Egg Passage Research Articles

Sperm ejection as a possible cryptic female choice mechanism in Odonata (Insecta)

Abstract. The few odonate studies of sperm use suggest that females spend apparently more sperm than appears necessary during oviposition (sometimes females may have their sperm stores reduced to 50% after a single oviposition episode). Furthermore, some studies document that females eject sperm during and after copulation. This raises the question of whether sperm reduction may be interpreted as a cryptic female choice mechanism. Using two zygopterans, Ischnura denticollis Burmeister and Enallagma praevarum Hagen, and one anisopteran, Pantala flavescens Fabricius, it is shown that females mate more than once, show a marked reduction in stored sperm, and that this is by ejection of sperm before to oviposition. The extent of sperm reduction is inversely related to the number of eggs laid. When mated to the same male, females show similar reductions in sperm stores and egg load and only rarely does the vaginal duct contain sperm. This suggests that marked sperm reduction is common in this insect order and is not explained by an excess of sperm obstructing the egg passage. It is suggested that female's sperm shortage is better explained as a cryptic female choice mechanism aimed at favouring the sperm of some males. This study provides exciting research avenues for future studies of female choice in an animal taxa whose sexual biology is otherwise regarded as controlled by males.

Long-term egg-yolk adaptation of the Orientia tsutsugamushi for preparation of a formalinized immunogen

To assess the feasibility of preparing a killed rickettsial vaccine against the scrub typhus, the Karp and Gilliam strains of the Orientia tsutsugamushi were adapted through sequential passages in eggs for more than 100 times over 9 years to produce approximately five times more antigen than the unadapted rickettsia. The egg-grown rickettsia was purified by differential centrifugation and batch-type ion-exchange chromatography, and inactivated by formalin treatment. Strong protective immunity was acquired against lethal challenges of the homologous strains and lasted fully for longer than 8 months in the C3H/He mice immunized with either of the single-strain immunogen or in combination of the two strain immuinogens. However, neither immunogen protected animals from the challenges with the two heterologous strains or Boryong, the prevalent strain in Korea, despite that three antigenic proteins of 47, 56, and 110 kDa were eminent in both preparations. IgM, IgG, and neutralizing antibody were induced in the immunized mice in a level and pattern comparable to that in animals infected with live rickettsiae.

The influence of homologous vs. heterologous challenge virus strains on the serological test results of rabies virus neutralizing assays

The effect that the relatedness of the viral seed strain used to produce rabies vaccines has to the strain of challenge virus used to measure rabies virus neutralizing antibodies after vaccination was evaluated. Serum samples from 173 subjects vaccinated with either purified Vero cell rabies vaccine (PVRV), produced from the Pittman Moore (PM) seed strain of rabies virus, or purified chick embryo cell rabies vaccine (PCECV), produced from the Flury low egg passage (Flury-LEP) seed strain of rabies virus, were tested in parallel assays by RFFIT using a homologous and a heterologous testing system. In the homologous system, CVS-11 was used as the challenge virus in the assay to evaluate the humoral immune response in subjects vaccinated with PVRV and Flury-LEP was used for subjects vaccinated with PCECV. In the heterologous system, CVS-11 was used as the challenge virus in the assay to evaluate subjects vaccinated with PCECV and Flury-LEP was used for subjects vaccinated with PVRV. Although the difference in G protein homology between the CVS-11 and Flury-LEP rabies virus strains has been reported to be only 5.8%, the use of a homologous testing system resulted in approximately 30% higher titers for nearly two-thirds of the samples from both vaccine groups compared to a heterologous testing system. The evaluation of equivalence of the immune response after vaccination with the two different vaccines was dependent upon the type of testing system, homologous or heterologous, used to evaluate the level of rabies virus neutralizing antibodies. Equivalence between the vaccines was achieved when a homologous testing system was used but not when a heterologous testing system was used. The results of this study indicate that the strain of virus used in the biological assays to measure the level of rabies virus neutralizing antibodies after vaccination could profoundly influence the evaluation of rabies vaccines.

