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K Krawczynski

Publications and source records attributed to K Krawczynski.

At least 19 recordsLinked to original sources
PubMed2006

Quantitative detection of hepatitis E virus RNA and dynamics of viral replication in experimental infection.

Hepatitis E virus (HEV) RNA has been detected in the stool and serum of patients with HEV infection and experimentally infected nonhuman primates. However, dynamics of HEV levels in the stool and serum during clinical and subclinical infections have not been determined. A real-time reverse transcription polymerase chain reaction assay, using SYBR Green I in a LightCycler, was developed and optimized to allow quantification of HEV RNA in the stool and serum of both genotype 1 and 2 isolates. The specificity of the assay was confirmed by testing known HEV-RNA-positive and -negative stool and serum specimens and the sensitivity was evaluated using a synthetic HEV RNA standard. Profiles of viraemia and faecal shedding in two chimpanzees inoculated with an isolate of HEV genotype 1 showed the appearance of virus in the stools on day 4 postinoculation (5.65-6.85 log copies/mg) and in the serum on day 7 postinoculation (6.0-6.93 log copies/mL). Peak HEV RNA levels in the stool and serum coincided with peak alanine aminotransferase (ALT) levels observed on day 22 postinoculation in the two chimpanzees. At the time of detection of IgG anti-HEV in serum, viral RNA was no longer detectable in the stool or serum and ALT values had returned to normal levels in both chimpanzees, suggesting the efficacy of the immune response in terminating viral replication. Quantitative evaluation of HEV RNA in humans may allow determining the role of virus levels in the pathogenesis and transmission of HEV.

Animals↗
PubMed2001

Experimental studies on subclinical hepatitis E virus infection in cynomolgus macaques.

Serial subclinical transmission among susceptible humans may serve as a reservoir of hepatitis E virus (HEV) in areas in which HEV is endemic. This hypothesis was investigated in an experimental primate model. Four groups of 4 cynomolgus macaques each were inoculated intravenously with 10(4)-10(5) (group 1), 10-100 (group 2), and 1-10 (group 3) cynomolgus macaque HEV infectious doses. All 4 animals in group 1 had clinical disease marked by alanine aminotransferase (ALT) elevation, fecal virus excretion, viremia, and seroconversion. Of the animals in groups 2 and 3, only 1 had evidence of biochemical hepatitis, although most had virus excretion and viremia (3 animals each in groups 2 and 3), and evidence of seroconversion (1 animal in group 2 and 3 animals in group 3). Viral genomic titers in stool specimens of animals with or without ALT elevation were similar. Infectivity studies confirmed the viability and transmission potential of the virus excreted by animals without ALT elevation. These data suggest that subclinical HEV infection may represent an HEV reservoir.

Alanine Transaminase↗
PubMed2001

Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients.

One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.

Amino Acid Sequence↗
PubMed2000

Duration of viraemia and faecal viral excretion in acute hepatitis E.

Data on duration of viral excretion and viraemia during hepatitis E virus (HEV) infection are limited. We tested serial stool and serum samples from 20 patients with acute hepatitis E for HEV RNA. Faecal excretion and viraemia in these patients were found to be short lived. In 19 patients, all samples obtained after biochemical resolution of hepatitis tested negative; in the remaining patient, HEV RNA was detected in the serum samples but not in stool after biochemical resolution. Long-term persistence of HEV in body fluids of infected individuals seems to be an unlikely reservoir for transmission of HEV.

Acute Disease↗
PubMed2000

Related TT viruses in chimpanzees.

A series of serum specimens obtained from two chimpanzees experimentally infected with hepatitis A virus (HAV), hepatitis C virus, and hepatitis G/GB-C virus were tested for TT virus (TTV) by polymerase chain reaction (PCR). All PCR fragments obtained from both animals were directly sequenced, and the nucleotide sequences were compared to each other and to all known TTV sequences. This comparison showed that both animals were infected simultaneously with four new TTV variants designated A, M1, M2, and M3. One chimpanzee was found to be infected with TTV only after HAV inoculation, whereas the other animal was infected with TTV before any experimental procedure was performed. A set of PCR primers specific for these four new TTV variants was used to amplify TTV-like sequences from nine naive chimpanzees. None of these animals was infected with the prototype TTV variant. Two of these animals, however, were infected with one of the new TTV variants, while one animal was infected with an additional new TTV variant designated T. Among 99 hepatitis patients, 29 were found to be infected with the prototype TTV variant. None of these human specimens was found to be positive by PCR specific for TTV variants A, M1, M2, and M3. Similarly, not a single specimen from a smaller subset of human serum samples was found to be positive for the TTV variant T. Phylogenetic analysis performed on all known TTV sequences demonstrated that TTV can be classified into 13 different, yet closely related TTV species, designated as TTV-I for the prototype variant through TTV-XIII. The new variants M1 and M2 were classified as two different genotypes of TTV-VI, variant M3 was classified as TTV-VII, variant A was classified as TTV-VIII, and variant T was classified as TTV-IX. Thus, the data obtained in this study suggest that TTV represents a large swarm of TTV-like species, some of which have not been detected in humans and circulate predominantly among chimpanzees.

