Molecular epidemiological characteristics of echovirus 6 in mainland China: extensive circulation of genotype F from 2007 to 2018.

Echovirus 6 (E6) is associated with various clinical diseases and is frequently detected in environmental sewage. Despite its high prevalence in humans and the environment, little is known about its molecular phylogeography in mainland China. In this study, 114 of 21,539 (0.53%) clinical specimens from hand, foot, and mouth disease (HFMD) cases collected between 2007 and 2018 were positive for E6. The complete VP1 sequences of 87 representative E6 strains, including 24 strains from this study, were used to investigate the evolutionary genetic characteristics and geographical spread of E6 strains. Phylogenetic analysis based on VP1 nucleotide sequence divergence showed that, globally, E6 strains can be grouped into six genotypes, designated A to F. Chinese E6 strains collected between 1988 and 2018 were found to belong to genotypes C, E, and F, with genotype F being predominant from 2007 to 2018. There was no significant difference in the geographical distribution of each genotype. The evolutionary rate of E6 was estimated to be 3.631 × 10-3 substitutions site-1 year-1 (95% highest posterior density [HPD]: 3.2406 × 10-3-4.031 × 10-3 substitutions site-1 year-1) by Bayesian MCMC analysis. The most recent common ancestor of the E6 genotypes was traced back to 1863, whereas their common ancestor in China was traced back to around 1962. A small genetic shift was detected in the Chinese E6 population size in 2009 according to Bayesian skyline analysis, which indicated that there might have been an epidemic around that year.

Complete genome sequence of a multi-recombinant echovirus 6 strain isolated from CSF in Ahvaz, Southwestern Iran.

BACKGROUND: Echovirus 6 (E6), is one of the main enteroviral serotypes, was initially isolated from patients with aseptic meningitis (AM) and is a major cause of hospitalization among children and adults worldwide. METHODS: A cerebrospinal fluid (CSF) sample was collected from patient with clinically suspected aseptic meningitis (AM) in August 2011. Following detection of a virus and subsequent virus serotyping, the whole genome sequence was determined. The sequence of the VP1 region of the isolated strain E6 RA/E6/Ahvaz/Iran/2011 showed 79% (>75%) nucleotide and 94% (>85%) amino acid homology with prototype strain D'Amori. The isolated strain was identified as an E6 serotype. A specimen was cultured in a human rhabdomyosarcoma (RD) cell line. Following propagation, the virus was further analyzed using the plaque assay technique, reverse transcription PCR (RT-PCR), rapid amplification of CDNA ends (RACE), TA cloning, sequencing, phylogenetic analysis, Simplot and boot scanning analyses (ver. 3.5) were applied to find evidence of recombination in the isolated strain. RESULTS: The isolated Echo6 strain RA/E6/Ahvaz/Iran/2011 has been recorded in GenBank with a partial and complete genome accession numbers (KX619440) (KX198605), respectively. The complete genomic sequence was 7435 nt, with a 742 bp 5' UTR, 117 bp 3' UTR, and an open reading frame (ORF) encoding a polypeptide of 2191 amino acids. The nucleotide analysis of the VP1 and structural genomic regions of the isolated strain showed high similarity with strain E6-10887-99 isolated from patient with facial nerve paresis in Russia in 1999. The recombinations evidence were observed in the isolated strain E6 RA/E6/Ahvaz/Iran/2011 and found to have a high levels of inter-serotypic exchanges in 2C and 3A-3C genomic regions with Echovirus13 and Echovirus14, respectively. CONCLUSION: Full genome sequence analysis of enteroviral is required to understand the epidemiological pattern and to evaluate the new enterovirus circulating in community.

Molecular evolution of two asymptomatic echovirus 6 strains that constitute a novel branch of recently epidemic echovirus 6 in China.

