Natural genetic exchanges between vaccine and wild poliovirus strains in humans

In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3' moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.     

Mutation and recombination variability of vaccine polioviruses isolated in Belarus (1960-1999)

One hundred and eight vaccine-derived strains of types 1, 2, and 3 poliovirus (25 PV1, 34 PV2, and 48 PV3) isolated in Belarus in 1960-1999 were analyzed by double restriction fragment length polymorphism assay (RFLP-1, -3D1). Forty-four (40.7%) of strains were genetically modified. Eight (7.4%) PV were modified by mutation, 16 (14.8%) by recombination, and 20 (18.5%) by both mutation and recombination. The genomes of 16 PV were analyzed by multiple RFLP technique covering VP1-, VP1/VP2A-, P2-, 3AC-, and 3D1-coding regions. The majority of recombinants were "simple" (with one crossing over site). One strain was "double" recombinant (two crossings over sites) and one more "multiple" recombinant (three crossing over sites). Partial nucleotide sequencing of some recombinant strains showed that the degree of these strains' divergence was less than 1% in comparison with the original vaccine viruses.     

Environmental Poliovirus Surveillance during Oral Poliovirus Vaccine and Inactivated Poliovirus Vaccine Use in Córdoba Province,Argentina

This study compares the presence of environmental poliovirus in two Argentinean populations using oral poliovirus vaccine (OPV) or inactivated poliovirus vaccine (IPV). From January 2003 to December 2005, Córdoba City used IPV in routine infant immunizations, with the exception of intermittent OPV use in August 2005. Between May 2005 and April 2006, we collected weekly wastewater samples in Córdoba City and the province''s three major towns, which continued OPV use at all times. Wastewater samples were processed and analyzed for the presence of poliovirus according to WHO guidelines. During the months of IPV use in Córdoba City, the overall proportion of poliovirus-positive samples was 19%. During an intermittent switch from IPV to OPV, this proportion increased to 100% within 2 months. During the 3 months when IPV was reintroduced to replace OPV, a substantial proportion of samples (25%) remained positive for poliovirus. In the OPV-using sites, on average, 54% of samples were poliovirus positive. Seventy-seven percent of poliovirus isolates showed at least one mutation in the VP1-encoding sequence; the maximum genetic divergence from the Sabin strain was 0.7%. Several isolates showed mutations on attenuation markers in the VP1-encoding sequence. The frequency or type of virus mutation did not differ between periods of IPV and OPV use or by virus serotypes. This study indicates that the sustained transmission of OPV viruses was limited during IPV use in a middle-income country with a temperate climate. The continued importation of poliovirus and genetic instability of vaccine strains even in the absence of sustained circulation suggest that high poliovirus vaccine coverage has to be maintained for all countries until the risk of reintroduction of either wild or vaccine-derived poliovirus is close to zero worldwide.In the context of the near achievement of poliomyelitis eradication and anticipated cessation of oral poliovirus (PV) vaccine (OPV), the World Health Organization (WHO) has recommended the use of inactivated PV vaccine (IPV) in countries that have IPV production facilities or other countries where immunization programs fulfill certain financial and logistic criteria (37). IPV has been shown to be safe and immunogenic in children in both developed and developing countries.(34) IPV diminishes the excretion of PV by children challenged with the Sabin strain of PV only moderately. The questions of whether and to which extent Sabin PV that is reintroduced into a population immunized with IPV could establish circulation, mutate to vaccine-derived PV (VDPV), and consequently cause poliomyelitis remain important. No such emergence of VDPV in developed countries using IPV has been reported. However, suboptimal hygienic conditions and insufficient vaccine coverage in middle- or low-income countries could favor the establishment of PV circulation after reintroduction, as indicated by recent VDPV outbreaks in populations with low OPV coverage (27, 38).Argentina currently uses OPV in the childhood immunization program according to recommendations from the Pan-American Health Organization. The last case of poliomyelitis due to wild-type PV was reported in Argentina in 1984 and in Córdoba Province in 1971 (24). In Córdoba City, the capital of Córdoba Province, standalone IPV (Imovax Polio; Sanofi Pasteur) replaced OPV (Polioral; Novartis Vaccines) in the routine childhood immunization program (2, 4, and 6 months of age plus a booster at 18 months age) from 1 January 2003 to 31 December 2005, while the surrounding provinces continued to use OPV. Due to an IPV shortage between 10 August and 7 September 2005, OPV was used in the capital during this period. We conducted environmental PV surveillance in Córdoba Province from May 2005 to April 2006 to describe environmental PV circulation and molecular characteristics of PV depending on the vaccine used. In the present evaluation, we also describe the dynamic of PV circulation around the change of IPV-OPV-IPV-OPV in the capital. This observation can contribute evidence regarding the dynamics of PV circulation and its implication for global immunization policy after polio eradication.     

