Detection of human metapneumovirus in respiratory secretions by reverse-transcriptase polymerase chain reaction, indirect immunofluorescence, and virus isolation in human bronchial epithelial cells

Over two winters in Newcastle upon Tyne, respiratory secretions, negative by immunofluorescence staining for other respiratory viruses, were tested for the presence of human metapneumovirus (HMPV) by RT-PCR. In the second winter, specimens were also tested by immunofluorescence staining with an anti-HMPV polyclonal rabbit antiserum and immunofluorescence positive specimens were inoculated into a line of human bronchiolar cells, 16HBE140. Overall, 55 of 549 (10%) specimens tested were positive for HMPV by RT-PCR. Of 162 specimens tested by both RT/PCR and immunofluorescence staining, 23 were positive by both techniques. Of five specimens positive by RT-PCR alone, only one was confirmed with a second set of primers. Of three specimens positive by immunofluorescence alone, only one was confirmed by virus culture. All four previously recognized sub-genotypes of the virus were identified by both RT-PCR and immunofluorescence staining. Sub-genotype A1 was prevalent in the first winter and B1 prevalent in the second. HMPV replication and virus isolation rates were higher in 16HBE140 cells than in monkey kidney cells and did not require exogenous trypsin. Low passage isolates of both sub-genotypes A2 and B1 replicated slowly reaching peak titers only 12 days after inoculation. In summary, single round RT/PCR and immunofluorescence staining with a polyclonal rabbit antiserum proved of equal sensitivity in the diagnosis of HMPV infection in respiratory secretions both detecting 96% of confirmed positive specimens. 16HBE40 cells provided a significant improvement on monkey kidney cells for the isolation and propagation of the virus.     

北京新发现的人偏肺病毒N蛋白编码基因的序列分析

目的　了解新近从北京地区发现的人类偏肺病毒 (humanmetapneumovirus ,HMPV)N蛋白编码基因的特征。方法　从经RT PCR检测HMPV阳性的临床鼻咽洗液标本中进行HMPVN蛋白全基因扩增、克隆至pUCm T载体中并进行测序 ,与GenBank中的多株HMPVN蛋白基因序列进行比较和进化树分析。结果　经扩增并测序 ,标本BJ1816和BJ1887N蛋白基因全长为 1185bp ,编码 394个氨基酸。BJ1816N蛋白基因与国际上第一株HMPV分离株NDL0 0 1和GenBank中其它的多株HMPV的核苷酸和氨基酸同源性分别为 86 .2 %～ 99.0 %和 96 .2 %～ 99.7%。BJ1887N蛋白基因与以上进行比较的各株HMPV的核苷酸和氨基酸同源性分别为 86 .6 %～ 97.0 %和 96 .4 %～ 99.5 %。BJ1816和BJ1887之间N基因的核苷酸和氨基酸同源性分别为 86 .6 %和 96 .4 %。提示BJ1816和BJ1887分属于HMPV的两个不同的基因进化簇。结论　从基因水平进一步证明新近发现的婴幼儿肺炎的新病原确为HMPV ,提示北京地区婴幼儿中同时存在两个不同进化簇 (即基因型 )的HMPV感染。     

A study of the genetic variability of human respiratory syncytial virus in Croatia, 2006–2008

Human respiratory syncytial virus (HRSV) is a common etiological agent of acute lower respiratory tract disease in infants. The molecular epidemiology of HRSV in Croatia over four consecutive seasons (from 2006 to 2008) was investigated. A total of 72 HRSV samples were chosen from 696 screened cases in a pediatric clinic in Zagreb. Molecular characterization of HRSV revealed the predominance of HRSV group B viruses in the first two epidemic seasons and HRSV group A viruses in the next two seasons. According to the phylogenetic analysis, NA1 and BA9 were the predominant circulating HRSV genotypes detected during the study. Overall, 82.9% of all HRSV A strains belonged to the NA1 genotype. The HRSV B genotype BA9, detected in two consecutive seasons (2006 and 2007), was the predominant circulating HRSV B genotype, accounting for 80.6% of all HRSV B strains. This study provides data on the circulation pattern of HRSV genotypes in Croatia and their molecular characterization. J. Med. Virol. 84:1985–1992, 2012. © 2012 Wiley Periodicals, Inc.     

