Isolation of arboviruses in Kenya, 1966-1971

The isolation in Kenya of arboviruses from mosquitoes and patients is reported. Middelburg and Semliki Forest virus (group A), Banzi virus (group B), Bunyamwera and Beliefe virus (Bunyamwera group), Sango virus (Simbu group), Pongola virus (Bwamba group), Tahyna virus (California group), ungrouped AR 1169/64 and a number of viruses still to be identified were isolated from mosquitoes. Of the latter some may be hitherto undescribed viruses. Bunyamwera, Rift Valley Fever and 2 unidentified viruses were isolated from patients. The isolates are discussed against the background of information from isolations and serology performed elsewhere. Attempts are made to define their rôle in human disease. Some superficial suggestions about the ecology are made.     

Genetic diversity of serotype A foot-and-mouth disease viruses in Kenya from 1964 to 2013; implications for control strategies in eastern Africa

Serotype A is the most genetically and antigenically diverse of the foot-and-mouth disease virus (FMDV) serotypes. Records of its occurrence in Kenya date back to 1952 and the antigenic diversity of the outbreak viruses in this region is reflected by the current use of two different vaccine strains (K5/1980 and K35/1980) and previous use of two other strains (K18/66 and K179/71). This study aimed at enhancing the understanding of the patterns of genetic variation of serotype A FMDV in Kenya. The complete VP1 coding region sequences of 38 field isolates, identified as serotype A FMDV, collected between 1964 and 2013 were determined. Coalescent-based methods were used to infer times of divergence of the virus strains and the evolutionary rates alongside 27 other serotype A FMDV sequences from Genbank and the World Reference Laboratory (WRL). This study represents the first comprehensive genetic analysis of serotype A FMDVs from Kenya. The study detected four previously defined genotypes/clusters (termed G-I, G-III, G-VII and G-VIII), within the Africa topotype, together with a fifth lineage that has apparently emerged from within G-I; these different lineages have each had a countrywide distribution. Genotypes G-III and G-VIII that were first isolated in 1964 are now apparently extinct; G-VII was last recorded in 2005, while G-I (including the new lineage) is currently in widespread circulation. High genetic diversity, widespread distribution and transboundary spread of serotype A FMDVs across the region of eastern Africa was apparent. Continuous surveillance for the virus, coupled to genetic and antigenic characterization is recommended for improved regional control strategies.     

深圳市2005-2007年H1N1亚型流感病毒HA1基因分子流行病学研究

目的 探讨2005-2007年深圳市H1N1流感病毒HA1基因变异特征.方法 选取深圳市2005-2007年分离的H1N1流感毒株,提取病毒RNA,用RT-PCR扩增HA1区基因片段,产物纯化后测序并进行基因序列分析.结果 2005-2007年流感病毒分离率平均为7.16%,H1N1流感病毒在2005年和2006年的分离数占总分离数的比例分别为56.14%和66.03%,而2007年仅占3.61%.核苷酸同源性和基因进化树结果一致,2005年4月份之前分离株与A/New Caledonia/20/1999为同一分支,2005年5月份之后的分离株与A/Solomon Island/3/2006为一支,而2006-2007年分离株又与国家代表株A/GDLH/219/2006在一个分支.氨基酸序列分析显示,绝大多数的毒株均在第130位点缺失一个赖氨酸;2005年5月以后的大部分毒株出现以下氨基酸变异:T82K、Y94H、R146K、R209K、T267N,2006年5月份之后的毒株在抗原决定簇B区发生了A190T、H193Y、E195D氨基酸变异,同时也发生A区R146K的置换.但所有毒株的潜在糖基化和受体结合位点均比较保守.发现1株病毒A/SZ/68/2007具特殊性,经与参照毒株比较,其326个氨基酸中有50个发生变化,其中有11个位于抗原决定簇位点、6个位于受体结合位点,且有4个氨基酸变化导致糖基化位点丢失.结论 2005-2007年深圳市人群中至少有3个类型HA1基因不同的H1N1流感病毒株;由于氨基酸变异引起病毒发生抗原漂移,其代表株为A/GDLH/219/2006;发现的A/SZ/68/2007病毒毒株具有特殊性,其抗原特性和流行病学意义还有待探讨.     

