Contributions of neurotropic human herpesviruses herpes simplex virus 1 and human herpesvirus 6 to neurodegenerative disease pathology

Human herpesviruses （HVs） have developed ingenious mechanisms that enable them to traverse the defenses of the central nervous system （CNS）. The ability of HVs to enter a state of latency, a defining char- acteristic of this viral family, allows them to persist in the human host indefinitely. As such, HVs represent the most frequently detected pathogens in the brain. Under constant immune pressure, these infections are largely asymptomatic in healthy hosts. However, many neurotropic HVs have been directly connected with CNS pathology in the context of other stressors and genetic risk factors. In this review, we discuss the potential mechanisms by which neurotropic HVs contribute to neurodegenerative disease （NDD） patholo- gy by highlighting two prominent members of the HV family, herpes simplex virus 1 （HSV-1） and human herpesvirus 6 （HHV-6）. We （i） introduce the infectious pathways and replicative cycles of HSV-1 and HHV-6 and then （ii） review the clinical evidence supporting associations between these viruses and the NDDs Alzheimer＇s disease （AD） and multiple sclerosis （MS）, respectively. We then （iii） highlight and dis- cuss potential mechanisms by which these viruses exert negative effects on neurons and glia. Finally, we （iv） discuss how these viruses could interact with other disease-modifying factors to contribute to the initiation and/or progression of NDDs.

Human herpesvirus 7 meningitis in an adolescent with normal immune function:A case report

BACKGROUND Human herpesvirus type 7(HHV-7)is a less common herpes virus that usually causes mild,self-limiting illnesses.However,in recent years,there have been increasing reports of HHV-7 causing serious central nervous system infections,especially meningitis.The pathogenesis and clinical features of HHV-7 meningitis,particularly in adolescents with normal immune function,remain incompletely studied.Therefore,the purpose of this report is to share a case of HHV-7 meningitis in an immunocompetent adolescent with a view to deepening our understanding of the disease.CASE SUMMARY A 12-year-old female was admitted with fever,headache,and vomiting.4 d before admission,the patient developed a fever without obvious induction,with a temperature up to 39.5℃,no convulsions,accompanied by chills,headaches,fatigue,and no muscle aches.The patient was treated with fever reduction,which could be reduced to 38℃;repeated high fever,accompanied by vomiting 7-8 times;and no abdominal pain or diarrhea.The patient was diagnosed with"acute suppurative tonsillitis"in a local hospital,and the blood routine was generally normal.The patient was given symptomatic support treatment such as"ceftriaxone sodium"and antiemetic rehydration for 2 d,and his condition did not improve.The patient's physical examination showed pharyngeal congestion,bilateral tonsil grade I hypertrophy,regression of purulent secretions,and cervical resistance.Ocular B-ultrasound:Opacity of the vitreous body and edema of the optic disc in both eyes.Optical coherence tomography examination showed that the macular fovea was generally normal in both eyes,with edema of the optic disc.DNA virus monitoring results:HHV-7.We gave ganciclovir antiviral therapy,dexamethasone anti-inflammatory treatment,mannitol to reduce cranial pressure,omeprazole to protect gastrointestinal mucosa,and calcium and potassium supplementation.CONCLUSION This study reports a case of HHV-7 meningitis in an adolescent with normal immune function.Through comprehensive analysis of the clinical manifestations,laboratory tests,and treatment methods of the patient,it is found that early identification and antiviral treatment are essential for the outcome of the disease.This case suggests that despite normal immune function,adolescents may still suffer from herpes virus type 7 meningitis,so clinicians should be vigilant and take effective treatment measures in time.