Animals↗
PubMed2000

Hepatitis E virus infection in chimpanzees: a retrospective analysis.

Different patterns of disease were observed among 11 chimpanzees who were inoculated intravenously with hepatitis E virus (HEV) positive fecal specimens from four different outbreaks (Nepal 1981, Uzbekistan 1981, Pakistan 1985, and Mexico 1986). Five chimpanzees had marginal or no liver enzyme elevations within 70 days of inoculation. Two of the chimpanzees had limited viremia, but did not produce detectable antibody. The four remaining chimpanzees had liver enzyme elevations, viral shedding, viremia, seroconversion to anti-HEV, and detectable HEV antigen in liver biopsy specimens. These results may reflect the range of infection patterns that develop in humans after natural exposure to the HEV.

Animals↗
PubMed2000

Hepatitis E.

Hepatitis E, previously known as enterically transmitted non-A, non-B hepatitis, is an infectious viral disease with clinical and morphologic features of acute hepatitis. Its causative agent, hepatitis E virus, consists of small, 32- to 34-nm diameter, icosahedral, nonenveloped particles with a single-stranded, positive-sense, 7.5-kb RNA. The virus has two main geographically distinct strains, Asian and Mexican; recently, novel isolates from nonendemic areas and a genetically related swine HEV have been described. HEV is responsible for large epidemics of acute hepatitis and a proportion of sporadic hepatitis cases in the Indian subcontinent, southeast and central Asia, the Middle East, parts of Africa, and Mexico. The virus is excreted in feces and is transmitted predominantly by fecal-oral route, usually through contaminated water. Person-to-person transmission is uncommon. Clinical attack rates are the highest among young adults. Recent evidence suggests that humans with subclinical HEV infection and animals may represent reservoirs of HEV; however, further data are needed. Diagnosis of hepatitis E is usually made by detection of specific IgM antibody, which disappears rapidly over a few months; IgG anti-HEV persists for at least a few years. Clinical illness is similar to other forms of acute viral hepatitis except in pregnant women, in whom illness is particularly severe with a high mortality rate. Subclinical and unapparent infections may occur; however, chronic infection is unknown. No specific treatment is yet available. Use of clean drinking water and proper sanitation is currently the most effective method of prevention. Passive immunization has not been proved to be effective, and recombinant vaccines for travelers to disease-endemic areas and for pregnant women currently are being developed.

Age Factors↗
PubMed2000

Hepatitis E: an overview and recent advances in clinical and laboratory research.

Hepatitis E virus (HEV) is a non-enveloped RNA (7.5 kb) virus that is responsible for large epidemics of acute hepatitis and a proportion of sporadic hepatitis cases in southeast and central Asia, the Middle East, parts of Africa and Mexico. Hepatitis E virus infection spreads by the faecal-oral route (usually through contaminated water) and presents after an incubation period of 8-10 weeks with a clinical illness resembling other forms of acute viral hepatitis. Clinical attack rates are the highest among young adults. Asymptomatic and anicteric infections are known to occur. Chronic HEV infection is not observed. Although the mortality rate is usually low (0.07-0.6%), the illness may be particularly severe among pregnant women, with mortality rates reaching as high as 25%. Recent isolation of a swine virus resembling human HEV has opened the possibility of zoonotic HEV infection. Studies of pathogenetic events in humans and experimental animals reveal that viral excretion begins approximately 1 week prior to the onset of illness and persists for nearly 2 weks; viraemia can be detected during the late phase of the incubation period. Immunoglobulin M antibody to HEV (anti-HEV) appears early during clinical illness but disappears rapidly over a few months. Immunoglobulin G anti-HEV appears a few days later and persists for at least a few years. There is no specific treatment available for hepatitis E virus infection. Ensuring a clean drinking water supply remains the best preventive strategy. Recombinant vaccines are being developed that may be particularly useful for travellers to disease-endemic areas and for pregnant women.