Characterization of a highly evolved vaccine-derived poliovirus type 3 isolated from sewage in Estonia

Two types of vaccine-derived polioviruses have been recently designated to emphasize the different origins of the evolved viruses: circulating vaccine-derived polioviruses (cVDPV) associated with outbreaks of paralytic disease and strains isolated from chronically infected immunodeficient individuals (iVDPV). We describe here a type 3 VDPV (PV3/EST/02/E252; later E252) isolated from sewage collected in Tallinn, Estonia, in October 2002. Due to aberrant properties in subtyping, the virus was subjected to detailed characterization. Partial genomic sequencing suggested that the closest relative was the oral vaccine strain PV3/Sabin, but the two virus strains shared only 86.7% of the 900 nucleotides (nt) coding for the capsid protein VP1. Phylogenetic analysis of the nearly complete genome [nt 19 to poly(A)] revealed multiple nucleotide substitutions throughout the genome and a possible Sabin 3/Sabin 1-recombination junction site in the 2C coding region. A calculation based on the estimated mutation frequency of the P1 region of polioviruses suggested that the E252 virus might have replicated in one or more individuals for approximately 10 years. No persons chronically excreting poliovirus are known in Estonia. Amino acid substitutions were seen in all known antigenic sites, which was consistent with the observed aberrant antigenic properties of the virus demonstrated by both monoclonal antibodies and human sera from vaccinated children. In spite of the apparent transmission potential, no evidence was obtained for circulation of the virus in the Estonian population.     

Antigenic and biochemical characterization of poliovirus type 1 isolates

By the introduction of Sabin oral poliovirus vaccine, the circulation of wild type polioviruses has virtually disappeared in Japan. However, an outbreak of poliomyelitis associated with sporadic transmission of type 1 wild strain occurred in Nagano in 1980. Furthermore, we found that some type 1 wild strains were introduced into Japan from abroad in 1981. In recent surveys, the two poliovirus type 1 isolates which have non-vaccine-like antigenic character were detected in Aichi. Then, an investigation to trace the origin of these strains was performed, by using intratypic serodifferentiation and biochemical techniques. Electrophoretic migration patterns of their structural polypeptides were quite different from the vaccine virus. In the oligonucleotide mapping, however, one of them gave patterns very similar to those of the vaccine virus. We could conclude that one originated most probably from wild strains, and the other was an antigenic variant derived from the vaccine virus. It showed that oligonucleotide mapping was a very useful method for identification of antigenic modified Sabin type 1 derivatives.     

