Limited data exist regarding whether a high-risk human papillomavirus (HR-HPV) infection increases the risk of developing renal cell carcinoma. The aim of this study was to investigate whether HPV infection has a role in the pathogenesis or development of a certain histological subtype of renal cell carcinoma. Formalin-fixed paraffin-embedded (FFPE) specimens of 122 patients with histopathologically proven renal cell carcinoma and their respective peritumoral tissues were examined. The presence of HPV-DNA was determined by a combination of MY/GP+ consensus primers and HPV-16/18 type specific nested PCRs followed by direct sequencing. Catalyzed signal-amplified colorimetric in situ hybridization (CSAC-ISH) technique was applied to determine the physical status of viral genome. The expression of p16INK4a and HPV L1 capsid proteins was evaluated using immunohistochemistry. HPV genome was detected in 37 (30.3%) tumor specimens and their four (4.1%) corresponding peritumoral tissues. HPV-18 was the most common viral type identified followed by HPV-16 and 58. Immunoexpression of p16INK4a was detected in 24 (20.3%) cases. Data analysis showed a significant correlation between p16INK4a expression and the presence of HR-HPV DNA (P
Genetic characterization of measles viruses (MVs) combined with acquisition of epidemiologic information is essential for measles surveillance programs used in determining transmission pathways. This study describes the molecular characterization of 26 MV strains (3 from 2010, 23 from 2011) obtained from urine or throat swabs harvested from patients in Turkey. MV RNA samples (n = 26) were subjected to sequence analysis of 450 nucleotides comprising the most variable C-terminal region of the nucleoprotein (N) gene. Phylogenetic analysis revealed 20 strains from 2011 belonged to genotype D9, 3 to D4, 2 strains from 2010 to genotype D4 and 1 to genotype B3. This study represents the first report describing the involvement of MV genotype D9 in an outbreak in Turkey. The sequence of the majority of genotype D9 strains was identical to those identified in Russia, Malaysia, Japan, and the UK. Despite lack of sufficient epidemiologic information, the presence of variants observed following phylogenetic analysis suggested that exposure to genotype D9 might have occurred due to importation more than once. Phylogenetic analysis of five genotype D4 strains revealed the presence of four variants. Epidemiological information and phylogenetic analysis suggested that three genotype D4 strains and one genotype B3 strain were associated with importation. This study suggests the presence of pockets of unimmunized individuals making Turkey susceptible to outbreaks. Continuing molecular surveillance of measles strains in Turkey is essential as a means of acquiring epidemiologic information to define viral transmission patterns and determine the effectiveness of measles vaccination programs designed to eliminate this virus.
Expansion of antiretroviral treatment programs have led to the growing concern for the development of antiretroviral drug resistance. The aims were to assess the prevalence of drug resistant HIV-1 variants and to identify circulating subtypes among HAART-naïve patients. Plasma specimens from N = 100 HIV+ HAART-naïve adult were collected between March 2008 and August 2010 and viral RNA were extracted for nested PCR and sequenced. PR-RT sequences were protein aligned and checked for transmitted drug resistance mutations. Phylogenetic reconstruction and recombination analysis were performed to determine the genotypes. Based on the WHO consensus guidelines, none of the recruited patients had any transmitted drug resistance mutations. When analyzed against the Stanford guidelines, 35% of patients had at least one reported mutation that may reduce drug susceptibility to PI (24%), NRTI (5%), and NNRTI (14%). The commonly detected mutation that may affect current first line therapy was V179D (3%), which may lead to reduced susceptibility to NNRTI. The predominant circulating HIV-1 genotypes were CRF01_AE (51%) and CRF33_01B (17%). The prevalence of unique recombinant forms (URF) was 7%; five distinct recombinant structures involving CRF01_AE and subtype B' were observed, among them a cluster of three isolates that could form a novel circulating recombinant form (CRF) candidate. Transmitted drug resistance prevalence among HAART-naïve patients was low in this cohort of patients in Kuala Lumpur despite introduction of HAART 5 years ago. Owing to the high genetic diversity, continued molecular surveillance can identify the persistent emergence of HIV-1 URF and novel CRF with significant epidemiological impact.