Norovirus genotypes involved in the outbreaks of gastroenteritis in Croatia during the winter season 2004--2005

Seven outbreaks and four sporadic cases of the non-bacterial gastroenteritis caused by a norovirus (NoV) were detected in Croatia between November 2004 and February 2005. An enzyme immunoassay (EIA) and three different RT-PCRs for the viral polymerase (ORF1 RT-PCR) and genogroup I (GI) or II (GII) of capsid gene regions (GI-ORF2 RT-PCR; GII-ORF2 RT-PCR) were performed to detect NoV in 21 stool samples. To characterize NoVs, sequencing of the ORF1 region was performed on 12 RT-PCR positive samples, whereas the ORF2 region was sequenced for 5 cases. Four outbreaks were caused by the genotype GII.4 (Lordsdale) and one outbreak was caused by the genotype GI.1 (Norwalk). One of the outbreaks was characterized as potentially mixed GII.4 and GI.1 infection. In the monitored period, genotype GII.4 dominated as the cause of noroviral infections in adults.     

Detection of human cytomegalovirus DNA in perilymph of patients with sensorineural hearing loss using real-time PCR

Although cytomegalovirus (CMV) has been detected in the inner ear fluid of patients who succumbed to the complications of symptomatic congenital CMV infection, it has not been detected in the inner ear fluid of living patients. In this study, real-time polymerase chain reaction (PCR) was used to measure CMV DNA in clinical samples (including perilymph) collected from five patients with deafness. In case 1, diagnosed as a symptomatic congenital CMV infection, 3 copies/microl of CMV DNA were detected in perilymph, although no viral DNA was detected in peripheral blood mononuclear cells (PBMCs) or urine samples. In case 4, a suspected asymptomatic congenital CMV infection, 36 copies/microg of CMV DNA were detected in PBMCs, but neither perilymph nor urine contained viral DNA. Likewise, in case 5, a case of deafness of unknown origin, 48 copies/microg of CMV DNA were detected in the PBMCs, but none in the perilymph or urine. CMV DNA was not detected in the samples obtained from the remaining two cases with deafness of unknown etiology. To our knowledge, this is the first report to detect CMV DNA in an inner ear sample obtained from a living human subject.     

Limited Inter- and Intra-patient Sequence Diversity of the Genetic Lineage A human metapneumovirus fusion gene

Human metapneumovirus (hMPV) is associated with respiratory tract illness especially in young children. Two hMPV genetic lineages, A and B, and four sublineages A1, A2 and B1, B2 have been defined. Infection with hMPV occurs through membrane fusion mediated by the hMPV fusion (F) protein. In this study, the inter- and intra-patient genetic diversity of the lineage A hMPV F gene was investigated. Ten isolates were collected from 10 hMPV infected children. Viral RNA was isolated and amplified, and approximately 10 clones from each isolate were sequenced. Altogether 108 clones were successfully sequenced. The average interpatient sequence diversity was 1.68% and 1.64% at nucleotide and amino acid levels, respectively. The samples were divisible into two groups on the basis of intrapatient sequence diversity. In group 1 (4 children) the intra-patient sequence diversity was low (nt: 0.26–0.39%, aa: 0.51–0.94%) whereas group 2 (6 children) had a higher intra-patient sequence diversity (nt: 0.85–1.98%, aa: 1.08–2.22%). Phylogenetic analyses showed that the group 1 children harboured sublineage Al only, but interestingly group 2 children harboured both sublineages Al and A2, indicating they had been infected with at least two viruses. Several independent viruses contained premature stop codons in exactly identical positions resulting in truncated fusion proteins. Possibly this is a mechanism for immune system evasion. The F protein is a major antigenic determinant, and the limited sequence diversity observed lay emphasis on the hMPV F gene as a putative target for future vaccine development.     