SSR标记在毛木耳种质资源遗传多样性研究中的应用

本研究利用基于毛木耳全基因组开发的SSR标记对27份毛木耳菌株（野生14株、栽培13株）的遗传多样性进行分析。首先随机选取3个菌株（2个野生菌株、1个栽培菌株）的DNA为模板,从144对SSR引物中筛选出扩增条带清晰、稳定性强、多态性丰富的引物24对。24对SSR引物共检测到116个多态性SSR片段,每对引物的多态性片段有3-7个,引物平均检测效率为4.83个,Shannon’s遗传多样性指数范围是0.866-1.885,多态性位点比率100%。供试菌株遗传相似系数范围是0.618-0.971,说明毛木耳种质资源具有丰富的遗传多样性。野生菌株与栽培菌株间平均遗传相似系数分别为0.746、0.779,说明毛木耳野生菌株遗传多样性更为丰富。经聚类分析,在遗传相似系数为0.680时,可将供试菌株分为无色（白色）类群Ⅰ和有色（浅黄色到红褐色）类群Ⅱ。遗传相似系数为0.704时,可将供试菌株中栽培菌株和野生菌株明显区分（14株野生菌株均在类群Ⅱ-2中,13株栽培菌株分别在类群Ⅰ和Ⅱ-1中）。本研究表明基于全基因组的SSR标记能从分子水平上揭示各菌株间的遗传差异,丰富毛木耳遗传多样性的研究手段,并为进一步进行毛木耳的品种选育、遗传学研究等提供有力手段。     

The maintaining of the active laboratory-based surveillance of the acute flaccid paralysis (AFP) cases in Romania in the framework of the strategic plan of the global polio eradication initiative

Until 2008 poliomyelitis was controlled in Romania by predominantly using Oral Poliovirus Vaccine Sabin (OPV); the alternative vaccination schedule (IPV formalin Inactivated Poliovirus Vaccine/OPV) will be implemented starting September 2008. The vaccination coverage with 4 doses of TOPV (trivalent oral polio vaccine) in the first 14 months of life has been > 90% since 1980. In Romania, the risk of the Vaccine-Associated Paralytic Poliomyelitis cases (VAPP) decreased from less than 2 VAPP cases/year in the 1995-2006 interval to 0 VAPP cases in 2007. The serological study was performed in 2006-2007 only in cases with pair serum samples from 28 acute flaccid paralysis (AFP) cases (age = 3 months - 14 years) and from 45 facial paralysis (FP) cases (age -6 months - 4 years 9 months). A high level of vaccinal coverage was shown for all poliovirus serotypes: >95% in AFP serum samples investigated; and for FP serum samples investigated the levels of antibodies against poliovirus (PV) serotypes were 98% for PV type 1; 87% for PV type 2: and 89% for PV type 3. If the European region is polio free since 2002, the risk of wild PV importation from endemic region remains present. The laboratory capacity for the fast detection and molecular investigations of the emergence of the new epidemic strains and a high level of population immunity must be maintained. A national seroprevalence study concerning all three PV serotypes must be performed.     

Swedish inactivated polio vaccine: laboratory standardization and clinical experience over a 30-year period.

Swedish inactivated polio vaccines have contained per single human dose a mean amount of viral antigen equivalent to 1 x 10(7.5) CCID50 of type 1, 1 x 10(7.4) of type 2 and 1 x 10(7.8) of type 3 produced on primary monkey kidney cells. Potency tests were made in comparison with an equivalent amount of live virus suspension of all three types. Validation of tests has been based on the response to type 1 only. Based on clinical experience with vaccine lots from 1957 and the establishment of the second live reference virus suspension in 1966, the minimum limit of immune response in guinea-pigs--expressed in extinction values--was decided as 1.5 for type 1 and type 3, and 1.0 for type 2. The potency test method used since 1959 in Sweden was two subcutaneous injections 2 weeks apart using 10 guinea-pigs per dilution and blood collected 1 week thereafter. Potency tests made according to European Pharmacopoeia revealed a somewhat lower value for type 2. D-antigen content in Swedish vaccines was low, however, the Swedish vaccine has protected against many episodes or outbreaks of wild virus in Sweden and immunized individuals elsewhere in the world. For the Swedish population a clear-cut clinical motivation for requiring a higher potency for type 2 as required in the European Pharmacopoeia or increased levels of D-antigen in the final product has not been presented. It was concluded that the European Pharmacopoeia method did not distinguish between doses of 0.5-1.0 ml. The minimum limit extinction value for type 2, i.e. 2.0 seemed to high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experiments on the time of application of insecticide to decrease the spread of yellowing viruses of sugar beet, 1954-66