BACKGROUND: Echovirus 6 (E6) infections are associated with aseptic meningitis and acute flaccid paralysis (AFP). But some infections, sometimes most of them, are asymptomatic. The mechanism of E6 virulence is unknown. Analyses of the molecular evolution of asymptomatic E6 may help understand why the infections show different manifestations. METHODS: Ninety-six stool samples of healthy children in Yunnan, China were collected and two E6 strains were isolated from them. The whole genomes of these two E6 strains were sequenced, and their molecular evolution was analyzed. RESULTS: The results showed that the two E6 strains may be derived from KJ7724XX strains, which were predominant in AFP patients in Shangdong in 2011. The evolution was accelerated when the two E6 strains formed, although no positive selection site was found. The 11 exclusive mutations on which selection force significantly changed were found in the 2C, 3AB and 3C genes. CONCLUSION: There are some E6 strains which did not cause the disease in the children of Yunnan. These E6 strains maybe come from a recombinant E6 strain which was associated with the outbreak of AFP in Shangdong in 2011. However, some new mutations were found in the 2C, 3AB and 3C genes of these asymptomatic strains, and these mutations may be constraint by the natural selection and could be potentially responsible for clinical presentations.

Molecular characterization of environmental and clinical echovirus 6 isolates from Poland, 2006-2014.

The aim of this study was to investigate the genetic variability of echovirus 6 (E6) isolates from environmental samples and clinical cases of aseptic meningitis from 2006 to 2014. The analysis of the VP1 region showed the extensive diversity (up to 18.8%) and revealed that E6 circulating in Poland belong to four groups. Environmental strains clustered in three groups excepting the 2012 outbreak group, which shows the sudden introduction of new epidemic variant with Asiatic origin. Data from the study established relationships of E6 from Poland with previously characterized strains and confirmed the importance of both clinical and environmental surveillance. J. Med. Virol. 89:936-940, 2017. © 2016 Wiley Periodicals, Inc.

[Genetic Characteristics of Echovirus Type 6 Isolated from Hunan Province, China, 2009-2014].

We wished to understand the genetic characteristics of enteric cytopathic human orphan (ECHO) virus type 6 (ECHO6) circulating in China. First, the partial VP1 coding region of six strains of the ECH-O6 virus isolated from cases of hand, foot and mouth diseases during routine surveillance in Hunan Province (China) from 2009 to 2014 were sequenced. Those sequences were analyzed along with 138 sequences of ECHO viruses covering five provinces of China and countries outside China retrieved from the GenBank database. A phylogenetic tree based on partial VPI was constructed, and it indicated that Chinese strains of the ECHO virus could form two distinct evolutionary branches: branch 1 and branch 2. All isolates of the ECHO virus from Hunan Province belonged to the 2c subranch, which revealed that they may share a common evolutionary origin. ECHO strains in branch 2 may be the predominant strains in China due to their wide geographic distribution and long period of circulation. We used nucleotide differences of >30%o as the basis of cluster division. ECHO, viruses could be divided into four clusters (A-D). Cluster D could be divided further into ten subclusters on the basis of nucleotide differences of 15%-30%. All ECHO6 isolates from Hunan Province belonged to the D7 subcluster. These data showed that the ECHO6 strains that circulated in Hunan Province in 2009-2014 were closely related to each other, and probably shared a common evolutionary origin. In addition, at least four distinct lineages of ECHO viruses have circulated in China.

[Analysis of genetic characteristics of ECHO6 virus isolated from an epidemic outbreak of encephalitis in Longyan, China].

This study aimed to analyze the etiology of the encephalitis outbreak in Longyan, Fujian Province, China in 2010, in order to provide valuable information for this prevention and control of this disease. Pathogens were confirmed from cerebrospinal fluid samples with fluorescent RT-PCR, virus isolation (RD cells), and neutralization tests. Then, the VP1 fragments or whole genome nucleotide sequences were determined for four virus strains using PCR. Homology was assessed using the MegAlign software, and a phylogenetic evolutionary tree was drawn using Mega 4.0 software. The results confirmed that the etiology of the outbreak was the ECHO6 intestinal virus, and the nucleotide sequence of the VP1 segment indicated that the C2 subtype was responsible. The genome sequence consisted of 7407 nucleotides, and resembled the genome of other ECHO and CoxB viruses with homology levels of 78.5%-87.3%. The encephalitis outbreak in Longyan in 2010 was caused by the ECHO6 C2 subtype intestinal virus, and its complete genome sequence length is similar to the standard strain (U16283) with a sequence homology of 80.4%.

[Genetic characterization of echovirus 6 isolated from meningitis and encephalitis cases in Shandong Province, China].