Relatively little is known about the burden of influenza in tropical countries. The seroprevalence of pandemic influenza A (H1N1) 2009, seasonal H1N1 and H3N2 was determined in Kuala Lumpur, Malaysia. Pre- and post-pandemic residual laboratory sera were tested by hemagglutination-inhibition. The seroprevalence of A(H1N1)pdm09 increased from 3.7% pre-pandemic to 21.9% post-pandemic, giving an overall cumulative incidence of 18.1% (95% CI, 13.8-22.5%), mainly due to increases in those <5, 5-17, and 18-29 years old. In contrast with findings from USA, Europe, and Australia, pre-existing seroprevalence to A(H1N1)pdm09 was low at 5.6% in the elderly age group of >55 years. A(H1N1)pdm09 affected almost a third of those <30 years in Kuala Lumpur. Pre-pandemic seroprevalence was 14.7% for seasonal H1N1 and 21.0% for H3N2, and these rates did not change significantly after the pandemic. Seasonal and pandemic influenza cause a considerable burden in tropical Malaysia, particularly in children and young adults.
Phylogenetic analysis was performed on hepatitis B virus (HBV) strains obtained from 86 hepatitis B surface antigen (HBsAg) positive donors from Thailand originating throughout the country. Based on the S gene, 87.5% of strains were of genotype C while 10.5% were of genotype B, with all genotype B strains obtained from patients originating from the central or the south Thailand. No genotype B strains were found in the north of Thailand. Surprisingly, one patient was infected with a genotype H strain while another patient was infected with a genotype G strain. Complete genome sequencing and recombination analysis identified the latter as being a genotype G and C2 recombinant with the breakpoint around nucleotide position 700. The origin of the genotype G fragment was not identifiable while the genotype C2 fragment most likely came from strains circulating in Laos or Malaysia. The performance of different HBsAg diagnostic kits and HBV nucleic acid amplification technology (NAT) was evaluated. The genotype H and G/C2 recombination did not interfere with HBV detection.
Enterovirus 71 (EV71) infection may cause severe neurological complications, particularly in young children. Despite the risks, there are still no commercially available EV71 vaccines. Hence, a candidate vaccine construct, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP1(1-100) ) protein, was evaluated in a mouse model of EV71 infection. Previously, it was shown that this protein construct provoked a strong immune response in vaccinated adult rabbits. That study, however, did not address the issue of its effectiveness against EV71 infection in young animals. In the present study, EV71 viral challenge in vaccinated newborn mice resulted in more than 40% increase in survival rate. Significantly, half of the surviving mice fully recovered from their paralysis. Histological analysis of all of the surviving mice revealed a complete clearance of EV71 viral antigens from their brains and spinal cords. In hind limb muscles, the amounts of the antigens detected correlated with the degrees of tissue damage and paralysis. Findings from this study provide evidence that immunization with the NPt-VP1(1-100) immunogen in a newborn mouse model confers partial protection against EV71 infection, and also highlights the importance of NPt-VP1(1-100) as a possible candidate vaccine for protection against EV71 infections.
Hepatitis B virus (HBV) and high liver iron deposits have both been associated with the development of cirrhosis. Among HBV factors, genotype and mutations in the basal core promoter (BCP) and precore regions have been most frequently studied but the evidence for a positive association with cirrhosis has been inconsistent. In this study, sera from persons with chronic HBV infection with and without cirrhosis were used for whole HBV genome analysis and for the estimation of serum iron marker (serum iron or ferritin) levels. Single codon analysis showed that the precore wild-type, TGG (nt 1,895-1,897), gave the highest accuracy (77.5%) for the identification of cirrhosis compared to other codons. When TGG was analyzed together with the precore start codon wild-type, ATG (nt 1,814-1,816), the accuracy was improved to 80.0% (odds ratio=35.29; 95% confidence interval=3.87-321.93; Phi=0.629; P<0.001). When the serum iron marker was included for analysis, it was clear that a combination of a precore wild-type and high serum iron marker gave a better accuracy (90.0%) (odds ratio=107.67; 95% confidence interval=10.21-1,135.59; Phi=0.804; P<0.001) for the identification of cirrhosis than either biomarker alone. It appeared that a combined use of both these biomarkers might help to predict the development of cirrhosis in a person with chronic HBV infection, but longitudinal studies are required to test this hypothesis.