深圳市2005—2007年H1N1亚型流感病毒HAl基因分子流行病学研究

目的 探讨2005-2007年深圳市H1N1流感病毒HA1基因变异特征。方法 选取深圳市2005-2007年分离的H1N1流感毒株，提取病毒RNA，用RT-PCR扩增HA1区基因片段，产物纯化后测序并进行基因序列分析。结果 2005-2007年流感病毒分离率平均为7.16%，H1N1流感病毒在2005年和2006年的分离数占总分离数的比例分别为56.14%和66.03%，而2007年仅占3.61%。核苷酸同源性和基因进化树结果一致，2005年4月份之前分离株与A/New Caledonia/20/1999为同一分支，2005年5月份之后的分离株与A/Solomon Island/3/2006为一支，而2006-2007年分离株又与国家代表株A/GDLH/219/2006在一个分支。氨基酸序列分析显示，绝大多数的毒株均在第130位点缺失一个赖氨酸；2005年5月以后的大部分毒株出现以下氨基酸变异：T82K、Y94H、R146K、R209K、T267N，2006年5月份之后的毒株在抗原决定簇B区发生了A190T、H193Y、E195D氨基酸变异，同时也发生A区R146K的置换。但所有毒株的潜在糖基化和受体结合位点均比较保守。发现1株病毒A/SZ/68/2007具特殊性，经与参照毒株比较，其326个氨基酸中有50个发生变化，其中有11个位于抗原决定簇位点、6个位于受体结合位点，且有4个氨基酸变化导致糖基化位点丢失。结论 2005-2007年深圳市人群中至少有3个类型HA1基因不同的H1N1流感病毒株；由于氨基酸变异引起病毒发生抗原漂移，其代表株为A/GDLH/219/2006；发现的A/SZ/68/2007病毒毒株具有特殊性，其抗原特性和流行病学意义还有待探讨。     

Genetic and antigenic relationship of foot–and–mouth disease virus serotype O isolates with the vaccine strain O1/BFS

Foot–and–mouth disease serotype O viruses (FMDV/O) are responsible for the most outbreaks in FMD endemic countries. O1/BFS is one of the recommended FMD/O vaccine strains by World Reference Laboratory for FMD. In the current study, FMDV/O1 BFS vaccine strain and serotype O field isolates (45) were analyzed phylogenetically and antigenically to gain more insight into the genetic and antigenic characteristics of the vaccine strain and field isolates.O1/BFS showed similarity with 89% of the field isolates using a virus neutralization test (VNT). The P1 region encoding the FMDV capsid was sequenced and analysed for 46 strains of FMDV/O. Phylogenetic analysis showed these viruses originated from five continents and covered eight of 11 reported topotypes. Five isolates that demonstrated low antigenic similarities with O1/BFS were analyzed for their antigenic variation at the known neutralizing antigenic sites. Three of the five isolates demonstrated unique amino acid substitutions at various antigenic sites. No unique amino acid substitutions were observed for the other two unmatched isolates. Positively selected residues were identified on the surface of the FMD virus capsid supporting that it is important to continuously monitor field isolates for their antigenic and phenotypic changes.In conclusion, the vaccine strain O1/BFS is likely to confer protection against 89% of the 45 FMDV/O isolates based on VNT. Thus O1/BFS vaccine strain is still suitable for use in global FMD serotype O outbreak control. Combining data from phylogenetic, molecular and antigenic analysis can provide improvements in the process of vaccine selection.     

H3N2 influenza virus transmission from swine to turkeys, United States

In 1998, a novel H3N2 reassortant virus emerged in the United States swine population. We report the interspecies transmission of this virus to turkeys in two geographically distant farms in the United States in 2003. This event is of concern, considering the reassortment capacity of this virus and the susceptibility of turkey to infection by avian influenza viruses. Two H3N2 isolates, A/turkey/NC/16108/03 and A/turkey/MN/764/03, had 98.0% to 99.9% nucleotide sequence identity to each other in all eight gene segments. All protein components of the turkey isolates had 97% to 98% sequence identity to swine H3N2 viruses, thus demonstrating interspecies transmission from pigs to turkeys. The turkey isolates were better adapted to avian hosts than were their closest swine counterparts, which suggests that the viruses had already begun to evolve in the new host. The isolation of swine-like H3N2 influenza viruses from turkeys raises new concerns for the generation of novel viruses that could affect humans.     

Serological and biochemical analysis of some recent type A foot-and-mouth disease virus isolates from the Middle East

In 1986 and 1987 foot-and-mouth disease virus (FMDV) serotype A was isolated from outbreaks of disease in Saudi Arabia and Iran. Selected virus isolates were antigenically distinct from the prototype A22 virus strain (A22/Iraq/64), but were serologically related to each other. However, polyacrylamide gel electrophoresis showed that whilst the respective Saudi Arabian structural polypeptides were homogeneous, those from an Iran isolate were distinct. Direct sequencing of part of the P-1D (VP1) gene demonstrated considerable difference in nucleotide homology between the two groups of viruses; the Saudi Arabian viruses were closely related to each other but only distantly related to both the A22 prototype virus strain and the Iranian virus isolate. The latter viruses were only slightly more closely related to each other. Thus there appeared to be at least two distinct FMDV type A variants co-circulating in the Middle East, both of which differed considerably from the classical A22 subtype.     