Detection of human orthopoxvirus infections and differentiation of smallpox virus with real‐time PCR

We developed a real‐time PCR protocol to detect orthopoxviruses (OPVs) from different clinical specimens and to separate variola virus from other OPVs. In our protocol, we used automated nucleic acid extraction system together with real‐time PCR to create a simple, safe and fast procedure to obtain an initial result. The sensitivity was better by using designed hybridization probes as compared to SYBR green I for detection. The detection limit ranged from 13 to 1,300 copies per 20 µl reaction volume depending on the sample type. The PCR detected all OPVs pathogenic to human (variola, cowpox, monkeypox, vaccinia) as well as camelpox and ectromelia viruses. Amplification of variola virus sequences could be distinguished from other OPVs by melting curve analysis. We also demonstrated the applicability of the assay in human cases of cowpox and vaccinia virus infections. J. Med. Virol. 81:146–152, 2009. © 2008 Wiley‐Liss, Inc.     

Time course of hepatitis A viremia and viral load in the blood of human hepatitis A patients

The hepatitis A virus (HAV) is the most common etiological cause of acute hepatitis infections in humans in industrialized countries. Investigations into the viral load during HAV viremia, however, are rare. Therefore, correlation studies between viral load, biochemical, and specific serological markers have been undertaken. The group of sera comprised a series of multiple consecutive blood samples drawn from 11 patients at different times after onset of the disease. During the period up to 70 days after the onset of icterus, the individual range was at 1 x 10(3) to 3 x 10(4) HAV genome equivalents/ml. From day 75 until 120 after onset of the disease, the levels traced were at 10(3). In one case, it was possible to trace 1.25 x 10(4) genome equivalents/ml up to 180 days after onset of icterus and in two cases even up to 408 and 490 days viral load levels of 5 x 10(3) and 4 x 10(4) were detected, respectively. The same sera were used to measure IgM class antibodies to hepatitis A virus and the total anti-HAV. The results demonstrate that a direct correlation to peak levels of viral load exists with peak serum transaminase levels, but neither with peak anti-HAV IgM levels nor with total anti-HAV. Decreasing amounts of anti-HAV IgM tend to occur with decreasing amounts of HAV genome equivalents; and, vice versa, increasing amounts of total anti-HAV are accompanied by decreasing amounts of HAV genome equivalents. The longest duration of viremia was found in patients infected with HAV genotype IA.     

Monitoring of adenovirus from conjunctival scrapings in Japan during 2005--2006

A total of 189 conjunctival scrapings were collected from patients in Tokyo, Japan by monitoring adenovirus infection in community-based clinics during 2005 and 2006. Of the 189 samples, 155 (82%) had adenoviruses detected by polymerase chain reaction (PCR). The serotypes were determined by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, using a combination of endonucleases, such as HhaI, AluI, and HaeIII, and neutralization tests (NTs). PCR-RFLP identified five serotypes: serotype 3: 16.8%, serotype 4: 9.7%, serotype 8: 34.8%, serotype 11: 23.2%, and serotype 37: 15.5%. Adenovirus 8 was the most common serotype identified. A subset consisting of 25 isolates identified as adenovirus 8 from this study plus 25 isolates from Kawasaki were analyzed using PCR sequencing of the hexon gene. Compared with prototype adenovirus serotype 8 and serotype 9 derived in Tokyo and Kawasaki, these isolates shared 61.7-62.8% and 80.5-82.7% amino acid homology, respectively, suggesting that a variant adenovirus serotype 8 was involved in this outbreak, and is different from the prototype adenovirus 8 virus. This variant had not been detected in Japan prior to 1996 and appears to be the most common adenovirus type 8 involved in cases of epidemic keratoconjunctivitis in Japan at present.     

耐甲氧西林葡萄球菌荧光定量PCR检测方法的实验研究

目的建立一种简便、特异的mecA基因荧光定量PCR检测方法，用于耐甲氧西林葡萄球菌（MRS）的快速鉴定。方法以煮沸法快速制备DNA模板，采用SYBRGreenI随机参入法，建立mecA基因的实时荧光定量PCR检测体系。并对检测体系的敏感性、特异性和灵敏度进行评价。结果本法对纯菌落的检测敏感性和特异性分别为98．5％和96．9％，检测灵敏度可达10^1CFU／ml，最小检菌量约为3个菌／反应体系。结论本实验所设计的荧光定量PCR方法用于MRS的检测具有快捷、高敏感性、高特异性和高灵敏度的特点。适用于MRS的快速检测。     

Molecular characterization of influenza viruses circulating in Northern Italy during two seasons (2005/2006 and 2006/2007) of low influenza activity

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.     