A Novel 2-dimensional Multiplex qPCR Assay for Single-Tube Detection of Nine Human Herpesviruses

Human herpesviruses are double-stranded DNA viruses that are classified into nine species.More than 90%of adults are ever infected with one or more herpesviruses.The symptoms of infection with different herpesviruses are diverse ranging from mild or asymptomatic infections to deadly diseases such as aggressive lymphomas and sarcomas.Timely and accurate detection of herpesvirus infection is critical for clinical management and treatment.In this study,we established a single-tube nonuple qPCR assay for detection of all nine herpesviruses using a 2-D multiplex qPCR method with a house-keeping gene as the internal control.The novel assay can detect and distinguish different herpesviruses with 30 to 300 copies per 25µL single-tube reaction,and does not cross-react with 20 other human viruses,including DNA and RNA viruses.The robustness of the novel assay was evaluated using 170 clinical samples.The novel assay showed a high consistency(100%)with the single qPCR assay for HHVs detection.The features of simple,rapid,high sensitivity,specificity,and low cost make this assay a high potential to be widely used in clinical diagnosis and patient treatment.

Genotypic analysis on the ORF-K1 gene of human herpesvirus 8 from patients with Kaposi's sarcoma in Xinjiang,China

Human herpesvirus 8 （HHV-8） is thought to be essential for the development of all forms of Kaposi＇s sarcoma （KS）. HHV-8 DNA is present virtually in all KS tumor biopsy samples. Genes at both ends of the HHV-8 genome have been shown to vary considerably. Seven major molecular subtypes of HHV-8 were defined based on the amino acid sequence of the open reading frame K1 （ORF-K1）, generally known as A, B, C, D, E, F, and Z. Most strains collected worldwide were clustered into two subtypes （A and C）. Here, the K1/VRI region of HHV-8 was amplified by nested PCR in 22 （81.48%） of 27 cases from Xinjiang Uygur Autonomous Region, a province in northwestern China. Phylogenetic analysis on the basis of the K1/VR1 amino acid sequence indicated that the majority of these KS patients were infected by subtype C HHV-8 （n = 18, including 15 belonging to the C2 group）, and several by subtype A （n = 4, including 3 being the A1 group）. This is the first report of subtype A HHV-8 in China. Furthermore, the correlations between different forms and lesions of KS and different subtypes of HHV-8 were analyzed. The findings showed that subtype A HHV-8 resulted in significantly more frequent mucosal KS lesions than subtype C. However, there was no obvious correlation between different forms of KS and different subtypes of HHV-8.

Reactivation of Latent Infection of Human Herpesvirus 8 in BC-3 Cells from Primary Effusion Lymphoma by Recombinant CytokinesSimilar to that Produced by HIV-1-infected T Cells

Objective: To study and confirm that recombinant cytokines similar to those produced by HIV-1 infected T cells induced lytic cycle replication of human herpesvirus 8 (HHV-8) in BC-3 cells, another cell line from primary effusion lymphama(PEL). Methods: The persistent stimulation of BC-3 was conducted by several cytokines known to be produced by HIV-1-infected T cells and important in growth and proliferation of Kaposi's sarcoma(KS)cells in vitro, such as the interferon-γ (IFN-γ) , tlie hepatocyte growth factor/scatter factor (HGF / SF) , the Oncostain M(OSM) , and the tumor necrosis factor-α (TNF-α)which is not produced by HIV-1-infected T cells. Treated and untreated BC-3 cells were collected at the 3rd and 7th day of persistent stimulation, respectively. Immuno-histochemical (IHC) staining, Northern blot, quantitative PCR (real- time PCR ) and electron microscopy (EM) were carried out to detect the expression of immunogenic protein ORF59, messenger RNA (mRNA) of minor capsid protein ORF26, and the presence of viral particles of HHV-8 from treated and untreated BC-3 cells. Results: It showed that IFN-γ, HGF/SF, OSM, and TNF-α were found to induce an increase in mRNA expression of ORF26 when added individually to BC-3 cells. Particularly, ORF26 expression stimulated with IFN-γ and TNF-α respectively, increased 6. 1 and 2. 5-fold(from real-time PCR results)at the 7th day when compared with untreated BC-3 cells. Meanwhile, about 20% of IFN-γ stimulated BC-3 cells expressed ORF59 at the 7th day as compared with 1. 5% of untreated BC-3 cells when IHC staining was employed. In addition, viral particles of HHV-8 were readily identified in BC-3 cells stimulated with IFN-γ at the 7th day with EM analysis. Conclusion;TNF-α and recombinant cytokines being similar to those produced by HIV- 1 infected T Cells could really induce HHV- 8 lytic cycle replication in BC-3 cells, another cell line of PEL.