The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT-PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV-A, 40 HEV-B, and 10 HEV-C) tested, 100% concordance in HEV-A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV-B and HEV-C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV-A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV-A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease.
This study examined the temporal distribution of rotavirus genotypes in Malaysia. Rotaviruses from children with diarrhea admitted to hospitals in 1996 (n = 93) and 2007 (n = 12) in two different regions of Peninsular (West) Malaysia were analyzed for their G and P genotypes using a hemi-nested RT-PCR assay. In the 2007 samples, the dominant strain was G9P. It was identified in 42% of the samples. Different strains all possessing the G1 genotype were identified in the rest of the samples. In contrast, 81% of the samples collected in 1996 were the G1P strain. No strains with G9 genotype were detected in samples collected in 1996.
The influenza activity and circulation of influenza viruses in Lombardy (the most populous Italian region) were observed during two consecutive seasons (2005/2006 and 2006/2007) characterized by low influenza activity by the Italian Influenza Surveillance Network. The molecular characteristics of circulating viruses were analyzed to evaluate the introduction of new variants and emergence of vaccine-escape viruses. In both seasons, the epidemic in Lombardy was sustained almost exclusively by influenza A viruses, accounting for 80.5% and 93.6% of total detections, respectively, and the co-circulation of A/H3 viruses belonging to distinct phylogenetic groups was observed. The A/H1N1 viruses isolated during the 2005/2006 season were closely related to A/New Caledonia/20/99, while the hemagglutinin (HA) sequences of the A/H1N1 viruses from the 2006/2007 season exhibited a greater diversity. These viruses were A/Solomon Islands/3/2006-like and showed several variants. All B isolates were similar to B/Malaysia/2506/2004 belonging to the B/Victoria/2/87-lineage. Influenza B virus was the dominant virus in Europe in the 2005/2006 season and accounted for the 20% of total detections in Lombardy. Overall, the viruses studied presented heterogeneity in their HA sequences suggesting the circulation of a miscellaneous set of variants during the two seasons notwithstanding the medium-low activity of influenza. The importance of virological surveillance of influenza viruses is recognized widely and the molecular characterization of the viruses, especially in vaccinated subjects, is of particular importance to evaluate the introduction and circulation of new variants.
The complete VP1 protein of EV71 was truncated into six segments and fused to the C-terminal ends of full-length nucleocapsid protein (NPfl) and truncated NP (NPt; lacks 20% amino acid residues from its C-terminal end) of newcastle disease virus (NDV). Western blot analysis using anti-VP1 rabbit serum showed that the N-terminal region of the VP1 protein contains a major antigenic region. The recombinant proteins carrying the truncated VP1 protein, VP1(1-100), were expressed most efficiently in Escherichia coli as determined by Western blot analysis. Electron microscopic analysis of the purified recombinant protein, NPt-VP(1-100) revealed that it predominantly self-assembled into intact ring-like structures whereas NPfl-VP(1-100) recombinant proteins showed disrupted ring-like formations. Rabbits immunized with the purified NPt-VP(1-100) and NPfl-VP(1-100) exhibited a strong immune response against the complete VP1 protein. The antisera of these recombinant proteins also reacted positively with authentic enterovirus 71 and the closely related Coxsackievirus A16 when analyzed by an immunofluorescence assay suggesting their potential as immunological reagents for the detection of anti-enterovirus 71 antibodies in serum samples.