Adult↗
PubMed1999

Antigenic epitopes of the hepatitis A virus polyprotein.

Forty-two antigenic domains were identified across the hepatitis A virus (HAV) polyprotein by using a set of 237 overlapping 20-mer synthetic peptides spanning the entire HAV polyprotein and a panel of serum samples from acutely HAV-infected patients. The term "antigenic domain" is used in this study to define a protein region spanned with consecutive overlapping immunoreactive peptides. Nineteen antigenic domains were found within the structural proteins, and 22 were found within the nonstructural proteins, with 1 domain spanning the junction of VP1 and P2A proteins. Five of these domains were considered immunodominant, as judged by both the breadth and the strength of their immunoreactivity. One domain is located within the VP2 protein at position 57-90 aa. A second domain, located at position 767-842 aa, contains the C-terminal part of the VP1 protein and the entire P2A protein. A third domain, located at position 1403-1456 aa, comprises the C-terminal part of the P2C protein and the N-terminal half of the P3A protein. The fourth domain, located at position 1500-1519 aa, includes almost the entire P3B, and the last domain, located at position 1719-1764 aa, contains the C-terminal region of the P3C protein and the N-terminal region of the P3D protein. It is interesting to note that four of the five most immunoreactive domains are derived from small HAV proteins and/or encompass protein cleavage sites separating different HAV proteins. The HAV-specific immunoreactivity of each antigenically reactive peptide was confirmed by using seven HAV seroconversion panels. Collectively, these data demonstrate that HAV structural and nonstructural proteins contain antigenic epitopes that can be efficiently modeled with short synthetic peptides.

Amino Acid Sequence↗
PubMed1999

Detection of genomic- and minus-strand of hepatitis C virus RNA in the liver of chronic hepatitis C patients by strand-specific semiquantitative reverse-transcriptase polymerase chain reaction.

Studies aimed at correlating the intrahepatic hepatitis C virus (HCV)-RNA level and anatomo-clinical features have been difficult because of sensitivity and specificity shortcomings of available techniques. We titered the genomic- and minus-strand HCV RNAs by a strand-specific, semiquantitative, genotype-independent reverse-transcriptase polymerase chain reaction (RT-PCR) in the liver tissue of 61 patients with chronic hepatitis C. Findings were correlated with the levels of HCV RNA in the serum, the HCV genotype, the expression of intrahepatic HCV antigens, the histological activity (using separate scores for the lobular and the portal/periportal necroinflammatory activity and for the fibrosis), and the response to interferon alfa (IFN-alpha) treatment. Genomic- and minus-strand HCV RNA were detected in 59 and 57 liver specimens, respectively. The HCV-RNA level in the serum correlated with the genomic-strand, but not with the minus-strand, HCV-RNA titer in the liver. No correlations were found between either strand of the intrahepatic HCV RNA and the level of expression of HCV antigens in the liver, or with the grading/staging of the underlying liver disease. The response to IFN-alpha treatment could be predicted by the serum HCV-RNA level and genotype, but not by the intrahepatic level of genomic- or minus-strand HCV RNA. These results suggest that, although the detection of the minus-strand HCV RNA reliably identifies the presence of replicating HCV in its target organ, the quantitative measurement of viremia remains the clinically meaningful "golden standard" for assessing the level of HCV replication.

Adult↗
PubMed1999

Excretion of hepatitis A virus (HAV) in adults: comparison of immunologic and molecular detection methods and relationship between HAV positivity and infectivity in tamarins.

Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enzyme immunoassay (EIA) to detect viral antigen and by reverse transcription-PCR amplification followed by ethidium bromide staining (PCR-ETBr) or nucleic acid hybridization (PCR-NA) to detect viral genetic material. A gradation of sensitivity was observed in the detection of virus by the three methods. In persons who had detectable virus, serial stool samples were found to be positive by EIA for up to 24 days after the peak elevation of liver enzymes. Viral genetic material could be detected by PCR-ETBr for up to 34 days and by PCR-NA for up to 54 days after the peak elevation of liver enzymes. After intravenous inoculation of tamarins with stool suspensions categorized as highly reactive for HAV (positive by EIA, PCR-ETBr, and PCR-NA), moderately reactive (positive by PCR-ETBr and PCR-NA), or weakly reactive (positive by PCR-NA), only tamarins infected with highly reactive stool suspensions (EIA positive) developed HAV infection. We conclude that positivity of stool specimens for HAV by PCR-ETBr or PCR-NA indicates a lower potential for infectivity, compared to that of EIA-positive stools.