Human herpesvirus 6A induces apoptosis of HSB-2 cellsvia a mitochondrion-related caspase pathway

Apoptosis plays an important role in the pathogenesis of viral infections.In this study,we investigated the cell death processes during productive HHV-6A infection and the underlying mechanisms.Annexin V-PI staining and electron microscopy indicated that HHV-6A is a strong inducer of apoptosis.HHV-6A infection decreased mitochondrial transmembrane potential and led to morphological changes of mitochondria.The cell death was associated with activation of caspase-3 and cleavage of DNA repair enzyme poly （ADP-ribose） polymerase,which is known to be an important substrate for activated caspase-3.Caspase-9 was activated significantly in HHV-6A-infected cells,whereas caspase-8 was not activated obviously.Moreover,HHV-6A infection upregulated Bax and downregulated Bcl-2.This is the first demonstration of mitochondrion-mediated,caspase-dependent apoptosis in HHV-6A-infected cells.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

Effectof the Local Strain CN5 of Human Herpesvirus 6 on CD Molecule Expression and PHA-Induced Proliferation of Cord Blood or Peripheral Blood Mononuclear Cells

Cord blood mononuclear cells (CBMC's) and adult peripheral blood mononuclear cells (PBMCs), cultured with RPMI 1640 medium containing 20% fetal calf serum plus 5 mg/L PHA and 10 mg/L IL-Z, were inoculated with CN5 strain of human herpesvirus 6 (HHV 6 ) isolated from a Chinese patient with exanthem subitum and the CN5 infected cell lysates were added to cultures of CBMCs and PBMCs, so as to observe the effects of the local strain CN5 on expression of CD molecule or on proliferation of mononuclear cells by the methods of APAAP staining and MTT assay.The results were as follows: ①Expressions of some CD antigens of CBMCs and PBMCs could change after CN5 strain infection. In both cases, CD3 expresion was down-regulated while CD4 expression was up-regulated. There were no significant differences of CD2, CD8 and CD45RA expressions between the two groups with and without CN5 infection. But the ratio of CD4 to CD8 sigmificantly rose because of the increasing of CD4 positiveity. ②The lysates of CB5-infected CBMCs inhibited the liferation of PBMCs, not of CBMCs, in a protein concentration-dependent pattern. This inhibition was partially neutralized by specific antiserum to CN5, not by antisera to INF-α and TNF-α.

Establishment of method for seroepidermiological detection of human herpes virus 8 infection by using the fusion protein in the prokaryotic expression system as antigen for testing

To establish a sensitive and specific method for seroepidermiological detection of human her- pes virus 8(HHV-8)infection,three potent antigenic proteins encoded by open reading frames(ORFs) K8.1,65 and 73C in genome of HHV-8 were produced as glutathione S-transferase fusion protein in the prokaryotic expression system and was used as antigen for testing.The recombinant fusion protein ex- pressed in the prokaryotic expression vector E.coli BL21 was purified by glutathione Sepharose 4B affin- ity chromatography and was quantitated with SDS-PAGE.All these 3 fusion proteins produced in the pro- karyotic expression system showed good immunogenicity as demonstrated by Western blotting and could be recognized by mixed sera of patients with Kaposi′s sarcoma(KS).The immuno-reactivities of the single or compound fusion protein were determined by means of ELISA and compared with the traditional immu- nofluorescence assay(IFA)to determine their sensitivity and specificity of the test.It was demonstrated that the sensitivity of mixed-antigen ELISA method was significantly higher than that of IFA(81.8% vs 34.4%),while the specificity of the former was demonstrated to be 97.9%.The coincidence of the de- tection rate between these two methods was considerably high,approaching up to 90.0%.These results suggest that the mixed antigen ELISA assay appears to be a sensitive and specific method for sero-epide- miological detection of human herpesvirus 8 infection.

