Clinical and epidemiological relevance of quantitating hepatitis E virus-specific immunoglobulin M

Diagnosis of acute hepatitis E by detection of hepatitis E virus (HEV)-specific immunoglobulin M (IgM) is an established procedure. We investigated whether quantitation of HEV IgM and its ratio to HEV total Ig furnished more information than conventional IgM tests that are interpreted as positive or negative. A previously described indirect immunoassay for total Ig against a baculovirus-expressed HEV capsid protein was modified to quantitate HEV-specific IgM in Walter Reed (WR) antibody units by using a reference antiserum and the four-parameter logistic model. A receiver-operating characteristics curve derived from 197 true-positive specimens and 449 true-negative specimens identified 30 WR units/ml as an optimum cut point. The median HEV IgM level in 36 patients with acute hepatitis E fell from 3,000 to 100 WR units/ml over 6 months, suggesting that 100 WR units/ml would be a more appropriate cut point for distinguishing recent from remote IgM responses. Among three hepatitis E case series, determination of the HEV IgM-to-total-Ig ratio in acute-phase serum revealed that most patients had high ratios consistent with primary infections whereas a few had low ratios, suggesting that they had sustained reinfections that elicited anamnestic antibody responses. The diagnostic utility of the new IgM test was similar to that of a commercially available test that uses different HEV antigens. In conclusion, we found that HEV IgM can be detected specifically in >95% of acute hepatitis E cases defined by detection of the virus genome in serum and that quantitation of HEV IgM and its ratio to total Ig provides insight into infection timing and prior immunity.     

Quantitation of immunoglobulin to hepatitis E virus by enzyme immunoassay

We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and inter-test coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.     

Quantitation of Immunoglobulin to Hepatitis E Virus by Enzyme Immunoassay

We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and intertest coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.     

Evaluation of diagnostic assays for hepatitis E virus in outbreak settings

Hepatitis E virus (HEV) is a major cause of hepatitis. We evaluated five HEV antibody diagnostic assays by using outbreak specimens. The Abbott immunoglobulin G (IgG), Genelabs IgG, and Walter Reed Army Institute of Research (WRAIR) IgM assays were about 90% sensitive; the Abbott IgG and WRAIR total Ig and IgM assays were more than 90% specific.     

Hepatitis E virus seroprevalence in acute viral hepatitis in a developed country confirmed by a supplemental assay

Hepatitis E virus (HEV) infection is prevalent among cases of acute viral hepatitis in young adults in developing countries. HEV infection is not restricted to endemic areas, but would appear to be worldwide in distribution. In order to document the incidence of HEV infection in acute hepatitis cases in a developed country, IgG and IgM anti-HEV antibodies and HEV RNA were tested in 101 Caucasian patients with acute viral hepatitis; 92 of these cases had markers of acute viral hepatitis other than HEV. Forty-seven (46.5%) cases had IgG anti-HEV; IgM anti-HEV and HEV viremia were not detected. As the incidence of anti-HEV was higher than would be expected, the possibility of the occurrence of false positive results was subsequently investigated. Supplemental antibody testing, using a broadly reactive epitope region, reduced the frequency of anti-HEV to 17%. Therefore, supplemental antibody testing confirms the hepatitis E virus seroprevalence in a developed country. Since IgM anti-HEV and HEV viremia were not detected, persons with IgG anti-HEV may be “subclinical HEV cases,” or have long-lived antibodies in their circulation. © 1996 Wiley-Liss, Inc.     

A Valuable Antigen Detection Method for Diagnosis of Acute Hepatitis E

Hepatitis E virus (HEV) is a serious public health problem. The commonly used tests that are specific for current HEV infection diagnosis include the detection of anti-HEV IgM and HEV RNA. Here, we report an improved enzyme-linked immunosorbent assay (ELISA) method for HEV antigen detection with a linear range equivalent to 6.3 × 10³ to 9.2 × 10⁵ RNA copies per ml. The monoclonal antibody (MAb) 12F12, a high-ability MAb that binds HEV virus, was selected as the capture antibody from a panel of 95 MAbs. The positive period of HEV antigenemia in infected monkeys using this test was, on average, 3 weeks longer than previously reported and covered the majority of the acute phase. The positive detection rates of IgM, RNA, and new antigen from the first serum samples collected from 16 confirmed acute hepatitis E patients were 81% (13/16), 81% (13/16), and 100% (16/16), respectively. In three patients, the initial serum specimens that tested negative for IgM, despite the presence of symptoms of acute hepatitis and elevated alanine aminotransferase (ALT) levels, were positive for HEV antigen and HEV RNA. In contrast, the serum samples of the three RNA-negative patients were antigen positive (and IgM positive), possibly due to the degradation of HEV nucleic acids. Our results suggest that this new antigen detection method has acceptable concordance with RNA detection and could serve as an important tool for diagnosing acute hepatitis E.     

