Hepatoviruses promote very-long-chain fatty acid and sphingolipid synthesis for viral RNA replication and quasi-enveloped virus release.

Virus-induced changes in host lipid metabolism are an important but poorly understood aspect of viral pathogenesis. By combining nontargeted lipidomics analyses of infected cells and purified extracellular quasi-enveloped virions with high-throughput RNA sequencing and genetic depletion studies, we show that hepatitis A virus, an hepatotropic picornavirus, broadly manipulates the host cell lipid environment, enhancing synthesis of ceramides and other sphingolipids and transcriptionally activating acyl-coenzyme A synthetases and fatty acid elongases to import and activate long-chain fatty acids for entry into the fatty acid elongation cycle. Phospholipids with very-long-chain acyl tails (>C22) are essential for genome replication, whereas increases in sphingolipids support assembly and release of quasi-enveloped virions wrapped in membranes highly enriched for sphingomyelin and very-long-chain ceramides. Our data provide insight into how a pathogenic virus alters lipid flux in infected hepatocytes and demonstrate a distinction between lipid species required for viral RNA synthesis versus nonlytic quasi-enveloped virus release.

Macrophage Depletion Reactivates Fecal Virus Shedding following Resolution of Acute Hepatitis A in Ifnar1-/- Mice.

Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1-/- mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm3 in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4+ or CD8+ T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifnγ, Tnfα, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1-/- mice and that macrophages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1-/- mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1-/- mice and may have relevance to relapsing hepatitis A in humans.

Nonlytic Quasi-Enveloped Hepatovirus Release Is Facilitated by pX Protein Interaction with the E3 Ubiquitin Ligase ITCH.

Hepatoviruses are atypical hepatotropic picornaviruses that are released from infected cells without lysis in small membranous vesicles. These exosome-like, quasi-enveloped virions (eHAV) are infectious and the only form of hepatitis A virus (HAV) found circulating in blood during acute infection. eHAV is released through multivesicular endosomes in a process dependent on endosomal sorting complexes required for transport (ESCRT). Capsid protein interactions with the ESCRT-associated Bro1 domain proteins, ALG-2-interacting protein X (ALIX) and His domain-containing protein tyrosine phosphatase (HD-PTP), which are both recruited to the pX domain of 1D (VP1pX), are critical for this process. Previous proteomics studies suggest pX also binds the HECT domain, NEDD4 family E3 ubiquitin ligase, ITCH. Here, we confirm this interaction and show ITCH binds directly to the carboxy-terminal half of pX from both human and bat hepatoviruses independently of ALIX. A small chemical compound (compound 5) designed to disrupt interactions between WW domains of NEDD4 ligases and substrate molecules blocked ITCH binding to pX and demonstrated substantial antiviral activity against HAV. CRISPR deletion or small interfering RNA (siRNA) knockdown of ITCH expression inhibited the release of a self-assembling nanocage protein fused to pX and also impaired the release of eHAV from infected cells. The release could be rescued by overexpression of wild-type ITCH, but not a catalytically inactive ITCH mutant. Despite this, we found no evidence that ITCH ubiquitylates pX or that eHAV release is strongly dependent upon Lys residues in pX. These data indicate ITCH plays an important role in the ESCRT-dependent release of quasi-enveloped hepatovirus, although the substrate molecule targeted for ubiquitylation remains to be determined. IMPORTANCE Mechanisms underlying the cellular release of quasi-enveloped hepatoviruses are only partially understood, yet play a crucial role in the pathogenesis of this common agent of viral hepatitis. Multiple NEDD4 family E3 ubiquitin ligases, including ITCH, have been reported to promote the budding of conventional enveloped viruses but are not known to function in the release of HAV or other picornaviruses from infected cells. Here, we show that the unique C-terminal pX extension of the VP1 capsid protein of HAV interacts directly with ITCH and that ITCH promotes eHAV release in a manner analogous to its role in budding of some conventional enveloped viruses. The catalytic activity of ITCH is required for efficient eHAV release and may potentially function to ubiquitylate the viral capsid or activate ESCRT components.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

Isolation and Genome Sequencing of Hepatitis E Virus Genotype 1 Imported from India to Japan.