Partial Sequence Analysis of the Genome of Human Herpesvirus 7 YY5 Isolated from Saliva Samples

Objective To isolate and identify Nanjing local strains of Human Herpesvirus 7 (HHV 7), and to analyze their partial genome characteristic. Methods The saliva specimens were collected from 2 healthy adults and 5 children with kidney disease. After treatment with antibiotics and filtering. they were inoculated on to the phytohemagglutin stimulated umbilical cord blood mononuclear cells (CBMCs). When the infected cells presented the typical ballooning and polykaryotic cytopathic effects (CPE), we identified them by transmission electron microscopy and polymerase chain reaction. PCR product was also sequenced. Results Four strains were isolated from the seven saliva specimens. The 186 base pair fragment of the isolated strain YY5 PCR products was sequenced, which encoded part of the HHV 7 U10 gene. The DNA sequence revealed an identity of 57.5% and 36 0%, respectively with HHV 6 and human cytomegalovirus (HCMV). At the amino acid level, the similarity of 51.6% was found between HHV 7 and HHV 6, and that of 25.8% between HHV 7 and HCMV. Conclusion The isolated viruses were HHV 7, and 186 bp fragments revealed an identity with HHV 7 RK and JI of 100%.

DETECTION OF HUMAN HERPESVIRUS 6(HHV-6) DNA IN SALIVARY GLANDS BY THE POLYMERASE CHAIN REACTION

To assess the presence of HHV-6-Specific DNA in human salivary glands, eighteen specimens of salivary gland tissue were investigated using the polymerase chain reaction. Eight of nine parotid glands, five of seven submandibular glands and one of two sublingual glands were found to have amplification of the HHV-6-specific sequence. The findings suggest that salivary gland tissue is one of the potential sites for HHV-6 persistence following primary infection and that saliva is a vehicle for transmission of the virus.

Pityriasis Rosea-Like Eruption by Covid-19 Infection: A New Case Report

Background: Coronavirus disease (Covid-19) becomes a pandemic worldwide in 2020. Different dermatological manifestations associated with Covid-19 infection like maculopapular, morbilliform, urticarial, vesicular, chilblain-like, petechiae, purpura, and livedoid rashes. Pityriasis rosea (PR) and PR-like eruptions were recently reported to increase in coronavirus cases. Aim: To evaluate and review the literature on PR and PR-like eruption associated with Covid-19 disease. Case Report: A 24-year-old female patient presented with a history of asymptomatic PR-like eruption that started during infection with the Covid-19 virus and resolved spontaneously over 14 weeks without therapy. Conclusion: PR and PR-like eruptions are not uncommon with Covid-19 disease and could be the early manifestation of SARS-CoV-2 virus infection.

