copies per mL)

in HBeAg-negative patients is 36% at 6 months post treatment.101,102 I mproved response rates are achieved in patients with low serum viral DNA, raised ALT and low HBeAg concentrations at baseline, and low HBeAg after 24 weeks’ treatment.102–104 HBsAg seroconversion occurs in 3% of the pegylated interferon treated patients,96,97,101 and the rate increases to 10% at 4 years after therapy.105 These response ?gures are better than those of lamivudine monotherapy. Results from follow-up studies suggest that interferon alfa therapy has long term bene? ts by promoting cumulative HBeAg seroconversion, increasing HBsAg loss, reducing development of cirrhosis and hepatocellular carcinoma, and extending survival—especially in responders.70,77,98–100 Further advantages of interferon-based therapy are the ? nite treatment duration and that patients do not develop drug resistance. Eventually, pegylated interferon treatment will replace interferon alfa because of its improved e?ectiveness and convenient once a week administration.36,95 Interferon-based therapy is associated with many adverse events; including in? uenza-like symptoms, fatigue, anorexia, weight loss, hair loss, thyroid dysfunction, emotional instability, and bone

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marrow suppression. Patients might need symptomatic treatment, and some require dose modi? cation or discontinuation of therapy.95 Most patients are able to complete 48 weeks of pegylated interferon treatment despite the side-e? ects.96,97,101

Nucleoside or nucleotide analogues

Oral antiviral agents have fast and potent inhibitory e?ects on hepatitis B virus polymerase and reverse transcriptase activity, and are safe and e? ective for hepatitis B virus DNA suppression, ALT normalisation, and histological improvement. Results from cross-trial 1 year treatment data suggest entecavir is the most potent of the drugs, followed by tenofovir, telbivudine, lamivudine, and adefovir. However, the antiviral potency of these drugs does not result in an increase in HBeAg seroconversion—which was seen at a rate of around 20% after 1 year of treatment (see table)—and HBsAg loss is very rare.106–110 After 1 year of lamivudine treatment an HBeAg seroconversion was achieved in more than half of patients with ALT values more than ? ve times the upper limit of normal, but in only 5% of patients with ALT values less than twice the upper limit of normal.111,112 Thus, patients with a stronger endogenous immune response to the hepatitis B virus might have an improved chance of reducing cccDNA and HBeAg translation.111 Additionally, patients with ALT values more than ? ve times the upper limit of normal could be developing severe hepatitis or hepatic decompensation, especially if they have advanced ? brosis.43 These patients should be monitored every week or every two weeks so that fast-acting antiviral drugs can be started to arrest the development, or deterioration, of hepatic de-compensation.113 Similarly, nucleoside and nucleotide analogues might rescue patients with decompensated cirrhosis by increasing serum albumin concentrations, and stabilising bilirubin values and prothrombin time, removing the need for transplantation and prolonging survival.114,115

20–50% of patients who have HBeAg seroconversion will relapse with HBeAg reversion within 6–12 months o? therapy, and more patients may relapse if the duration of consolidation therapy after HBeAg seroconversion is less than 6 months.25,36

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n HBeAg-negative patients, there is no end point such as HBeAg seroconversion, so treatment duration is inde?nite, and stopping drug therapy after 1 year is associated with a 90% relapse rate.116 Relapses are characterised by increasing hepatitis B virus DNA level followed by rising ALT or a hepatitis ? are which could be life threatening.117 Therefore, continuing long-term drug therapy is usually necessary to maintain a virological response. Results of a large randomised controlled trial in patients with advanced ? brosis or cirrhosis showed the bene? ts of long-term therapy after 3 years of lamivudine therapy were reduced dis-ease progression and hepatocellular carcinoma development.118

The emergence of drug-resistant genotypic mutations of hepatitis B virus in long term therapy is a major problem. Clinically, emergence of drug resistance is indicated by viral breakthrough—an increase of serum hepatitis B virus DNA to more than ten-fold increase in HBV concentrations from the nadir of initial response—with subsequent biochemical breakthrough or raised ALT values in more than 90% of patients.119 Hepatitis ?ares develop frequently, sometimes associated with hepatic decompensation during continued antiviral therapy,120 and, the initial clinical and histological bene?ts of antiviral therapy diminish.118,121,122

The occurrence of drug resistance rises with increasing therapy duration, although the generation of nucleoside and nucleotide analogues has improved potency and raised the genetic barrier to resistant mutations (see table). Drug resistance occurs most frequently with lamivudine, followed by telbivudine, adefovir and tenofovir, and is very low with entecavir.106–110

Figure 4: Management of patients with chronic hepatitis B virus (HBV) infection

HBeAg=hepatitis B e antigen. ULN=upper limit of normal. NA=nucleoside or nucleotide analogues. IFN=interferon-based therapy. HCC=hepatocellular carcinoma.