To analyze the genetic characteristics of echovirus 6 (E6) isolated from meningitis and encephalitis cases in Shandong Province, China, we collected cerebrospinal fluid samples from meningitis and encephalitis cases in Shandong Province from 2007 to 2012 for virus isolation. Viral RNAs were extracted from positive isolates, and complete VP1 coding regions were amplified by RT-PCR and sequenced. Homology comparison and phylogenetic analysis were performed. Six isolates were identified as E6 by microneutralization assay and molecular typing. The homology analysis showed that the six isolates had 78. 6%-99. 8% nucleotide and 95. 5%-100. 0% amino acid identities with each other, as well as 76. 9%-78. 4% nucleotide and 92. 3%-95. 1% amino acid identities with the prototype strain (D' Amori). The phylogenetic analysis based on the integrated VP1 sequences indicated that all Shandong E6 isolates could be separated into four clusters, designated as A, B, C, and D. The six E6 isolates belonged to clusters A, B, and D. Our study reveals high genetic differences between Shandong E6 isolates and suggests different transmission lineages of E6 co-circulated in Shandong Province.

Molecular evolution and epidemiology of echovirus 6 in Finland.

Echovirus 6 (E-6) (family Picornaviridae, genus Enterovirus) is one of the most commonly detected enteroviruses worldwide. The aim of this study was to determine molecular evolutionary and epidemiologic patterns of E-6. A complete genome of one E-6 strain and the partial VP1 coding regions of 169 strains were sequenced and analyzed along with sequences retrieved from the GenBank. The complete genome sequence analysis suggested complex recombination history for the Finnish E-6 strain. In VP1 region, the phylogenetic analysis suggested three major clusters that were further divided to several subclusters. The evolution of VP1 coding region was dominated by negative selection suggesting that the phylogeny of E-6 VP1 gene is predominantly a result of synonymous substitutions (i.e. neutral genetic drift). The partial VP1 sequence analysis suggested wide geographical distribution for some E-6 lineages. In Finland, multiple different E-6 lineages have circulated at the same time.

Genomic characterization of echovirus 6 causing aseptic meningitis in Hokkaido, Japan: a novel cluster in the nonstructural protein coding region of human enterovirus B.

We determined four complete nucleotide sequences of echovirus 6 (E6) isolated from an epidemic of aseptic meningitis (AM) in Hokkaido, Japan, in 2011. Phylogenetic analysis of the genes encoding viral capsid protein 1 revealed that the strains were closely related to E6 strains isolated in China in recent years, but they were distantly related to E6 strains isolated from patients with AM in Osaka Prefecture, Japan, in 2011. The genes encoding the viral protease and RNA-dependent RNA polymerase (3CD) were closely related to those of several non-E6 strains of the species Human enterovirus B isolated in China, South Korea, and Australia from 1999 to 2010, resulting in a novel cluster in the phylogenetic tree. These results suggest that the incidence of AM in Japan in 2011 was caused by at least two lineages of E6 strains, and a lineage of the 3CD gene was interspersed among different serotypic strains isolated in Western Pacific countries.

Intercity spread of echovirus 6 in Shandong Province, China: application of environmental surveillance in tracing circulating enteroviruses.

Environmental surveillance is an effective approach in investigating circulating enteroviruses and had been conducted in the cities of Jinan and Linyi since February 2008 and April 2010, respectively. This study analyzed 46 sewage samples collected in the two cities in 2011 and found that echovirus 6 (E6) was the predominant serotype, with 134 isolates (65 in Jinan and 69 in Linyi) from 23 (50%) samples. This differs from the 2010 data that found 29 E6 isolates in Jinan and only 3 in Linyi. Phylogenetic analysis of the VP1 coding region showed that all environmental E6 samples from 2008 to 2011 (n = 167) segregated into two lineages and revealed an increase in VP1 gene diversity in 2011, suggesting that the increased number of E6 detections reflects a real epidemic in the two cities. Most Linyi isolates (n = 61, or 88%) in 2011 segregated into sublineage 1a, together with 18 Jinan isolates in 2011. Interestingly, the ancestral VP1 sequence of sublineage 1a inferred using the maximum-likelihood method had 100% identity with the sequence of one environmental isolate from Jinan in August 2010, suggesting an intercity spread from Jinan to Linyi. By Bayesian phylodynamic methods, the most recent common ancestor of Linyi isolates in sublineage 1a dated back to 24 December 2010, revealing that this sublineage was likely imported into Linyi from August to December in 2010. This study demonstrates that environmental surveillance is a sensitive method in tracing transmission pathways of circulating enteroviruses among different regions and reveals that E6-associated aseptic meningitis is an emerging concern in China.