Serological response to hepatitis E virus genotype 3 infection: IgG quantitation, avidity, and IgM response

Sequential sera were collected from 18 acute cases of UK-acquired hepatitis E. The virus strains in all cases were of genotype 3. The IgM and IgG response to acute infection were documented over time using EIA kits based on a peptide antigen, pE2, which is derived from a genotype 1 strain of hepatitis E virus (HEV). Ninety-five percentage of acute sera were IgM positive; after 6 months or more only 12% remained positive. The kit was adapted to quantify the IgG response (in WHO U/ml) and to determine antibody avidity. Following acute infection, anti-HEV IgG concentrations rose between 6.9- and 90-fold. IgG avidity was low (<25%) in most acute sera. After 6 months IgG avidity was greater than 50% in all cases. One patient with a poor IgM response and high avidity antibody in acute sera may have had a second HEV infection. Taken together, these results confirm that the pE2-based EIA kits are suitable for diagnosing acute HEV genotype 3 infection. With simple modifications the IgG kit can measure anti-HEV concentration and avidity, which can be used to confirm acute infection.     

Acute sporadic hepatitis E in children living in Cairo, Egypt.

Seventy-three pediatric patients with acute hepatitis and 19 control patients without liver disease living in Cairo, Egypt, were evaluated with a newly developed Western blot assay for IgM antibody to hepatitis E virus (IgM anti-HEV). The mean age of acute hepatitis patients was 6.4 years (range, 1-13 years); 56% were male. Among the 73 acute cases, hepatitis A was diagnosed in 30 (41%), possible acute hepatitis B in three (4%), hepatitis E in nine (12%), and by exclusion, non-A, non-B hepatitis in 29 (40%). Two additional acute cases were positive for both IgM anti-HAV and IgM anti-HEV. None of the 19 control subjects had IgM anti-HEV. Parenteral risk factors were associated with cases of non-A, non-B hepatitis but were not associated with acute hepatitis E. Contact with a family member with jaundice was associated with acute hepatitis A. In contrast to prior epidemics of enterically-transmitted non-A, non-B hepatitis, HEV was found to be a common cause of acute hepatitis in a pediatric population. This study provides additional evidence that HEV may be a frequent cause of acute sporadic hepatitis among children living in some developing countries.     

Seroepidemiology of hepatitis E virus in patients with non-A, non-B, non-C hepatitis in Hungary

Many cases of acute hepatitis remain undiagnosed and the hepatitis E virus (HEV) is emerging in industrialized countries. The aim of this study was to assess the role HEV as causative agent in acute non-A, non-B, and non-C hepatitis patients in Hungary. 10.5% of the 264 acute non-A, non-B, and non-C hepatitis patients tested had anti-HEV IgG and 1.9% had anti-HEV IgM as tested by ELISA. After confirmation by Western blot 6.1% of the acute non-A, non-B, and non-C hepatitis patients had anti-HEV IgG antibodies only and 1.1% of the patients had both IgG and IgM. All 19 patients that were positive for anti-HEV IgG and/or IgM tested negative for HEV RNA by PCR. Only a small proportion of the acute hepatitis cases in the southwest of Hungary are assumed to be attributed to HEV infection, however, hepatitis E should be considered along with hepatitis A, B, and C in the diagnosis of acute hepatitis.     

Acute sporadic hepatitis E in Kuwait

Fifty-seven adult patients with acute hepatitis and 34 comparison patients without liver disease were evaluated using a newly developed Western blot assay for IgM antibody to hepatitis E virus. The mean age of patients with hepatitis was 32 years (range, 18-55 years); 88% were male. Among patients with acute hepatitis, hepatitis A (anti-HAV IgM positive) was diagnosed in two (4%), hepatitis B (anti-HBc IgM positive) in three (5%), and hepatitis E (anti-HEV IgM positive) in 34 (60%). One hepatitis patient had CMV IgM, another had EBV IgM, and 16 others (28%) were negative for all serologic markers of acute viral hepatitis. No patient with acute hepatitis A or B and none of the comparison patients without acute hepatitis had anti-HEV IgM. All but one case of acute hepatitis E were found among expatriates of Asian origin, and acute hepatitis E was associated significantly with recent travel to the Indian subcontinent. These data suggest that acute hepatitis E is common among foreign workers in Kuwait but that little HEV transmission is occurring directly in Kuwait. © 1994 Wiley-Liss, Inc.     