Variation in the spike protein of the 793/B type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs

The degree of variation exhibited within the 793/B serotype (also known as 4/91 and CR88 serotypes) was investigated with nine French and 10 British isolates, collected between 1985 and 1994. The S1 part (1644 nucleotides) of the spike protein gene of the first known isolate of this serotype, FR/CR85131/85, had 95.9% to 97% nucleotide identity with the other isolates. Partial sequencing of isolates from Iran and Saudi Arabia, isolated in 2000, revealed approximately 95% nucleotide identity with European isolates, including the two live 793/B vaccinal strains, showing that they were not re-isolations of vaccinal virus. The data indicates that strains within the 793/B serotype have ≥96% nucleotide identity within the whole S1 gene and ≥93% nucleotide identity within the first 560 nucleotides, and ≥92% and ≥86% amino acid identities in the corresponding protein regions. This is similar to the identities exhibited within the Massachusetts serotype. Sequence analysis of a 793/B field isolate after passage in embryonated eggs, then in chickens and then again in eggs revealed selection for a serine and alanine at S1 amino acid position 95 in chicken-passaged and egg-passaged virus, respectively. There was no change in pathogenicity. This is the first demonstration at gene sequence level of host-driven selection for infectious bronchitis virus.

Identification of Immunoreactive Ghrelin and its mRNA in the Oviduct of Laying Japanese Quail, Coturnix japonica

The aim of this study was to determine whether ghrelin is produced in the oviduct of Japanese quail. Japanese quail laying hens and 20-days-old immature ones were used. The matured birds were examined at two different times of ovulation cycle, namely just after oviposition (preovulation group) and 6h after oviposition (5-h-postovulation group). The oviductal tissues were processed for paraffin sections and immunostained for ghrelin. The expression of ghrelin mRNA in the oviduct of preovulation group was examined by RT-PCR. In the preovulation group, we observed positive immunostaining of ghrelin in the epithelial cells of the mucosal folds in the infundibulum (tubular region) and magnum. The density of immunoreactive products was stronger in the cephalic and middle parts than in the caudal part in the magnum. No immunoreactivity was observed in the tissues of isthmus, uterus and vagina. RT-PCR analysis confirmed that mucosal tissue of the infundibulum and magnum, but not other oviductal segments, expressed mRNA for ghrelin. The intensity of immunostaining for ghrelin in the infundibulum, cephalic and middle parts of magnum was reduced in 5-h-postovulation group compared with preovulation group. In immature birds, no immunoreactivity for ghrelin was observed in both magnum and uterus tissues. These results suggest that the mucosal epithelium in the oviduct of quail hens produces ghrelin, and it may be secreted during the passage of eggs through oviduct. Ghrelin may not be produced in the oviduct of immature birds, and the production may occur in association with a development of oviduct.

Venereal Colibacillosis (Acute Vaginitis) in Turkey Breeder Hens

Two flocks of turkey breeders experienced an increased mortality and high culling rate in the first weeks of egg production. The majority of dead and culled hens had cheesy core in the cloaca and vagina. Postmortem examination revealed fibrinous pseudomembranes in the vagina and cloaca. The thickness of these membranes posed an obstruction to egg passage leading to internal laying and egg peritonitis. Swabs from cloaca and vagina produced numerous colonies of only E. coli. Investigations of this unusual vaginitis showed that these two flocks had a higher number of immature hens with present hymens, and insemination crews mistakenly inseminated all hens in which they were able to evert the cloaca. Breaking the hymen with an insemination pipette created a wound and developed extensive infection with E. coli bacteria.