[Complete nucleotide sequence of a human echovirus 6 strain KM57-09 isolated in Yunnan, China, in 2009].

OBJECTIVE: To analyze the genetic characterization of the complete genome from a human echovirus 6 (Echo6) strain KM57-09 isolated in Yunnan, China, in 2009. METHODS: Using the RT-PCR, eight fragments containing about 1000 nucleotides which covered the whole viral genome were sequenced. The sequences were aligned with other reference enterovirus sequences downloaded from the GenBank, using Mega 5.05, RDP 3 and SimPlot 3.5.1 softwares. RESULTS: Similar to the other human enterovirus, KM57-09 isolate genome appeared to have 7419 nucleotides in length, encoding for 2191 amino acids. In the complete genome, the rates of homology on nucleotide and amino acid among the seven Echo6 isolates were 79.3% - 80.2% and 93.3% - 94.4%, respectively as well as 79.3% and 93.6% of the rates of homology when compared with that of D' Amor prototype strain. In different segment of genome. The 2C-3A genome region was most similar to the HN-2-E25 strain, the 5' UTR, VP4, 3D and 3' UTR genome region were most similar to the CoxB5-Henan-2010. In the VP1 gene, the rates of homology on nucleotide and amino acid among the China isolates were 80.0% - 96.0% and 95.8% - 99.0%, respectively, and showed 77.6% - 96.0% and 95.2% - 99.0% of the rates on homology when compared to the other Echo6 reference strains isolated from other countries or areas, respectively. RESULTS: from phylogenetic analysis showed that the Echo6 formed five distinct groups, A-E. The KM57-09 strain belonged to clade E. The nucleotide divergence between clades was 15.6% - 23.3%. The putative recombinant event for KM57-09 was detected with RDP 3, SimPlot 3.5.1 and 3D sequence phylogenetic analysis. CONCLUSION: All the Echo6 isolates could be divided into five clades, the KM57-09 strain belonged to Clade E. The Echo6 strains isolated in China were contributed to several different chains of transmission.

Genome analysis of two type 6 echovirus (E6) strains recovered from sewage specimens in Greece in 2006.

Echovirus 6 (E6) is one of the main enteroviral serotypes that was isolated from cases of aseptic meningitis and encephalitis during the last years in Greece. Two E6 (LR51A5 and LR61G3) were isolated from the sewage treatment plant unit in Larissa, Greece, in May 2006, 1 year before their characterization from aseptic meningitis cases. The two isolates were initially found to be intra-serotypic recombinants in the genomic region VP1, a finding that initiated a full genome sequence analysis. In the present study, nucleotide, amino acid, and phylogenetic analyses for all genomic regions were conducted. For the detection of recombination events, Simplot and bootscan analyses were carried out. The continuous phylogenetic relationship in 2C-3D genomic region of strains LR51A5 and LR61G3 with E30 isolated in France in 2002-2005 indicated that the two strains were recombinants. SimPlot and Bootscan analyses confirmed that LR51A5 and LR61G3 carry an inter-serotypic recombination in the 2C genomic region. The present study provide evidence that recombination events occurred in the regions VP1 (intraserotypic) and non-capsid (interserotypic) during the evolution of LR51A5 and LR61G3, supporting the statement that the genomes of circulating enteroviruses are a mosaic of genomic regions of viral strains of the same or different serotypes. In conclusion, full genome sequence analysis of circulating enteroviral strains is a prerequisite to understand the complexity of enterovirus evolution.

Cocirculation of two transmission lineages of echovirus 6 in jinan, china, as revealed by environmental surveillance and sequence analysis.