Primary Effusion Lymphoma in a HIV-1/2-Infected Patient

Background: Primary effusion lymphoma (PEL) is a lymphoid proliferation related to Kaposi sarcoma herpesvirus 8/human herpesvirus 8 (KSHV/HHV8) that affects mainly human immunodeficiency virus (HIV) infected individuals but can also occur in other immunodeficiency settings. It is characterized by lymphomatous effusions in different serous body cavities without the presence of a detectable tumor mass. The diagnosis is challenging and the clinical outcomes are poor. Aim: The aim of this paper is to report a rare case of PEL in a man who have sex with women (MSW) with HIV-1/2 infection, history of visceral Kaposi sarcoma (KS) and the development of a seronegative arthritis previous to the lymphoproliferative disease diagnosis. PEL presented with ascites, was treated with high-dose chemotherapy and autologous stem cell transplantation, with a good clinical outcome. Case Presentation: We describe a case of a 48-year-old HIV-1/2-infected patient from a high HHV8 seroprevalent country, hospitalized following a three-month history of increased abdominal volume and general constitutional symptoms. Laboratory data revealed normocytic normochromic anemia and a high level of lactate dehydrogenase. A diagnostic paracentesis was performed with cytology compatible with high-grade B-cell lymphoma. Peritoneal fluid cytology showed large lymphoid cells expressing leucocyte-common antigen CD45 without expression of the CD20 antigen (B-lymphocytes) and positivity for HHV8 by immunocytochemical staining, compatible with the diagnosis of PEL.

Human Herpesvirus 6 Infection Complicated by Viral Encephalitis Following Liver Transplantation:A Case Report

Human herpesvirus 6(HHV-6)is a significant pathogen following solid organ transplantation.Here,we report a 64-year-old female with cirrhosis related to nonalcoholic fatty liver disease who underwent orthotopic allogeneic liver transplantation and was ultimately diagnosed with HHV-6 encephalitis through cerebrospinal fluid analysis and MRI.Empirical treatment with daily ganciclovir was initiated according to characteristics indicative of viral encephalitis 3 days before confirmed diagnosis.Subsequent improvement in symptoms was observed,with clearance of HHV-6 from the blood.The complex diagnosis and management of this case accentuate the possibility of serious consequences of HHV-6 infection in postoperative liver transplant patients.Clinicians must maintain a high index of suspicion for HHV-6 reactivation,especially given its association with immunosuppressive drug regimens.Prompt recognition and initiation of antiviral therapy are paramount,particularly when patients present with fever or psychiatric symptoms,as these may indicate HHV-6 encephalitis.

AIDS and associated malignancies

AIDS associated malignancies （ARL） is a major complication associated with AIDS patients upon irnmunosuppression. Chronically immunocompromised patients have a markedly increased risk of developing lymphoproliferative disease. In the era of potent antiretrovirals therapy （ARV）, the malignant complications due to HIV-1 infection have decreased in developed nations where ARV is administered, but still poses a major problem in developing countries where HIV-1 incidence is high and ARV is still not yet widely available. Even in ARV treated individuals there is a concern that the prolonged survival of many HIV-1 carriers is likely to eventually result in an increased number of malignancies diagnosed. Malignancies that were found to have high incidence in HIV-infected individuals are Kaposi＇s sarcoma （KS）, Hodgkin＇s disease （HD） and non-Hodgkin＇s lymphoma （NHL）. The incidence of NHL has increased nearly 200 fold in HIV-positive patients, and accounts for a greater percentage of AIDS defining illness in the US and Europe since the advent of HAART therapy. These AIDS related lymphomas are distinct from their counterparts seen in HIV- 1 seronegative patients. For example nearly haft of all cases of ARL are associated with the presence of a gamma herpesvirus, Epstein Barr virus （EBV） or human herpesvirus-8 （HHV-8）/Kaposi＇s sarcoma associated herpesvirus （KSHV）. The pathogenesis of ARLs is complex. B-cell proliferation driven by chronic antigenemia resulting in the induction of polyclonal and ultimately monoclonal lymphoproliferation may occur in the setting of severe immunosuppression.

Isolated ileal perforation due to cytomegalovirus reactivation during management of terbinafine hypersensitivity

We report a case of 71-year-old man who developed a hypersensitivity syndrome associated with terbinafine. He was placed on terbinafine (250 mg/d) for the treatment of tinea pedis due to diabetes mellitus. Following the treatment with terbinafine, he developed druginduced hypersensitivity syndrome (DIHS). Systemic corticosteroid led to transient improvement of his clinical manifestations. Three months after disease onset, he presented with panperitonitis due to ileal perforation, and underwent an emergency operation. The affected ileum was resected and ileostomy was performed in the terminal ileum. Cytomegalovirus (CMV)-specific IgG antibodies were significantly increased, high-titer CMV antigenemia was detected, and pathological examination of the resected ileum confirmed CMV infection. Based onthese observations, we strongly recommend that physicians monitor reactivation of the family of herpesvirus other than herpesvirus 6, to manage DIHS properly.