Sprays of demeton-methyl insecticide decreased the spread of yellowing viruses by aphids in sugar-beet crops in England. Between 1957 and 1960, when yellows was prevalent, the incidence, assessed as ‘infected-plant-weeks’, was decreased by 36–41 % by one spray, depending on when it was applied, and by 55 % by two sprays, giving average yield increases of 1½ and 2 ton/acre of roots respectively. Between 1962 and 1966, when yellows spread less, a spray at the time when growers were advised to spray by the British Sugar Corporation decreased yellows incidence by 37 %, whereas sprays 2 weeks earlier or later decreased it by 24 % and 25 % respectively. Between 1958 and 1966 an annual average of 160000 of the country's 440000 acres of sugar beet has been sprayed, often to control Aphis fabae as well as to check the spread of yellows. A spray gives a profitable yield increase when yellows incidence in unsprayed plots is 20 % at the end of August.     

Development and Pre-Clinical Evaluation of Two LAIV Strains against Potentially Pandemic H2N2 Influenza Virus

H2N2 Influenza A caused the Asian flu pandemic in 1957, circulated for more than 10 years and disappeared from the human population after 1968. Given that people born after 1968 are naïve to H2N2, that the virus still circulates in wild birds and that this influenza subtype has a proven pandemic track record, H2N2 is regarded as a potential pandemic threat. To prepare for an H2N2 pandemic, here we developed and tested in mice and ferrets two live attenuated influenza vaccines based on the haemagglutinins of the two different H2N2 lineages that circulated at the end of the cycle, using the well characterized A/Leningrad/134/17/57 (H2N2) master donor virus as the backbone. The vaccine strains containing the HA and NA of A/California/1/66 (clade 1) or A/Tokyo/3/67 (clade 2) showed a temperature sensitive and cold adapted phenotype and a reduced reproduction that was limited to the respiratory tract of mice, suggesting that the vaccines may be safe for use in humans. Both vaccine strains induced haemagglutination inhibition titers in mice. Vaccination abolished virus replication in the nose and lung and protected mice from weight loss after homologous and heterologous challenge with the respective donor wild type strains. In ferrets, the live attenuated vaccines induced high virus neutralizing, haemagglutination and neuraminidase inhibition titers, however; the vaccine based on the A/California/1/66 wt virus induced higher homologous and better cross-reactive antibody responses than the A/Tokyo/3/67 based vaccine. In line with this observation, was the higher virus reduction observed in the throat and nose of ferrets vaccinated with this vaccine after challenge with either of the wild type donor viruses. Moreover, both vaccines clearly reduced the infection-induced rhinitis observed in placebo-vaccinated ferrets. The results favor the vaccine based on the A/California/1/66 isolate, which will be evaluated in a clinical study.     

Characterization of CHAT and Cox type 1 live-attenuated poliovirus vaccine strains

CHAT and Cox type 1 live-attenuated poliovirus strains were developed in the 1950s to be used as vaccines for humans. This paper describes their characterization with respect to virulence, sensitivity for growth at high temperatures, and complete nucleotide and amino acid sequences. The results are compared to those for their common parental wild virus, the Mahoney strain, and to those for two other poliovirus strains derived from Mahoney, the Sabin 1 vaccine strain and the mouse-adapted LS-a virus. Analysis of four isolates from cases of vaccine-associated paralytic poliomyelitis related to the CHAT vaccine revealed genetic and phenotypic properties of the CHAT strain following replication in the human gut. CHAT-VAPP strain 134 contained a genome highly evolved from that of CHAT (1.1% nucleotide differences), suggesting long-term circulation of a vaccine-derived strain in the human population. The molecular mechanisms of attenuation and evolution of poliovirus in humans are discussed in the context of the global polio eradication initiative.     

Mouse neurovirulence determinants of poliovirus type 1 strain LS-a map to the coding regions of capsid protein VP1 and proteinase 2Apro.