Phenotypic and genetic analyses of the heterogeneous population present in the cold-adapted master donor strain: A/Leningrad/134/17/57 (H2N2)

For the past three decades the cold-adapted (ca) and temperature sensitive (ts) master donor strain, A/Leningrad/134/17/57 (H2N2) has been successfully used as the basis for the live attenuated reassortant influenza A vaccine. This donor strain was developed from A/Leningrad/134/57 (H2N2) wild-type (wt) virus following 17 passages in eggs at 25°C. Our detailed investigation has revealed that the A/Leningrad/134/17/57 (Len/17) master donor stock is a mixed population comprised of numerous variants of the ca/ts Len/17 influenza virus. We have identified these variants to exhibit a broad range in their temperature sensitive phenotype when assayed on Madin–Darby canine kidney (MDCK) cells at 37°C. A selection of these variant clones has been fully characterized by sequencing in order to understand the variability in the ts phenotype. This study has also addressed the feasibility of using cell culture technology for the propagation and subsequent manufacturing of the cold-adapted influenza vaccine (CAIV), particularly with respect to retaining the defined mutations that contribute toward the ca/ts phenotype.

On the origins and relationships of Newcastle disease virus vaccine strains Hertfordshire andMukteswar, and virulent strain Herts'33

The origins and relationships of Newcastle disease virus (NDV) vaccine strains Hertfordshire OI) andMukteswar, and the virulent Herts'33 were studied using partial sequence analysis of the fusion protein gene.The mesogenic strain H was obtained by egg passages of a field virus isolated in England in 1933 (later knownas Herts'33). Different lines of the strain Herts'33, however, divided into two distinct groups: genotype IV, and ahitherto undescribed lineage, which comprised the Weybridge line (Herts'33/56). Vaccine strain H and the twoclusters comprising viruses designated Herts'33 displayed 6.5 to 6.8% and 15.6 to 16.3% mutational distances,respectively, which precluded parent-offspring relationships with either of them. In contrast, the different linesof the vaccine strain Mukteswar, which was reportedly derived from an Indian field isolate in the mid-1940s,showed 98.9 to 100% sequence similarity to strain H. It is therefore probable that the two vaccines were derivedfrom the same virus stock.

Characterization of a recombinant Newcastle disease virus expressing the green fluorescent protein

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the fusion (F)- and hemagglutinin-neuraminidase genes in a full-length cDNA clone of NDV. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in embryonated eggs and the allantoic fluid was used to infect cells. Northern blot analysis of RNA isolated from infected cells demonstrated the proper transcription of the introduced GFP-mRNA. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDVGFP1) expressing the reporter gene. The expression of the heterologous gene was maintained stably for at least five passages in embryonated eggs. The replication kinetics in embryonated eggs and pathogenicity in chickens of rNDVGFP1 did not differ significantly from that of the parent virus. Using GFP autofluorescence, virus infected cells could be tracked easily in native preparations, organ explants and primary tracheal cell cultures. Taken together, these data demonstrate the use of GFP-expressing recombinant NDV for analysis of NDV dissemination and pathogenesis and indicate the potential usefulness of NDV as a vaccine vector.

Defective influenza A virus generated entirely from plasmids: its RNA is expressed in infected mouse lung and modulates disease

Naturally produced defective influenza virus has antiviral activity and, in sufficient amount, can protect mice from lethal influenza, irrespective of the virus subtype causing the disease. However, such defective virus preparations contain many undefined defective RNA sequences, and it is thus not possible to establish dose-response relationships. To address this situation, we have transfected DNA encoding a cloned defective RNA into Vero cells along with the 17 A/WSN (H1N1) plasmids required for infectious helper virus, and produced molecularly cloned defective virus. Here we used POLI-220 that expresses a 445 nt defective RNA isolated from a mouse-protective defective equine H3N8 virus, and POLI-317 that expresses a 585 nt defective RNA from an avian H7N7 virus. Both originate from genomic segment 1. Virus preparations were UV-irradiated selectively to destroy virus infectivity but not the activity of the defective RNAs, and adult mice were inoculated intranasally with defective virus and WSN (H1N1) challenge virus (10 LD 50). Defective POLI-220 and POLI-317 RNAs were detected readily in infected lung tissue by RT-PCR, but these Vero cell preparations did not modulate disease. However, after a single passage in embryonated eggs, defective POLI-220 and POLI-317 viruses significantly delayed the onset of disease and death in WSN-infected mice, although did not affect final mortality. Direct PCR sequencing confirmed the identity of mouse-passaged defective RNAs and showed that none had undergone any sequence changes. With this advance it will now be possible to study the interference phenomenon in vivo with defective viruses carrying a defined defective RNA.