Intestinal Kaposi's sarcoma may mimic gastrointestinal stromal tumor in HIV infection

Diffuse intestinal Kaposi＇s sarcoma shares macroscopic and histopathologic features with gastrointestinal stromal tumors. Correct diagnosis may pose a clinical challenge. We describe the case of a young HIV-1-infected African lady without advanced immunodeficiency, who presented with a diffuse spindle cell tumor of the gut. Initial diagnosis was of a gastrointestinal stromal tumor, based on endoscopy and histopathology. Further evaluation revealed evidence for human herpesvirus 8 （HHV8） and the diagnosis had to be changed to diffuse intestinal Kaposi＇s sarcoma. Antiretroviral triple therapy together with chemotherapy was commenced, and has led to the rapid remission of intestinal lesions. With a background of HIV infection, the presence of HHV8 as the causative agent of Kaposi＇s sarcoma should be determined, as distinct treatment is indicated.

Effects of HHV-6 Infection in Vitro on Cytokines Induction and NK Activity of Peripherial Blood Mononuclear Cells

Three Nanjing local strains (CN5, CN8, CN10) of human herpesvirus 6 (HHV-6] had been isolated and identified by electronmicroscopy, antibody reactions with IFA and specific PCR. The purpose of this paper was to study effects on cytokines synthesis and NK activity of peripheral blood mononuclear cells(PBMC) in vitro post infection of human herpesvirus 6. PBMCs cultural supernatants were collected at different hours post infection. Cytokines such as TNF-α, IFN-α and IL-8 could be detected as early as 24 h post infection, plateaued at 48h, and then decreased gradually. The levels of these cytokines in infected PBMCs supernatants were markedly higher than those in uninfected ones, but the IL-6 level was lower than that of uninfected one. These differences between infected and uninfected groups were all significant(P<0.05). There were no differences in the induction of TNF-α, IFN-α and IL-8 between the local strain CN8 and GS strain (all P>0.05), while the inhibition of IL-6 production induced by GS strain was more prominant than that induced by CN8 (P<0.05). It was also found that NK activity was augmented at 24h post infection, which was more striking in CN8 strain infection group than in GS strain infection group (P<0.05), after then, it was gradually decreased. From these reults, it could be inferred that the increase of cytokines synthesis and augmentation of NK activity were associated with convalescence and pathogenicity of the HHV-6 infection. GS strain, which belongs to groups A, disturbed the function of human immunity more remarkably than the local strain CN8, which belongs to group B.

Epstein-Barr virus induces human nasopharyngeal epithelial cells to escape from the replicative senescence

OBJECTIVE: To observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization. METHODS: The morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining. RESULTS: Morphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture. CONCLUSIONS: Nasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells.Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.

Detection of human herpesvirus 8 DNA in acute leukemia patients

Objective To determine the prevalence of human herpesvirus 8 (HHV-8) DNA in acute leukemia (AL) patients. Methods The presence of HHV-8 DNA sequences in peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC) from 50 AL patients was examined using polymerase chain reaction (PCR). Nine human hematopoietic cell lines and PBMC from 30 normal donors were also included. Results HHV-8 DNA sequences were detected in one case of acute myelogenous leukemia (AML). The specimens from the bone marrow aspirate, peripheral blood and serum of this patient were all positive. None of the normal donors and human hematopoietic cell lines showed evidence of HHV-8 DNA. Conclusion The results suggest that the prevalence of HHV-8 is low in AL in China.