Adult↗
PubMed1999

Hepatitis E: an overview.

Hepatitis E is an enterically transmitted, acute, self-limited, icteric viral disease that occurs in large numbers in countries of the Indian subcontinent, Asia, and Africa. The frequency of epidemics and the high mortality rate among infected pregnant women are strong indicators that hepatitis E is an important cause of morbidity and mortality in humans. Several isolates of hepatitis E virus (HEV) derived from infected humans and experimental animals have recently been cloned and sequenced, allowing investigators to determine the molecular structure of the HEV genome. Laboratory diagnosis of HEV infection is done by detection of HEV antibodies, HEV RNA in stool and serum samples, HEV particles in stool specimens, and HEV antigen in hepatocytes and stool specimens. The detection of anti-HEV by enzyme immunoassay, with the use of several recombinant HEV proteins or synthetic peptides, is the most frequently applied method for the diagnosis of the infection and characterization of its epidemiologic features. Laboratory determination of HEV replication, immune response, and liver pathologic features in patients with hepatitis E and in infected primates has facilitated studies of the disease. Preventive measures against HEV infection include the passive transfer of protective antibodies or active immunization. In efforts to develop HEV vaccines, various recombinant proteins have been used. Although a range of protective immune responses have been induced in primates, further modifications of immunogen, adjuvant, and immunization schedules are necessary to prevent HEV infection. Much remains to be learned about epidemiology of HEV infection, reservoir(s) of the virus, and protective immunity in order to develop effective strategies to prevent hepatitis E.

Journal Article↗
PubMed1998

Recurrent hepatitis C virus infection after liver transplantation: immunohistochemical assessment of the viral antigen.

BACKGROUND: The value of immunohistochemical methods to identify hepatitis C virus antigen (HCVAg) in liver tissue has not been established. We have evaluated the significance of HCVAg expression in livers of patients with transplants and recurrent hepatitis C virus (HCV) infection. METHODS: Forty-two liver biopsy specimens from 32 liver-transplant recipients with recurrent HCV infection were tested for HCVAg using fluorescein isothiocyanate-labeled polyclonal, polyreactive human immunoglobulin. Histologic assessment of liver and quantitation of HCV RNA in sera were carried out in specimens obtained simultaneously with biopsies. RESULTS: HCVAg was found in 33% of the liver specimens obtained during the first month after transplantation and in all liver specimens obtained between 1 and 18 months after transplantation. Amounts of the antigen were significantly greater in specimens obtained more than 1 month after transplantation. A statistically significant increase of the average HCV RNA level in serum was observed in samples tested after the first month after the transplantation, and some decrease in the HCV RNA level was found in those obtained between 6 and 18 months after transplantation. Larger amounts of HCVAg were observed in specimens corresponding to episodes of acute or chronic hepatitis than in those associated with minimal parenchymal evidence of rejection. CONCLUSIONS: OBSERVATIONS of HCVAg expression in liver biopsy specimens indicated that the presence of viral antigens in hepatocytes is a constant finding in specimens obtained 1 month or longer after transplantation. Although large amounts of HCVAg correlated with acute or chronic hepatitis, the nature of this association with the development of pathologic changes remains to be established.

Adult↗
PubMed1998

Detection of intrahepatic hepatitis C virus replication by strand-specific semi-quantitative RT-PCR: preliminary application to the liver transplantation model.