Poliovirus type 1 strain LS-a [PV1(LS-a)] is a OV variant adapted to mice by multiple passages through mouse and monkey tissues. To investigate the molecular basis underlying mouse neurovirulence of PV1(LS-a), a cDNA of the viral genome containing nucleotides 112 to 7441 was cloned, and the nucleotide sequence was determined. Compared with that of the mouse avirulent progenitor PV1(Mahoney), 54 nucleotide changes were found in the genome of the PV1(LS-a) virus, resulting in 20 amino acid substitutions in the virus polyprotein. Whereas the nucleotide changes were scattered throughout the genome, the amino acid substitutions were largely clustered in the capsid proteins and, to a certain extent, in the virus proteinase 2Apro. By in vitro mutagenesis, PV1(LS-a)-specific capsid mutations were introduced into a cDNA clone of PV1(Mahoney). We show that neither the individual amino acid mutations nor combinations of mutations in the region encoding VP1 conferred to PV1(Mahoney) the mouse-adapted phenotype of PV1(LS-a). Chimeric cDNA studies demonstrated that a recombinant type 1 virus containing the PV1(LS-a) sequence from nucleotide 2470 to nucleotide 3625 displayed a neurovirulent phenotype in mice. Further dissection of this region revealed that mouse neurovirulence of PV1(LS-a) was determined by multiple mutations in regions encoding both viral proteinase 2Apro and capsid protein VP1. The mouse neurovirulent viruses, PV1(LS-a), W1-M/LS-Pf [nucleotides 496 to 3625 from PV1(LS-a)], and W1-M/LS-NP [nucleotides 2470 to 3625 from PV1(LS-a)], showed increased sensitivity to heat treatment at 45 degrees C for 1 h. Surprisingly, the thermolabile phenotype was also displayed by a recombinant of PV1(Mahoney) carrying a PV1(LS-a) DNA fragment encoding the N-terminal portion of 2Apro. This suggests that base substitutions in the region encoding 2Apro affected capsid stability, thereby contributing to the neurovirulence of the virus in mice.     

鸡西市1957-2003年麻疹流行特征分析

目的:了解鸡西市麻疹流行病学特征,探讨控制麻疹的策略。方法:采用描述流行病学,对鸡西市1957～2003年麻疹发病及死亡情况进行分析。结果:1957～1966年麻疹发病呈高发状态,年平均发病率为1280.03/10万,每年1～6月份发病占病例总数的83.09%,使用麻疹疫苗前具有明显的周期性,每隔3～4年出现1个发病高峰,使用麻疹疫苗后整个流行呈明显下降态势;发病主要集中在1～7岁年龄组,占84.09%,男女性别比为1.27:1。结论:实施计划免疫对控制麻疹疫情效果显著,为控制麻疹疫情应继续加强麻疹疫苗的常规免疫,适时开展强化免疫,加强麻疹监测与报告。     

Genetic Characterisation of Malawian Pneumococci Prior to the Roll-Out of the PCV13 Vaccine Using a High-Throughput Whole Genome Sequencing Approach

Background

Malawi commenced the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the routine infant immunisation schedule in November 2011. Here we have tested the utility of high throughput whole genome sequencing to provide a high-resolution view of pre-vaccine pneumococcal epidemiology and population evolutionary trends to predict potential future change in population structure post introduction.

Methods

One hundred and twenty seven (127) archived pneumococcal isolates from randomly selected adults and children presenting to the Queen Elizabeth Central Hospital, Blantyre, Malawi underwent whole genome sequencing.

Results

The pneumococcal population was dominated by serotype 1 (20.5% of invasive isolates) prior to vaccine introduction. PCV13 is likely to protect against 62.9% of all circulating invasive pneumococci (78.3% in under-5-year-olds). Several Pneumococcal Molecular Epidemiology Network (PMEN) clones are now in circulation in Malawi which were previously undetected but the pandemic multidrug resistant PMEN1 lineage was not identified. Genome analysis identified a number of novel sequence types and serotype switching.