Genetic and antigenic analysis of epidemic influenza viruses isolated during 2006-2007 season in Taiwan

Influenza viruses are some of the most active pathogens in Taiwan. The monitoring influenza activity has been coordinated by the Centers for Diseases Control, Taiwan, and the surveillance is based on integrated clinical and virological surveillance components. Data from sentinel physician networks and other sources, mainly hospitals were collected. During 2006-07 season, a total of 1724 cases of laboratory-confirmed influenza were reported by collaborating laboratories and sentinels, which was five fold higher than during the corresponding part of the 2005-06 season. Of the Taiwan isolates analyzed using post-infection ferret antisera, 1.5% were H1N1 (A/Hi), 21.5% H3N2 (A/H3), and 77.0% influenza B viruses. This reflects the predominance of influenza B viruses during 2006-07 season. In addition, continued antigenic drift was seen with the A/I-B viruses compared with the previous season's reference strains. However, an increasing number of recent A/H3 isolates characterized in our report were amantadine sensitive. Preparation for an influenza pandemic is presently a high priority in Taiwan. Laboratory-based surveillance systems must be timely in order to be effective. The data presented here highlights the need to characterize the circulating strains both antigenically and genetically during regular surveillance. Any contribution of individual genes or gene combinations to usual or unusual epidemic characteristics might thus be identified ensuring that virus strains can be selected for vaccine formulation that will most closely match the circulating viruses.     

北京地区人偏肺病毒融合蛋白F编码基因的序列分析

目的进一步了解北京地区人偏肺病毒（hMPV）编码基因的特征。方法在原先工作的基础上，从分属于hMPV的两个不同基因进化簇（即基因型）的两份临床标本BJ1816和BJ1887中扩增F蛋白全基因，克隆至pBS-T载体中并进行测序，与GenBank中hMPV的基因序列进行比较分析和种系进化分析。结果BJ1816和BJ1887的F基因全长均为1620个核苷酸，编码539个氨基酸。BJ1816和BJ1887F蛋白基因之间的核苷酸和推导的氨基酸同源性分别为83．8％和94．4％，与同一基因簇内的hMPV有相当高的氨基酸同源性（98．3％～99．6％）。BJ1816和BJ1887的F蛋白是典型的I型糖蛋白，具有与迄今已发现的hMPV相同的裂解位点（RQSR）。BJ1816和BJ1887之间F2亚单位的氨基酸同源性高于F1亚单位的同源性（96．9％vs93．8％）；除位于羧基末端的跨膜区和胞质尾区外，F1亚单位的其余部分较保守（95．4％）；糖基化位点保守。种系进化分析显示BJ1816和BJ1887属于不同的进化簇。结论F蛋白的序列分析进一步证明BJ1816和BJ1887分属于不同的基因型，其F蛋白的基因特征与其他国外文献中所报道的hMPV相似，是病毒的膜表面糖蛋白，提示其在病毒的感染与免疫中起到重要的作用。     

Epidemiology and genetic variability of human metapneumovirus during a 4‐year‐long study in Southeastern Brazil

Epidemiological and molecular characteristics of human metapneumovirus (hMPV) were compared with human respiratory syncytial virus (hRSV) in infants and young children admitted for acute lower respiratory tract infections in a prospective study during four consecutive years in subtropical Brazil. GeneScan polymerase chain assays (GeneScan RT‐PCR) were used to detect hMPV and hRSV in nasopharyngeal aspirates of 1,670 children during January 2003 to December 2006. hMPV and hRSV were detected, respectively, in 191 (11.4%) and in 702 (42%) of the children admitted with acute lower respiratory tract infections at the Sao Paulo University Hospital. Sequencing data of the hMPV F gene revealed that two groups of the virus, each divided into two subgroups, co‐circulated during three consecutive years. It was also shown that a clear dominance of genotype B1 occurred during the years 2004 and 2005, followed by genotype A2 during 2006. J. Med. Virol. 81:915–921, 2009. © 2009 Wiley‐Liss, Inc.