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Studies have shown that suppression of the hepatitis B virus to undetectable level at 24 weeks of telbivudine and lamivudine therapy is associated with low incidence of drug resistance at week 48.110 Rescue therapy for drug resistance is now available and, therefore, monitoring hepatitis B virus DNA levels during drug treatment to

detect drug resistance early enough to start rescue

therapy before serum hepatitis B virus DNA increases over 1×10? copies per mL 123,124 is very important. I n patients with drug resistance, add-on therapy without cross-resistance (adefovir and tenofovir vs lamivudine, telbivudine and entecavir) should be instituted.123,125 Switching to interferon-based therapy can be a further option.126

Drug treatment strategies

I n view of the array of drugs available, physician and patient should decide which drug should be used, and when to start and stop treatment. Oral antiviral agents with fast and potent hepatitis B virus-suppressive e? ects are preferred for patients with ensuing or overt hepatic decompensation. Interferon-based therapy is preferred in patients with compensated liver disease—particularly in young patients, women of childbearing age, and those with low ALT values—because of the ? nite

duration of treatment, sustained response, and long

term bene? ts including prevention of hepatocellular carcinoma.36,127

For oral antiviral agents, the bene? t of long-term therapy should be weighed against possible

drug resistance and durability of treatment response. If long term therapy is anticipated, the drug with the lowest rate of resistance is preferable, although cost

may a? ect choice. A combination of two or more antiviral drugs, as in H I V therapy, reduces drug resistance but does not enhance e? cacy.96,101,128,129 Figure 4

shows an algorithm for the management of patients

with chronic hepatitis B, designed on the basis of published guidelines.

25,26Drug therapy is not usually recommended for children and women of childbearing age—unless there is an absolute indication such as ensuing or overt hepatic decompensation—because of potential problems

associated with long-term therapy.36

When treatment is absolutely indicated, interferon-based therapy is preferred for non-pregnant women, and pregnancy is discouraged during treatment. Women who become pregnant while receiving oral antiviral agents can continue treatment with telbivudine or tenofovir, which are category B agents.36,130

I n patients with concurrent hepatitis C or D virus

infection, the dominant virus should be determined and treated accordingly. I n patients with HI V coinfection,

adefovir or interferon (if CD4 is greater than 500) is preferred if HI V infection does not need treatment. I f HI V treatment is required, tenofovir or combination lamivudine and tenofovir should be included in the active

antiretroviral therapy.25,36

Hepatitis B virus re-activation is a serious complication in patients undergoing transplantation, immuno-suppression, or chemotherapy. Patients should be screened for HBsAg and, if positive, prophylactic therapy with a direct antiviral agent should be started before the beginning of and at least 12 weeks after the end of the immunosuppressive treatment or chemotherapy.36,131 For all patients with hepatitis B virus-associated liver failure who are listed for transplantation, nucleoside or nucleotide analogues should be started and continued after transplantation as prophylaxis against re-infection of the allograft.25,36

Con? ict of interest statement

Yun-Fan Liaw has been involved in clinical trials or served as a global advisory board member of Roche, BMS, GSK, Novartis, Gilead Sciences. Chia-Ming Chu declares that he has no con? ict of interest.

Acknowledgments

We thank Chang-Gung Medical Research Fund and the Prosperous

Foundation, Taipei, Taiwan, for long term grant support; Ellen Donaldson and Eric Matluck for English editing; and Su-Chiung Chu for secretarial assistance.References

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XX公司 蠕虫和病毒传播处置预案 2019年12月

文档控制 更改记录

一、总则 第一条目的 本预案为蠕虫和病毒攻击和传播的安全事件处理专项预案，其目的主要是为了进一步规范对蠕虫和病毒攻击和传播安全事件的处理方法和处理程序，提高对此类安全事件的反应速度。 第二条基本原则 1．防范为主，加强监控。通过加强信息安全防范意识，提高网络系统的安全性。完善信息安全事件的日常监测、发现机制，及时采取有效的应对措施，迅速控制事件影响范围，力争将损失降到最低程度，从而缓解或抵御病毒爆发事件的安全威胁。 2．以人为本，协同作战。把保障公共利益以及本单位和其他组织的合法权益的安全作为首要任务。相关部门协同配合、具体实施，及时获取充分而准确的信息。通过跟踪研判，果断决策，迅速处置，以最大程度地减少危害和影响。 3．规范操作，常备不懈。加强防病毒技术储备，规范应急处置措施与操作流程，确保应急预案切实有效，实现信息安全突发事件应急处置的科学化、程序化与规范化。 第三条适用范围 本预案适用于本单位中遇到病毒攻击情况下的应急响应工作。