Arbovirus isolations from mosquitoes: Kano Plain, Kenya.

Arbovirus isolation attempts on 324,486 mosquitoes captured over a four-year period on the Kano Plain, Kenya, yielded 15 isolates including Pongola (six strains), Ilesha (three strains), Germiston (two strains), Sindbis (one strain), Barur (one strain) and two viruses which could not be characterized. Mansonia uniformis, Anopheles gambiae and Culex antennatus constituted 70% of the total collection and accounted for all of the isolates except one, which came from Anopheles funestus.     

Genome analysis of Rift Valley fever virus, Mayotte

As further confirmation of a first human case of Rift Valley fever in 2007 in Comoros, we isolated Rift Valley fever virus in suspected human cases. These viruses are genetically closely linked to the 2006-2007 isolates from Kenya.     

Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94–101, 93–101, and 93–102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93–102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93–102 residues to 8 aa residues at positions 94–101 in 3A protein.     

Systematic evaluation of suspension MDCK cells,adherent MDCK cells,and LLC-MK2 cells for preparing influenza vaccine seed virus

Suspension Madin–Darby canine kidney (MDCK) cells (MDCK-N), adherent MDCK cells (MDCK-C), and adherent rhesus monkey kidney LLC-MK2 cells (LLC-MK2D) were systematically evaluated for the preparation of influenza vaccine seed viruses for humans on the basis of primary virus isolation efficiency, growth ability, genetic stability of the hemagglutinin (HA) and neuraminidase (NA) genes, and antigenic properties in hemagglutination inhibition (HI) test of each virus isolate upon further passages. All the subtypes/lineages of influenza viruses (A(H1N1), A(H1N1)pdm09, A(H3N2), B-Victoria, and B-Yamagata) were successfully isolated from clinical specimens by using MDCK-N and MDCK-C, whereas LLC-MK2D did not support virus replication well. Serial passages of A(H1N1) viruses in MDCK-N and MDCK-C induced genetic mutations of HA that resulted in moderate antigenic changes in the HI test. All A(H1N1)pdm09 isolates from MDCK-C acquired amino acid substitutions at the site from K153 to N156 of the HA protein, which resulted in striking antigenic alteration. In contrast, only 30% of MDCK-N isolates showed amino acid changes at this site. The frequency of MDCK-N isolates with less than two-fold reduction in the HI titer was as high as 70%. A(H3N2) and B-Yamagata isolates showed high antigenic stability and no specific amino acid substitution during passages in MDCK-N and MDCK-C. B-Victoria isolates from MDCK-N and MDCK-C acquired genetic changes at HA glycosylation sites that greatly affected their antigenicity. When these cell isolates were applied to passages in hen eggs, A(H1N1), B-Victoria, and B-Yamagata viruses grew well in eggs, while none of the cell isolates of A(H3N2) viruses did. Thus, we demonstrate that MDCK-N might be useful for the preparation of influenza vaccine seed viruses.     

Coltiviruses and seadornaviruses in North America, Europe, and Asia

Coltiviruses are tickborne viruses of the genus Coltivirus. The type species, Colorado tick fever virus (from North America), has been isolated from patients with flulike syndromes, meningitis, encephalitis, and other severe complications. Another coltivirus, Eyach virus, has been isolated from ticks in France and Germany and incriminated in febrile illnesses and neurologic syndromes. Seadornaviruses are endemic in Southeast Asia, particularly Indonesia and China. The prototype virus of the genus, Banna virus (BAV), has been isolated from many mosquito species, humans with encephalitis, pigs, and cattle. Two other seadornaviruses, Kadipiro and Liao Ning, were isolated only from mosquitoes. The epidemiology of seadornaviruses remains poorly documented. Evidence suggests that BAV is responsible for encephalitis in humans. Infection with BAV may be underreported because it circulates in regions with a high incidence of Japanese encephalitis and could be misdiagnosed as this disease.     