Diagnosis of acute hepatitis E by antibody and molecular testing: A study on 277 suspected cases

BackgroundAcute hepatitis due to hepatitis E virus (HEV) infection is both indigenous and imported to Europe. Few studies provide information about the role of HEV as an agent for acute hepatitis in Spain.ObjectivesTo investigate the frequency of the HEV infection among patients displaying acute hepatitis of unexplained origin in Spain, comparing the performance of two different diagnostic approaches.Study designSpecific IgM antibody and HEV RNA tests were used to study samples from 277 patients with acute hepatitis of unknown aetiology received during a six-year period. Samples were sent by 52 hospitals from almost all regions of Spain.ResultsEvidence of acute infection by HEV was obtained for 30 patients in total (10.8%), and 16 cases were unrelated to recent international travel. On samples from 158 patients tested for both anti-HEV IgM and HEV RNA at admission, the yield of IgM antibody testing (11.4%) was higher than the yield of HEV RNA testing (9.5%).ConclusionsHEV could be responsible in Spain of about 11% of cases of acute hepatitis of unknown origin overall, and of about 8% of cases unrelated to international travel or immigration. India and neighbour countries represent the highest risk for import of epidemic HEV strains into Spain. Both antibody assays and molecular tests are required to optimise the final yield of laboratory diagnosis.     

ELISA for IgG-class antibody to hepatitis E virus based on a highly conserved, conformational epitope expressed in Escherichia coli.

In assays based on most recombinant hepatitis E virus (HEV) antigens, the IgG antibody responses to HEV are observed commonly to wane or disappear after the acute phase of infection. Such IgG assays have therefore been used for the diagnosis of acute HEV infection, but they have limited usefulness in seroepidemiological studies. Using western immunoblotting, it was shown previously that the open reading frame (ORF) 2.1 antigen, representing the carboxy-terminal 267 amino acids (aa) of the capsid protein, exposes a conformational epitope which allows optimal detection of convalescent antibody compared to other proteins expressed in Escherichia coli. This conformational epitope is shown to be highly conserved between divergent human HEV isolates, and the development of a sensitive and highly specific enzyme immunoassay (ELISA) based on this recombinant antigen is described. The ORF2.1 ELISA allows the detection and quantitation of both acute- and convalescent phase HEV-specific IgG, and will help to define better the antibody responses to the virus and the prevalence of HEV infection worldwide.     

Detection of immunoglobulin M antibodies to hepatitis E virus by class capture enzyme immunoassay

The measurement of antibodies to hepatitis E virus (anti-HEV) has been essential for understanding the epidemiology of hepatitis E. Studies to determine the prevalence of HEV infections require a reliable serologic assay that is sensitive and specific. It is also important to distinguish the acute from the convalescent phase of an infection; this usually requires the detection of the immunoglobulin M (IgM) class of antibody. Few enzyme immunoassays (EIAs) that measure IgM anti-HEV have been described, and most have utilized the sandwich method. The present study describes an EIA that detects IgM anti-HEV by antibody class capture methodology. The assay was validated by using serum and/or plasma panels from experimentally infected nonhuman primates. It was used to demonstrate an anamnestic response and the reappearance of IgM anti-HEV in a chimpanzee experimentally challenged with HEV at two different times 45 months apart. The class capture method was more sensitive than the sandwich EIA when used to test clinical samples from two hepatitis E epidemics in Pakistan; it also had the advantage of distinguishing IgM anti-HEV in the presence of high titers of IgG anti-HEV.     

Empty virus-like particle-based enzyme-linked immunosorbent assay for antibodies to hepatitis E virus

Hepatitis E, an enterically transmitted non-A, non-B hepatitis, is a serious viral infection that occasionally causes large epidemics in developing countries. In developed countries, the disease only appears sporadically due to the transmission routes, and it is considered to be less important. The hepatitis E virus (HEV) cannot grow in cultured cells and no reliable assay system has ever been developed. In addition, the present diagnostic are not perfect, and actual rates of HEV infection may be underestimated. Highly purified empty virus-like particles (VLPs) of HEV have been produced by the use of a recombinant baculovirus vector in insect cells. Using these VLPs as an antigen, an enzyme-linked immunosorbent assay (ELISA) for antibodies to HEV was developed. A panel of 164 sera that were randomized and coded, and sera collected periodically from three patients with hepatitis E were used for the evaluation. The sensitivity of the assay was shown to be equal to or better than that obtained in previous research that used the same serum panel. The ELISA demonstrated that the serum IgM level of the patients was highest at the onset of the clinical illness and then rapidly decreased. In contrast, a high level of circulating IgG antibody titers lasted for more than 4 years. In Japan, a non-endemic country, the prevalence of the IgG class antibody to HEV in healthy individuals was found to range from 1.9% to 14.1%, depending on the geographical area. Only one out of 900 (0.1%) serum samples was IgM-positive. The IgM class antibody to HEV was detected in 10.8% of non-A, non-B, and non-C acute hepatitis patients in northeast China, whereas none of the patients in Korea had the IgM antibody. The ELISA utilizing the VLPs is sensitive and specific in its detection of the IgM and IgG antibodies to HEV. The ELISA is therefore useful for diagnosing HEV infection and for seroepidemiological study of hepatitis E.     