Hepatitis E virus (HEV) is the causative agent of viral hepatitis E. In Japan, HEV genotype 3 (G3) and G4 are predominantly detected, while G1, mainly imported from countries in continental Asia, is rare. In the present study, we detected a G1 HEV strain in a patient who visited Japan from India. When PLC/PRF/5 cells (subclone 4-21) were inoculated with a stool suspension from this patient, accumulation of HEV RNA was observed in the spent culture medium, indicating that HEV had been successfully isolated from this specimen. A nearly complete HEV genome was obtained by RT-PCR amplification. Phylogenetic analyses revealed that the newly isolated HEV strain, designated 9HE36c, belongs to subtype 1g of HEV G1.

Novel flavonoid hybrids as potent antiviral agents against hepatitis A: Design, synthesis and biological evaluation.

Two series of flavonoid hybrids, totaling 42 compounds, were designed, synthesized and evaluated to develop antiviral compounds effective against hepatitis A virus (HAV). A recombinant viral screening system revealed that most of the synthesized derivatives exhibited significant anti-HAV activity, and compounds B2, B3, B5 and B27 were identified as potential inhibitors of HAV. Post-treatment of cells with B2, B3, B5 and B27 after HAV infection strongly suppressed HAV infection, whereas pretreatment or simultaneous treatment were ineffective. Furthermore, these four compounds significantly inhibited HAV (HM175/18f strain) production in a dose-dependent manner. Analyses using HAV subgenomic replicon systems indicated that these compounds specifically inhibit HAV RNA replication. More importantly, the most potent compounds B2 and B27 also showed clear inhibitory effects on two other HAV strains, KRM031 and TKM005, which also isolated from clinical patients. Our study is the first to report these newly designed flavonoid hybrids as lead compounds for the development of novel anti-HAV drugs.

Iminosugar Glucosidase Inhibitors Reduce Hepatic Inflammation in Hepatitis A Virus-Infected Ifnar1-/- Mice.

Iminosugar compounds are monosaccharide mimetics with broad but generally weak antiviral activities related to inhibition of enzymes involved in glycobiology. Miglustat (N-butyl-1-deoxynojirimycin), which is approved for the treatment of lipid storage diseases in humans, and UV-4 [N-(9-methoxynonyl)-1-deoxynojirimycin] inhibit the replication of hepatitis A virus (HAV) in cell culture (50% inhibitory concentrations [IC50s] of 32.13 µM and 8.05 µM, respectively) by blocking the synthesis of gangliosides essential for HAV cell entry. We used a murine model of hepatitis A and targeted mass spectrometry to assess the capacity of these compounds to deplete hepatic gangliosides and modify the course of HAV infection in vivo. Miglustat, given by gavage to Ifnar1-/- mice (4,800 mg/kg of body weight/day) depleted hepatic gangliosides by 69 to 75% but caused substantial gastrointestinal toxicity and failed to prevent viral infection. UV-4, similarly administered in high doses (400 mg/kg/day), was well tolerated but depleted hepatic gangliosides by only 20% after 14 days. UV-4 depletion of gangliosides varied by class. Several GM2 species were paradoxically increased, likely due to inhibition of ß-glucosidases that degrade gangliosides. Both compounds enhanced, rather than reduced, virus replication. Nonetheless, both iminosugars had surprising anti-inflammatory effects, blocking the accumulation of inflammatory cells within the liver. UV-4 treatment also resulted in a decrease in serum alanine aminotransferase (ALT) elevations associated with acute hepatitis A. These anti-inflammatory effects may result from iminosugar inhibition of cellular α-glucosidases, leading to impaired maturation of glycan moieties of chemokine and cytokine receptors, and point to the potential importance of paracrine signaling in the pathogenesis of acute hepatitis A. IMPORTANCE Hepatitis A virus (HAV) is a common cause of viral hepatitis. Iminosugar compounds block its replication in cultured cells by inhibiting the synthesis of gangliosides required for HAV cell entry but have not been tested for their ability to prevent or treat hepatitis A in vivo. We show that high doses of the iminosugars miglustat and UV-4 fail to deplete gangliosides sufficiently to block HAV infection in mice lacking a key interferon receptor. These compounds nonetheless have striking anti-inflammatory effects on the HAV-infected liver, reducing the severity of hepatitis despite enhancing chemokine and cytokine expression resulting from hepatocyte-intrinsic antiviral responses. We propose that iminosugar inhibition of cellular α-glucosidases impairs the maturation of glycan moieties of chemokine and cytokine receptors required for effective signaling. These data highlight the potential importance of paracrine signaling pathways in the inflammatory response to HAV and add to our understanding of HAV pathogenesis in mice.

Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus.

The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens1. Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and is released from hepatocytes without lysis in small vesicles that resemble exosomes2,3. These quasi-enveloped virions are infectious and are the only form of virus that can be detected in the blood during acute infection2. By contrast, non-enveloped naked virions are shed in faeces and stripped of membranes by bile salts during passage through the bile ducts to the gut4. How these two distinct types of infectious hepatoviruses enter cells to initiate infection is unclear. Here, we describe a genome-wide forward screen that shows that glucosylceramide synthase and other components of the ganglioside synthetic pathway are crucial host factors that are required for cellular entry by hepatoviruses. We show that gangliosides-preferentially disialogangliosides-function as essential endolysosome receptors that are required for infection by both naked and quasi-enveloped virions. In the absence of gangliosides, both virion types are efficiently internalized through endocytosis, but capsids fail to uncoat and accumulate within LAMP1+ endolysosomes. Gangliosides relieve this block, binding to the capsid at low pH and facilitating a late step in entry involving uncoating and delivery of the RNA genome to the cytoplasm. These results reveal an atypical cellular entry pathway for hepatoviruses that is unique among picornaviruses.

Integrin α3 is involved in non-enveloped hepatitis E virus infection.

Hepatitis E virus (HEV) causes acute and fulminant hepatitis worldwide. Although enveloped (e) and non-enveloped (ne) forms of HEV have been discovered, host factors involved in infection, including receptors, remain to be elucidated. Here, we identified integrin α3 (encoded by ITGA3), a protein that binds and responds to the extracellular matrix, as an essential host factor for HEV infection. Integrin α3 expression was lower in four HEV-non-permissive cell subclones than in an HEV-permissive subclone. ITGA3 knockout cells lost HEV permissibility, suggesting that integrin α3 is critical for HEV infection. Stable expression of integrin α3 in an HEV-non-permissive subclone provided permissibility only to infection by neHEV; expression of integrin α3 lacking the ectodomain did not. Direct interaction between neHEV and the integrin α3 ectodomain was confirmed by co-precipitation using a soluble integrin α3-Fc. These results strongly suggest that integrin α3 is a key molecule for cellular attachment and entry of neHEV.

Detection of Subgenotype IA and IIIA Hepatitis A Viruses in Rivers Flowing through Metro Manila, the Philippines.

Hepatitis A virus (HAV) is a common infectious etiology of acute hepatitis worldwide. The Philippines remains highly endemic for hepatitis A, but there is still a lack of information about HAV in the country. To evaluate the HAV contamination in environmental water in the Philippines, we conducted the detection and genetic analyses of HAV RNA in samples from river water. Twelve water samples were collected at 6 sampling sites of 3 rivers in Metro Manila, in both the dry and wet seasons in 2012 and 2013. The HAV RNA was detected in all the 6 samples collected in the dry season, and in one sample from the wet season. Phylogenetic analysis confirmed that the HAV strains detected in the river water included multiple sequences belonging to subgenotypes IA and IIIA. This indicates that at least 2 genotypes of the HAV strains are circulating in the environment in the Philippines, posing a risk of HAV infection to not only residents, but also tourists, especially in the dry season.