Changes in the Type I Collagen mRNA Expression in the Isthmus During the Passage of Egg Through the Oviduct in Japanese Quail

Type I collagen is one of the major components contained in the eggshell membrane. The aim of this study was to determine whether the expression of type I collagen mRNA is changed during the passage of the egg through the oviduct in Japanese quail. Immunofluorescent cytochemistry to localize type I collagen in the oviduct and semi-quantitative RT-PCR to examine the changes in its mRNA expression were carried out. Immunoreactive type I collagens were localized in the tubular gland cells of the isthmus as well as in the interglandular connective tissue of all the oviductal regions. The expression of type I collagen (α2) mRNA was significantly greater when the egg was in the isthmus, compared when the eggs were in the other regions. We suggest that the synthesis of type I collagen by the glandular cells of the isthmus is enhanced in association with the egg passage through that region.

Involvement of the leader sequence in Sendai virus pathogenesis revealed by recovery of a pathogenic field isolate from cDNA.

We previously demonstrated that a systematic passage of a pathogenic field isolate of Sendai virus (SeV), the Hamamatsu strain, in embryonated eggs caused attenuation of virulence to mice, and we isolated viral clones of distinct virulence (K. Kiyotani et al. Arch. Virol. 146:893-908, 2001). One of the clones, E15cl2, which was obtained from the virus at the 15th egg passage of E0, the parental Hamamatsu clone for egg passage, had 165-fold-attenuated virulence to mice and possessed only four mutations in the entire 15,384-base genome: in an antigenomic sense, U to A at position 20 (U20A) and U to A at position 24 (U24A) in the leader sequence, the promoter for transcription and replication, and A to G at position 9346 (silent) and A to U at position 12174 (Ser to Cys) in the L gene. To examine the possibility that leader mutations affect virus pathogenesis, we recovered live viruses from cDNA derived from the Hamamatsu strain. A mutant virus possessing either a mutation of U20A or U24A in the leader sequence showed a slightly lower pathogenicity than that of the parental virus, whereas a double mutant virus possessing both of the mutations showed 25-fold-attenuated virulence, accompanying a significantly lower virus replication in the mouse lung. Replications of the leader mutant viruses were also impaired in a primary culture of mouse pulmonary epithelial cells but not in chicken embryo fibroblasts. These findings suggest that leader mutations of SeV affect virus pathogenesis by altering virus replication in a host-dependent manner.

Molecular and enzymatic characterisation of Schistosoma mansoni thioredoxin

Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilising systems. Here, we report the identification and characterisation of a thioredoxin from Schistosoma mansoni. The predicted protein has similarity to previously characterised thioredoxins including conservation of the redox active site. Recombinant six-histidine tagged schistosome thioredoxin had insulin reduction activity and supported the enzymatic function of thioredoxin reductase and thioredoxin peroxidase. By Western blotting, all mammalian stages of the schistosome lifecycle expresses thioredoxin. Thioredoxin is present in egg secretory products and antibodies against the recombinant protein produce the circumoval precipitin reaction. This is the first identification of defined antigen producing this reaction. Furthermore, thioredoxin is a novel egg immunogen as it elicits an antibody response in schistosome-infected mice. The most significant IgG production against thioredoxin occurs after parasite oviposition commences. These observations suggest that thioredoxin participates in processes vital to the parasite and may facilitate the passage and survival of eggs across inflamed host tissues.

Ambisense sendai viruses are inherently unstable but are useful to study viral RNA synthesis.