Enterovirus environmental surveillance on sewage from the city of Jinan, Shandong Province, China, was initiated in 2008. Thirty echovirus 6 (E6) strains-1 in 2008 and 29 in 2010-were isolated and identified. Most E6 isolates (n = 21) came from the sewage collected on August 2010, revealing high local E6 activity at that time. Interestingly, the VP1 sequences of most isolates, even from the same sewage, were not identical. Phylogenetic analysis of VP1 sequences revealed two lineages for these isolates, with 78.0 to 80.0% nucleotide identities with one another, 94.8 to 100.0% identity within the major lineage, and 92.7 to 98.5% identity within the minor one. The VP1 sequences of environmental isolates, clinical isolates from 1998 to 2010, and global E6 were subjected to evolutionary analysis using Bayesian phylodynamic methods. The inferred E6 VP1 ancestral sequence dated back to 1901 (range, 1873 to 1928) and evolved with 7.047 × 10(-3) substitutions per site per year. Shandong E6 segregated into three clusters, and the two environmental lineages belonged to clusters A and C, which originated in 2003 and 1992, respectively. The antigenicity analysis via neutralization assay confirmed great antigenic differences between Shandong isolates and a prototype strain. These findings underscore the value of continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses in the population and give further insight into E6 evolution.

Complete nucleotide sequence analysis of the VP1 genomic region of Echoviruses 6 isolated from sewage in Greece revealed 98% similarity with Echoviruses 6 that were characterized from an aseptic meningitis outbreak 1 year later.

The molecular characterization of two enterovirus strains (LR51A5 and LR61G3) isolated from the sewage treatment plant unit in Larissa, Greece, in May and June 2006 and the investigation of their relationship with enteroviruses of the same serotype isolated in Greece in 2001 and 2007 were performed by complete VP1 sequence analysis of the isolates. The close phylogenetic relationship and the high nucleotide similarity (98%) led to the conclusion that the virus isolated from sewage in 2006 was associated with that isolated from an aseptic meningitis outbreak 1 year later. Bootscan analysis of the VP1 genomic region revealed that intraserotypic multi-recombination events might have been involved in the evolutionary past history of the LR51A5 and LR61G3 isolates.

Molecular epidemiology of Echovirus 6 in Greece.

The objective was to investigate the genetic relationships among Echovirus 6 (E6) strains circulating in Greece and to compare them with the respective strains from other geographic regions. Cerebrospinal fluid samples collected during the period 2006-2007 from 84 patients with aseptic meningitis or encephalitis were tested for a probable enteroviral infection. Two RT-PCRs amplifying overlapping regions of the VP1 gene were performed, while isolation procedures were applied in one third of cases. All PCR products were sequenced, and further phylogenetic analysis was performed for E6 strains. Enteroviruses were detected in 27 out of 84 cases (32.14%) and E6 was the predominant serotype (11 out of 27, 40.74%). Three distinct clades of Greek E6 sequences were seen in the phylogenetic tree: sequences of the present study were placed in clades A and B, while sequences of a former study in Greece were clustered in clade C. Sequences of clades A and C presented high genetic homology (>95%) with sequences from other countries, while sequences of clade B were unique, differing by more than 15% from all known E6 sequences. The most prevalent enterovirus in Greece during the period 2006-2007 was E6, and was associated with aseptic meningitis. A high degree of heterogeneity was observed among Greek E6 strains.

[Genotypes of echovirus 6 in Monastir region]. / Génotypes d'échovirus 6 circulant dans la région de Monastir.

UNLABELLED: OBJECTIVE OF THE WORK: Echoviruses of serotype 6 were reported to be endemic in Tunisia and even in other country over the world. they are associated with many outbreak meningitis. The Objective of this study was to genetically characterize echovirus 6 fields isolates. It gives a first approach on the molecular epidemiology of this serotype. MATERIAL AND METHODS: Phylogenetic analysis of partial sequence in the 3'half of the VP1 region (2874-3529) from 25 strains of echovirus 6. RESULTS: 9 genotypes of echovirus 6 were individualized. Study area was Monastir, a touristic tunisian city. Strains were isolated from wastewater during one year, may correspond to three genotypes. CONCLUSION: Many genotype could circulating during the same time and in the same region. This phenomena was reported to be atypic in the case of poliovirus.

Molecular phylogeny of VP1, 2A, and 2B genes of echovirus isolates: epidemiological linkage and observations on genetic variation.