Evaluation of Heating Conditions for Inactivation of Hepatitis E Virus Genotypes 3 and 4.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis throughout the world. HEV genotypes 1 through 4 infect humans, whereas genotypes 3 and 4 (Gt3 and Gt4) also infect other animals. In developed countries, the main HEV infection route is by foodborne transmission, resulting from the consumption of undercooked meat. It is important to know the criteria for HEV control in daily cooking. In this study, we assessed the heat conditions required to inactivate HEV Gt3 and Gt4 in culture supernatants and spiked minced pork meat. HEV inactivation was determined by measuring viral RNA amplification in PLC/PRF/5 cell culture. In our cell culture assay, an inoculum containing HEV titer that is equivalent to >105 genome RNA copies can be determined as infectious. The internal temperature of pork during heating was measured to represent that achieved during cooking. Both HEV Gt3 and Gt4 were inactivated in culture supernatants heated at >65°C for 5 min and at >80°C for 1 min and in minced meat at 70°C for 5 min. Inoculated culture supernatant contained 108 HEV genome RNA copies (103 infectious units [IU]); therefore, it was indicated that HEV titer decreased >3 log IU after heating. In a comparison of Gt3 and Gt4, Gt4 showed slightly greater heat stability than Gt3. Boiling showed superior heating efficacy compared with roasting, and pork liver was slightly easier to heat than pork loin. Heating for 5 min by both boiling and roasting increased the internal temperature of pork products to more than 70°C. Although our data revealed that HEV Gt4 was slightly more heat stable than Gt3, both genotypes were inactivated by the appropriate heating conditions. Therefore, the risk of HEV foodborne infection could be mitigated by the appropriate cooking of pork meat. It is also important that both the supplier and the consumer are cognizant of the risk of HEV foodborne infection from livestock products.

Establishment of hepatitis E virus infection-permissive and -non-permissive human hepatoma PLC/PRF/5 subclones.

PLC/PRF/5 cells show limited permissiveness, meaning that almost all subclones are permissive; however, some subclones do not exhibit permissiveness for hepatitis E virus (HEV) infection. In this study, the single-cell cloning of PLC/PRF/5 was performed and heterogeneous subclones characterized. Notably, the efficiency of intracellular virus replication did not correlate with the permissiveness for HEV infection. However, as well as binding permissive subclones, virus-like particles bound non-permissive subclones on various levels, suggesting that these subclones have some deficiencies in the attachment and entry steps of infection. Our data would be useful for investigating the HEV life cycle.

Molecular detection of hepatitis E virus in rivers in the Philippines.

To understand the hepatitis E virus (HEV)-pollution status in the environment in the Philippines, a total of 12 water samples were collected from rivers in Manila City for detection of HEV RNA. Three of 12 samples were positive for HEV RNA indicating that HEV is circulating in the Philippines. Phylogenetic analysis classified all of the HEV sequences into genotype 3.

The hepatitis E virus capsid C-terminal region is essential for the viral life cycle: implication for viral genome encapsidation and particle stabilization.

Although the C-terminal 52 amino acids (C52aa) of hepatitis E virus (HEV) capsid are not essential for morphology, the C52aa-encoding region is required for replication. Transfection of a C52aa knockdown mutant showed transient growth, and the earliest population included a majority of noninfectious (possibly empty) particles and a minority of infectious particles with C-terminal capsid degradation. Finally, the complete revertant was generated reproducibly. C52aa is essential for the viral life cycle, promoting accurate encapsidation and stabilizing encapsidated particles.

Expression of herpes simplex virus type 1 recombinant thymidine kinase and its application to a rapid antiviral sensitivity assay.