Ambisense Sendai virus (SeV) was prepared in order to study the control of viral RNA synthesis. In these studies, we found that the relative ratios of genomes/antigenomes formed during infection are largely determined by the relative strengths of the replication promoters, independent of the presence of a functional mRNA start site. We also found that the ability of the viral polymerase (vRdRP) to respond to an mRNA editing site requires prior (re)initiation at an mRNA start site, similar to the acquisition of vRdRP processivity in the absence of nascent chain coassembly. During these studies, the inherent instability of ambisense SeV upon passage in embryonated chicken eggs was noted and was found to be associated with a point mutation in the ambisense mRNA (ambi-mRNA) start site that severely limited its expression. Since the interferon (IFN)-induced antiviral state is mediated in part via double-stranded RNA (dsRNA), the efficiency of the ambi-mRNA poly(A)/stop site was examined. This site was found to operate in a manner similar to that of other SeV mRNA poly(A)/stop sites, i.e., at approximately 95% efficiency. This modest level of vRdRP read-through is apparently tolerable for natural SeV because the potential to form dsRNA during infection remains limited. However, when mRNAs are expressed from ambisense SeV antigenomes, vRdRP read-through of the ambi-mRNA poly(A)/stop site creates a capped transcript that can potentially extend the entire length of the antigenome, since there are no further poly(A)/stop sites here. In support of this hypothesis, loss of ambi-mRNA expression during passage of ambisense SeV stocks in eggs is also characterized by conversion of virus that grows poorly in IFN-sensitive cultures and is relatively IFN sensitive to virus that grows well even in IFN-pretreated cells that restrict vesicular stomatitis virus replication, i.e., the wild-type SeV phenotype. The selection of mutants unable to express ambi-mRNA on passage in chicken eggs is presumably due to increased levels of dsRNA during infection. How natural ambisense viruses may deal with this dilemma is discussed.

Identification of mutations associated with attenuation of virulence of a field Sendai virus isolate by egg passage.

Abstract. We have reported that attenuation of the virulence of a field Sendai virus (SeV) isolated by egg passage is associated with an impediment of viral genome replication in mouse respiratory cells (Kiyotani et al., Arch Virol 146, 893-908, 2001). To determine the molecular basis for the attenuation, we sequenced entire genomes of representative SeV clones isolated during egg passages and compared those with that of the parental SeV clone E0. E15c2, a 165-fold attenuated clone in 50% mouse lethal dose (MLD50) isolated at the 15th egg passage, possessed only four mutations in the entire genome: U to A at position 20 (U20A) and U24A in the leader promoter region and A9362G and A12174U in the L gene from the 5'-end of antigenome. The former mutation in the L gene was silent and the latter changed deduced amino acid Ser at position 1207 to Cys (Serl207Cys) in the L protein, a catalytic subunit of viral polymerase. E30c12, a further 6-fold attenuated clone isolated at the 30th egg passage, had an additional four mutations: A8074G (Glu461Gly) and A8077G (Asp462Gly) in the hemagglutinin-neuraminidase (HN) gene and A13598C (silent) and G13927A (Ser1791Asn) in the L gene. On the other hand, a virulent revertant clone, E30M15c15, which was obtained by 15 mouse passages of E30c12 and had 250-fold mouse virulence compared to E30c12, possessed eight mutaions: A24U in the leader, C1325U (silent) in the nucleocapsid gene, G8074A (Gly461Glu) in the HN gene, G10433U (Lys626Asn), C13598A (silent), A13927G (Asn1791Ser), C14626U (Thr2024Ile) and A15272C in the L gene. Among these, the mutations in the leader and the HN gene and two of the mutations in the L gene (C13598A and A13927G) were true reversions to E0. The significance of the mutations detected in the leader as well as in the L and HN genes was discussed in the context of attenuation of SeV pathogenicity by egg passage.