二、术语与定义 第四条计算机病毒 计算机病毒（Computer Virus）是编制者在计算机程序中插入的破坏计算机功能或者数据的代码，能影响计算机使用，能自我复制的一组计算机指令或者程序代码。计算机病毒有独特的复制能力，能够快速蔓延，并难以根除。 第五条蠕虫病毒 蠕虫病毒（worm）和一般的病毒有着很大的区别。对于蠕虫，现在还没有一个成套的理论体系。一般认为：蠕虫是一种通过网络传播的恶性病毒，它具有病毒的一些共性，如传播性、隐蔽性、破坏性等等，同时具有自己的一些特征，如不利用文件寄生（有的只存在于内存中），对网络造成拒绝服务，以及和黑客技术相结合等。在产生的破坏性上，蠕虫病毒也不是普通病毒所能比拟的，网络快速发展使得蠕虫可以在短短的时间内蔓延整个网络，造成整个网络瘫痪！ 三、病毒分析阶段 第六条事件分析 （1）使用netstat –ano命令查看操作系统是否存在异常连接。 （2）查看windows、recycle目录下是否存在异常文件。 （3）通过Pchunter等进程查看工具，查看是否存在异常进程在运行，定位至程序存放目录。 （4）分析病毒传播机制，如通过文件共享传播、通过邮件或文

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3、提高office软件自身对宏的审核与限制。如：Word菜单\工具\宏\安全性\有两个选项：安全级应为“中及其以上”， 4、可靠发行商应不信任对VB项目的访问。 5、提高office软件自身对宏的审核与限制。 6、对于已染毒的模板文件（Normal.dot），应先将其中的自动宏清除，然后将其置成只读方式。 7、对于其他已染毒的文件均应将自动宏清除。 8、对于在另存菜单中只能以模板方式存盘.或者无法使用“另存为（Save As）”，或不能再被转存为其它格式的文件。 9、平时使用时要加强预防。对来历不明的宏最好删除。 10、安装反病毒软件。 脚本病毒是用脚本语言（如VBscript、Javascript、PHP等）编写而成的恶意代码。 脚本语言的特点： 1.语法结构简单。 2.学习使用容易。 3.以解释方式执行。 4.开发较快，广泛使用。 5.执行较慢。 一般防范： 1.卸载VB脚本依赖的WSH。 2.增强IE安全设置。

肿瘤的免疫逃避

肿瘤的免疫逃避 正常机体每天有许多细胞可能发生突变，并产生有恶性表型的瘤细胞，但一般都不会发生肿瘤。对此，Burner提出了免疫监视学说，认为机体免疫系统通过细胞免疫机制能识别并特异地杀伤突变细胞，使突变细胞在未形成肿瘤之前即被清除。但当机体免疫监视功能不能清除突变细胞时，则可形成肿瘤。 然而，尽管机体有如此多样的抗肿瘤免疫效应，但肿瘤仍可在体内发生、发展，并且随着肿瘤的增长，肿瘤细胞能产生一些细胞因子。这些因子可以诱导机体产生一些抑制免疫的细胞，并诱导机体产生体液抑制因子，从而抑制抗肿瘤的免疫功能。肿瘤细胞自身也可以分泌一些具有免疫抑制作用的产物，侵犯局部转移的淋巴结，导致机体局部乃至全身的免疫功能低下。提示肿瘤具有一系列的免疫逃避机制来对抗机体的免疫系统。 1、肿瘤抗原调变作用 在肿瘤免疫过程中，T细胞的免疫功能起着关键性的作用。可溶性肿瘤抗原经抗原提呈细胞（APC）摄入后经加工处理，然后将处理后的抗原提呈给辅助性T细胞，后者通过分泌细胞因子促进杀伤性T细胞增殖，并产生特异杀伤作用。然而许多肿瘤细胞并没有死于机体的免疫杀伤，可以通过多种机制逃逸T细胞的免疫监视作用。 大多数肿瘤抗原的免疫原性很弱，不能诱发有效的抗肿瘤免疫应答。机体对肿瘤抗原的免疫应答导致肿瘤细胞表面抗原减少或丢失，从而肿瘤细胞不被免疫系统识别，逃避了机体的免疫攻击，这种现象称为“抗原调变”（Antigen Modulation）。这一理论在小鼠白血病细胞系中得到证实，该细胞系经抗体、补体处理后，丧失了细胞表面的胸腺白细胞抗原（TL抗原）。抗TL抗原的抗体与肿瘤细胞结合后，细胞表面出现斑点样的抗原分布改变，以后该抗原逐渐消失。在其他实验中也发现抗肿瘤抗体导致肿瘤抗原消失的类似现象。将肿瘤细胞与特异抗体或者细胞毒性淋巴细胞（CTLs）共同培养，也可以迅速诱导肿瘤抗原的丢失。抗原调变这一现象在生长快速的肿瘤普遍存在，抗原调变可由于细胞自身的机制或抗原抗体复合物脱落所致。这种肿瘤细胞表面抗原丢失仅反映肿瘤细胞表型的改变，经调变的细胞再次进入原宿主，抗原将再次出现，并重新诱发抗体产生。