Nipah virus infection of porcine stable kidney cells (PS), human neuronal cells (SK-N-MC), human lung fibroblasts cells (MRC-5), and human monocytes (THP-1) were examined. Rapid progression of cytopathic effects (CPE) and cell death were noted in PS cell cultures treated with Nipah virus, followed by MRC-5, SK-N-MC, and THP-1 cell cultures, in descending order of rapidity. Significant increase in the intracellular Nipah virus RNA occurred beginning at 24 hr PI in all the infected cells. Whereas, the extracellular release of Nipah virus RNA increased significantly beginning at 48 and 72 hr PI for the infected MRC-5 cells and PS cells, respectively. No significant release of extracellular Nipah virus RNA was detected from the Nipah virus-infected SK-N-MC and THP-1 cells. At its peak, approximately 6.6 log PFU/microl of extracellular Nipah virus RNA was released from the Nipah virus-infected PS cells, with at least a 100-fold less virus RNA was recorded in the Nipah virus-infected SK-N-MC and THP-1. Approximately 15.2% (+/-0.1%) of the released virus from the infected PS cell cultures was infectious in contrast to approximately 5.5% (+/-0.7%) from the infected SK-N-MC cells. The findings suggest that there are no differences in the capacity to support Nipah virus replication between pigs and humans in fully susceptible PS and MRC-5 cells. However, there are differences between these cells and human neuronal cells and monocytes in the ability to support Nipah virus replication and virus release.
An outbreak of acute hemorrhagic conjunctivitis occurred in French Guiana between April and July 2003, with approximately 6,000 cases in the two major cities Kourou and Cayenne. Since acute hemorrhagic conjunctivitis is not a notifiable disease in France, there was no registration of the number of cases. Therefore, these were estimated by comparing the consumption of antibiotic eye drops and ophthalmic ointments during 2002 and 2003. The outbreak rapidly spread into the Caribbean Islands, causing an outbreak in Guadeloupe in October. Viral isolates from conjunctival swabs of 16 patients were confirmed to be enterovirus by PCR directed to the 5' UTR of the genome. The isolates could not be neutralized by the Melnick intersecting pools, but were shown to be CV-A24 variant by limited sequencing within the VP1 and 3C regions of 12 strains. Phylogenetic analysis revealed that they were similar to the genotype III strains causing outbreaks in Korea 2002 and Malaysia 2003. The previous outbreak of conjunctivitis caused by CV-A24 in the Caribbean in the 1980s was also introduced from Asia, and disappeared after 3 years. This new introduction from Asia and its rapid spread into the Caribbean, where the infection disappeared after a few months, indicates that the CV-A24 variant has a different epidemiological pattern in this region compared to South East Asia, since it has not established an endemic infection. It had to be reintroduced from Asia, where it has been circulating since the 1970s.
A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.
Rubella has been listed as a mandatory notifiable disease in Taiwan since 1988. Because of high coverage rates with an effective vaccine, rubella cases have decreased dramatically in Taiwan since 1994. However, rubella outbreaks still occur due to imported transmission. Five large clusters were detected in Taiwan from 2007 to 2011. In 2007, one cluster was caused by rubella genotype 1E viruses that were imported from Vietnam, whereas another cluster was caused by genotype 2B viruses and was untraceable. In 2008, two clusters were caused by different lineages of genotype 1E viruses that were imported from Malaysia. In 2009, a cluster that was caused by genotype 2B viruses was associated with imported cases from Vietnam. The rubella viruses from 124 confirmed cases from 2005 to 2011 were characterized, and the data revealed that these viruses were distributed in the following four genotypes: 1E (n = 56), 1h (n = 1), 1j (n = 4), and 2B (n = 63). Of these viruses, 93 (75%) were associated with imported cases, and 43 of 56 genotype 1E viruses were associated with imported cases from China, Vietnam, Malaysia, and Indonesia. One genotype 1h virus was imported from Belarus, and three of four genotype 1j viruses were imported from the Philippines. Of 63 rubella genotype 2B viruses, 46 were imported from Vietnam, Thailand, Malaysia, China, Germany, and South Africa. Molecular surveillance allows for the differentiation of circulating rubella viruses and can be used to investigate transmission pathways, which are important to identify the interruption of endemic virus transmission.