Analyses of evolutionary and virulence divergency of Hong Kong H5N1 influenza A viruses isolated from humans

Nucleotide and amino acid sequence homologies of the entire RNA segments of six H5N1 human influenza viruses were divided into two or three variant groups. This result was compatible with that of phylogenetic analysis, which showed that trees of the eight genes were composed of two or three minor branch clusters. The PB2, PA, NP and M proteins of human H5N1 viruses were further characterized by the presence of previously reported amino acid sequences peculiar to those of human strains. The virulence of A/Hong Kong/483/97 (HK483) in mice exhibited a sharp contrast to that of A/Hong Kong/156/97 (HK156). Virulence of the former even remained after constant repeated passage in eggs, while the latter rapidly decreased in virulence for mice after only a few passages in eggs. It became apparent that Madin–Darby canine kidney (MDCK) cell-grown virus (HK156-CK) and its clones derived from mouse brain produced a fatal infection in mice through intranasal (i.n.) or intracerebral (i.c.) routes. Conversely, the pathogenicity shown by the egg-derived parental virus (HK156-E3) and its clones were considerably lower in mice after i.n. or i.c. infection. A series of experimental infections revealed that the marked differences in neurovirulence among viruses could be attributed to whether or not viruses were transmitted from the lung to the brain. In amino acid sequence comparison of the entire proteins, it was suggested that a total of six amino acid differences in the PB1(residues 456 and 712), PA (residue 631), HA (residue 211), NP (residue 127) and NS1 (residue101) proteins related closely to changes in pathogenicity or neurovirulence of the HK156 virus in mice. As a result, it was evident that less pathogenic and neurovirulent strains appeared through adaptation and selection of variants during passage in eggs.

Genes coding for polymerase proteins are essential for attenuation of the cold-adapted A/Leningrad/134/17/57 (H2N2) influenza virus

Background: The A/Leningrad/134/17/57 (H2N2) (Len/17) cold-adapted (ca) master strain used as a donor of attenuation for preparing live attenuated influenza vaccine was derived from the A/Leningrad/134/57 wild-type virus (Len/wt) after 17 passages in eggs at 25 °C. In contrast to Len/wt, the Len/17 virus is sensitive to high (40 °C) temperature ( ts), is able to grow at low (25 °C) temperature ( ca), and is attenuated ( att) for humans. Len/17 is different from Len/wt by eight coding mutations in internal genes PB2 (V-478-L), PB1 (K-265-N, V-591-I), PA (L-28-P, V-341-L), M1 (I-15-V), M2 (A-86-T) and NS2 (M-100-I). However, the role of individual mutant genes in the manifestation of the ts, ca, and att phenotypes was not established. To evaluate this, single gene reassortants (SGRs) with one mutated gene in the background of the Len/wt genome were studied. Methods: Since heterogeneity (A- or T-86 in the M2 gene) was noted in Len/17, the ts and ca clone 28 of this donor (Len/17-cl28) that has A-86 in M2 was used to prepare the SGRs. Preservation of mutations in SGRs was monitored by PCR-restriction (RFLP) analysis. Virus replication in ferret lungs was observed to evaluate the att phenotype of SGRs. Results: PB2- and PB1-SGRs each manifested the ts phenotype, and PB2-SGR demonstrated the ca phenotype as well. PA-SGR was ca, but not ts. Mutations in the M1 and NS2 genes did not affect the ts or ca phenotypes of corresponding SGRs. Ferrets infected with Len/17-cl28, Len/wt and PB2-, PB1- or PA-SGRs demonstrated comparable titers in nasal turbinates on the third day, while infectious virus was found only in the lungs of ferrets infected with Len/wt and PA-SGR. Conclusion: These data suggest that mutations in the PB2 (V-478-L) and PB1 (K-265-N, V-591-I) genes play a critical role in the attenuation of the ca Len/17 vaccine donor strain.