辽宁省部分地区2008年虫媒病毒分离鉴定

目的在辽宁省采集蚊虫标本进行病毒分离,了解虫媒病毒分布情况。方法2008年夏季在辽宁省丹东和锦州市采集蚊虫标本,利用C6/36和BHK-21等细胞分离病毒;对新分离病毒利用血清学、分子生物学和生物信息学方法进行鉴定。结果在辽宁省丹东和锦州市共采集蚊虫标本3属5种9296只,包括库蚊属的三带喙库蚊、淡色库蚊,伊蚊属的刺扰伊蚊、背点伊蚊和按蚊属的中华按蚊。从蚊虫标本中共得到4株病毒分离物,其中1株为乙型脑炎(乙脑)病毒(丹东市的三带喙库蚊),2株为版纳病毒(锦州市的中华按蚊),1株为辽宁病毒和版纳病毒混合(锦州市的中华按蚊)。新分离乙脑病毒属于基因Ⅰ型乙脑病毒,E基因编码病毒毒力和抗原表位的位点未发生改变。结论在辽宁省再次分离到乙脑病毒,并首次证明辽宁省存在辽宁病毒。     

Protective immunity against foot-and-mouth disease virus induced by a recombinant vaccinia virus

We report the construction of a recombinant vaccinia virus expressing the precursor for the four structural proteins of FMD virus (FMDV) (P1) strain C3Arg85 using a procedure for isolation of recombinant vaccinia viruses based solely on plaque formation. Adult mice vaccinated with this recombinant vaccinia virus elicited high titers of neutralizing antibodies against both the homologous FMDV and vaccinia virus, measured by neutralization assays. Liquid phase blocking sandwich enzyme-linked immunosorbent assays (ELISAs) using whole virus as antigen showed high total antibody titers against homologous FMDV, similar to those induced by the conventional inactivated vaccine. When ELISAs were carried out with heterologous strains A79 or O1Caseros as antigens, sera from animals vaccinated with the recombinant virus cross-reacted. Mice boosted once with the recombinant vaccinia virus were protected against challenge with infectious homologous virus. These results indicate that recombinant vaccinia viruses are efficient immunogens against FMDV when used as a live vaccine in a mouse model.     

云南省西双版纳地区2011年蚊虫及虫媒病毒调查

目的了解云南省西双版纳地区蚊虫媒介的分布特点及当地虫媒病毒情况,为虫媒病毒病防治提供科学依据。方法在云南省西双版纳州采集蚊虫标本,用细胞培养法分离病毒,并用RTPCR法检测常见虫媒病毒核酸;在西双版纳州采集发热患者血清及脑脊液标本,并用ELISA法检测常见病毒性脑炎IgM抗体。结果共采获蚊虫5属29种13337只,其中三带喙库蚊、中华按蚊、带足按蚊分别占蚊虫标本总数的79.98%(10667/13337)、7.95%(1060/13337)和7.38%(984/13337),三带喙库蚊为当地优势蚊种。采用流行性乙型脑炎(乙脑)病毒、版纳病毒、甲病毒属、环状病毒等多种虫媒病毒引物对214批蚊虫标本进行PCR检测,结果均为阴性;采用多种细胞对蚊虫标本进行病毒分离,结果也为阴性。用相关脑炎病毒试剂盒对采集到的52份急性期血清标本及54份脑脊液标本进行ELISA检测,发现乙脑病毒IgM阳性16例,单纯疱疹病毒IgM抗体阳性4例,腮腺炎病毒IgM抗体阳性13例,埃可病毒IgM抗体阳性3例,登革热病毒IgM抗体阳性1例。结论 2011年西双版纳地区采集到的蚊虫标本中未检测到乙脑、版纳及环状病毒等虫媒病毒,但血清学检测结果表明当地发热患者存在乙脑等多种病毒性脑炎感染。     

Multiple Origins of Foot-and-Mouth Disease Virus Serotype Asia 1 Outbreaks, 2003�C2007

We investigated the molecular epidemiology of foot-and-mouth disease virus (FMDV) serotype Asia 1, which caused outbreaks of disease in Asia during 2003–2007. Since 2004, the region affected by outbreaks of this serotype has increased from disease-endemic countries in southern Asia (Afghanistan, India, Iran, Nepal, Pakistan) northward to encompass Kyrgyzstan, Tajikistan, Uzbekistan, several regions of the People’s Republic of China, Mongolia, Eastern Russia, and North Korea. Phylogenetic analysis of complete virus capsid protein 1 (VP1) gene sequences demonstrated that the FMDV isolates responsible for these outbreaks belonged to 6 groups within the Asia 1 serotype. Some contemporary strains were genetically closely related to isolates collected historically from the region as far back as 25 years ago. Our analyses also indicated that some viruses have spread large distances between countries in Asia within a short time.     