Antiviral-resistant herpesvirus infection has become a great concern for immunocompromised patients. Herpes simplex virus type 1 (HSV-1) infections are treated with viral thymidine kinase (vTK)-associated drugs such as acyclovir (ACV), and most ACV-resistance (ACV(r)) is due to mutations in the vTK. The standard drug sensitivity test is usually carried out by the plaque reduction assay-based method, which requires over 10 days. To shorten the time required, a novel system was developed by the concept, in which 293T cells transiently expressing recombinant vTK derived from the test sample by transfection of the cells with an expression vector were infected with vTK-deficient and ACV(r) HSV-1 (TAR), and then cultured in a maintenance medium with or without designated concentrations of ACV, ganciclovir (GCV) and brivudine (BVdU). The replication of TAR was strongly inhibited by ACV, GCV and BVdU in 293T cells expressing recombinant vTK of the ACV-sensitive HSV-1, whereas replication was not or slightly inhibited in cells expressing the recombinant vTK of highly resistant or intermediately resistant HSV-1, respectively. An inverse correlation was demonstrated in the 50% effective concentrations (EC(50)s) and inhibitory effects of these compounds on the replication of TAR among ACV(s) and ACV(r) HSV-1 clones. These results indicate that the EC(50)s of the vTK-associated drugs including ACV can be assumed by measuring the inhibitory effect of drugs in 293T cells expressing recombinant vTK of the target virus. The newly developed antiviral sensitivity assay system for HSV-1 makes it possible to estimate EC(50) for vTK-associated drugs, when whole vTK gene is available for use by gene amplification directly from lesion's samples or from virus isolates.

Long-term observation of herpes simplex virus type 1 (HSV-1) infection in a child with Wiskott-Aldrich syndrome and a possible reactivation mechanism for thymidine kinase-negative HSV-1 in humans.

Herpes simplex virus type 1 (HSV-1) infections in a child with congenital immunodeficiency syndrome were observed over a 10-year period. The child suffered from recurrent and severe HSV-1 mucocutaneous infections. He frequently suffered from acyclovir (ACV)-resistant (ACV(r)) HSV-1 infection in the later phase of his life, especially after the episode of ACV(r) HSV-1 infection. Virological analyses on the HSV-1 isolates recovered from this patient revealed that all the ACV(r) HSV-1 isolates were thymidine kinase (TK)-negative (TK(-)) due to a single cytosine (C) deletion within the 4-C residues (positions 1061 to 1064) in the TK gene, indicating that the recurrent TK(-)/ACV(r) HSV-1 infections throughout the patient's life were due to the identical ACV(r) HSV-1 strain. Furthermore, it was found that the ACV-sensitive (ACV(s)) isolate recovered from the skin lesions that appeared between the episodes of ACV(r) infection at the ages of 8 and 9 contained ACV(r) HSV-1 with the same mutation in the TK gene. These results indicate that, although TK activity is required for reactivation of TK(+)/ACV(s) HSV-1 from latency and TK(-)/ACV(r) HSV-1 is unable to reactivate from latency, the TK(-)/ACV(r) HSV-1 strain isolated herein reactivated in this patient, possibly by using the TK activity induced by the latently co-infected TK(+)/ACV(s) HSV-1.

Virulence and pathophysiology of the Congo Basin and West African strains of monkeypox virus in non-human primates.

Monkeypox virus is divided into Congo Basin and West African strains. The virulence and pathophysiology of two strains, Zr-599 (a Congo Basin monkeypox virus) and Liberia (a West African monkeypox virus), were evaluated in non-human primates. Four monkeys were infected by the subcutaneous (SC) and two by the intranasal (IN) inoculation routes for Zr-599 and Liberia at a dose of 10(6) p.f.u. One monkey in the Liberia/SC group was demonstrated to be co-infected with Gram-positive cocci and was excluded from analyses. Infections in three of the four Zr-599/SC monkeys and in one of the three Liberia/SC monkeys were fatal. Virus genome levels in blood in the Zr-599/SC monkeys were approximately 10 times higher than those in the Liberia/SC monkeys. Zr-599 affected respiratory, genito-urinary and gastrointestinal tract organs more severely than Liberia. Zr-599 was more virulent than Liberia and one of the factors might be the difference in organ tropism.

Loop-mediated isothermal amplification-based diagnostic assay for monkeypox virus infections.