Live attenuated reassortant influenza vaccine prepared using A/Leningrad/134/17/57 (H2N2) donor strain is genetically stable after replication in children 3-6 years of age

Background: Live influenza A vaccines (LIVs) for adults and children are prepared in Russia by reassorting current epidemic strains with cold-adapted (ca) attenuated donor viruses A/Leningrad/134/17/57 (H2N2) (Len/17) and A/Leningrad/134/47/57 (H2N2) (Len/47), respectively. Len/17 and Len/47 were derived from the A/Leningrad/134/57 (H2N2) wild-type virus after 17 and 47 passages in eggs at 25 °C. Two doses of Len/47-based vaccine have been used for vaccinating children. In a recent study, children were vaccinated with the Len/17-based LIV in order to determine if this vaccine is safe and immunogenic after a single dose vaccination. As part of this study, type A isolates obtained from children 3–6 years of age on the third day after vaccination were analyzed for genetic stability. Methods: PCR-restriction (RFLP) analysis was used to detect mutations in genomes of isolates. Results: Internal genes of Len/17 contain eight coding mutations in the PB2 (1), PB1 (2), PA (2), M1 (1), M2 (1) and NS (1) genes. All of these mutations, except one, were conserved in the genomes of all 28 strains studied. Similar to other studies with Len/17, the A-86-T change in the M2 protein, which was shown to be heterogeneous in the parent donor strain, was absent in 13 isolates. Since it was shown that mutations in polymerase genes play a leading role in the attenuation of Len/17, it is important to emphasize their conservation in all of the isolates. Moreover, an additional mutation in the PB2 protein (S-478-R), previously known only for the more attenuated Len/47 ca donor strain, was detected in 9 of 28 isolates, indicating that this mutation may also be heterogeneous in the donor virus. Conclusion: The Len/17-based LIV is genetically stable in children.

Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes.

To identify the molecular determinants contributing to the inability of recent human influenza A(H3N2) viruses to agglutinate chicken erythrocytes, phenotypic revertants were selected upon passage in eggs or MDCK cells. The Leu194Ile or Val226Ile substitutions were detected in their hemagglutinin (HA) sequence concomitantly with the phenotypic reversion. Remarkably, as little as 3.5% of variants bearing a Val226Ile substitution was found to confer the ability to agglutinate chicken erythrocytes to the virus population. Hemadsorption assays following transient expression of mutated HA proteins showed that the successive Gln226 --> Leu --> Ile --> Val changes observed on natural isolates resulted in a progressive loss of the ability of the HA to bind chicken erythrocytes. The Val226Ile change maintained the preference of the HA for SAalpha2,6Gal over SAalpha2,3Gal and enhanced binding of the HA to alpha2,6Gal receptors present on chicken erythrocytes. In contrast, simultaneous Ser193Arg and Leu194Ile substitutions that were found to confer the ability to agglutinate sheep erythrocytes increased the affinity of the HA for SAalpha2,3Gal.

The female genital system of Ooperipatellus decoratus (Onychophora, Peripatopsidae): An ultrastructural study

The female genital system of the oviparous peripatopsid Ooperipatellus decoratus consists of an ovary, oviducts equipped with receptacula seminis and additional pouches, uteri, and a vagina. It is examined using transmission and scanning electron microscopy. The ovary is made up of paired ovarian tubes united anteriorly and posteriorly and differentiated into a sterile dorsal part and a fertile ventral part with exogenous oocytes. Fertilization presumably occurs in the oviducts once the oocytes pass the receptaculum seminis. Although the receptacula seminis have been reported to occur in juvenile O. decoratus females only, the present study reveals that they are present in adult females as well. Their wall consists of a cuboidal epithelium covered with a thin collagen-muscle layer. The additional pouches are projections of the oviducts facing the receptacula seminis. They are distally closed to the haemocoel by a flattened epithelium and lack external muscle cells. A thin collagen layer is only found proximally. The uteri are characterized by a columnar epithelium with folded cell membranes allowing extension of the uteri, thus facilitating the passage of the large uterine eggs towards the vagina. Another dominating feature of the uteri is a distally increasing secretory production, which probably contributes to chorion development. Cilia occurring along the entire length of the uteri are considered to assist in the transport of eggs towards the vagina.