Conclusions

High throughput genome sequencing is now feasible and has the capacity to simultaneously elucidate serotype, sequence type and as well as detailed genetic information. It enables population level characterization, providing a detailed picture of population structure and genome evolution relevant to disease control. Post-vaccine introduction surveillance supported by genome sequencing is essential to providing a comprehensive picture of the impact of PCV13 on pneumococcal population structure and informing future public health interventions.     

The Effect of Vaccination on the Evolution and Population Dynamics of Avian Paramyxovirus-1

Newcastle Disease Virus (NDV) is a pathogenic strain of avian paramyxovirus (aPMV-1) that is among the most serious of disease threats to the poultry industry worldwide. Viral diversity is high in aPMV-1; eight genotypes are recognized based on phylogenetic reconstruction of gene sequences. Modified live vaccines have been developed to decrease the economic losses caused by this virus. Vaccines derived from avirulent genotype II strains were developed in the 1950s and are in use globally, whereas Australian strains belonging to genotype I were developed as vaccines in the 1970s and are used mainly in Asia. In this study, we evaluated the consequences of attenuated live virus vaccination on the evolution of aPMV-1 genotypes. There was phylogenetic incongruence among trees based on individual genes and complete coding region of 54 full length aPMV-1 genomes, suggesting that recombinant sequences were present in the data set. Subsequently, five recombinant genomes were identified, four of which contained sequences from either genotype I or II. The population history of vaccine-related genotype II strains was distinct from other aPMV-1 genotypes; genotype II emerged in the late 19^th century and is evolving more slowly than other genotypes, which emerged in the 1960s. Despite vaccination efforts, genotype II viruses have experienced constant population growth to the present. In contrast, other contemporary genotypes showed population declines in the late 1990s. Additionally, genotype I and II viruses, which are circulating in the presence of homotypic vaccine pressure, have unique selection profiles compared to nonvaccine-related strains. Collectively, these data show that vaccination with live attenuated viruses has changed the evolution of aPMV-1 by maintaining a large effective population size of a vaccine-related genotype, allowing for coinfection and recombination of vaccine and wild type strains, and by applying unique selective pressures on viral glycoproteins.     

A high variability of mixed infections and recent recombinations of hepatitis B virus in Laos

In Lao PDR, where more than 8% of the population are chronic carriers of HBsAg, multiple genotypes and subgenotypes co-circulate and are prone to generate recombinant viruses. Phylogenetic analyses of multiple clones per donor revealed mixed infections of subgenotypes B1, B2, B4, C1, C5, I1 and I2 in almost 6% of HBsAg positive rejected blood donors. Recombination analyses and distance calculations furthermore showed that about 65% (17/26) of the mixed infected donors showed recombinations in the S-gene alone, involving the predominant genotypes B and C. These results suggest that, at least in Laos, hepatitis B virus (HBV) mixed infections lead to frequent recombinations. In many donors with recombinant strains, the recombinant fragment and a non-recombinant strain of the same genotype co-existed (127/185 analysed recombinant fragments). For a large proportion of these (60/127), the most closely related known virus was found, although not always exclusively, in the same donor. Recombinant virus strains are largely distinct. This is reflected in an unexpected diversity in recombination breakpoints and the relatively rare recombinations with identical recombination patterns of the same genotypes in different donors. Recent recombination events would explain the limited spread of each of the recombinants. Using a published mutation rate of 4.2 × 10(-5) mutations per site and year, the observed minimum genetic distances of 0-0.60% between parent strain and recombinant fragment would correspond to 0-71 years of evolution from a most recent common ancestor (MRCA). Thus several lines of evidence are suggestive of recent independent recombination events, a proportion of these even occurring within the same donors. In conclusion, our analyses revealed a high variability of mixed infections as a very probable breeding ground of multiple variable recombination events in Laos that so far have not led to new dominant strains.     

Epstein-Barr virus recombinants from overlapping cosmid fragments.

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)