实用指导(二)：慢病毒感染贴壁细胞实验步骤

实用指导（二）：慢病毒感染贴壁细胞实验步骤 和元生物|2016-03-1513:51 慢病毒慢病毒（Lentivirus）是一类改造自人免疫缺陷病毒(HIV)的病毒载体，基因组为RNA，对分裂细胞和非分裂细胞均具有感染能力。慢病毒基因组进入细胞后，在细胞浆中反转录为DNA，形成DNA整合前复合体，进入细胞核后，DNA整合到细胞基因组中。整合后的DNA转录成mRNA，回到细胞浆中，表达目的蛋白；或产生小RNA。慢病毒介导的基因表达或小RNA干扰作用持续且稳定，并随细胞基因组的分裂而分裂。 以24孔为例： 第一天：准备细胞将状态良好的目的细胞接种到24孔板中，细胞计数后，进行铺板，每孔加入500ul培养基。保证第二天感染病毒时融合效率达到30-40%。通常，24孔板内以 5-8*10^4cells/孔的密度铺板。 第二天：病毒感染，换液1计算病毒加药量 选择合适MOI值，进行病毒感染。 加药量计算方法：（细胞数×MOI值/病毒原液滴度）×10^3=病毒加药量（μl）。 2弃去部分培养基 将每组中加入的病毒及感染增强剂的体积去掉，保证加入病毒和感染增强剂后，每孔中仍保持500ul的液体量。 3加入慢病毒加药量计算方法

（细胞数×MOI值/病毒原液滴度）×10^3=病毒加药量（μl），培养基的总量必须充分覆盖住细胞。 4添加感染增强剂polybrene,保证终浓度为5ug/ml Polybrene是一种聚阳离子，可以中和电荷以促进假病毒外壳与细胞膜的作用。Polybrene 最佳浓度因不同细胞株而异并应根据经验而定（通常范围为2-10μg/ml）。过长时间暴露于Polybrene（>12小时）可对某些细胞产生毒性作用。 5病毒感染8~12小时后，观察细胞状态。 如细胞状态差，尽快换新鲜培养基；如细胞状态良好，可以在24小时内换液，但不宜超过24小时。弃去培养基后每孔加入500μl新鲜的完全培养基。5%CO2培养箱培养。 第五天：观察荧光表达情况病毒感染细胞72h后，在荧光显微镜下观察细胞，估计慢病毒感染目的细胞的效率。若荧光弱，可到96h后观察。如果96h仍无荧光，即感染实验失败。

计算机病毒与防范基础知识测试题

计算机病毒知识测试题（单选） 姓名得分： 注：每题5分，满分100分 1．下面是关于计算机病毒的两种论断，经判断______ (1)计算机病毒也是一种程序，它在某些条件上激活，起干扰破坏作用，并能传染到其他程序中去；(2)计算机病毒只会破坏磁盘上的数据. A)只有(1)正确B)只有(2)正确 C)(1)和(2)都正确D)(1)和(2)都不正确 2.通常所说的“计算机病毒”是指______ A)细菌感染B)生物病毒感染 C)被损坏的程序D)特制的具有破坏性的程序 3.对于已感染了病毒的U盘，最彻底的清除病毒的方法是_____ A)用酒精将U盘消毒B)放在高压锅里煮 C)将感染病毒的程序删除D)对U盘进行格式化 4.计算机病毒造成的危害是_____ A)使磁盘发霉B)破坏计算机系统 C)使计算机内存芯片损坏D)使计算机系统突然掉电 5.计算机病毒的危害性表现在______ A)能造成计算机器件永久性失效 B)影响程序的执行，破坏用户数据与程序 C)不影响计算机的运行速度 D)不影响计算机的运算结果，不必采取措施 6.计算机病毒对于操作计算机的人，______ A)只会感染，不会致病B)会感染致病 C)不会感染D)会有厄运 7.以下措施不能防止计算机病毒的是_____ A)保持计算机清洁 B)先用杀病毒软件将从别人机器上拷来的文件清查病毒 C)不用来历不明的U盘 D)经常关注防病毒软件的版本升级情况，并尽量取得最高版本的防毒软件 8.下列4项中，不属于计算机病毒特征的是______ A)潜伏性B)传染性C)激发性D)免疫性 9.下列关于计算机病毒的叙述中，正确的一条是______ A)反病毒软件可以查、杀任何种类的病毒