Phylogenetic relationships between 37 echovirus clinical isolates, most of them originating from an aseptic meningitis outbreak during 2001 in Greece, were investigated by RT-PCR and sequencing. The generic primers 292 and 222 were used to amplify about 300 bp of the 5' end of VP1 while primers EUG3a, 3b, 3c, and EUC2 amplified the entire coding sequence of the 2A and 2B genes. Phylogenetic trees were constructed for each genomic region using the clinical isolates' sequences and those of the prototype echoviruses in order to investigate the correlation of part of VP1 with the serotype as well as the genetic variation of the echovirus genome in 2A and 2B. The phylogenetic grouping pattern of the clinical isolates revealed that there is a correlation of serotype and genotype in the part of VP1 that was investigated, while this pattern is disrupted in the adjacent genomic regions that were sequenced. Sequence analysis of the adjacent 2A and 2B genes provided a different pattern of phylogenetic relationships and strong evidence of epidemiological linkage of most of the clinical isolates.

Characterization of nonpolio enteroviruses recovered from patients with aseptic meningitis in Korea.

OBJECTIVES: We attempted to characterize nonpolio enteroviruses recovered from Korean patients with aseptic meningitis. METHODS: We performed RT-PCR on the 5'-nontranslated region using clinical specimens. Infectious clinical isolates were amplified by infecting Vero cells with RT-PCR-positive clinical specimens. We then investigated the direct effect in primary neuronal cells or cardiomyocytes following virus infection. RESULTS: Total 12 clinical isolates were subtypically analyzed by both RT-PCR/sequencing comparison of the VP-1 region and neutralization assay. 43-2, 43-2S, 57 and 58 were found to be coxsackievirus B1 (CVB1), 312 to be CVB5, 14-2S and 327 to be echovirus 6, 165 to be echovirus 9, 337 to be echovirus 11, and 270 to be echovirus 30. All the clinical isolates tested showed profound cytotoxicity to various degrees in the primary neuronal cells within 24 h postinfection at 10 MOI. By contrast, a significant cytopathic effect was observed in the primary cardiomyocytes at 3-5 days postinfection at 50 MOI. CONCLUSIONS: The present study suggests that the clinical isolates recovered from Korean patients belonged to different CVB or echovirus serotypes and that these viruses showed diversities in their virulence in primary neuronal cells and cardiomyocytes.

Fatal neonatal echovirus 6 infection: autopsy case report and review of the literature.

A full-term, healthy male neonate was delivered by caesarian section to a 26-year-old primigravida woman who had a history of fever and upper respiratory tract infection. On the fourth day of life, the neonate developed a sepsis-like syndrome, acute respiratory and renal failure, and disseminated intravascular coagulopathy. He died 13 days after birth. Postmortem examination revealed jaundice, anasarca, massive hepatic necrosis, adrenal hemorrhagic necrosis, renal medullary hemorrhage, hemorrhagic noninflammatory pneumonia, and severe encephalomalacia. Echovirus type 6 was isolated from blood, liver, and lungs. Although uncommon, echovirus type 6 infection may produce a spectrum of pathologic findings similar to those seen with the more commonly virulent echovirus type 11.

Factors involved in enzyme-linked immunoassay of viruses and evaluation of the method for identification of enteroviruses.

A quantitative enzyme-linked immunosorbent assay was used for identification of selected enteroviruses: poliovirus type 1, echovirus type 6, coxsackievirus A type 9, and coxsackievirus B types 1 through 6. Partially purified viral antigens or virus-specific antibodies were adsorbed to polystyrene spectrophotometer cuvettes, which permitted the assays to be reported and compared in terms of enzyme units specifically reacting. Both the adsorbed antigen and the adsorbed antibody methods were approximately equal in terms of sensitivity and specificity of reaction. By use [14C]leucine-labeled enteroviruses, the amount of virus that bound to the plastics used was shown to be dependent on the purity of the virus preparation used, but it was higher than the amount that was bound by plastics coated with viral antibody. Diluents which contained 0.15% (vol/vol) Tween 20 and 2.0% (wt/vol) bovine serum albumin in phosphate-buffered saline, pH 7.2, were found to be the most effective in inhibiting nonspecific adsorption of immunoreagents. However, the presence of these inhibitors in phosphate-buffered saline solutions also caused desorption of virus or viral antibody during immunoassays; the amount of virus desorption varied with the type of preparation used, and antibody desorption was dependent on the concentration of antibody initially used for adsorption. For specific identification of a given enterovirus type by the enzyme-linked immunosorbent assay method used, approximately 10(5) plaque-forming units of virus per assay tube were required.