BACKGROUND/AIMS: Although the hepatitis C virus infection recurs in virtually all patients after liver transplantation, up to 50% of patients may not have histological recurrent hepatitis 1 year after liver transplantation. To study the relationship between hepatitis C virus infection and liver disease after liver transplantation, we compared the intrahepatic hepatitis C virus replication levels with the liver histopathology among liver transplant recipients. METHODS: The intrahepatic negative-strand HCV RNA (i.e. the putative hepatitis C virus replication intermediate RNA) was evaluated by a semi-quantitative, strand-specific reverse transcriptase-polymerase chain reaction in 44 liver specimens from 23 patients with hepatitis C virus reinfection after liver transplantation. Results were compared with the time from liver transplantation, presence, grading and staging of the recurrent hepatitis, amount of hepatitis C virus antigens in the liver and serum HCV RNA levels. RESULTS: Negative-strand HCV RNA was detected in 42 liver specimens as early as 7 days after liver transplantation. Its titers correlated with the amount of intrahepatic hepatitis C virus antigens, but not with HCV RNA levels in serum. Levels of negative-strand HCV RNA in 19 specimens without hepatitis were comparable to those seen in 25 specimens with hepatitis (p=0.492), and were unrelated to the liver disease grading and staging scores. The intrahepatic hepatitis C virus replication could occasionally precede the recurrence of the hepatitis by several months. CONCLUSIONS: Molecular evidence has been obtained for intrahepatic hepatitis C virus replication occurring early after liver transplantation. The level of replication is not correlated with the development of recurrent hepatitis, suggesting that hepatitis C virus may replicate without inducing morphological evidence of liver damage.

Adult↗
PubMed1997

Acute non-A-E hepatitis in the United States and the role of hepatitis G virus infection. Sentinel Counties Viral Hepatitis Study Team.

BACKGROUND: Little is known about the relation of the newly discovered hepatitis G virus (HGV) to the cause and clinical course of acute and chronic viral hepatitis. METHODS: We selected patients from a surveillance study of acute viral hepatitis in four U.S. counties who had acute disease during 1985 to 1986 or 1991 to 1995. Serum samples were tested for HGV RNA by the polymerase chain reaction. RESULTS: HGV RNA was detected in 4 of 45 patients with a diagnosis of non-A-E hepatitis (9 percent), 23 of 116 patients with hepatitis C (20 percent), 25 of 100 patients with hepatitis A (25 percent), and 32 of 100 patients with hepatitis B (32 percent) (P<0.05 for the comparison of hepatitis B with hepatitis non-A-E or C). The clinical characteristics of the acute illness were similar for patients with HGV alone and those with hepatitis A, B, or C with or without HGV infection. During a follow-up period of one to nine years, chronic hepatitis did not develop in any of the patients with HGV alone, but 75 percent were persistently positive for HGV RNA, as were 87 percent of those with both hepatitis C and HGV infection. The rates of chronic hepatitis were similar in patients with hepatitis C alone (60 percent) and those with both hepatitis C and HGV infection (61 percent). CONCLUSIONS: The evidence from this surveillance study does not implicate HGV as an etiologic agent of non-A-E hepatitis. Persistent infection with HGV was common, but it did not lead to chronic disease and did not affect the clinical course in patients with hepatitis A, B, or C.

Acute Disease↗
PubMed1997

Novel hepatitis agents: the significance of clinical and experimental studies. An overview.

A new virus with genomic organization similar to that of the family Flaviviridae was identified in patients with viral hepatitis and designated hepatitis G virus (HGV) or hepatitis GB virus C (GBV-C). HGV/GBV-C can be transmitted by blood and results in persistent infection, as shown in patients with posttransfusion non-A, non-B hepatitis, in transfusion recipients, and in donors of blood received by HGV-positive patients. The parenteral route of transmission of HGV/GBV-C infection was confirmed in experimentally infected chimpanzees. Epidemiologic studies of sporadic, community-acquired viral hepatitis have not indicated an association between HGV/GBV-C and acute non A-E hepatitis. Thus, the disease association of this new virus remains unconfirmed and its role in the etiology of acute and chronic hepatitis is unclear. The experimental model of HGV/GBV-C infection may define the biology of the virus replication.

Animals↗
PubMed1997

Acute hepatitis E by a new isolate acquired in the United States.

OBJECTIVE: To report the first case of acute hepatitis E by a novel isolate acquired in the United States and confirmed by nucleotide sequencing. MATERIAL AND METHODS: We describe the clinical manifestations and the results of associated laboratory studies in a man who was found to have acute hepatitis E infection. RESULTS: A 62-year-old man was hospitalized because of fever, abdominal pain, and jaundice. After an initial evaluation did not provide a cause, his serum was found to be positive for IgG anti-hepatitis E virus (HEV) by three antibody assays. Serum was also positive for HEV RNA by reverse transcriptase polymerase chain reaction (PCR). Sequencing results from the PCR products demonstrated substantial differences at the nucleotide level between this strain and the known Mexican and Burmese strains. CONCLUSION: On the basis of this initial report, HEV should be considered an etiologic agent in patients with acute non-ABC hepatitis in the United States.

Acute Disease↗