Acute hepatitis A is the chief etiology of acute hepatitis in Egyptian children: a single-center study

Acute hepatic illness is an important health issue in children. Our work aimed to determine the prevalence of viral hepatitis in symptomatic children. It is a prospective cohort study of 268 children presented with acute hepatitis. Complete blood count, liver panel, and anti-hepatitis A virus (HAV) IgM were done initially. Cases negative for HAV were tested for anti-hepatitis E (HEV) IgM, anti-Epstein-Barr virus viral capsid antigen (EBV VCA) IgM, anti-cytomegalovirus virus IgM, hepatitis B surface antigen, anti-hepatitis B core IgM antibody, and anti-HCV antibody. Anti-HCV was repeated after 12 weeks to exclude seroconversion. In cases with negative viral serology, ceruloplasmin, total immunoglobulin G, antinuclear antibody, and abdominal ultrasound were done. Follow-up visits were bimonthly till recovery, then after 6 months. The mean age?±?SD was 7.1?±?3.7 years (1.5–18), and 56% were males. Acute HAV infection was diagnosed in 260 (97%) of cases and acute EBV infection in one case (0.4%). HAV/HEV coinfection was excluded in 70 HAV-positive cases. Six (2.2%) children remain undiagnosed and one child lost follow-up. About 80% of HAV-cases had normal laboratory results within 45 days. Unusual presentation of HAV infection was noticed in six children: four (1.5%) were relapsing, one had a cholestatic course, and one case had severe hemolytic anemia. Acute HAV infection was the chief etiology of acute hepatitis in our Egyptian children. The majority of the presentations were mild and children recover within a few weeks. An unusual pattern of HAV in children can be observed in endemic areas.     

Acute hepatitis E virus infection in Taiwan 2002–2006 revisited: PCR shows frequent co‐infection with multiple hepatitis viruses

Sporadic cases of acute hepatitis E virus (HEV) infection with production of anti‐HEV IgM have been reported occasionally in Taiwan despite no reported outbreaks in the past. This study was undertaken to determine whether serological markers correlated with virus detection. From 2002 to 2006, 72 reported cases of acute hepatitis E seropositive for anti‐HEV IgM in Taiwan were enrolled for investigation. Acute phase serum samples were collected for detection of HEV RNA, HBV DNA, HCV RNA, and GBV‐C RNA by PCR. The results showed that viral sequences of HEV, HBV, HCV and GBV‐C were detected in 54 (75%), 21 (29.2%), 9 (12.5%), and 22 (30.6%) of cases, respectively. Acute hepatitis A co‐infection was excluded in all patients because none were seropositive for anti‐HAV IgM and, nine patients (12.5%) did not seroconvert to anti‐HEV IgG. These results suggest that serum markers did not correlate completely with viremia in the diagnosis of acute HEV infection. Multiple viruses may co‐infect with acute hepatitis E virus in Taiwan. Detection of hepatitis E viremia together with seropositivity for anti‐HEV IgM and followed by seroconversion to anti‐HEV IgG should be included in the diagnostic criteria for HEV infection. J. Med. Virol. 81:1734–1742, 2009. © 2009 Wiley‐Liss, Inc.     

Novel approach for detection of hepatitis E virus infection in German blood donors

The risk of transfusion-transmitted hepatitis E virus (HEV) infections by contaminated blood products remains unknown. In the present study, we evaluated and compared different nucleic acid amplification technique (NAT) methods for the detection of HEV in blood components. Minipools of a total of 16,125 individual blood donors were screened for the presence of HEV RNA using the highly sensitive RealStar HEV RT-PCR kit, revealing a minimum detection limit of 4.66 IU/ml. Thirteen donors were HEV RNA positive (0.08%), and of these donors, only three already showed reactive IgM antibody titers. The detected HEV strains all belonged to genotype 3 and were most closely related to German HEV strains from wild boars and pigs as well as from human hepatitis E cases. Furthermore, HEV RNA and HEV-specific IgM and IgG titers were determined in 136 blood donors with elevated alanine aminotransferase (ALT) levels and in 200 donors without pathological findings. HEV RNA was not detectable, but 8.08% (elevated ALT) and 0.5% (nonelevated ALT) of donors showed reactive HEV IgM titers. The overall seroprevalence rate of HEV IgG amounted to 5.94% (elevated ALT, 5.88%; nonelevated ALT, 6.0%). The clinical relevance of transfusion-associated hepatitis E infection still requires further investigation. However, in connection with raising concerns regarding blood safety, our NAT method provides a sensitive possibility for HEV testing.     