Infection with the hepatitis B virus (HBV) may lead to an acute or chronic infection. It is generally accepted that the clinical outcome of infection depends on the balance between host immunity and viral survival strategies. In order to persist, the virus needs to have a high rate of replication and some immune-escape capabilities. Hence, HBVs lacking these properties are likely to be eliminated more rapidly by the host, leading to a lower rate of chronicity. To test this hypothesis, 177 HBV genomes from acute non-fulminant cases and 1,149 from chronic cases were retrieved from GenBank for comparative analysis. Selection of candidate nucleotides associated with the disease state was done using random guess cut-off and the Bonferroni correction. Five significant nucleotides were detected using this filtering step. Their predictive values were assessed using the support vector machine classification with five-fold cross-validation. The average prediction accuracy was 61% ± 1%, with a sensitivity of 24% ± 1%, specificity of 98% ± 1%, positive predictive value of 92% ± 4% and negative predictive value of 56% ± 1%. BCP/X, enhancer I and surface/polymerase variants were found to be associated almost exclusively with acute hepatitis. These HBV variants are novel potential markers for non-progression to chronic hepatitis.
The immunodominant region of hepatitis B virus (HBV) located in the viral small surface antigen (S-HBsAg) elicits virus-neutralizing and protective antibodies. In order to develop an easy and inexpensive method to produce this region without the need for extensive purification, amino acid residues 111-156 of S-HBsAg were fused to the C-terminal end of the 10B capsid protein of T7 phage. Western blotting and ELISA confirmed the expression of the recombinant protein on the surface of the phage particles. The recombinant phage exhibited the antigenic and immunogenic characteristics of HBsAg, illustrating its potential as an immunological reagent and vaccine.
Suboptimal viral suppression and CD4 response to antiretroviral treatment (HAART) is known to cause poor outcomes with the increase cost of treatment. We aimed to assess factors associated with such control among HIV/AIDS patients in Malaysia. Four hundred and six HIV/AIDS patients, using Antiretroviral Therapy (ART) for at least the past three months, treated as outpatients at medication therapy adherence clinics (MTAC) were recruited. CD4 cell counts, viral load readings along with co-variants such as socio-demographic factors, adverse drug reactions, comorbidities, and medication record were obtained. Statistical Package for Social Sciences (SPSS(®) ) version 18 and STATA IC(®) version 12 were used for data analysis. CD4 counts were found highest among those within the age category 41-50 years (390.43 ± 272.28), female (402.64 ± 276.14), other ethnicities (400.20 ± 278.04), and participants with no formal education (414.87 ± 290.90). Patients experiencing adverse effects had a 2.28 (95%CI:1.25-4.18) fold greater risk of poor CD4 control, while patients with comorbidities had 2.46 (95%CI:1.02-5.91) fold greater risk of mild viral suppression. Adverse drug reactions, co-morbidities were found to be significantly associated with poor immunological and virological outcomes in HIV/AIDS patients. However, a comprehensive evaluation is needed to better understand other confounders.
Study site: Medication therapy adherence clinic, Hospital Sungai Buloh, Selangor, Malaysia
According to WHO, Malaysia has been classified as a concentrated epidemic country due to progression of HIV infection in the population of injecting drug users. The main objectives of current study are to determine the prevalence of HBV among HIV-positive individuals in a tertiary care hospital of Malaysia and to assess the predictors involved in the outcomes of HIV-HBV co-infected patients. A retrospective, cross-sectional study is conducted at Hospital Palau Pinang, Malaysia. The collection of socio-demographic data as well as clinical data is done with the help of data collection form. Data were analyzed after putting the collected values of required data by using statistical software SPSS version 20.0 and P > 0.05 is considered as significant. Results show that the overall prevalence of HBV was 86 (13%) including 495 (74.5%) males and 169 (25.5%) females among a total of 664 HIV-infected patients. It was observed that there is a high prevalence of HIV-HBV co-infection in males 76 (11.4%) as compared to females 10 (1.5%) (P = 0.002). The median age of the study population was 39 years. The statistical significant risk factors involved in the outcomes of HIV-HBV co-infected patients were observed in the variables of gender, age groups, and injecting drug users. The findings of the present study shows that the prevalence of HBV infection among HIV-positive patients was 13% and the risk factors involved in the outcomes of HIV-HBV co-infected patients were gender, age, and intravenous drug users.