Genetic and antigenic characterisation of serotype A FMD viruses from East Africa to select new vaccine strains

Vaccine strain selection for emerging foot-and-mouth disease virus (FMDV) outbreaks in enzootic countries can be addressed through antigenic and genetic characterisation of recently circulating viruses. A total of 56 serotype A FMDVs isolated between 1998 and 2012, from Central, East and North African countries were characterised antigenically by virus neutralisation test using antisera to three existing and four candidate vaccine strains and, genetically by characterising the full capsid sequence data. A Bayesian analysis of the capsid sequence data revealed the viruses to be of either African or Asian topotypes with subdivision of the African topotype viruses into four genotypes (Genotypes I, II, IV and VII). The existing vaccine strains were found to be least cross-reactive (good matches observed for only 5.4–46.4% of the sampled viruses). Three bovine antisera, raised against A-EA-2007, A-EA-1981 and A-EA-1984 viruses, exhibited broad cross-neutralisation, towards more than 85% of the circulating viruses. Of the three vaccines, A-EA-2007 was the best showing more than 90% in-vitro cross-protection, as well as being the most recent amongst the vaccine strains used in this study. It therefore appears antigenically suitable as a vaccine strain to be used in the region in FMD control programmes.     

Which influenza vaccine formulation should be used in Kenya? A comparison of influenza isolates from Kenya to vaccine strains, 2007–2013

IntroductionEvery year the World Health Organization (WHO) recommends which influenza virus strains should be included in a northern hemisphere (NH) and a southern hemisphere (SH) influenza vaccine. To determine the best vaccine formulation for Kenya, we compared influenza viruses collected in Kenya from April 2007 to May 2013 to WHO vaccine strains.MethodsWe collected nasopharyngeal and oropharyngeal (NP/OP) specimens from patients with respiratory illness, tested them for influenza, isolated influenza viruses from a proportion of positive specimens, tested the isolates for antigenic relatedness to vaccine strains, and determined the percentage match between circulating viruses and SH or NH influenza vaccine composition and schedule.ResultsDuring the six years, 7.336 of the 60,072 (12.2%) NP/OP specimens we collected were positive for influenza: 30,167 specimens were collected during the SH seasons and 3717 (12.3%) were positive for influenza; 2903 (78.1%) influenza A, 902 (24.2%) influenza B, and 88 (2.4%) influenza A and B positive specimens. We collected 30,131 specimens during the NH seasons and 3978 (13.2%) were positive for influenza; 3181 (80.0%) influenza A, 851 (21.4%) influenza B, and 54 (1.4%) influenza A and B positive specimens. Overall, 362/460 (78.7%) isolates from the SH seasons and 316/338 (93.5%) isolates from the NH seasons were matched to the SH and the NH vaccine strains, respectively (p < 0.001). Overall, 53.6% and 46.4% SH and NH vaccines, respectively, matched circulating strains in terms of vaccine strains and timing.ConclusionIn six years of surveillance in Kenya, influenza circulated at nearly equal levels during the SH and the NH influenza seasons. Circulating viruses were matched to vaccine strains. The vaccine match decreased when both vaccine strains and timing were taken into consideration. Either vaccine formulation could be suitable for use in Kenya but the optimal timing for influenza vaccination needs to be determined.     

天津市儿童流行性感冒病原学检测分析

目的：对天津市2001－2002年流行性感冒（流感）患儿进行病原学分析。方法：用狗肾传代（MDCK）细胞和鸡胚双腔法进行流感病毒分离。用鸡红细胞和人O型红细胞凝集试验证实病毒的存在,用红细胞凝集抑制试验进行型和亚型的鉴定。结果：2001年10月至2002年3月共采集14岁以下流感样患儿咽拭子标本238份,发离出64株流感病毒,检出率为26．9％;其中A（H3N2）亚型42株,占检出数的65．6％;A（H1N1）亚型13株,占20．3％;B型9株,占14．1％,所分离的64株流感病毒都具有适应于MDCK细胞株生长及与人O型红细胞凝集良好的生物学特性,转种鸡胚后,绝大多数（62／64）能适应鸡胚生长;但64份阳性咽拭子标本液直接接种鸡胚,仅有3份血凝阳性,此外,96．4％（53／55株）A型流感病毒均可由O相转为D相,有2株A（H3N2）为O相特征,B型流感病毒均为O相特征,结论：天津地区流感病毒存在A（H3N2）、A（H1A1）和B三个型,以A（H3N2）为优势流行型。