1995至2000年北京地区散发性急性肝炎患者肝炎病毒抗体的检测与分析

目的 了解北京市地区散发性肝炎患者甲型、乙型、丙型和戊型肝炎病毒感染型别分布及重叠感染。方法 用EIA法检测1995斫2月至2000年12月北京地区散发性急性肝炎患者抗－HAVIgM、HBsAg／抗－HBcIgM、抗－HCVIgM/IgG和抗－HEVIgM/IgG。结果 214例散发性急性肝炎患者血清,抗甲、乙、内和戊型肝炎病毒IgM总阳性数155例,戊型肝炎76例。有9名患者检出2种肝炎病毒抗原或抗体阳性,其中在3名肝硬化患者和2名静脉吸毒者同时检测到HBV和HCV抗原或抗体,1例HBsAg阳性者检测到抗－HAVIgM,3例－HCVIgG阳性者中分别检测到2例抗－HBVIgM和1例抗－HEVIgM。肝炎病毒重叠感染的9名患者年龄在31岁至49岁之间。结论 北京地区散发性急性肝炎78％是由消化道传染的甲戊型肝炎病毒引起,戊型肝炎在四种肝炎中位居首位,其次为甲型肝炎、乙型肝炎和丙型肝炎。肝炎病毒重叠感染多见乙、丙型肝炎病毒合并感染或慢性乙、丙型肝炎患者合并甲型或戊型肝炎病毒感染。在我国对散发性病毒性肝炎的预防应引起高度重视。     

散发性急性戊型肝炎血清抗体动态变化和肝脏超微结构的病理观察

为探讨散发性戊型肝炎血清抗体动态变化，应用酶联免疫法（ＥＩＡ）检测了７例急性戊型肝炎患者抗戊型肝炎病毒（ＨＥＶ）ＩｇＧ和ＩｇＭ抗体，并对１例患者进行了肝超微结构病理检测。结果表明，发病后１０天至４５天内抗ＨＥＶ－ＩｇＧ和ＩｇＭ滴度最高，发病第４０天仍有肝细胞肿胀，胞浆空化和线粒体固缩等病理变化。患者血清抗－ＨＥＶＩｇＭ滴度在４５天后逐渐下降，２个月内全部消失，抗－ＨＥＶＩｇＧ消长情况与ＩｇＭ相似，但在７个月时仍有３例（４３％）抗体阳性。     

Serum immunoglobulin G subclass responses in different phases of hepatitis E virus infection

To investigate the specific immunoglobulin (Ig) G subclass responses in patients with hepatitis E virus (HEV) infection, an open reading frame 2 (ORF2) protein based enzyme‐linked immunosorbant assay was used to measure antibody levels in sera obtained at different phases of infection. Sera were collected at 2–31 days and at 6 months after the onset of symptoms corresponding to the acute (n = 48, 100% IgM‐positive) and convalescent (n = 17/48, 53% IgM‐positive) phases of infection, respectively. IgM‐negative sera from 61 individuals infected at least ≥6 months ago (prior exposure) were also tested. IgG1, IgG2, IgG3, and IgG4 antibodies were detected in 100%, 6%, 56%, and 4% of acute phase sera, respectively, and in 100%, 0%, 0%, and 65% of convalescent phase sera, respectively. IgG1 antibody levels were significantly higher than those of the other detectable subclasses of IgG in the acute and convalescent sera (P < 0.05). The IgG3 antibodies in six acute phase patients were replaced by IgG4 antibodies in the convalescent phase of infection. Patients with prior exposure to HEV had low total IgG antibody titers and decreased IgG1 seropositivity compared with those in the acute and convalescent phases. IgG1 was the only major subclass of antibody to be detected in all the three phases of infection. Other than IgG1 antibodies, the subclass antibody response was restricted to IgG3 and IgG4 antibodies in the acute and convalescent phases of infection, respectively. J. Med. Virol. 85:828–832, 2013. © 2013 Wiley Periodicals, Inc.