Monkeypox virus (MPXV) causes a smallpox-like disease in non-human primates and humans. This infection is endemic to central and western Africa. MPXV is divided into two genetically different groups, Congo Basin and West African MPXV, with the former being the more virulent. A real-time quantitative MPXV genome amplification system was developed for the diagnosis of MPXV infections using loop-mediated isothermal amplification (LAMP) technology. Primers used for genome amplification of Congo Basin (C-LAMP), West African (W-LAMP), and both Congo Basin and West African (COM-LAMP) MPXV by LAMP were designed according to the nucleotide sequences of the Congo Basin-specific D14L gene, the West African-specific partial ATI gene, and the partial ATI gene that is shared by both groups, respectively. The sensitivity and specificity of the LAMP were evaluated with nested PCR using peripheral blood and throat swab specimens collected from Congo Basin MPXV or West African MPXV-infected monkeys. The sensitivity and specificity of COM-LAMP, C-LAMP, and W-LAMP were 80% (45/56) and 100% (64/64); 79% (19/24) and 100% (24/24); and 72% (23/32) and 100% (40/40), respectively. The viremia level determined by LAMP assays increased with increases in the severity of the monkeypox-associated symptoms. The newly developed LAMP assay was confirmed to be a rapid, quantifiable, and highly sensitive and specific system effective in the diagnosis of MPXV infections. The LAMP assays made it possible to discriminate between Congo Basin and West African MPXV. The LAMP developed in this study is useful not only for diagnosis of but also for the assessment of MPXV infections.

Detection, genetic characterization, and quantification of norovirus RNA from sera of children with gastroenteritis.

BACKGROUND: Norovirus (NoV) infection is thought to be confined to the intestines, whereas many reports suggest antigenemia and viremia occur during rotavirus gastroenteritis. OBJECTIVES: To detect NoV RNA in sera and cerebrospinal fluids (CSF) from NoV-infected children, and to quantify and genetically characterize the NoV found in these compartments. STUDY DESIGN: Semi-nested PCR was conducted on stool, serum and CSF samples from 56 patients with acute gastroenteritis. Positive samples for NoV were analyzed further by sequencing and real-time PCR. RESULTS: From 39 patients with NoV RNA in stools, 6 also had NoV RNA in sera and none had NoV RNA in CSF. Genotypes of the NoV in stool and serum from the same patient matched completely. The strains in this study had high homology (98.1-100%) with registered strains in the database. The median viral load in stools of the serum-positive patients was greater than that of the serum-negative patients, but this difference was not statistically significant (9.8 x 10(9)copies/g versus 1.1 x 10(9)copies/g (p=0.117)). CONCLUSIONS: NoV RNA appeared in the blood stream in 15% of the patients of NoV gastroenteritis. Although the viral load in stool was not statistically correlated with NoV appearance in serum, genetic analysis indicated that NoV RNA in sera originated from the NoV gastroenteritis.

Development of a rapid immunochromatographic test for noroviruses genogroups I and II.

Norovirus (NoV) is known to cause acute gastroenteritis in children worldwide. Although reverse transcription-PCR (RT-PCR) method is considered to be the "gold standard" for diagnosis of this viral infection, it requires skillful personnel and well-equipped laboratory. In this study, a rapid and easily performable diagnostic kit was developed using immunochromatographic method with rabbit polyclonal antibodies raised against recombinant virus-like particles (rVLPs) of most prevalent genotypes, genogroup II genotypes 3 and 4. This kit was evaluated for reactivity to rVLPs and detection of natural viruses in stool samples collected from children with diarrhea in comparison to the results obtained by RT-PCR. In the prospective assessment, the kit showed agreement rate of 84.1%, sensitivity of 69.8% and specificity of 93.7%. Genotyping of the RT-PCR positive samples by sequence analysis revealed that some heterogeneous genotypes were also detected while some in homogeneous genotypes occasionally showed false negative records resulting in lower sensitivity. No cross-reactivity with other common viral pathogens was observed. Taken together with the result of the detection limit of viral load as small as approximately 10(6-7)copies/g of stool, the current immunochromatography test is justified for screening for NoV infection with simple laboratory support.